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					Adv Biochem Engin/Biotechnol (2005) 97: 1–28
DOI 10.1007/b135821
© Springer-Verlag Berlin Heidelberg 2005
Published online: 8 August 2005

Aquaculture of “Non-Food Organisms”
for Natural Substance Production
Gerd Liebezeit
Research Centre Terramare, Schleusenstrasse 1, 26382 Wilhelmshaven, Germany
gerd.liebezeit@terramare.de

1       Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .                                                                        2

2       Culture Aspects . . . . . . . . . .                          .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .    5
2.1     Medium . . . . . . . . . . . . . .                           .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .    5
2.2     Food . . . . . . . . . . . . . . . .                         .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .    6
2.3     Currents . . . . . . . . . . . . . .                         .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .    7
2.4     Larval Production and Settlement                             .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .    7
2.5     Example Flustra foliacea . . . . .                           .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .    7

3       Organisms . . .      .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .    9
3.1     Porifera . . . . .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .    9
3.2     Bryozoa . . . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   11
3.3     Molluscs . . . .     .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   11
3.3.1   Ophistobranchs       .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   12
3.4     Others . . . . .     .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   12

4       Applications Other than Pharmaceutical                                       .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   13
4.1     Marine Cements . . . . . . . . . . . . . .                                   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   13
4.2     Biominerals . . . . . . . . . . . . . . . .                                  .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   13
4.3     Antifouling Compounds . . . . . . . . .                                      .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   14
4.4     Other Applications . . . . . . . . . . . .                                   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   14

5       Interactions with Microautotrophs and -heterotrophs . . . . . . . . . . . .                                                                              16

6       Further Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . .                                                                         17

7       Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .                                                                        18

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .                                                                         18

Abstract Marine invertebrates are already sources of commercially important secondary
metabolites and may become even more so as knowledge on marine natural products
and chemical ecology develops. Among the producers of these compounds predominantly
sponges, bryozoa and molluscs have received the attention of academic and industrial
research and development. For all these invertebrate groups culture techniques have
been developed encompassing in situ, laboratory and cell culture approaches for the
production of natural products. Potential applications of these are not restricted to phar-
maceuticals but include marine cements, biominerals and antifouling compounds. In
addition, markets exist for ornamental species. All culture approaches require sound eco-
logical knowledge about the organisms to be cultured and possible symbiotic interactions
between host invertebrates and microheterotrophs.
2                                                                           G. Liebezeit

Keywords Aquaculture · Porifera · Bryozoa · Molluscs · Natural product



1
Introduction

Invertebrates are defined as any animal lacking a backbone. The invertebrates
include the tunicates and lancelets of the phylum Chordata, as well as all
animal phyla other than Chordata including members of the phyla Porifera
(sponges), Cnidaria (coelenterates), Ctenophora, Platyhelmintes (flatworms),
Nematoda (roundworms), annelida (segmented worms), arthropoda, mol-
lusca, echinodermata, endo- and ectoprocta and protochordates. Approxi-
mately 95% of all the earth’s animal species are invertebrates; of these the vast
majority are insects and other arthropods. Invertebrates are important as par-
asites and are key players in all ecological communities, e.g. [1–6]. According
to Brusca and Brusca [7] more than 151 000 species of invertebrates have been
reported in the aquatic environment.
   Services and natural products from marine organisms have elicited con-
siderable interest (Fig. 1), e.g. in cancer research and treatment [8–13]. These
and other aspects of marine natural products have been reviewed by a.o.
Baslow [14], Scheuer [15–19], Bohlin [20], Faulkner [21–51], Guyot [52],
Cart [53], Olson [54], Abad and Bermejo [55], Blunden [56, 57], Proksch
et al. [58–60] and Jha and Zi-rong [61]. Invertebrates provide the vast major-
ity of active marine metabolites [25]. To further illustrate this point from 1969
to 1995 approximately 200 new patents were issued worldwide for marine-
derived biochemicals with potential therapeutic activities. Between 1996 and
1999 the rate of discovery and patenting increased considerably with close




Fig. 1 Diagram illustrating the services rendered by various classes of marine organisms
to man
Aquaculture of “Non-Food Organisms”for Natural Substance Production            3

to 100 new compounds patented in just these 3 years [62]. The rate of new
discoveries will certainly be increasing in the future as marine biomedical
research matures and more and more researchers and companies turn their
attention to the seas [63–65].
   Nevertheless, as mankind negatively influences the oceanic ecosystems
through for example pollution, species introduction, overfishing and destruc-
tive fishing methods, concerns have been expressed that opportunities to
learn more about marine organisms and their commercial potential may be-
come limited in the future [63, 66]. Thus, increased research and development
efforts are necessary.
   Reviews of the natural product chemistry of bryozoa have been given by
Cristophersen [67] and Blackman and Walls [68] while information on nat-
ural products from Porifera can be found in Faulkner [23, 38, 41, 47], Sarma
et al. [69], Proksch [59, 70], Engel and Pawlik [71], Guyot [72] as well as
Kobayashi and Ishibashi [73]. While the first publications on chemical aspects
of sponges date from 1882 onwards describing pigments, steroids, guani-
dines, amines, and related compounds [74–81] an important break-through
in the discovery of medicinal properties of natural products of sponge ori-
gin was made with the isolation of sponge arabinose nucleosides such as
spongouridine from Tethya crypta [82, 83]. A synthetic modification of this
compound is now clinically used against Hodgkins lymphoma, acute myelo-
cytic leukaemia, and the herpes virus [84] providing an early example of the
applicability and commercial success of marine-derived pharmaceuticals.
   Molluscs and here especially ophistobranchs have also provoked consider-
able interest. More than 400 compounds which might be of pharmacological
value have been described in the literature [85, 86]. These include hypoten-
sive agents, cardioactive substances, muscle relaxants, antibiotics, antiviral
and antitumour agents. Toxins of marine snails are also of interest, espe-
cially conotoxins in signal transmission research and due to their analgetic
properties [87, 88]. Tuncates and other invertebrate phyla have received less
attention [89–91].
   Despite these considerable academic efforts, only a few metabolites found
in marine invertebrates have so far entered into any commercial activities
(Table 1).
   In addition, a few more metabolites are presently under clinical investiga-
tions (Table 2, see also the compilation by Haefner [92]).
   Due to the wide-ranging potential applications of marine bioactive com-
pounds (Tables 1,2) the need for a reliable and continuous supply of these and
other compounds from marine invertebrates arises. Providing an adequate
supply of raw material for marine pharmaceutical compounds has so far
been solved by in situ collection of large quantities of invertebrates [113, 114]
although limitations in available biomass [77–81] and its usually patchy dis-
tribution may impede the permanent success of this approach. Furthermore,
bioactive compounds are normally present in minor quantities, e.g. produc-
4                                                                              G. Liebezeit

Table 1 Examples of commercially available compounds from marine invertebrates

    Product                        Source                           Application

    Ara-A                          Cryptotethya crypta, sponge      antiviral
    Ara-C                          Cryptotethya crypta, sponge      anticancer
    Manoalide                      Luffariella variabilis, sponge   phospholipase-A
                                                                    inhibition
    Aequorin                       Aequora victoria, jellyfish       bioluminescent
                                                                    calcium indicator
    Green Fluorescent Protein      Aequora victoria, jellyfish       reporter gene
    Resilience® (Estée Lauder)     Pseudopterogorgia                additive
                                   elisabethae,                     to skin creams
                                   gorgonian

Table 2 Selected examples of compounds from marine invertebrates presently in clinical
trials (see also [10, 12, 58, 93])

    Compound                          Source                                  References

    ecteinascidin 743                 Ecteinascidia turbinata, tunicate       [94–96]
    bryostatin-1                      Bugula neritina, bryozoan               [97–100]
    aplidine (dehydrodidemnin B)      Aplidium albicans, tunicate             [101, 102]
    kahalalide F                      Elysia rufescens, mollusc               [103, 104]∗
    didemnin B                        Trididemnum solidum, tunicate           [105–107]
    discodermolide                    Discodermia dissoluta, sponge           [108–112]
∗   This appears to be one example of dietary uptake of bioactive metabolites, see 5

tion of 1 g of ecteinascidin-743 or E-743, an antitumour agent from a tunicate,
would require collection of about 1 ton of organism [115, 116]. Given the fact
that annual demands for secondary metabolites from marine sources which
have passed all clinical trials and are ready to enter the market will fall in the
range of 1 to 5 kg [59] an amount impossible to obtain from natural sources,
alternative techniques for obtaining these amounts are required.
   Thus, aquaculture, either under controlled or natural conditions, and
chemical synthesis [117, 118] may develop into (commercially) attractive al-
ternatives [113]. Furthermore, application of the latter techniques would
also protect natural resources and biodiversity. Chemosynthesis is, however,
both a challenging and difficult task as the complex molecular structure of
marine metabolites usually gives rise to complex synthesis pathways and
low yields. Marine peptides such as the conotoxins are a noticeable ex-
ception and can be produced in virtually unlimited amounts [119]. Thus
“marine organisms should probably be used as inspiration [for] and not as
the source of the chemicals, [...]” (Faulkner in [120]). Hence, present ap-
proaches attempt to characterise the pharmaceutically active parts of the
Aquaculture of “Non-Food Organisms”for Natural Substance Production           5

molecules of interest and to synthesise these [118]. In fact, for the commer-
cial production of the above mentioned E-743 such an approach was taken.
Part of the molecule is produced by fermentation using Pseudomonas flu-
orescens, another part is synthesised chemically [121]. For discodermolide,
a microtubule-stabilising compound from the sponge Discoderma disso-
lute [108, 112], routes for total synthesis with reasonable yields in the gram-
range have been developed [122–132].
   Toonen [133] reviews historical aspects of marine invertebrate culture dat-
ing back to the beginning of the 20th century when first successful attempts
were made to culture corals and polychaetes. A detailed treatment of in-
vertebrate culture, although mostly restricted to laboratory scale, has been
provided by the Committee on Marine Invertebrates [134]. Here, a total of
86 species have been considered. Also, information may be found in the
proceedings volume edited by Smith and Chanley [135] and in treatises on
maintaining invertebrates in ornamental aquaria [136, 137]. Recent advances
in invertebrate culture have been reviewed by Vasta and Marsh [138] while
rearing of invertebrate larvae has been treated by McEdward [139]. Details on
methods for obtaining and handling marine, especially invertebrate, eggs and
embryos can be found in Costello and Henley [140].


2
Culture Aspects

In culturing marine invertebrates for pharmaceutical purposes basically three
different approaches can be followed: in situ, laboratory, i.e. bioreactor, and
cell cultures. For laboratory culture a number of basic prerequisites exists
which need to be taken into consideration:

1.   composition of the medium for laboratory and cell cultures;
2.   source and composition of the food for whole animals;
3.   feeding inducing currents;
4.   larval production and settlement.

2.1
Medium

Natural seawater is a complex mixture and contains besides the main in-
organic ions a vast array of dissolved and particulate organic compounds,
bacteria, fungi, invertebrate and vertebrate larvae, phytoplankton and zoo-
plankton. Furthermore, in coastal waters a high load of inorganic particles is
present. While the larvae of sessile organisms may compete with the organism
of interest for space and food inorganic particles increase the metabolic effort
of the cultured organisms as they have to select and reject non-food particles.
6                                                                     G. Liebezeit

   Thus, removal of these interfering substances and organisms becomes ne-
cessary. Although techniques such as continuous centrifugation, UV or ozone
oxidation and sterilisation are available these are, in terms of time and energy
consumption, highly inefficient for larger scale cultures.
   In addition, success of cultivation using the in situ techniques method
strongly depends on the unpredictable and often suboptimal supply of food
in the natural environment. Especially in temperate climates phytoplankton
abundance is seasonally variable and leads to pronounced growth rate vari-
ability of invertebrates over the course of the year. Furthermore, interannual
variability also adds to the uncertainties in culture success when natural en-
vironments are used.
   Artificial culture media are thus a suitable alternative and have been de-
scribed for invertebrates [141] while numerous recipes for artificial seawater
can be found in the literature [142–145].

2.2
Food

Although transepidermal uptake of dissolved organic compounds has been
described for some marine invertebrate species the vast majority depend on
particulate organic matter, in most cases phytoplankton, as a source of nutri-
tion [146]. This requires the establishment of food preferences for the species
to be cultured, e.g. Jebram [147–155] reports extensively on this aspect for
various bryozoan species. O’Dea and Okamura [156] note a marked influence
of temperature and ambient chlorophyll a concentrations on zooid size and
colony growth rate, respectively, of the bryozoan species Conopeum seurati.
   Numerous recipes for phytoplankton media have been given in the litera-
ture the most generally applied being those provided by Guillard [157–159].
   Photobioreactor design has also been extensively worked on and various
types of reactors have been described [160–164]. Despite this, so far no large
scale commercial systems are available for other purposes than cultivation
of microalgae such as Spirulina spp. or Chlorella vulgaris for nutritional or
aquaculture purposes [165–169]. In this context it should be pointed out that
optimum growth conditions in terms of light, temperature or nutrient re-
quirements need also be established for those phytoplankton species that are
intended as food for cultured invertebrates.
   Phytoplankton, due to its usually small size, is particularly suitable for the
rearing of filter-feeding species or invertebrate larvae only. For adult animals
larger size food is required. These may include rotifers and other zooplankton
species such as brine shrimps. However, these again require phytoplankton
for their own growth. Brachionus plicatilis, Artemia salina or Daphnia magna
have been tested successfully with a variety of invertebrates [134, 170, 171]. As
these species are also used as food in for example shrimp aquaculture systems
they are usually easily available in large quantities from commercial sources.
Aquaculture of “Non-Food Organisms”for Natural Substance Production          7

Again with only the fish aquaculture industry in mind copepods are receiv-
ing increasing attention [172] although these zooplankters may be employed
in other applications as well.

2.3
Currents

For a number of invertebrate species the influence of currents on feeding in-
duction and behaviour has been described [173–177]. Currents are not only
necessary to transport food particles but also to activate feeding responses
such as shell opening in bivalves or extension of the lophophore in bryozoa.
Generally, at high current velocities feeding activity is reduced or even com-
pletely stopped although adaptations have been described [178].

2.4
Larval Production and Settlement

Larvae of most sessile marine invertebrates can be easily obtained by field
sampling of gravid specimen [116, 179] while mollusc larvae may be derived
from laboratory-reared individuals. Larvae of some species may be concen-
trated by their attraction to a light source or by careful sieving.
   For sessile invertebrate species knowledge on factors inducing and influ-
encing the settlement of planktonic larvae is a prerequisite for successful
culture. Larval settlement is controlled by both exogenous and endogenous
factors [180–187]. These include but may not alone be restricted to age of
competent larvae, cues from adult conspecifics, surface texture, the presence
of (bacterial) biofilms, proximity to a food source etc.

2.5
Example Flustra foliacea

As an example for the elaborate interplay of the parameters described above
culture experiments with Flustra foliacea, a cheilostome bryozoan species,
will be described briefly in the following. These were carried out in our
laboratory from 1998 to 2003. Fig. 2 summarises the basic requirements for
a successful culture.
   F. foliacea is an erect growing species which in its first year exhibits en-
crusting growth. In the natural environment, larvae occur regularly in Febru-
ary and can be sampled by divers. As they are rather delicate collection by net
sampling is usually not particularly successful.
   The larvae will settle on almost any surface provided it has been condi-
tioned in seawater, i.e. a biofilm must be present. After metamorphosis food
for the growing colony consists of a mixture of Dunaliella tertiolecta, Phaeo-
dactylum tricornutum and Cryptomonas spec. This mixture was chosen after
8                                                                               G. Liebezeit




Fig. 2 Flustra foliacea—from larva to initial colony. Bar sizes: left: 100 µm, right: 500 µm.
Ancestrula—settled larva and starting point of the growing colony


a large variety of different microalgal species had been tested for their suit-
ability as food. Monospecific food proved to be less acceptable. Attempts were
also made to “predigest” the phytoplankton partly by feeding it to Oxyrrhis
marina, a heterotrophic flagellate, and then using a mixture of O. marina and
the three phytoplankton species to supply F. foliacea colonies. However, in the
long term no significant differences in growth rates were observed and hence
only pure phytoplankton food was regularly used.
   Temperature proved to be a critical parameter and had to be kept below
16 ◦ C. This is about the maximum temperature the organism will experience
in its natural environment. At temperatures between 16 and 18 ◦ C feeding ac-
tivities and growth stopped while at temperatures above 18 ◦ C the colonies
usually died. Salinity, on the other hand, exerted an influence not as pro-
nounced as temperature. The bryozoa tolerated salinities as low as 28 PSU.
   As in the North Sea F. foliacea is found in water depths around 14 to 16 m
and it tolerates dim light only, i.e. exposure to direct sunlight is to be avoided.
During growth experiments in outdoor tanks an almost 100% mortality of the
colonies was noted when the tanks were not shaded.
   Near bottom current measurements in the natural environment gave cur-
rent velocities of 0.05 to 1.8 m/sec. This is in accordance with values pro-
vided by Stebbing [188] who gave a range from 1 to 1.8 m/sec. Accordingly,
in the culture basins current velocities around 1 m/sec were continuously
maintained.
   While growth rates comparable to those observed in nature were eventu-
ally achieved after considerable experimentation it proved extremely difficult
to instigate the organisms to produce sperm and eggs for laboratory-reared
larvae. This was eventually and reproducibly achieved after about four years
of culture of colonies collected in the wild. As culture conditions were not
changed it can be assumed that a complete adaptation to the laboratory envi-
ronment took several years. Growth rates based either on biomass or colony
height increase were lower than those recorded for wild colonies.
   This example illustrates the difficulties in maintaining and adapting a ma-
rine invertebrate species to controlled conditions in the laboratory. Establish-
Aquaculture of “Non-Food Organisms”for Natural Substance Production           9

ment of optimum conditions, both in terms of the physical environment and
food, for the survival of specimens collected in nature is accompanied by at-
tempts to encourage reproduction. This may, as in the case of Flustra foliacea,
take several years.


3
Organisms

3.1
Porifera

Attempts in sponge culture had been made as early 1862 [189] although more
intensive work was carried out at the beginning of the 20th century [190, 191]
and later by Arndt [192] mainly for the production of bathing sponges. How-
ever, extensive work in this field started only after the discovery of secondary
metabolites with pharmacological potential.
   At present, the most commonly applied technique is in situ culture. Osinga
et al. [193] and Brümmer and Nickel [194] review the state of the art of whole
sponge culture. For this type of culture intact specimens are collected from
the wild, cut, fixed to an artificial support [195, 196] and re-introduced as
explants to the natural environment, i.e. basically the same technique that
had been employed already some hundred years ago [190, 191]. Alternatively,
more control on these cultures can be exerted when flow-through systems
utilising natural seawater are employed. While this approach circumvents the
necessity to provide food for the cultured organism it also depends on the
vagaries of nature—the high variability of planktonic biomass both season-
ally and interannually, the presence of predators and the risk of bacterial
infections of freshly cut sponge pieces. This approach is based on the highly
developed potential of sponges for regeneration [197] which results from the
high telomerase activity of sponge cells [198].
   As an example for the in situ approach, the detailed study of Duckworth
and Battershill [199] using three sponge species with different growth forms
may be considered. The authors farmed sponge explants (1) inside mesh
support structures, (2) on rope threaded through explants and (3) on rope
wrapped around explants. Explants attached to substrate were used as con-
trols. Only two of these techniques showed some potential for larger-scale
application in terms of growth, explant survival and metabolite production.
   Laboratory systems have been developed but so far no reports of a suc-
cessful long-term operation are available [194]. Both open aquaria and closed
bioreactor systems [200–202] have been used. Operation of laboratory sys-
tems requires a detailed knowledge of the natural environment and the living
conditions of sponges (e.g. [203]). Even then, success in maintaining the spe-
cimen alive or observing growth appears to be limited of 22 species in culture
10                                                                    G. Liebezeit

as whole sponges only 14 survived for longer periods of time and only five
showed growth [194, 200].
    Armstrong and Goldsworthy [204] note that in cultures of Pecten maximus
no biofouling occurred when the clams are covered by the sponge species
Suberites ficus ssp. rubrus. Although primary cell cultures were successfully
established no cell lines were achieved. A number of techniques were investi-
gated for establishing cells and pieces of sponge tissue onto scallop shells but
with none of these satisfactory results were obtained. Mechanical methods,
i.e. sticking small pieces of sponge to scallop shells, proved also impractical
even where they resulted in subsequent sponge growth. Improving natural
settlement of sponges onto the scallops would thus be the only economically
practical method for increasing sponge yield.
    Providing artificial substrates for sponge explants has been the aim of sev-
eral studies [165, 175]. Here calcium carbonate and magnesium hydroxide are
co-precipitated by a DC current onto a structured cathode, e.g. a metal mesh,
which then serves as a substrate to which sponges can be attached.
    Thus, the observations of Armstrong and Goldsworthy [204] together with
the experiments made by Müller et al. [195, 205] might provide clues for the
laboratory production of sponge seedlings from larvae which then might be
transplanted for growth into the natural environment.
    While in situ culture has been demonstrated to be a suitable alternative to
in situ collection [204, 206, 207] it is still beset with a number of difficulties
as discussed above. Cell cultures of marine sponges might thus be considered
as an alternative to in situ techniques. The advantage of cell cultures is that
they can be completely controlled and easily manipulated for optimal pro-
duction of the target metabolites. The first report on sponge cell culture dates
back only to 1994 [208]. Thus, this technique is still in its infancy and a con-
tinuous cell line has yet to be established. Possible approaches to tackle this
problem have been recently discussed in detail [194, 209]. Nevertheless, cell
culture approaches have been extensively worked on as reviewed by Pomponi
et al. [210–212] and de Rosa et al. [213].
    Axenic cultures of sponge aggregates (primmorphs) may provide an al-
ternative to cell culture. Primmorphs are defined as three-dimensionally
arranged cell aggregates that are composed of both proliferating and differen-
tiating cells. Detailed studies on the formation of these aggregates have been
provided by a number of authors [214–218] and reviews have been presented
by Müller and co-workers [209, 215, 219].
    An important aspect in primmorph culture is the development of suitable
media. The necessity of amending natural seawater with silicate and iron has
been established (e.g. [215, 220]). It might similarly be important to provide
suitable growth substrates as well as to control initial cell density and culture
temperature [221].
Aquaculture of “Non-Food Organisms”for Natural Substance Production          11

3.2
Bryozoa

Of the moss animals only Bugula neritina has received attention as this
species produces bryostatin 1, a potent antineoplastic agent [99]. Men-
dola [179] describes in detail larger scale culture techniques. Initial labo-
ratory tests failed, largely due to difficulties associated with providing an
adequate food supply. In situ techniques were more successful and proved
even economically feasible. Mendola [116, 179] also discusses shortcomings
and problems associated with the in situ-approach such as for example exclu-
sion of larvae of nudibranchs that prey on developing Bugula colonies or the
effects of weather and climate.
   For other bryozoan species only laboratory scale cultures have been de-
scribed [148–151, 153–155, 222, 223]. Emschermann [224] developed a 30 L
tank incorporating a water circulation system (see also [225]). Cell cultures of
bryozoa have so far only been investigated in relation to morphological and
ultrastructural features [226] and biofouling aspects [227].
   So far, reproducible laboratory production of larvae, not only of bryozoan
species, has not been achieved and collection of gravid colonies in the natural
environment appears to be the method of choice [179]. This will, however, not
affect natural populations as only a very limited amount of biomass needs to
be collected to obtain a large number of larvae.
   Kahle et al. [228] describe another technique for culture of erect growing
bryozoan species employing “cuttings”, i.e. pieces of tissues that were re-
moved from the mother colony and fixed in specially designed PVC/silicone
rubber holdings. These secondary colonies exhibited growth comparable to
that determined in nature.

3.3
Molluscs

Marine molluscs and here especially shell-bearing species have been used
as a resource since historical time for example as food, currency, utensils
or musical instruments. The probably best known application example of
a molluscan-derived chemical is Tyrian purple extensively used and highly
valued in for example ancient Rome [229].
   In some cases potentially interesting compounds have been isolated from
molluscs, e.g. a HIV virus-inhibiting compound from the green mussel Perna
viridis has been patented [230].
   Within the phylum molluscs the gastropoda and within this class especially
the ophistobranchs have received considerable attention. Nevertheless, of the
about 6000 known species of ophistobranchs only about 250 have so far been
investigated with respect to their natural products chemistry [91].
12                                                                   G. Liebezeit

3.3.1
Ophistobranchs

The ophistobranchs include the sea slugs and their relatives the sea hares,
sea butterflies, canoe shells, and others. Shells may or may not be present;
if present they may be reduced and/or internally located. This implies that
these organisms must have other means of preventing predator attack which
are usually of a chemical nature [25, 35, 58, 84, 87, 89, 91]. These compounds
are usually highly active and have thus elicited interest in a.o. pharmaceutical
research.
   The sea hare Aplysia spp., due to its simple brain containing only some
20.000 nerve cells, has been extensively used in studies of the molecular
basis of learning and memory in mammals [231]. In addition, a large num-
ber other species have been used in biomedical research ([231], p. 85). Due
to the large demand on these invertebrates culture efforts have been made
(e.g. [232–236]).
   Both macroalgal (cf. [232, 237]) and artificial food [238, 239] has been
tested for Aplysia adults. Larval metamorphosis appears to be influenced
by macroalgal extracts with those from red algae being the most effi-
cient [234, 240].
   Bayne [241] discusses technical aspects of a.o. mollusc cell culture al-
though so far no continuous cell lines have been established [242]. The
majority of attempts to provide these have been made in food production
research although applications in environmental research have also been
reported [243].

3.4
Others

The horseshoe crab Limulus polyphemus (Merostomata, Arthropoda) is used
extensively in the biomedical and pharmaceutical industries. Horseshoe crabs
have blue, copper-based blood that clots when exposed to endotoxins re-
leased by certain gram-negative bacteria. The clotting feature of Limulus
blood serves as commercially important in tests of the sterility of fluids and
artificial implants intended for use on human patients. The blood enzyme re-
sponsible for clotting is called Limulus Amoebocyte Lysate (LAL) and occurs
exclusively in L. polyphemus [244, 245].
   This compound can be easily obtained from specimens collected in the
wild with a survival rate of about 90% [246]. As this species is also employed
for animal consuming biomedical purposes [247] leading to the development
of conservation and management plans (e.g. [248]), laboratory protocols for
Limulus culture have also been described [134, 249, 250]. For the in vitro pro-
duction of amoebocytes a US patent has already been issued [251] although
no evidence of large-scale use of this technique was found in the literature.
Aquaculture of “Non-Food Organisms”for Natural Substance Production         13

4
Applications Other than Pharmaceutical

While aspects presented above of the culture of various organism classes have
been considered with a view to pharmaceutical applications the following
deals largely with other products of these organisms. While some of these
such as mussel silk from Pinna nobilis [252, 253] or dyes may be of historic
interest others may well develop into commercially attractive compounds.

4.1
Marine Cements

Barnacles, bryozoa and mussels secrete a cement in order to attach to solid
surfaces in the sea. Van den Spiegel [254] and DeMoor et al. [255] de-
scribe a defensive glue produced by the holothurian Holothuria forskalii while
Grebel-Köhler [256] analysed bryozoan cements. Polychaete worms such as
Lanice conchilega or Phragmatopoma californica build tubes by cementing to-
gether material such as sand and shells [257, 258]. Apparently most of these
marine cements have a common general structure being polyphenolic pro-
teins which act as “glue” (e.g. [259–263]). Mussel adhesive proteins can be
subdivided into three types depending on the function they serve in byssal
threads: (1) fibrous proteins which form the load-bearing cables in the core
of the threads; (2) cuticular proteins forming a protective coat around the
cables; and (3) adhesive proteins connecting the cables to a hard surface.
The fibrous proteins can be collagenous, silk-like, elastic, or any combina-
tion. Covering these are the cuticular proteins, which are distinguished by
their surface coupling properties, repeated primary sequence, and their high
content of lysine and the non-essential amino acid 3,4-dihydroxyphenyl-l-
alanine (DOPA). The adhesive proteins are of low molecular weight, contain
DOPA, and assemble to form microcellular solids [264].
   Several of these proteins have attracted biotechnological attention as
cell and tissue attachment factors, anticorrosives, or metal-sequestering
reagents [264–266]. They may serve as glues in clinical or underwater ap-
plications. This requires intense work on culture of these sessile and mobile
organisms.

4.2
Biominerals

Most sponges have siliceous spicules that serve as skeletal elements. They
may also, in combination with chemical compounds, act as feeding deter-
rents (e.g. [267]). Similar to baciallariophyceae and radiolaria sponges em-
ploy protein matrices to precipitate biogenic opal in a highly ordered fashion.
Thus, isolation, characterisation and eventual encoding of these proteins,
14                                                                    G. Liebezeit

silicateins, cathepsins etc. (e.g. [268, 269]), might lead to commercially inter-
esting nanostructures (e.g. [270, 271], see also [272]).
    Marine carbonates as highly interconnected microporous materials are
similarly receiving attention mainly in medical applications [273]. Three-
dimensional microporous skeletons are found in echinoderms and certain
species of coral. The sizes of pores are uniform and range from 15 to 500 µm,
depending on the species. The carbonate framework may be used as a tem-
plate for the deposition of metals, ceramics, or polymers which, after removal
of the carbonate by a mild acid treatment, provides an interconnected porous
composite structure [274].
    The intricate skeletal architecture of marine carbonates and silicates thus
offers a whole new vista of applications, especially as these can be tailored
through control of the growth conditions to a considerable extent.

4.3
Antifouling Compounds

Due to environmental considerations the use of the highly effective organ-
otin compounds will be phased out by 2008 [275]. Copper-based alterna-
tives might similarly prove to be deleterious in the long term [276, 277].
Some marine organisms are able to maintain a surface that is essen-
tially free of epibionts. Hence research efforts have been made to find
natural compounds that prevent settlement or survival of fouling species
(cf. [278, 279]). Sponges and bryozoa have been found to contain such
metabolites (e.g. [204, 280–283]). Again, in some cases associated bacteria
were found to be the actual producing organisms [284].
   Thus, despite progress towards characterising compounds that inhibit
micro- and/or macrofouling, evidence for an ecological role of these com-
pounds is still poor. So far broad spectrum antifoulants of sufficient promise
have not been discovered. Even then major obstacles have to be faced before
these resources can be commercially exploited among them the need to pro-
cure sufficient material and the cost of registration. Nevertheless, structural
identification of natural antifouling compounds might, in addition to gaining
information on the relative importance of chemical versus physical defences
of marine organisms, provide a conceptual framework for the development of
synthetic novel coatings.

4.4
Other Applications

Ornamental fish and crustaceans are in large demand on the world market.
Winfree [285] gives a total of 1500 species which are sold world-wide. Of these
about 80% may be taken into culture. Thus, pressure from collecting free-
living specimens can be enormous potentially endangering natural stocks.
Aquaculture of “Non-Food Organisms”for Natural Substance Production            15

Florida Agricultural Statistics Service alone gives a market value for orna-
mental fish from aquaculture sources of 43.2 mill. US$ in 1999 [286] while
global estimates range between 4 and 15 bill. US$ [287]. Sport fishery also has
a considerable demand for bait organisms, e.g. the polychaete Nereis virens
has been farmed in NE England and the Netherlands since 1984 for this pur-
pose [288].
   A detailed discussion of the requirements for successful culture of poly-
chaetes, albeit largely with the bait supply in mind, has been provided
by Olive [288]. He argues that five major prerequisites must be fulfilled:
(a) efficient protocols for mass fertilisation and production of larvae and
juveniles; (b) maintenance of a brood stock thus alleviating the need
for inputs from natural populations; (c) simple and inexpensive systems
for the rearing of juveniles; (d) extension of the breeding season and/or
cryo-preservation of larvae to maintain an all year round larval supply
and (e) optimal growth conditions through control of nutrition, tempera-
ture and, where appropriate, photoperiod. Further technical information,
especially on culture of polychaete larvae, may be found in Irvin and
Martindale [289].
   Rinkevich and Shafir [290] discuss strategies to avoid large-scale collec-
tion of ornamental animals. These involve besides direct culture, collection
of larvae, fragmentation (for colonial species) and cryopreservation of larvae.
Calado et al. [291] describe the use of the planktonkreisel for the complete
larval development of ornamentous shrimps and crabs. Again with the aquar-
ium trade in mind Ellis and Sharron [292] provide a detailed manual for the
culture of soft corals while Rippingale and Payne [293] present similar infor-
mation for calanoid copepods.
   In environmental research invertebrates have been used to study uptake,
bioaccumulation and metabolic fate of both inorganic and organic com-
pounds [294], e.g. Goerke [295] describes culture methods for the polychaete
Nereis virens for uptake studies of 14 C-labelled 2,4,6,2 ,4 -pentachlorobiphenyl.
Further examples may be found in Kaiser [296].
   Another interesting use of marine invertebrates although at present not
directly associated with their culture is the use in biofiltration systems to
remove surplus particulate organic matter from culture systems of other ma-
rine organisms such as fish or shrimp (e.g. [297, 298]). This approach may,
however, eventually be combined with the production of natural compounds
from invertebrates and will then require controlled species composition of the
biofilters.
   As with decreasing global fisheries yields the demand for alternative food
supplies in shrimp and fish aquaculture increases a number of attempts have
been made to use marine invertebrates for this purpose. Especially annelid
worms are of interest as a number of commercially interesting fish species
such as for example sole (Solea spp.) require these as food (e.g. [299]). This
includes also production of feed for ornamental species.
16                                                                   G. Liebezeit

   Lubzens et al. [300] reviewed the methodologies devised for reliable supply
of rotifers in large quantities and problems associated with rotifer produc-
tion, nutritional quality and effect on fish health and nutrition. Rotifers,
however, may also be used as a source for bioactive metabolites [301].


5
Interactions with Microautotrophs and -heterotrophs

Another important aspect to be considered when marine invertebrates are to
be used as a source of for example pharmaceuticals is the widely discussed
but still insufficiently researched area of interactions between the invertebrate
and associated bacteria, fungi or autotrophic microalgae (e.g. [57–59, 119,
206, 207, 302–310]). Highly diverse bacterial communities have been found in
sponges (e.g. [311]). Already in 1928 Arndt [192] discussed the possibility of
symbiotic bacteria being the producers of toxic compounds extractable from
sponges.
   Unless inhibited by the active production and excretion of compounds
preventing settlement, biofilms are a common occurrence on any marine sur-
face (cf. [312–314]). The main research emphasis has, however, been placed
on fouling aspects (e.g. [315–318]) rather than on the production of natural
compounds from these complex assemblages.
   Burgess et al. [319] isolated over 400 strains of surface-associated bac-
teria from various seaweeds and invertebrates from Scottish coastal waters
of which about one third produced antimicrobial compounds. Antimicrobial
compounds produced by bacteria isolated from marine invertebrates appar-
ently are a common occurrence (e.g. [59, 320]). Osinga et al. [321] review
these aspects for sponges. For a number of pharmacologically active com-
pounds the actual source organisms have been identified as bacteria, e.g.
bryostatin is synthesised by the bacterial symbiont of the bryozoa Bugula
neritina, Candidatus Endobugula sertula [322].
   On the other hand, this association is not always straightforward. Swin-
holide A for example has been isolated from a marine sponge [323, 324]
but was later assumed to be of cyanobacterial origin due to structural sim-
ilarities to tolytoxins and scypophycins [323, 325]. Nevertheless, this com-
pound was found in the heterotrophic eubacterial fraction of the sponge/
cyanobacteria/bacteria complex [326] indicating complex interspecies rela-
tionships in associations of organisms.
   The close association between corals and autotrophic micro-organisms is
well known. Sea slugs also contain phytoplankton-derived chloroplasts [327]
and symbiotic autotrophic organisms may also eventually become a major
target of marine drug or energy-related research and culture development.
   Another question in this context is whether the association between ma-
rine invertebrates and microheterotrophs is of obligate or facultative nature,
Aquaculture of “Non-Food Organisms”for Natural Substance Production          17

i.e. whether microbial symbionts produce any or all of the natural products
attributed to their hosts and hence contribute to for example the chemical de-
fence of the association or whether “accidentally” attached micro-organisms
are responsible. So far, all compounds presently being investigated in greater
detail apparently can be attributed to microbial symbionts. Many associations
between marine invertebrates and micro-organisms appear to be quite spe-
cific. Nevertheless, a number of marine natural products, particularly those
from sponges, have been attributed to undefined “symbionts” (e.g. [38, 328]).
    Although it might be tempting to use the straightforward approach and
isolate the “real” producers which as bacteria are—at least theoretically—
amenable to traditional culture and larger scale fermentation techniques it
should be pointed out that the vast majority of marine bacteria cannot be
cultured at present. Especially for symbiotic microheterotrophs this will cer-
tainly also hold true.
    Another difficulty arising even after successful culture of the microbial
symbiont is the possibility that symbionts may not produce the same sec-
ondary metabolites when grown in culture (e.g. [329]). Furthermore, it is
possible that exchange of nutrients, biochemical stimuli, or metabolic inter-
mediates between the symbiotic partners is required for the production of
a particular natural product [330].


6
Further Considerations

A fact which has been largely overlooked so far in all attempts to develop
culture techniques for either marine invertebrates, cells or cell aggregates
is the fact that in nature the production of secondary metabolites is usu-
ally connected to a specific purpose, be it defence, attraction of the opposite
sex or other ways of communication. Such stimuli are normally not present
in cultures with the possible exception of sex pheromones. Although Müller
et al. [331] report that a.o. primmorph contrary to cell cultures of Dysidea
avara produced avarol, in the long term, a decrease and eventual stop in
metabolite production in culture might occur. This is a well known occur-
rence in the culture of toxic phytoplankton species where toxicity is lost after
a number of cell divisions. Hence, this might necessitate the development of
culture techniques for which production-instigating factors have to be identi-
fied and supplied.
    As discussed above the invertebrates originally identified as metabolite
source may actually not be the responsible producers. In addition, the pos-
sibility exists that these compounds are acquired from external sources,
i.e. through food uptake. Fahey and Garson [332] provide an example for
the carnivorous nudibranch Asteronotus cespitosus. From this species halo-
genated metabolites have been isolated that had previously been considered
18                                                                             G. Liebezeit

to be characteristic for the sponge Dysidea herbacea. This implies a di-
etary origin rather than de novo synthesis. Again, due to the different feed-
ing types of sponges and nudibranchs it can be assumed that the ultimate
source of bioactive compounds might be microalgae and/or associated mi-
croheterotrophs [306, 333, 334].
   Chemical defence compounds are also frequently acquired via the food.
For Aplysia species it has been long known that the brominated aplysiatoxin
is derived from its cyanobacterial food [335, 336]. Another example is the
marine snail Glossodoris quadricolor that feeds on the sponge Latrunculia
magnifica [337]. The 2-thiazolium macrolides latrunculin A and B provoke
a flight reflex in fishes.


7
Conclusions

Although the interest in developing marine invertebrates as sources of bioac-
tive compounds to larger scales than the laboratory is huge and significant
research efforts both by academia and industry are made to achieve this, this
goal will certainly not be reached within a few years. It should also be pointed
out that the identification and structural elucidation of bioactive compounds
should always be accompanied by a detailed study of the ecological situation
the producing organism finds itself in. This will then enable better definitions
of growth conditions under artificial conditions.
   On the other hand, synthesis chemistry might aim at identifying the active
key structures of marine secondary metabolites rather than rebuilding whole
molecular structures [105]. This may result in reduced efforts and possibly
better yields in synthesis chemistry.
   Another similarly challenging avenue of research is the development of
techniques to culture symbiotic microheterotrophs or to identify and eventu-
ally clone the genetic information responsible for the production of bioactive
compounds into bacteria that can be easily maintained and grown to a large
scale. As yet, this has not been achieved for any marine drug but may be
accomplished within the years to come.
   Despite all these present difficulties and shortcomings the ocean still holds
a large number of compounds to be discovered and great challenges for the
development of sustainable production techniques for marine invertebrates.


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Adv Biochem Engin/Biotechnol (2005) 97: 29–62
DOI 10.1007/b135822
© Springer-Verlag Berlin Heidelberg 2005
Published online: 8 August 2005

Bioprocess Engineering Data on the Cultivation
of Marine Prokaryotes and Fungi
Siegmund Lang (u) · Marén Hüners · Verena Lurtz
Technische Universität Braunschweig, Institut für Biochemie und Biotechnologie,
Spielmannstr. 7, 38106 Braunschweig, Germany
s.lang@tu-bs.de

Dedicated to Prof. Dr. Axel Zeeck on the occasion of his 65th birthday

1       Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     30

2       Isolation of Marine Microorganisms . . . . . . . . . . . . . . . . . . . . .           31

3       Media Used in the Cultivation of Marine Microorganisms . . . . . . . . .               32

4     Overview on Bioprocess Engineering Data . . . . . . . . . . . . . . . .          .   .   34
4.1   Bioprocess Engineering Data on Marine Mesophilic Bacteria and Fungi              .   .   35
4.1.1 Metabolite Production . . . . . . . . . . . . . . . . . . . . . . . . . . .      .   .   35
4.1.2 Biopolymer Production . . . . . . . . . . . . . . . . . . . . . . . . . . .      .   .   45
4.2   Bioprocess Engineering Data
      on Marine Psychrophilic/Psychrotrophic Microbes . . . . . . . . . . . .          . .     50
4.2.1 Enzyme and Metabolite Production . . . . . . . . . . . . . . . . . . . .         . .     50
4.2.2 Barophilic Strains: A Special Challenge . . . . . . . . . . . . . . . . . .      . .     53
4.3 Bioprocess Engineering Data
      on Marine Hyperthermophilic and Barophilic Archaea . . . . . . . . .             . .     56

5       Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .         58

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .       59

Abstract The temperature/pressure dependency of marine prokaryotes and fungi, in
terms of their growth behaviour as well as their potential to produce new metabolites or
enzymes, is evaluated. Advanced shake-flask cultivations and controlled bioreactor culti-
vations following the batch-type, fed-batch-type and/or continuous-type procedures are
summarized. After a summary of the fermentation data available so far, values on max-
imal biomass, specific growth rates, and (sub)optimal production yields are presented.
The application of mesophilic microbes, especially bioactive metabolites, to intensify bio-
process engineering studies, is the goal. Cold-active enzymes and thermostable enzymes
are the targets of experiments with psychrophilic and hyperthermophilic enzymes. A spe-
cial challenge to bioengineers is also provided by barophilic strains originating from
depths of, say, nearly 11 000 m, or from hydrothermal vents.

Keywords Mesophilic · Psychrophilic · Hyperthermophilic microbes ·
Barophilic microbes · Bioreactor design · Bioactive metabolite and enzyme production
30                                                                  S. Lang et al.

Abbreviations
ASW           artificial sea water
Cfu           colony forming units
D             dilution rate
HPLC          high-pressure liquid chromatography
LB medium Luria-Bertani medium
MB            marine broth (Difco 2216)
MPa           megapascals
OD            optical density
Pa            pascal
pO2           oxygen partial pressure
QO2           oxygen consumption rate
QCO2          carbon dioxide production rate
rpm           rotations per minute
RQ            respiratory coefficient
td            doubling time
v/vm          volume per volume per minute
YE            yeast extract
µmax          maximal growth rate




1
Introduction

For marine biotechnology, like biopharmaceutical biotechnology, bioprocess
engineering represents the path from discovery to commercialisation. As an
example, the many hundreds of bioactive compounds discovered and isolated
from various marine organisms have led to only minimal potential commer-
cialization due to the limited availability of the compounds for clinical trials
or further modification by chemical or biocatalytical means. In terms of the
biopharmaceuticals found in marine systems, sponges are the most studied
organisms, followed by tunicates and and coelenterates [1]. However, the pro-
duction of sponge biomass is still one of the main outstanding goals of marine
biotechnology. At the moment only minor progress is visible [2]. Summaris-
ing the recent publications on marine natural products, Faulkner observed
a significant increase in the number of papers reporting studies on marine
bacteria and fungi [3–7]. In comparison to higher eukaryotes, these microor-
ganisms are easier to cultivate and, additionally, there is a lot of successful
experience with terrestrial species [8].
   Despite these encouraging observations, it is still true to say that there
is a lack of research into bioreactor engineering and fermentation protocol
design in the field of cultivating marine microorganisms to produce high-
value products [9, 10]. One of the principal problems is the fact that less than
5% of all bacteria observed by microscopic methods are found to be cultur-
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi   31

able under standard conditions [11–13]. This fact greatly limits our ability to
isolate and cultivate the majority of the interesting and new bacterial forms
present. When mesophilic species, for example, have been found to grow suf-
ficiently, in general they have been found to like salt concentrations of 2% to
4% and temperatures of 18 to 30 ◦ C. Although they grow in in the presence
of minute concentrations of nutrients, they appear to have a requirement for
complex carbon sources, including proteins or polysaccharides.
   The goal of this publication is therefore to give an overview on charac-
teristic successfully-applied culture conditions, including media and biopro-
cess engineering data, 1) in relation to the differeny biodiversities of marine
prokaryotes and fungi, and 2) with respect to their product classes (low- and
high-molecular weight substances).


2
Isolation of Marine Microorganisms

Based on estimates of culturable microbes, 80% to 95% of marine bacteria
are gram-negative rather than gram-positive. They are widely distributed in
free suspension, attached to flocculated material (bacterioplankton), in sed-
iments, on animate and inanimate surfaces, and as partners in symbiosis or
in commensalism with other marine organisms [11–13]. Marine fungi grow
on a variety of substrates ranging from wood to sediments, muds, soils, sand,
algae, corals, calcareous tubes of molluscs, decaying leaves of mangroves, in-
tertidal grasses and living animals [14].
   Depending on the local origins of above microorganisms in the sea—
including surface layers, deep-sea, hydrothermal vents—many of them need
special cultivation conditions with respect to temperature and pressure, re-
spectively. Therefore, in terms of promoting a successful isolation, a variety
of parameters may influence the results of initial lab-cultivation experiments.
These include the sampling conditions, the pre-treatment of the sample (siev-
ing, mixing, cooling, transport, storage, heating, and so on), and the enrich-
ment procedure [15]. In the case of extremophiles, the isolation procedure
needs more expenditure. For instance, deep-sea sediment samples were col-
lected by means of sterilized mud samplers from unmanned submarines and,
additionally, pressurized at approximately 100 MPa in a pressure vessel in
order to harvest barophilic bacteria [16].
   Classification of isolates into the main taxa is usually performed by dot
blot hybridization of extracted DNA, whole cell hybridization using fluor-
escent probes, signature polymerase chain reactions [15], and additionally,
by chemo-taxonomic, morphological, cytological and physiological studies.
Table 1 shows some examples of new microbial species from different marine
environments.
32                                                                       S. Lang et al.

Table 1 Some examples of new microbial species isolated from different marine environ-
ments: seawater, sediment, plants, animals, cold and hot areas, and deep-sea regions

Strain               Marine Origin                                              Ref(s)

Bacteria:
Oceanibulbus         Seawater/Picoplankton, depth of 10 m, North German Sea     [51]
indolifex
Streptomyces sp.   Mangrove sediment near Pohoiki, Hawaii, Pacific Ocean         [41]
Rhodovulum sp.     Sea sediment mud, Thailand                                   [67]
Bacillus sp.       Schizymenia dubyi (macroalga), Omaezaki coast, Japan         [40]
Haliangium sp.     Seaweed of a sandy beach, Miura Peninsula, Japan             [34]
Micromonaspora sp. Soft coral, Indian Ocean                                     [28]
Actinomadura sp.   Polychaete, Northern coast of Spain                          [37]
Alteromonas sp.    Polychaete, hydrothermal vent, East Pacific Rise,             [66]
                   2600 m depth
Agrobacterium sp. Ecteinascidia turbinata (tunicate),                           [44]
                   Formentera Island, Spain
Microbacterium sp. Halichondria panicea (sponge), Adriatic coast, Croatia       [47]
Pseudo-            Crella rosea (sponge), Adriatic coast, Croatia               new
alteromonas sp.
Moraxella sp.      Antarctic seawater, French Ant. base J.S. Dumont d’Urville   [94]
Bacillus sp.       Antarctic seawater, French Ant. base J.S. Dumont d’Urville   [96]
Shewanella sp.     Deep-sea sediment of Mariana Trench, depth of 10 898 m       [16]
Moritella sp.      Deep-sea sediment of Mariana Trench, depth of 10 898 m       [16]
Pseudomonas sp.    Deep-sea sediment of Japan Trench, depth of 4418 m           [106]
Thermococcus       Hydrothermal vent, depth of 1380 m,                          [118]
peptonophilus      Western Pacific Ocean
Fungi:
Penicillium sp.      Sea bottom of Uchiura Bay, Japan                           [55]
Keissleriella sp.    Sea bottom of Yellow Sea, Sheyang Port, China              [58]
Hypoxylon            Mangrove wood, intertidal zone, Shenzen, China             [60]
oceanicum




3
Media Used in the Cultivation of Marine Microorganisms

Sea water contains about 3% sodium chloride plus small amounts of many
other minerals and elements. Microorganisms found in the sea usually have
a specific requirement for the sodium ion in addition to growing optimally
at the water activity of seawater (aw = 0.980). Such organisms are called
halophiles. Mild halophiles and moderate halophiles need distinct NaCl con-
centrations: 1–6% and 6–15%, respectively. This requirement is usually ex-
plained by their smf-dependent (sodium motive force) active transport and
flagellar rotation, and the need for stability or activation of membrane and
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi    33

Table 2 Typical media used for the cultivation of marine prokaryotes [18, 19]

Component                  Marine broth (MB) Luria-Bertani (LB) Artificial sea water
                           (g L–1 )          medium (g L–1 )a   medium (g L–1 )b

Supply line
Peptone                     5.00               10.00                 3.50
Yeast extract               1.00                5.00                 3.50
Main elements
NaCl                       19.45               19.45                23.00
MgCl2                       5.90                5.90                 5.08
MgSO4                                                                6.16
Fe(III) citrate             0.10                0.10
Fe2 (SO4 )3                                                          0.03
Na2 SO4                     3.24                3.24
CaCl2                       1.80                1.80                 1.47
KCl                         0.55                0.55                 0.75
Na2 CO3                     0.16                0.16
Na2 HPO4                    0.008               0.008                0.89
(NH4 )NO3                   0.0016              0.0016
NH4 Cl                                                               5.00
Trace elements
KBr                         0.08                0.08
SrCl2                       0.034               0.034
NaF                         0.0024              0.0024
NaSiO3                      0.004               0.004
H3 BO3                                          6.11 × 10–4
MnCl2                                           3.89 × 10–4
CuSO4                                           5.60 × 10–5
ZnSO4 · 7H2 O                                   5.60 × 10–5
Al2 (SO4 )3 · 18H2 O                            5.60 × 10–5
NiSO4 · 6H2 O                                   5.60 × 10–5
Co(NO3 )3 · H2 O                                5.60 × 10–5
TiO2                                            5.60 × 10–5
LiCl                                            2.80 × 10–5
SnCl2                                           2.80 × 10–5
KJ                                              2.80 × 10–5
(NH4 )6 Mo7 O24 · 4H2 O                         5.60 × 10–5
a   see [18], b see [19]


periplasmic components [17]. But salinity is not the general rule for the
successful cultivation of all marine microorganisms; alternative culture con-
ditions are presented in Chapter 4.
   Concerning the main growth sources, the carbon and nitrogen sources and
the inorganic ion components, some typical and frequently-used examples
are presented in Table 2. Marine broth (MB) and Luria-Bertani (LB) medium
34                                                                   S. Lang et al.

differ only in the amount of peptone/yeast extract and in the trace element
composition. Artificial sea water (ASW1) consists of a lower content of ingre-
dients and has to be supplemented by further components depending on the
individual purpose.
   In terms of the purchase of special marine nutrients or salts, the fol-
lowing addresses are of interest: Difco Laboratories, Detroit, MI, USA or
Nordwald, Hamburg, Germany (for Marine Broth 2216); Carolina Biological
Supply Company, Burlington, NC, USA (for filtrated sea water); Instant Ocean
Salts (found in stores for aquarium accessories).
   In 1993, Fenical and Jensen [11] stated that unfortunately very little infor-
mation is known about the specific nutrients and growth factors required by
most marine microorganisms. Common media constituents such as peptone,
simple sugars, and so on, would be unrealistic marine nutrients, and in the
marine environment are apparently replaced with complex carbon sources
such as chitin, sulfated polysaccharides, and marine proteins. In addition, vir-
tually nothing is known of the effects of uncommon inorganic elements, such
as lithium, silicon, and so on, which are also abundant in marine habitats.
This statement also appears to be valid ten years later.
   In relation to the control of antibiotic synthesis, in 1999 Marwick et al. [9]
stated that the following growth conditions often favour—but this is no gen-
eral rule—the production of marine microbes:
a) carbon source: slowly utilizable galactose instead of glucose (causing
   catabolite repression)
b) nitrogen source: ammonia; specific amino acids (including cysteine, me-
   thionine)
c) phosphate source: should be limited
d) trace elements: bromide ions, iron ions
e) oxygenation: increase of oxygen partial pressure or O2 limitation
f) salinity: natural marine salinity not necessary
g) induction: signal molecules such as homoserine lactones
h) immobilization: viable cells attached to polymeric surfaces.



4
Overview on Bioprocess Engineering Data

As we cover the main topics associated with bioprocess engineering stud-
ies on the lab-scale cultivation of selected marine microbes, it makes sense
to distinguish the following in terms of their temperature- and/or pressure-
dependencies:

 • Mesophilic microbes (20–45 ◦ C)
 • Psychrophilic microbes (≥ 0–20 ◦ C) including barophilic species
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi   35

 • Thermophilic (45–65 ◦ C) and hyperthermophilic microbes (66–110 ◦ C)
   including barophilic species
    In addition to growth, the efficiency of synthesizing the following products
is considered:
 • Low-molecular weight metabolites
 • Non-proteinogenic polymers
 • Enzymes, including extremophilic ones

4.1
Bioprocess Engineering Data on Marine Mesophilic Bacteria and Fungi

4.1.1
Metabolite Production

Compared to the large number of publications on structural analyses of ma-
rine microbial metabolites, including antibiotics, those on quantitative bio-
chemical engineering studies are virtually non-existent in the literature. Most
fermentations reported were carried out at the shake-flask level and, addi-
tionally, have been described very briefly without details provided on growth
and production over time, and predominantly focusing on isolation and elu-
cidation of new molecular structures [20–27]. In 1999, Marwick et al [9] crit-
icised this lack of knowledge and they found that they could only highlight a
few areas in which empirical and mechanistic knowledge of terrestrial antibi-
otic fermentations could be applied to marine microbes. After giving advice
on growth media, physical parameters and regulation tricks, the authors rec-
ommended intensifying studies on bioprocess engineering such as bioreactor
configuration, oxygen-media mass transfer and reducing shear stress.
   In contrast to the above article, we will give an overview on initial biopro-
cess engineering studies, including not only lab-scale bioreactor cultivations,
but also advanced shake-flask fermentations. With a few exceptions, we only
cite publications containing an extensive protocol on growth and metabolite
production.

Metabolite Production using Bacteria (Shake-Flask Scale)

In terms of shake-flask protocols that are useful starting points to biopro-
cess engineering, for example, they present the time courses of growth, pH
and metabolite production, recent publications are summarised in Table 3.
For three species of Micromonaspora isolated from different marine habitats,
the time courses of growth (measured as packed cell volume), pH, and of
bioactive metabolites (determined by HPLC) were well documented [28–33],
which allows up-scaling to bioreactor cultivations. Also, initial biotechnolog-
ical protocols have been recently established for four other bacteria of marine
36                                                                           S. Lang et al.

Table 3 Advanced shake-flask cultivations of marine mesophilic bacteria with respect to
important bioprocess conditions and metabolites. Bioactivities of these metabolites: cy-
totoxic, antibacterial, antifungal, antitumoural, respectively. PCV indicates packed cell
volume

Strain (origin)                                Growth         Product           Ref(s)
Cultivation conditions                                        (mg L–1 )

Micromonaspora sp. (coral)                     PCV:12%        Thiocoraline      [28, 29]
0.25 L, glucose, starch, soybean meal, pH 7,                  (9)
28 ◦ C, 250 rpm, 96 h
Micromonaspora sp. (sponge)                    PCV: 4%        Macrolide         [30, 31]
0.25 L, glucose, starch, soybean meal, pH 7,                  (4)
28 ◦ C, 250 rpm, 120 h
Micromonaspora sp. (seawater)                  PCV: 30%       Arisostatins      [32, 33]
0.10 L, glucose, protein, L-leu, pH 7,                        A, B (22)
30 ◦ C, 200 rpm, 8 d
Haliangium sp. (seaweed)                       pH course:     Haliangicin       [34, 35]
0.10 L, casitone, YE, pH 7, adsorber resin;    7–7.8%         (10)
28 ◦ C, 180 rpm, 17 d
Bacillus sp. (marine)                          OD620 : 0.47   Selenohomo-       [36]
0.10 L, ASW, glucose, seleno-D, L-met,                        cystine (1)
27 ◦ C, 5 d
Actinomadura sp. (polychaete)                  PCV: 16%       Polycyclic        [37]
0.25 L, glucose, tryptone, pH 7,                              xanthone
28 ◦ C, 250 rpm, 96 h                                         (16a )
a   Activity (IC–1 )
                50



origin: for the cultivations of species of Haliangium, Bacillus and Actino-
madura, producing very interesting bioactive metabolites [34–37].

Metabolite Production using Bacteria (Bioreactor Scale)

Batch cultivations on a scale ranging from 3 L to 1000 L have been reported
for the production of the bioactive compounds thiomarinol, hydroxyakalone,
macrolactins, and chalcomycin B by Alteromonas sp., Agrobacterium auran-
tiacum, Bacillus sp. and Streptomyces sp., respectively [38–41]. The technical
details on agitation and aeration are partially given, but in other cases, such
as for the 1000-L bioreactor culture of Agrobacterium aurantiacum, this infor-
mation is lacking (Table 4). No values have been noticed for all four bacteria
concerning the time courses on growth and metabolite. On the other hand,
the final concentrations of the compounds were exactly documented, ranging
from 0.16 mg L–1 (hydroxyakalone) to 12.5 mg L–1 (thiomarinol). For a Strep-
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi              37

Table 4 Bioreactor cultivations of marine mesophilic bacteria with respect to import-
ant bioprocess conditions and metabolites. Bioactivities of these metabolites: cytotoxic,
antibacterial, antifungal, antitumoural, xanthine oxidase inhibiting, respectively

Strain (origin)                                Growth                Product         Ref(s)
Cultivation conditions                         behaviour             (mg L–1 )

Alteromonas rava (sea water)                   No details            Thiomarinol     [38]
15-L Batch, MB,                                                      (12.5)
23 ◦ C, 100 rpm, 0.5 v/vm, 23 h
Agrobacterium aurantiacum (marine)             No details            Hydroxyakalone [39]
1000-L Batch, ASW, peptone, YE, glucose,                             (0.16)
20 ◦ C, 4 d
Bacillus sp. (macroalga)                       No details            Macrolactins  [40]
3-L Batch, MB, 30 ◦ C, 3–7 d                                         G-M (7)
Streptomyces sp. (mangrove sediment)           No details            Chalcomycin B [41]
18-L Batch, YE – malt extract medium,                                (0.43)
pH 6.5, 28 ◦ C, 120 rpm, 0.28 v/vm, 3 d
Streptomyces sp. (sediment)                    Packed cell           Bioxalomycins   [42]
300-L Batch, glucose, dextrin, NZ-amine,       volume: 3%a           (9)a
YE, 28 ◦ C, 200 rpm, 0.67 v/vm, 50 h
Alteromonas putrefaciens (fish)                 Biomass (g L–1 ):     C20:5           [43]
2-L Fed-batch, peptone (220 g L–1 ),           29 (A)                (100/A-C)
YE (110 gL–1 ), pH 7;
feeding strategies A, B, Cb                    43 (B)
20 ◦ C, 500 rpm, 15–25 h                       41 (C)
Agrobacterium sp. (tunicate)                   pH:7.2 → 11;          Agrochelin      [44]
50-L Batch, instant ocean salts, glucose,      pO2 (0 → 10 h):       (5)
pH 7.2, 28 ◦ C, 250 rpm, 1 v/vm, 1.5 bar,      95% → 82%
25–35 h
Bacillus cereus (sponge)                       pH: 7.5 → 8.2         Thiopeptides    [45, 46]
18-L Batch, oatmeal, YE, pH 7.5,                                     (1.2)
28 ◦ C, 250 rpm, 1 v/vm, 48 h
Microbacterium sp. (sponge)
(a) 40-L Batch, ASW, glucose, peptone, YE,     Biomass: 12 g L–1     GGL.2c (200)    [47]
     pH 7.5, 30 ◦ C, 800 rpm, 0.4 v/vm, 27 h   µmax = 0.10 h–1
(b) 40-L Batch, ASW, glycerol, peptone, YE,    Biomass: 11 g L–1 ;   GGL.2c (300)    new
     pH 6.5, 30 ◦ C, 500 rpm, 0.4 v/vm, 33 h   µmax = 0.13 h–1
Bacillus pumilus (sponge)                      Biomass: 10 g L–1 ; Diglucosyl        [48]
30-L Batch, ASW, glucose, YE, pH 7.5,          µmax = 0.20 h–1     glycerolipid
30 ◦ C, 500 rpm, 0.4 v/vm, 11 h                                    (90)
Pseudoalteromonas sp. MH-Cr6.3 (sponge):       Biomass: 16 g L–1 ; C16:1 (380)       new
30-L Batch, MB, ad. YE, peptone, glucose,      µmax = 0.62 h–1     C17:1 (50)
pH 7.6, 27 ◦ C, 520 rpm, 0.2 v/vm, 10–25 h
a relative units; b A: total cell mass; B: total cell mass + cell yield; C: growth rate, for
control; c glucosylmannosyl glycerolipid
38                                                                  S. Lang et al.

tomyces sp. from a mangrove sediment, bioactive bioxalomycins were de-
tected in both the supernatant and cell extract samples from 30-L and 300-L
fermentations, repectively. Production of antibiotic activity peaked at 48–50 h
(measured by HPLC) and closely paralleled cell growth, during which time
glucose was more rapidly assimilated than dextrin. Unfortunately the time
course values were expressed in relative units [42].
    For five other bacterial metabolite productions the information was suffi-
cient (Table 4): Alteromonas putrefaciens, accumulating eicosapentaenoic acid
(EPA) intracellularly, requires peptone and yeast extract for growth. Hib-
ino et al [43] tried to raise the cell yield from the nutrients supplied to
the bioreactor. For this purpose a comparison of three automated feeding
strategies for peptone (220 g L–1 ) and yeast extract (110 g L–1 ) solutions was
performed in 2-L fed-batch cultures. Control indices were (a) total mass,
(b) total mass and estimated cell yield, and (b) growth rate. In the case
of (a), a considerable amount of peptide accumulated in the later phase,
indicating overfeeding of the complex nutrients. Strategies (b) and (c) en-
abled the overfeeding to be avoided, resulting in more effective utilization
of the nutrients. Overall, the maximum cell concentrations were increased
by 1.5 times, while the total amount of nutrients fed were reduced by half.
Agrochelin, a new alkaloid cytotoxic substance, was produced by the fermen-
tation of Agrobacterium sp. isolated from the marine tunicate Ecteinascidia
turbinata. Using a bioreactor of capacity 75 litres with 50 litres of the pro-
duction medium, the cultivation was carried out under well-documented
physical conditions, and was additionally recorded quantitatively over time
with respect to pH, oxygen partial pressure and product concentration [44].
The minimal partial pressure of oxygen was registered at 10 h. The produc-
tion of the active compound started at 20 h, and the maximum production
rate occurred at between 25 and 35 h. Recently novel thiopeptides have been
found in the culture broth of Bacillus cereus, which was isolated from the
marine sponge Halichondria japonica [45, 46]. They exhibited potent an-
tibacterial activities against staphylococci and enterococci, including multiple
drug resistant strains. The graph on the time course of fermentation indi-
cated that the production of thiopeptide 1 started 24 h after inoculation, then
increased and reached a maximum (5.6 mg L–1 ; HPLC data) at 96 h. In con-
trast, the production of thiopeptide 2 showed a maximum (9.6 mg L–1 ; HPLC
data) at 24 h, and then decreased. After collecting 100 litres of fermentation
broth (from several 18-L cultivations, each lasting 48 h), the subsequent iso-
lation and purification procedure resulted in 120 mg thiopeptides 1 and 2
(= 1.2 mg L–1 ).
    Slightly higher contents of bioactive metabolites, new glycoglycerolipids,
were reported for cultivations with Microbacterium sp., isolated from the
sponge Halichondria panicea, and Bacillus pumilus strain AAS3, isolated
from the sponge Acanthella acuta. In general, 10-L bioreactors (Fig. 1) and
50-L bioreactors were used for these experiments. The production of the
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi    39




Fig. 1 Top view of an open 10-L bioreactor (stirred tank) used for the cultivation of
metabolite-producing marine mesophilic bacteria. Foto: V. Lurtz


glucosylmannosyl-glycerolipid GGL.2 was favoured when the former strain
was grown on artificial seawater medium supplemented with 20 g L–1 glu-
cose [47]. Glycoglycerolipid production was correlated with growth and
reached a maximum value of 200 mg L–1 at a biomass of 12 g L–1 . The time
course data on pO2 , QO2 , and QCO2 illustrate the different growth phases of
the bacterium very clearly. Recently, optimisation of the carbon sources using
shake-flask experiments has shown that glycerol affords the highest specific
glucosylmannosyl-glycerolipid production. After scaling up in a 40-l bioreac-
tor volume, the product yield approached 300 mg L–1 or 25 mg per g biomass
(Fig. 2). The native product shows interesting bioactivities [49].
   Bacillus pumilus produced a diglucosyl-glycerolipid (GGL11) with 14-
methylhexadecanoic acid and 12-methyltetradecanoic acid as the main fatty
acid moieties. On a 30-L scale, using artificial seawater supplemented with
glucose (20 g L–1 ), yeast extract (10 g L–1 ), and suitable nitrogen/phosphate
sources, the growth-associated glycoglycerolipid production reached its max-
imum yield of 90 mg L–1 after 11 h [48]. The physiological activity indicated
by the pO2 electrode data, as well as QO2 and QCO2 , were well correlated
with cell growth. Anti-tumor-promoting studies of both classes of glyco-
glycerolipids showed that the carbohydrate/glycerol backbones had a more
potent inhibitory activity than the native acylated compounds. In inhibi-
tion studies towards the tumour cell lines HMO2 and Hep G2, the native
glucosylmannosyl-glycerolipid was very effective [48].
40                                                                            S. Lang et al.




Fig. 2 40-L Batch cultivation of Microbacterium sp. HP2. Conditions: 50 L bioreactor; ar-
tificial seawater, 20 g L–1 glycerol, 3.5 g L–1 peptone, 3.5 g L–1 yeast extract; pH adjusted
to 7.5; 30 ◦ C; 500 rpm; aeration rate 0.4 v/vm. GGL.2: 1-O-acyl-3-[α-glucopyranosyl-
(1-3)-(6-O-acyl-α-mannopyranosyl)] glycerol, with 14-methyl-hexadecanoic acid and
12-methyl-tetradecanoic acid as lipid moieties

   In other recent studies by our group, a yellow-pigmented bacterium was
isolated from the marine sponge Crella rosea (collected from the Adriatic
coast in Croatia at a depth of 3–30 m). The strain was gram-negative, aero-
bic and polar flagellated. The nearest phylogenetic neighbour was determined
by 16 S rDNA sequence analysis as Pseudoalteromonas piscicida (99.8%) [50].
Solvent extract of this Pseudoalteromonas sp. strain MH-Cr6.3 was found to
inhibit the growth of different microorganisms such as Bacillus megaterium,
Staphylococcus aureus, Pseudomonas aeruginosa, and Ustilago violacea. The
biologically-active agent was identified as a mixture of C16:1 and C17:1 acids.
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi            41

Despite the well-known structures of these compounds, a bioprocess en-
gineering approach to this marine bacterium was of interest. After initial
shake-flask and bioreactor studies leading to maximum specific growth rates




Fig. 3 30-L Batch cultivation of Pseudoalteromonas sp. MH-Cr6.3. Conditions: 50 L biore-
actor; marine broth, additionally 30 g L–1 yeast extract, 5 g L–1 peptone, 5.5 g L–1 glucose;
pH adjusted to 7.6; 10% of inoculum; 27 ◦ C; 520 rpm; aeration rate 0.2 v/vm
42                                                                        S. Lang et al.

of 0.21 h–1 and fatty acid contents of 60 mg L–1 , the basis medium and physi-
cal parameters were optimised. Finally, after supplementing the marine broth
with 30 g L–1 yeast extract, 5 g L–1 peptone and 5.5 g L–1 glucose, the 30-L cul-
tivation was carried out at 27 ◦ C, pH 7.6 ± 0.2, 520 rpm, and at an aeration
rate of 0.2 v/vm. Figure 3 shows that the biomass increased to 16 g L–1 at 8.5 h
which was followed by a slow decrease down to 12 g L–1 after 25 h. The maxi-
mal growth rate of 0.62 h–1 was reached after 4–5 h. Antimicrobial fatty acids
C16:1 and C17:1 were first detected (determined as an inhibition zone towards
B. megaterium) after 3–5 h and giving a maximum between 10 and 15 h. After
chromatographic isolation, maximum concentrations of 380 mg L–1 of C16:1
and 50 mg L–1 of C17:1 were detected. Figure 4 indicates that the metabolites
are mainly cell-associated. In terms of the individual actual concentrations of
free amino acids (0.15 to 2.3 g L–1 at t = 0) detectable in the medium, Fig. 5
shows that asparagine, arginine and histidine were consumed totally after
4–5 h, and serine, threonine and phenylalanine after 6–7 h. After 10 h the
content of all free amino acids was zero. Considering the diversity of the car-
bon and nitrogen substrates, ranging from single amino acids (very quickly
utilized) via peptides to glucose, the reason for the multiphasic course of pO2
in Fig. 3 becomes clear.
   In 2000, within the network of Marine Biotechnology in Lower Saxony
(Germany), with special focus on bacteria from the German North Sea,
our group started to transfer the 100-mL flask experiments to 8- to 40-L-




Fig. 4 TLC-based spectrum of the cell-associated products (solvent extract) obtained
during a 30-l batch cultivation of Pseudoalteromonas sp. MH-Cr6.3. Conditions: see leg-
end of Fig. 3. TLC: thin layer chromatography. Developing system: CHCl3 /CH3 OH/H2 O,
(65/15/2; v/v/v). Detecting agent: anisaldehyde/sulfuric acid reagent. Products at RF
values of 0.03–0.20: desoxythymidine, desoxyadenosine, adenosine, uridine
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi   43




Fig. 5 Amino acid concentration profiles during the batch-cultivation of Pseudoal-
teromonas sp. MH-Cr6.3. Conditions: see legend of Fig. 3


scale glass or stainless steel bioreactors. To facilitate cooperative studies by
chemists, large amounts of crude natural products were made available for
isolating pure metabolites. The growth and metabolite production of strain
Hel45, identified as Oceanibulbus indolifex [51], was presented recently [15].
Publications on additional strains and bioreactor cultivations will follow in
the near future.

Metabolite Production using Fungi

An interesting overview, discussing mainly the various molecular structures
of metabolites derived from cultivations of marine fungi, has been given by
Biabani and Laatsch [52]. Despite the relatively large number of metabolites
found, the number of publications presenting extra biotechnological informa-
tion is small. With some exceptions, shake-flask experiments and bioreactor-
based studies unfortunately contain no graphs on fungal growth curves, on
specific growth rate, productivity of metabolite synthesis, and so on. On the
other hand, the downstream processing, beginning with the separation of
mycelia and supernatant and including the extraction up to the chromato-
graphic purification of single metabolites, is always well described. Table 5
confirms this fact. Except for the data on nutrients, working volumes of culti-
vation vessels, temperatures and incubation times, in many cases the authors
44                                                                           S. Lang et al.

Table 5 Shake-flask and bioreactor cultivations of mesophilic fungi with respect to
important bioprocess conditions and metabolites. Bioactivities of these metabolites:
antibacterial, antifungal, antimicroalgal, neuritogenic, PAF (platelet activating factor)-
antagonistic

Strain (origin)                            Growth            Product             Ref(s)
Cultivation conditions                     behaviour         (mg L–1 )

Shake-flask cultures:                      No details         Diketopiperazine    [53]
Strain M-3 (laver) 0.3 L, potato dextrose                    (0.27)
broth, 20 ◦ C, 21 d
Fusarium sp. (alga)                        Biomass:          Halymecins A-C      [54]
0.4 L, seawater, glucose, YE, pH 8.0,      5.5 g L–1         (16)
30 ◦ C, 100 rpm, 96 h
Bioreactor cultures:                       No details        Epolactaene         [55]
Penicillium sp. (sediment)                                   (0.55)
18 L, glucose, starch, soybean meal,
pH 5.8, 27 ◦ C, 300 rpm, aeration:
0.39 v/vm, 50 h
Phoma sp. (crab)                           No details        Phomactins          [56, 57]
300 L, sucrose, peptone, potato, pH 8.5,                     (1.90)
23 ◦ C, 80 rpm, 12 d
Keissleriella sp. (sediment)               No details        Naphthalenone       [58]
15 L, potato dextrose broth,                                 (0.05)
25 ◦ C, 200 rpm, 7 d
Exophiala pisciphila (sponge)              OD600 = 2.2       Exophilin A         [59]
15 L, ASW, glucose, L-asparagine, pH 7,    µmax = 0.03 h–1   (2.4)
25 ◦ C, 0 rpm, 0.67 v/vm, 10 d
Hypoxylon oceanicum (mangrove wood) Wet weight:              Lipodepsipeptide/ [60–62]
300 L, seawater, glycerol, soy peptone, 30 g L–1             Polylactones
pH 7, 22 ◦ C, 250 rpm, 1 v/vm, 6–8 d    µmax = 0.08 h–1      (300/30)



only focus on the isolation of a particular bioactive product. This is the case
for cultivations of a non-identified fungus M-3 [53], Penicillium sp. [54],
Phoma sp. [56, 57], and Keissleriella sp. [58], although the scales ranged from
0.3-L to 300-L working volume. Two fungal metabolite productions have been
reported on a broader basis. In the case of Exophilia pisciphila, a member of
the so-called “black yeasts”, a 15-L cultivation in a 20-litre glass bottle fer-
menter was carried out at 25 ◦ C for ten days without agitation under aeration
of 0.67 v/vm [59]. The time course of the pH of the medium, the optical dens-
ity at 600 nm, and the production of antibacterial substances as evaluated by
inhibition zone diameter are all presented. The antibacterial activity of the
broth drastically increased after six days and reached its maximum after ten
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi   45

days of cultivation. After the solvent-extraction of 15 L broth and purification,
the bioactive substance (36 mg) was identified as exophilin A. The cell wall-
targeted antifungal activity of Hypoxylon oceanicum extracts resulted from
the production of novel lipodepsipeptides and well-known macrocyclic poly-
lactones [60–62]. In an optimised medium, the growth in 30-L and 300-L
reactors was rapid, with cells reaching stationary phase by day 3. By day 8, the
extensive lysis of filaments was noted microscopically, and cell mass sharply
decreased. Production of metabolites thus occurred in the stationary phase of
growth. Titers of the lipodepsipeptides and the polylactones reached approxi-
mately 300 mg L–1 and 30 mg L–1 , respectively, determined by reverse-phase
HPLC.

4.1.2
Biopolymer Production

Non-Proteinogenic Biopolymers

Non-proteinogenic biopolymers from marine prokaryotes, both bacteria
and archaea, offer a number of novel material properties and commercial
opportunities. The characteristics of marine exopolysaccharides, melanins
and polyhydroxyalkanoates that enhance the survival abilities of the or-
ganisms producing them can be exploited for a number of products, rang-
ing from emulsifiers via adhesives to bioplastics. A summary was given
by Weiner [63].
   Concerning exopolysaccharides, Abu et al [64] reported on Shewanella
colwelliana, a marine bacterium isolated in association with the oyster Cras-
sostrea virginica, which produced an abundant exopolysaccharide with po-
tential adhesive properties. Immunosuppressive effects of a 70-kd polysac-
charide have been reported by Ohmori et al [65] when produced in small
amounts by the marine Aeromonas caviae (Table 6). Using Alteromonas sp.
in a 1.4-L batch cultivation on marine broth supplemented with glucose,
a polyelectrolyte-type polysaccharide was excreted during the stationary
phase of growth; it contained glucose, galactose, glucuronic acid, and galac-
turonic acid as major components. After 120 h, 11 g L–1 product could be
harvested. The cell dry weight, composed of the cells and firmly-bound
polysaccharide, was maximal after 72 h of culture and then decreased as the
polysaccharide was released into the medium [66].
   In terms of producing another biopolymer encountered inside every cell
but rarely found extracellularly—ribonucleic acid (RNA)—a special photo-
synthetic marine bacterium seems to be highly applicable. Photosynthetic
bacteria have been used for wastewater treatment because they have a rela-
tively high growth rate and can utilize a wide range of organic compounds.
However, difficulty in cell separation due to their low flocculating ability has
been the major disadvantage in this application. Now the new photosyn-
46                                                                             S. Lang et al.

Table 6 Shake-flask and bioreactor cultivations of mesophilic microbes with respect to
important bioprocess conditions and mainly non-proteinogenic biopolymer production.
MLSS = Mixed-liquor suspended solids (cells recovered by filtration and dried, also con-
taining surface-bound products). YXP/S = yield coefficient on g MLSS per g acetate

Strain (origin)                          Growth                Product                Ref(s)
Cultivation conditions                   behaviour

Aeromonas caviae (fish)                   OD650 = 1.4           Polysaccharide:        [65]
Shake-flask, peptone, YE, glucose                               72 mg L–1
25 ◦ C, 40 rpm, 24–48 h
Alteromonas sp. (polychaete)             Biomass:              Polysaccharide:        [66]
1.4-L bioreactor culture,                5–14 g L–1            11 g L–1
MB, glucose, pH 7.2,
25 ◦ C, 350–800 rpm, 0.67 v/vm, 120 h
Rhodovulum sp. (sediment)                1.1 g MLSS L–1        Flocculant:          [67]
0.1-L shake-flask culture,                (8 × 105 CFU mL–1 )   RNA (62 mg g–1 )
NaCl, glutamate, malate, pH 8.0,                               DNA (8 mg g–1 )
20 ◦ C, aerobic dark conditions, 120 h                         Protein (49 mg g–1 )
Rhodovulum sp. (sediment)                                      Flocculant:            [70]
0.66 l single-tower fermenter, NaCl,                           RNA
acetate, pH 8.0, 20 ◦ C;                                       DNA
gassing (N2 or O2 or air):                                     Protein
0.45–0.75 v/vm,
(a) Batch cultures, dissolved oxygen     1.1 g MLSS L–1
    content: 8 mgL–1 , 80 h              YXP/S = 0.32
(b) Continuous feeding of medium;        42.7 g MLSS L–1
    dissolved oxygen content:
    3–10 mgL–1 , 750 h
Rhodovulum sp. (sediment)                2.8 g MLSS L–1        Exo-/Intra-            [71]
0.5 L (1-L jar fermentor), NaCl,         YXP/S = 0.45–0.55     cellular RNA:
acetate, pH 8,                                                 460 mg L–1
continuous aerobic cultivation
in the dark, 20 ◦ C, 300 rpm, 1 v/vm,
D = 0.32–0.5 h–1


thetic bacterium Rhodovulum sp. PS88, recently isolated from sea sediment
mud from a shrimp farm, could fulfill the above requirements. Originally
enriched under anaerobic light conditions, its growth was better under aer-
obic dark than under anaerobic light conditions. 30 ◦ C was found to be
optimal for growth but flocculation at 30 ◦ C was weaker than that at 20 ◦ C.
When PS88 was grown in glutamate/malate medium containing 3.5% NaCl,
RNA, DNA and protein were produced exocellularly, causing obvious floc-
culation after 120 h [67, 68]. After finding that Ca2+ and Mg2+ enhance the
self-flocculation [69], high cell density cultures were achieved by continu-
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi   47

ously feeding the medium without performing cell recycling by filtration [70].
Using a single-tower fermentor, after initial batch cultivation for 180 h, con-
tinuous medium feeding (3% NaCl, 15 g L–1 sodium acetate) was started,
leading to 43 g L–1 of mixed-liquor suspended solids (including cells and ex-
ocellular RNA) at 750 h of culture with a high consumption rate of acetate
(22.5 g L–1 d–1 ). These flocculated cells exhibited good settling characteristics.
Later on, the feasibility of exocellular RNA production by PS88 was investi-
gated, focusing on the production for medical purposes of RNA using a con-
ventional continuous culture system [71]. At a dilution rate of 0.32–0.5 h–1 ,
the maximum RNA production was 460 mg RNA per L broth (200 mg RNA
per g suspended solids).

Enzymes and Other Proteins

Marine microbial enzymes have several advantages for industrial utiliza-
tion. The optimum activity of marine enzymes usually occurs at high salin-
ity, making these enzymes utilizable in many harsh industrial processes,
where using concentrated salt solutions would otherwise inhibit many en-
zymatic transformations. In addition, most marine microbial enzymes are
considerably thermotolerant, remaining stable at room temperature over long
periods [72, 73].
    Table 7 shows a selected list of recent advanced shake-flask studies, as well
as of controlled bioreactor investigations on this topic. To examine the degra-
dation and response of marine bacteria to chitin, Vibrio harveyi was grown
on either α-chitin (snow crab) or β-chitin (squid pen). Initial growth rates
were about fivefold higher in β-chitin than in α-chitin. Final cell yields were
5.5-fold higher in β-chitin than in α-chitin. Chitinase activity was over sixfold
higher in β-chitin than in α-chitin for five days (after the initial lag phase).
The faster degradation of β-chitin is the result of an increased number of
cells [74]. In order to find strains with α-1,4- and 1,6-glucosidase enzymes
with potential uses in shrimp feed production, Bacillus strains were isolated
from the marine environment [75]. Among them, strain LMM-12 produced
large amounts of an extracellular thermostable α-glucosidase that permitted
good growth on starch.
    Alkaline proteases are robust enzymes with considerable industrial po-
tential in detergents, leather processing, silver recovery, medical purposes,
food processing, feeds and chemical industry, as well as in waste treatment.
In terms of their microbial production, some recently published examples
have been found: Hyphomonas jannaschiana, isolated from shellfish beds
near hydrothermal vents, exhibits extracellular heat stable protease activ-
ity during the late-logarithmic and stationary phases of cell growth [76].
Teredinobacter turnirae cells were immobilized in calcium alginate beads
and used for alkaline protease production. The maximum activity was ob-
tained at 3% (w/v) sodium alginate and 3% CaCl2 concentrations with a 1/2
48                                                                            S. Lang et al.

Table 7 Shake-flask and bioreactor cultivations of mesophilic microbes with respect to
important bioprocess conditions and protein (mainly enzymes) production

Strain (origin)                        Growth                   Protein             Ref(s)
Cultivation conditions                 behaviour

Shake-flask cultures:
Vibrio harveyi (marine)                Cell densities:          Chitinase:          [74]
ASW, snow crab (α-chitin) or           < 109 cells mL–1 (α-ch.) < 10–100 µmol
squid pen (β-chitin), pH 7.5, 9 d      1010 cells mL–1 (β-ch.) MUFa mL–1 h–1
Bacillus sp. (sediment)                OD600 = 2.7              α-1,4 and α-1,6-    [75]
Mineral salts, glucose, starch,        µmax = 0.75 h–1          glucosidases:
37 ◦ C, 300 rpm, 16 h                                           690 U mg–1
Hyphomonas jannaschiana                OD550 => 1               Alkaline protease: [76]
(mussel shell) MB, 37 ◦ C, 17 h                                 0.36 U mL–1
Teredinobacter turnirae (shipworm) Cell/alginate ratio:         Alkaline            [77]
immobilized in Ca-alginate.        1/2 (w/v),                   protease:
0.05 L, NH4 Cl, sucrose, pH 8,     bead size: 2–6 mm            2400 U mL–1
30 ◦ C, 120 rpm,
repeated batches (for 72 h)
Bioreactor cultures:
Vibrio harveyi (seawater)              1.77 g L–1 biomass       Protease:           [78]
1.0 L, MB, skim milk, pH 7.2,          µmax = 0.507 h–1         4.28 U mg–1
30 ◦ C, 700 rpm, 0.8/0.5 v/vm, 8 h     (0.8 v/vm)               (0.5 v/vm)
Pseudomonas sp. (marine)               Cell/alginate ratio:     L-Glutaminase:      [79]
immobilized in Ca-alginate             1/3 (w/v),               36 U mL–1
Packed bed reactor (h = 45 cm,         bead size: 4 mm          (D = 0.64 h–1 ,
d = 3.6 cm); seawater, glutamine                                2% glutamine)
(0.5–2%, w/v), glucose, pH 6,
30 ◦ C, D = 0.64–1.48 h–1 /
continuous process
Vibrio sp. (marine), recombinant                                                    [80]
NaCl, tryptone, pH 6.6, 30 ◦ C                                  Recomb. EtxBb :
(a) 1.4-L chemostat,                   no details               3.0 mg L–1 h–1
    with carbenicillin, 1 v/vm,                                 (D = 0.14 h–1 )
    500 rpm, D = 0.14–0.42 h–1
(b) Hollow fibre reactor                no details               9.4 mg L–1 h–1
    (100 kd cut-off);
    medium flow: 10 mL h–1 ;
    aeration: 1 Lmin–1
a   Methylumbelliferone, b B-subunit pentamer of E. coli heat-labile enterotoxin
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi   49

cell/alginate ratio, i.e. 2400 U mL–1 . The beads were used for eight successive
fermentation batches, each lasting 72 h, without loss of volumetric protease
productivity [77]. Very recently, Vibrio harveyi, a ubiquitous luminous bac-
terium from warm marine waters, was cultured in a 1.5 L jar-fermentor [78].
Using marine broth supplemented with skim milk as medium, the agitation
and aeration rates were varied to obtain the highest extracellular protease
activity. Specific growth rate increased as a consequence of increasing aera-
tion rates. The maximum activity of 4.28 units per mg protein was achieved
with 700 rpm and 0.5 v/vm. For L-glutaminase production, a marine Pseu-
domonas sp. was immobilised by Ca-alginate gel entrapment and then used
under repeated batch process and a continuous process employing a packed
bed reactor (PBR) [79]. Immobilised cells could produce 25 U mL–1 of en-
zyme over 20 cycles of repeated batch operation and did not show any decline
in production upon reuse. Continuous production of L-glutaminase in PBR
was studied at different substrate concentrations and dilution rates. Enzyme
production decreased with increasing dilution rate, regardless of the sub-
strate concentrations employed. The maximal enzyme yield (36.05 U mL–1 )
was observed at a dilution rate of 0.64 h–1 in the medium containing 2%
w/v glutamine, and maximal productivity (30.56 U mL–1 h–1 ) was recorded at
a dilution rate of 1.27 h–1 with the same glutamine concentration. For a non-
enzymatically working protein, the non-toxic B-subunit pentamer of E. coli
heat-labile enterotoxin EtxB, cloned into a marine Vibrio sp., a comparison
was made between growth and recombinant protein synthesis in three types
of bioreactors [80]. Resistance to carbenicillin was used to select plasmid-
containing cells. In batch and continuous culture, volumetric productivities
were highest when cells were grown in the presence of carbenicillin. Without
antibiotic selection, the highest volumetric productivity (9.4 mg EtxB L–1 h–1 )
was observed in hollow-fibre bioreactors. The production phase could be
maintained for over 50 h.
   Solid-state (or -substrate) fermentation (SSF) is characterised by a fermen-
tation process on a solid support that has a low moisture content (lower limit
≈ 12%). The solid materials are classified into two main categories: inert
supports (synthetic materials) and non-inert supports (such as agricultural
residues, including wheat straw, rice hulls and corn cobs). The former act
as an attachment place, whereas the latter also function as a source of nu-
trients. Robinson et al [81] and Rodriguez Couto et al [82] propagated SSF
to produce a high product concentration that, additionally, has a relatively
low energy requirement. SSF has been exploited for the production of food,
feed, fuel and enzymes. It can be carried out on a variety of agricultural
residues. But the use of this technology for the production of commercially
valuable metabolites is at present under-utilized, with a strong preference
towards conventional and familiar liquid fermentations. The authors only
concede that problems arise when experiments are scaled-up [81]. In terms
of application of this cultivation technology to marine biotechnology, Table 8
50                                                                       S. Lang et al.

Table 8 Small-scale solid state fermentations with marine mesophilic microbes that are
performed to produce enzymes

Strain (origin)                             Solid          Enzyme              Ref(s)
Cultivation conditions                      substrate      (activity)

Vibrio costicola (marine)                   Polystyrene    L-Glutaminase       [83]
0.25 L-flask; moistening: mineral salts      beads          (88 U g–1 solid)
medium/L-glutamine; 37 ◦ C, 36 h            (5 g)
Vibrio costicola (marine)                   Polystyrene    L-Glutaminase       [84]
0.25 L-flask; moistening (80%): seawater/    beads          (157 U g–1 solid)
L-glutamine, pH 7; 35 ◦ C, 24 h             (5 g)
Beauveria sp. (sediment)                    Polystyrene    L-Glutaminase       [85]
0.5 L-flask; moistening (80%): seawater/     beads          (50 U mL–1 )
L-glutamine, glucose, pH 9; 27 ◦ C, 96 h    (10 g)
Beauveria bassiana (sediment)               Prawn waste    Chitinase           [86]
Petri plates; moistening (90%): seawater/   (5 g)          (248 U g–1 solid)
prawn waste, pH 9.5; 27 ◦ C, 5 d
Beauveria bassiana (sediment)               Wheat bran/    Chitinase           [87]
Petri plates; moistening (75%): seawater/   chitin         (246 U g–1 solid)
wheat bran, chitin, pH 9; 28 ◦ C, 48 h      (5 g)



presents some examples of the production of L-glutaminases and chitinases,
respectively. The experiments were performed at a very small volume level,
for example on petri plates or in flasks < 1 L. In the case of L-glutaminase
production with Vibrio costcola and Beauveria sp., the use of polystyrene as
the solid support was preferred [83–85]. Natural polymeric substrates, such
as prawn waste (waste from the shellfish processing industry, containing 23%
chitin) or colloidal chitin (mixed with wheat bran) were successful used to
overproduce chitinases [86, 87].

4.2
Bioprocess Engineering Data
on Marine Psychrophilic/Psychrotrophic Microbes

4.2.1
Enzyme and Metabolite Production

Psychrophilic marine microbes have been found in polar regions, especially
in the seas of the Antarctic, and in deep-sea sediments at temperatures lower
than 3 ◦ C. They have upper growth limits below 20 ◦ C. For comparison, psy-
chotrophic microbes are capable of growing at 0 ◦ C but show an upper limit
of 40 ◦ C [88]. In general, both types produce enzymes adapted to function-
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi   51

ing at low temperatures. These enzymes are characterised by a high catalytic
efficiency at low and moderate temperatures but are rather thermolabile.
Compared to their mesophilic counterparts, their molecular changes tend to
increase the flexibility of the protein structure by weakening the intramolec-
ular interactions and by increasing the interactions with the solvent [89–92].
The application of these enzymes offers considerable potential to the biotech-
nology industry, for example, in the detergent and food industries, for the
production of fine chemicals, and in bioremediation processes.
    Table 9 shows results from selected shake-flask and bioreactor experiments
in advanced cultivation studies on psychrophilic/psychrotrophic microbes.
For instance, the psychrotrophic yeast Candida humicola secretes an acidic
protease into the medium [93]. The secretion of the protease was greater dur-
ing exponential growth at low temperatures than during growth at higher
temperatures. Bacteria such as Moraxella sp., Bacillus sp., Alteromonas halo-
planktis, Pseudoalteromonas haloplanktis and Arthrobacter sp. were shown to
be able to excrete cold-active lipase, protease and polysaccharide-degrading
enzymes [94, 95]. In all cases the authors showed that the strains grew faster
at 17 to 25 ◦ C/28 ◦ C; but the enzyme production was maximal at tempera-
tures close to those of their original environment (2 ◦ C to 4 ◦ C). Two 10-L
cultivations were performed at 4 and 25 ◦ C for the protease (a subtilisin) of
the Antarctic Bacillus strain TA39 [96]. At 25 ◦ C, the production of cells at the
stationary phase was half that obtained at 4 ◦ C, whereas the protease secre-
tion hardly reached one third of that recorded at 4 ◦ C. After a lag phase of
about 20 h, the doubling time at 4 ◦ C during the exponential growth was 9 h
compared with 2 h at 25 ◦ C.
    Besides enzyme production, additional goals using cold-adapted microbes
have been attempted. For instance, the growth kinetics of two psychrotoler-
ant Antarctic bacteria, Hydrogenophaga pseudoflava and Brevibacterium sp.,
were examined over a range of temperatures in both batch culture and
glycerol-limited chemostat culture. In batch culture, the maximal growth
rates (µmax ) at 16 ◦ C and 2 ◦ C were 0.14 h–1 and 0.048 h–1 , respectively, for
Hydrogenophaga, and were 0.062 h–1 and 0.019 h–1 , respectively, for the other
strain. The values of µmax measured in the chemostat at 16 and 2 ◦ C corre-
sponded well with those measured in batch cultures at the same tempera-
tures. For example, with 0.5 g L–1 glycerol, for Hydrogenphaga at 16 ◦ C in
the chemostat, µmax was 0.139 h–1 , while at 2 ◦ C the µmax was 0.055 h–1 . For
Brevibacterium sp. at 16 ◦ C, the µmax was 0.095 h–1 and it was 0.024 h–1 at
2 ◦ C. For both bacteria, the specific affinity (µmax /KS ) for glycerol uptake was
lower at 2 than at 16 ◦ C, indicating a greater tendency to substrate limita-
tion at low temperature [98]. Additionally, the authors studied competition
between above strains in glycerol-limited chemostat experiments subjected
to non-steady-state conditions of temperature [99]. For metabolite produc-
tion at 5 ◦ C, the production of an aromatic amine, 2-phenylethylamine, by
the psychrophilic marine bacterium Psychroflexus torquis was reported [100],
52                                                                             S. Lang et al.

Table 9 Shake-flask and bioreactor cultivations of psychrophilic/psychrotrophic microbes
with respect to important bioprocess conditions and enzyme or metabolite production

Strain (origin)                            Growth              Product/Goal          Ref(s)
Cultivation conditions

Shake-flask cultures:
Candida humicola (Antarctic soil)          OD600 = 1           Acidic protease       [93]
Shake-flasks, yeast nitrogen base                               (15 U)
medium, glucose, BSA,
4 ◦ C (6 d), 15 ◦ C (4 d), 22 ◦ C (3 d),
Moraxella sp. (Antarctic seawater)                             Lipase:             [94]
0.1 L, NaCl, LB medium, pH 8.5,            td = 4.5 h (3 ◦ C) 1150 U mL–1 (3 ◦ C)
3 ◦ C; 17 ◦ C; 25 ◦ C; 250 rpm             td = 2.0 h (17 ◦ C) 380 U mL–1 (17 ◦ C)
Bacillus sp. (Antarctic seawater)                              Protease:           [94]
0.1 L, MB, pH 7.6                          td = 9.5 h (4 ◦ C) 6.8 U mL–1 (4 ◦ C)
4 ◦ C; 25 ◦ C; 250 rpm                     td = 2.0 h (25 ◦ C) 2.5 U mL–1 (25 ◦ C)
Alteromonas haloplanktis                                     α-Amylase:          [94]
(Antarctic seawater)                     td = 4.0 h (4 ◦ C) 1950 U mL–1 (4 ◦ C)
0.1 L, NaCl, LB medium, maltose, pH 7.6, td = 1.0 h (18 ◦ C) 259 U mL–1 (18 ◦ C)
4 ◦ C (80 h), 18 ◦ C (30 h); 250 rpm
Alteromonas haloplanktis (Antarctic)       Growth (28 ◦ C)     Cellulase (4 ◦ C)     [95]
Pseudoalteromonas haloplanktis             Growth (28 ◦ C)     Cellulase (17 ◦ C)
(Antarctic)
Arthrobacter sp. (Antarctic)               Growth (22 ◦ C)     Chitobiase (4 ◦ C)
Marine broth + inducers
Arthrobacter agilis (Antarctic sea ice)                        Carotinoids           [101]
0.1 L, MB, NaCl (0–10%),                   µmax (h–1 ):        (mg g–1 biomass):
5 ◦ C – 40 ◦ C, 150 rpm                    0.025 (5 ◦ C)       1.2 (5 ◦ C)
                                           0.230 (30 ◦ C)      0.2 (30 ◦ C)
Bioreactor Cultures:
Bacillus sp. (Antarctic seawater)                              Subtilisin:         [96]
10 L, marine broth, pH 7.6;                td = 9 h (4 ◦ C)    7 U mL–1 (4 ◦ C)
4 ◦ C; 25 ◦ C; 6 d                         td = 2 h (25 ◦ C)   2.6 U mL–1 (25 ◦ C)
Flavobacterium balustinum (salmon)         No details          Cp-70 protease:       [97]
3 L, peptone, YE, casein, pH 7.4,                              261 U mg–1
10 ◦ C, 150 rpm, 3 d                                           protein
Hydrogenophaga pseudoflava (Antarctic)      µmax (h–1 ):        Competition           [98, 99]
0.5 L chemostat, FC2 medium+glycerol,      0.139 (16 ◦ C)      studies
2 ◦ C, 16 ◦ C, D = 0.02 h–1                0.055 (2 ◦ C)
Brevibacterium sp. (Antarctic)             0.095 (16 ◦ C)
same conditions; D = 0.01 h–1              0.024 (2 ◦ C)
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi   53

but unfortunately without cultivation details. More information is available
from a report on carotinoid accumulation in the psychotrophic bacterium
Arthrobacter agilis in response to thermal and salt stress [101]. The maximum
growth rate occurred at 30 ◦ C, with a drastic decline as the cultivation tem-
peratures diverged. Lowering of the temperature resulted in a concomitant
increase in carotinoid production, which may contribute to membrane stabil-
isation at low temperature. Changes in growth rates were minimal in culture
media containing 0–2% NaCl, while a gradual decrease in growth rates oc-
curred at higher salinity.

4.2.2
Barophilic Strains: A Special Challenge

In most of the deep sea, microorganisms grow at 2–3 ◦ C and hundreds of bars
of hydrostatic pressure. At nearly 11 000 m, the Challenger Deep is the deep-
est known ocean site, and the microbes that are active there must be able to
function at pressures greater than 100 MPa. While the growth temperatures
of these organisms define them primarily as psychrophiles, their pressure op-
tima characterise them as barotolerant, barophilic, or obligately barophilic
strains [102]. Since the cultivation of microbes needing pressure for growth
does not permit the use of simple shake flasks, in this case advanced bio-
processes have been reported in the literature. In very early studies, the
formation of chitinases by psychrophilic and psychrotrophic marine Antarc-
tic bacteria were investigated under simulated deep-sea conditions [103]. Two
psychrophilic Vibrio sp. strains, isolated from depths below 2000 m, grew
well at 400 bars in culture bags deposited in pressure cylinders. Growth
rates and growth yields were similar or even higher than for the 1-bar cul-
tures; although the lag phases were somewhat prolonged in the pressurised
cultures. Corresponding to the retarded growth, chitinase formation in the
pressurised cultures lagged behind. Growth of the psychotrophic strains was
clearly restricted under simulated deep-sea conditions. Table 10 summarizes
some recent studies. For instance, using Shewanella sp. F1, belonging to the
major genera of cultivated barophiles, a continuous culture system that allows
growth in steady-state populations under pressures up to 71 MPa (700 atm)
was constructed and tested (Fig. 6). The bioreactor involved was a 500-mL
nylon-coated titanium reactor. The strain was grown at 0.1, 30.4 and 40.5 MPa
and dilution rates of 60 and 90% of the organism’s maximum growth rate
(determined at 0.1 MPa) in the required complex medium at levels of 3.3
and 0.33 mg of dissolved organic carbon per litre (10 mg L–1 yeast extract
and 1 mg L–1 yeast extract, resp.) in the reservoir. The data obtained at 3 ◦ C
show that at different growth rates and substrate concentrations this iso-
late maintained its overall barophilic character. The steady state populations
at elevated pressures were larger than the populations at 0.1 MPa (1 atm).
This was determined by both direct counting and viable counting. Similar
54                                                                            S. Lang et al.

Table 10 Bioreactor cultivations of psychrophilic/barophilic microbes with respect to im-
portant bioprocess conditions

Strain (origin)                           Growth                                  Ref(s)
Cultivation conditions                    behaviour

Shewanella sp.                           (a) 10 mg L–1 YE, D = 0.044 h–1 , 3 ◦ C: [102, 104]
(seawater; depth: 4900 m)                    106 cells mL–1 (0.1 MPa)
ASW, YE (10 mg L–1 ; 1 mg L–1 ), pH 7.3;     3 × 106 cells mL–1 (30.4 MPa)
3 ◦ C; 8 ◦ C.                            (b) 10 mg L–1 YE, D = 0.066 h–1 , 3 ◦ C:
Pressurised chemostat (0.5 L, titanium)      3 × 106 cells mL–1 (0.1 MPa)
Pressure: 0.1 MPa; 30.4 MPa, 40.5 MPa        7 × 106 cells mL–1 l (30.4 MPa)
Continuous culture:                      (c) 1 mg L–1 YE, D = 0.044 h–1 , 3 ◦ C:
D = 0.044 h–1 ; D = 0.066 h–1                8 × 104 cells mL–1 (0.1 MPa)
                                             9 × 104 cells mL–1 (30.4 MPa)
                                         (d) 10 mg L–1 YE, D = 0.066 h–1 , 3 ◦ C:
                                             3 × 104 cells mL–1 (0.1 MPa)
                                             9 × 104 cells mL–1 (30.4 MPa)
                                         (e) 10 mg L–1 YE, D = 0.044 h–1 , 8 ◦ C:
                                             5 × 105 cells mL–1 (0.1 MPa)
                                             3 × 106 cells mL–1 (40.5 MPa)
                                         (f) 10 mg L–1 YE, D = 0.066 h–1 , 8 ◦ C:
                                             2 × 105 cells mL–1 (0.1 MPa)
                                             6 × 105 cells mL–1 (30.4 MPa)
Moritella japonica                        µmax = 0.35(td )
                                                       –1                         [105]
(seawater, depth: 6356m):
MB, 10 ◦ C, 50 MPa (pressure vessel)
Shewanella sp.                            µmax = 0.15(td )
                                                       –1                         [16]
(sediment, depth of 10 898 m)
MB, 10 ◦ C, 70 MPa (pressure vessel)
Moritella sp.                             µmax = 0.20(td )
                                                       –1                         [16]
(sediment, depth of 10 898 m)
MB, 10 ◦ C, 80 MPa (pressure vessel)
Pseudomonas sp.                           Highest µmax values:                    [106]
(sediment, depth of 4418 m)               0.033 h–1 (10 ◦ C, 0.1 MPa)
KNO3 , MB, glucose, 10–37 ◦ C             0.074 h–1 (20 ◦ C, 0.1 MPa)
Pressure vessel, 0.1–60 MPa,              0.228 h–1 (30 ◦ C, 10 MPa)
without agitation                         0.205 h–1 (37 ◦ C, 20 MPa)
Strains KBRP1, KBRP4 (seaweed):           Survival (20 min at 120 MPa):           [107]
MB, 25 ◦ C                                96% (KBRP1)
High pressure batch reactor (120 MPa)     92% (KBRP4)



results were obtained at 8 ◦ C, which is the optimum temperature for this She-
wanella strain F1. With one exception (when optimum growth occurred at
40.5 MPa), optimum growth occurred at 30.4 MPa. When the number of vi-
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi        55




Fig. 6 Scheme of the pressurised chemostat used to cultivate Shewanella sp. isolated
at a depth of 4900 m. From [104]. R, medium reservoir; P, titanium pump head; DM,
HPLC drive module; PM, pressure module; V, injection valve; W, constant-temperature
water bath; CV, titanium culture vessel (0.5 L); M, Magnetic stirrer; SC, subsampling
chamber; BV, backpressure regulating valve; RV, receiving vessel. Heavy line: pressurized
part of the system


able cells was expressed as a percentage of the direct count, the value was
usually higher for elevated-pressure samples than for 0.1-MPa samples. The
percentage of viable cells was relatively low overall compared to the direct
cell count (about 25% of the maximum value) [102, 104]. Moritella japonica,
isolated from a Japan trench sediment, was able to grow in pressure vessels
under hydrostatic pressures ranging from 0.1 to 70 MPa and at temperatures
ranging from 4 to 15 ◦ C. The optimal temperature and pressure conditions for
growth were 15 ◦ C and 50 MPa, respectively [105]. Two extremely barophilic
bacteria, Shewanella sp. and Moritella sp., isolated from the Mariana trench,
Challenger Deep (about 11 000 m), gave their highest growth rates at pres-
sures of 70 and 80 MPa, respectively [16]. A barotolerant member of the genus
Pseudomonas (deep-sea isolate) was grown in a medium containing nitrate
ions without air or agitation [106]. The fastest specific growth rate at 30 ◦ C
was 0.228 h–1 at 10 MPa. The growth rate decreased as the hydrostatic pres-
sure was increased. The fastest growth rate at 37 ◦ C was 0.205 h–1 at 20 MPa.
At only 20 ◦ C and 10 ◦ C, the optimum hydrostatic pressure for growth was
0.1 MPa (1 atm) in each case, but with lower µmax values. Use of high pres-
sure as a stressing agent and/or an intensification tool was discussed, and its
potential was demonstrated by uncovering the existence of barotolerant (at
120 MPa) marine microbes obtained from seaweeds [107].
56                                                                   S. Lang et al.

4.3
Bioprocess Engineering Data
on Marine Hyperthermophilic and Barophilic Archaea

Thermophiles, extreme thermophiles, and hyperthermophiles have adapted
to environments with temperatures from 45 ◦ C to an upper limit of 113 ◦ C
[108, 109]. Submarine hydrothermal vents are particularly good sources of
these specialised prokaryotes, including archaea and bacteria. They consist
of anaerobic and aerobic chemolithoautotrophs and heterotrophs; the latter
are able to utilize various polymeric substrates such as starch, hemicellulose,
proteins and peptides. Metabolic processes and specific biological functions
of these microorganisms are mediated by enzymes and proteins that func-
tion under extreme conditions. These enzymes show unique features, are ex-
tremely thermostable and usually resistant against chemical denaturants. The
major stabilising interactions include increased salt-bridge content, increased
content of hydrophobic interactions, reductions in cavity size and reduced
content of thermolabile residues [110, 111]. The number of genes from ther-
mophiles that have been cloned and expressed in mesophiles is increasing
sharply. The majority of proteins produced in mesophilic hosts are able to
maintain their thermostabilities. Thermostable enzymes have considerable
potential in the food, chemical and pharmaceutical industries [112, 113].
   On the other hand, the benefits of whole cell applications of thermophiles/
hyperthermophiles, such as reduced viscosity of media or reduced contami-
nation by mesophilic microorganisms, respectively, have been relatively over-
looked. Therefore, Bustard et al [114] recommend that bioprocess intensifica-
tion studies should be performed with special emphasis on high temperature
bioreactor operation and corrosion reduction of materials. Reproding opti-
mum growth conditions may be difficult to achieve in conventional bioreactor
systems used for the cultivation of mesophilic microorganisms. Potentially,
therefore, novel reactor systems and solid state fermentation (such as immo-
bilisation onto porous glass, specific high-grade plastics) may be employed to
circumvent these difficulties [114]. The intention of the present publication is
to review some recent successful cultivations with hyperthermophilic archaea
(Table 11).
   Using the marine heterotrophic Pyrococcus furiosus, a system for continu-
ous cultivation in the absence of elemental sulphur has been developed [115].
An all-glass “gas-lift” bioreactor was used to provide high mass transfer at
low shear forces, whilst eliminating the potential for corrosion. The most suit-
able gas for optimal stripping was nitrogen. When the reactor was gassed with
0.5 v/vm, a cell density of 3 × 109 cells per mL could be maintained at 90 ◦ C
under chemostat conditions at a dilution rate of 0.2 h–1 . In addition, Krahe
et al [116] studied the fermentation of Pyrococcus furiosus in a stirred biore-
actor as well as in a membrane reactor. For the former, a cell density of 3 × 109
cells per mL was reached at a stirrer speed of 1800 rpm. Growth was neg-
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi           57

Table 11 Bioreactor cultivations of hyperthermophilic/barophilic archaea with respect to
important bioprocess conditions

Strain (origin)                                           Growth                    Ref(s)
Cultivation conditions                                    behaviour

Pyrococcus furiosus (marine):                             Maximal cell density:     [115]
NaCl, peptone, YE, pH 7, 90 ◦ C;                          3 × 109 cells mL–1
5-L all-glass “gas-lift” reactor; gassing: 0.5 v/vm
(N2 ); continuous culture, D = 0.2 h–1
Pyrococcus furiosus (marine):                                                       [116]
NaCl, amino acid cocktail, maltose, L-cysteine; 90 ◦ C;   Maximal cell density:
(a) stirred 2-L reactor, 1800 rpm,                        (a) 3 × 109 cells mL–1
    gassing: 0.4 v/vm (N2 /CO2 )
(b) Dialysis membrane reactor (1.2 L inner chamber,       (b) 35 × 109 cells mL–1
    4.5 L outer chamber),
    flow rate of the dialysate: 5 L h–1
Methanococcus jannaschii (hydrothermal vent):             Maximal biomass:          [117]
Mineral salts medium, YE, tryptone, pH 6, 85 ◦ C;         2 g L–1 ;
12 L stainless steel-constantly stirred tank reactor      µmax = 1.20 h–1
(3 six-bladed Rushton-type turbine impellers);
flow rates for H2 : 19.2 L min–1 , CO2 : 4.8 L min–1 ,
H2 S(in N2 ): 0.215 L min–1 ; 600 rpm; 1.25 × 105 Pa
Thermococcus peptonophilus (hydrothermal vent,            Maximal cell density:     [118]
                                                            8
depth of 1380 m): Peptides, anaerobic,                    10 cells mL–1
85–95 ◦ C; 0.1–60 MPa;                                    µmax = 0.67 h–1
DEEP-BATH (=high pressure/                                (90 ◦ C, 45 MPa)
high temperature bioreactor)



atively influenced above this stirrer speed. Performing the fermentation in
a dialysis membrane (cuprophane) reactor, the microorganism was cultivated
in the inner chamber(1.2-L working volume), the dialysing medium being in
the outer chamber (4.5-L working volume). This cultivation led to the for-
mation of 35 × 109 cells per mL, corresponding to 2.6 g L–1 dry biomass. The
authors suppose that this dramatic increase in cell density could be either
due to the dilution of inhibiting products or to the supply of one or several
essential nutrients for growth through the membrane.
   Methanococcus jannaschii is a hyperthermophilic (optimal growth tem-
perature, 85 ◦ C) and barophilic methanarchaeon isolated from surface ma-
terial collected from the base of a “white smoker” submarine hydrothermal
vent. Mukhopadhyay et al [117] reported the first media recipes and protocols
for mass culture of this organism in a 16-litre constantly stirred tank reac-
tor (12-L working volume) to relatively high cell densities, 2 g L–1 dry mass,
and provided guidelines for scaling up these cultures to higher volumes.
58                                                                             S. Lang et al.

Canganella et al [118] studied the effects of high temperatures and elevated
hydrostatic pressures on the physiological behaviour and viability of the ex-
tremely thermophilic deep-sea archaeon Thermococcus peptonophilus. High
hydrostatic pressure, up to 45 MPa, enhanced the growth rate and produced
a shift of the optimal temperature of growth from 85 to 90 ◦ C. Thus, barophily
was expressed with increasing cultivation temperature and it became evident
at 95 ◦ C when growth at 0.1 MPa (1 atm) was highly repressed. At high pres-
sure (60 MPa) and high temperature (90 ◦ C), the number of protein bands
(SDS-PAGE of total cell proteins) of T. peptonophilus seemed unchanged, but
cell growth was accompanied by overproduction of specific proteins.


5
Concluding Remarks

There is a lack of research data for bioreactor engineering and fermentation
protocol design in the field of marine prokaryotes and fungi. Most cultiva-
tion strategies used to produce low- or high-molecular-weight biochemicals
are carried out at the shake-flask level without an understanding of the pro-
duction process, offering poor prospects for successful scale-up. For instance,
data on specific growth rates are necessary to give recommendations for




Table 12 Maximum growth rates for marine microbes and their nutrients. Pressure con-
ditions: in general 1 atm (= 0.1 MPa). L-Asn: L-asparagine.

Marine microbe             Nutrient                Conditions          µmax (h–1 ) Ref(s)

Microbacterium sp.         Glycerol, YE, peptone   30 ◦ C              0.130        new
Bacillus pumilus           Glucose, YE             30 ◦ C              0.200        [48]
Pseudoalteromonas sp.      MB, glucose             27 ◦ C              0.620        new
Exophiala pisciphila       Glucose, L-Asn          25 ◦ C              0.030        [59]
Hypoxylon oceanicum        Glycerol, soy peptone   22 ◦ C              0.080        [60]
Vibrio harveyi             MB, skim milk           30 ◦ C              0.500        [78]
Hydrogenophaga             Glycerol                 2 ◦C               0.055        [98]
pseudoflava                                         16 ◦ C              0.139
Pseudomonas sp.            MB, glucose             10 ◦ C, 0.1 MPa     0.033        [106]
                                                   30 ◦ C, 0.1 MPa     0.220
                                                   10 ◦ C, 10 MPa      0.031
                                                   30 ◦ C, 10 MPa      0.228
Methanococcus jannaschii   YE, tryptone            85 ◦ C, 0.125 MPa   1.200        [107]
Thermococcus               Peptides                90 ◦ C, 45 MPa      0.670        [118]
peptonophilus
Bioprocess Engineering Data on the Cultivation of Marine Prokaryotes and Fungi       59

suitable dilution rates in continuous cultures. In general, it is necessary to
evaluate the following conditions:
 • Temperature dependency: mesophilic, psychrophilic, (hyper)thermophilic,
    and/or barophilic conditions
 • Ingredients of media for growth and/or production of biochemicals
 • Bioreactor types: well-known stirred tank reactors, pressurised vessels,
    and so on
 • Distinguishing growth and production phases
 • The production phase in the case of two or more biochemicals
   Table 12 presents a detailed overview of selected marine microbe cultiva-
tions reported so far, documenting, for example, specific growth rates. Low
values were calculated with mesophilic fungi; higher values up to 0.62 or
1.20 h–1 , respectively, were derived using metabolite-producing mesophilic
bacteria or hyperthermophilic species, respectively. Psychrophilic/psychro-
tolerant strains show growth rates that increase with temperature, but mostly
up to a limit of 20 ◦ C.

Acknowledgements The authors would like to thank R. Pukall (DSMZ, Braunschweig, Ger-
many) for strain identification, V. Wray and M. Nimtz (GBF, Braunschweig, Germany) for
molecular structure elucidations, D. Rasch and W. Graßl for technical assistance (biore-
actor equipment), the German Ministry for Education and Research (Bonn, Germany),
the Government of Lower Saxony (Hannover, Germany), and the VW foundation for
generous financial support.


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Adv Biochem Engin/Biotechnol (2005) 97: 63–103
DOI 10.1007/b135823
© Springer-Verlag Berlin Heidelberg 2005
Published online: 24 August 2005

Downstream Processing in Marine Biotechnology
Kai Muffler · Roland Ulber (u)
Institute of Technical Chemistry, University of Hannover, Callinstr. 3, 30167 Hannover,
Germany
ulber@mv.uni-kl.de

1       Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .                                           64
2       General Downstream Procedures . . .          .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   66
2.1     Sample Disruption . . . . . . . . . . .      .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   66
2.2     Solid–Liquid Separations . . . . . . . .     .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   69
2.3     Membranes for Ion Exchange . . . . . .       .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   73
2.4     Solvent Extraction . . . . . . . . . . . .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   77
2.5     Affinity Adsorption (Chromatography)          .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   80
3       Examples . . . . . . . . . . . . . . . . . . . . . . . . . .                     .   .   .   .   .   .   .   .   .   .   .   81
3.1     Isolation and Purification of Enzymes . . . . . . . . . .                         .   .   .   .   .   .   .   .   .   .   .   81
3.2     Downstream Processing of Oligo- and Polysaccharides                              .   .   .   .   .   .   .   .   .   .   .   85
3.2.1   Isolation and Purification of Chitin and Chitosan . . .                           .   .   .   .   .   .   .   .   .   .   .   85
3.2.2   Isolation of Fucoidan and Fucoidanases . . . . . . . . .                         .   .   .   .   .   .   .   .   .   .   .   89
3.3     Downstream Processing of Polyunsaturated Fatty Acids                             .   .   .   .   .   .   .   .   .   .   .   91
3.4     Downstream Processing of Bioactive Compounds
        With Low Molecular Weight . . . . . . . . . . . . . . .                          . . . . . . . . . . .                       96
4       Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .                                           99
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .                                             99

Abstract Downstream processing is one of the most underestimated steps in bioprocesses
and this is not only the case in marine biotechnology. However, it is well known, es-
pecially in the pharmaceutical industry, that downstreaming is the most expensive and
unfortunately the most ineffective part of a bioprocess. Thus, one might assume that new
developments are widely described in the literature. Unfortunately this is not the case.
Only a few working groups focus on new and more effective procedures to separate prod-
ucts from marine organisms. A major characteristic of marine biotechnology is the wide
variety of products. Due to this variety a broad spectrum of separation techniques must
be applied. In this chapter we will give an overview of existing general techniques for
downstream processing which are suitable for marine bioprocesses, with some examples
focussing on special products such as proteins (enzymes), polysaccharides, polyunsat-
urated fatty acids and other low molecular weight products. The application of a new
membrane adsorber is described as well as the use of solvent extraction in marine
biotechnology.

Keywords Downstream processing · Bioseparation · Membrane processes · Purification ·
Product recovery
64                                                          K. Muffler · R. Ulber

1
Introduction

Downstream processing is one of the most underestimated steps in biopro-
cesses and this is not only the case in marine biotechnology. However, it is
well known, especially in the pharmaceutical industry, that downstreaming is
the most expensive and unfortunately the most ineffective part of a biopro-
cess. Thus, one might assume that new developments are widely described in
the literature. Unfortunately this is not the case. Only a few working groups
focus on new and more effective procedures to separate products from ma-
rine organisms. In this chapter we will give an overview of existing general
techniques for downstream processing which are suitable for marine biopro-
cesses with some examples focussing on special products such as proteins
(enzymes), polysaccharides, polyunsaturated fatty acids (PUFAs) and other
low molecular weight products.
   A chief characteristic of marine biotechnology is the wide variety of prod-
ucts. Due to this variety a broad spectrum of separation techniques must be
applied. However, for nearly all products one starts with a dilute suspension
and tries to produce a highly purified dry product. In the case of extracel-
lular products the solids in this suspension may include intact organisms,
other insoluble fractions of the medium or natural sample, and perhaps insol-
uble products. For intracellular products the solids also include fragmented
mycelia caused by cell disruption, which is necessary to gain the products. Ac-
cording to this starting point nearly each downstream process consists of the
four following steps [1]:
 • Removal of insoluble particles
 • Isolation of the product
 • Purification
 • Polishing
With respect to laboratory-scale downstreaming one is normally only limited
by the available equipment. For up-scale processes one should have in mind
that the developed process should also be applicable in industry. Thus, Bel-
ter et al. [1] have formulated the following questions, which are also crucial
to marine biotechnology downstream processes:
 • What is the value of the product?
 • What is the acceptable product quality?
 • Where is the product in each process stream?
 • Where are the impurities in each process stream?
 • What are the unusual physicochemical properties of the product and the
    principal impurities?
 • What are the economics of various alternative separations?
In addition it is important to use materials which are available for all scale-
up processes, since the downstream behaviour of the product may alter while
Downstream Processing in Marine Biotechnology                              65

changing, for example, the type of chromatographic resin or membrane mate-
rial. Normally the recovery costs exceed the bioprocess costs; however, some
upstream parameters influence the downstream processing:

 •   Characteristic properties of the producing microorganism or cell line
 •   Location of the product
 •   Stability of the product
 •   By-products and impurities
 •   Concentration of the product in the medium from which it is to be recov-
     ered




Fig. 1 General downstream scheme in biotechnology
66                                                          K. Muffler · R. Ulber

A general downstream scheme based on these parameters is given in Fig. 1.
More detailed information about these various downstream procedures is
given in Sect. 2.
    The main aims of the primary separation step are to achieve a volume re-
duction and to make a first stage purification of the product by removing
dissimilar components from the broth. The most important techniques in use
are:
  • Membrane processing
  • Ion exchange chromatography
In the membrane process, ultrafiltration and reverse osmosis are often used
for separation, concentration and desalting. In addition, polar membranes
are used for ion exchange and desalting. Ion exchange chromatography is
applied in order to remove either major contaminants from the broth or
the desired product from the broth. Chromatographic procedures – based
on columns or membranes – are also often used in the purification step.
Other techniques in this part of the downstream process are precipitation and
liquid–liquid extraction. Recrystallization, lyophilization and drying are used
as final product treatments. In this review we will focus on techniques that are
suitable for primary separation and purification.


2
General Downstream Procedures

2.1
Sample Disruption

The influence of the sample disruption step in upstream and downstream
processes cannot be ignored. The disruption is dependent on the properties
of the sample. The main focus of the disruption is always to release as much as
possible of the product. However, depending on the mechanism, parts of the
product may be destroyed during the disruption process. Thus, very effective
and short procedures are required. A distinction can be made between me-
chanical, chemical and enzymatic procedures for product release. Mechanical
methods are often preferred because of short residence time, lower operating
costs, and contained operation [2]. The most common mechanical means of
disruption are:
  • Homogenizers [3, 4]
  • Bead mills [4]
A homogenizer consists of a positive-displacement pump, which supplies the
liquid sample at high pressure through a small nozzle or an orifice valve. The
disruption results from the combination of shear force and impingement on
the valve. The level of disruption depends on the upstream pressure and the
Downstream Processing in Marine Biotechnology                                67




Fig. 2 Schematic diagram of a homogenizer [4]


geometry of the nozzle or valve, but temperature and velocity of the sample
also influence the performance. Homogenizers show a high degree of flexibil-
ity with regard to sample type, they are easy to clean and to sterilize and they
do not require much maintenance. The disadvantage is that the homogenizers
produce aerosols, which can be harmful especially when handling unknown
microorganisms. During the disruption heat may be generated which may
lead to denaturation of products (e.g. proteins). Usually multiple-pass oper-
ation is required.
   In marine biotechnology a commonly used hand-driven homogenizer for
smaller samples is the Teflon glass homogenizer [5].
   Bead mills use horizontal grinding chambers filled with glass beads or
other resistant materials such as zirconium oxide, zirconium silicate, tita-
nium carbide, etc. [6]. The dispensed sample is introduced into the grinding
chamber on a continuous basis. The level of disruption depends on the vari-
able turning speed of the bead mill. The grinding beads agitate and stress
the product to be ground. This stress causes the solids to disperse and/or
break up. The resulting homogenate is separated from the grinding media
by mechanical means. By using bead mills, cell disruption can be achieved
in a single run with better temperature distribution and temperature con-
68                                                            K. Muffler · R. Ulber

trol. In addition, short residence time is possible but with broad residence
time distribution. Aerosol generation is minimized. However, bead mills are
difficult to clean and sterilize. Bead abrasion leads to contamination of the
homogenate and the performance and capacity vary greatly according to cell
type. Gray et al. describe the use of a bead mill for the phylogenetic analysis of
microbial communities present in marine sediments [7]. They report a proto-
col that can be used for efficient cell lysis and recovery of DNA from marine
sediments. Key steps in this procedure include the use of a bead mill ho-
mogenizer for matrix disruption and uniform cell lysis and then purification
of the released DNA by agarose gel electrophoresis. For sediments collected
from two sites in Puget Sound, over 96% of the cells present were lysed.
The method yields high-molecular-weight DNA that is suitable for molecular
studies, including amplification of 16S rRNA genes.
   Another possibility for cell disruption or at least product liberation is the
use of microwaves or ultrasound, which can be combined with sample ex-
traction by organic solvents. Microwave techniques are widely used in acid
digestion of solid samples. Their use in the extraction of organic analytes
from environmental samples is less widespread, despite the availability of
commercial devices for this purpose and their potential for reducing analy-
sis time and solvent consumption. Kornilova et al. describe the application of
microwave-assisted extraction to the analysis of biomarker climate proxies in
marine sediments [8]. Factorial design was applied to determine the influence
of temperature, volume of solvent and extraction time on the efficiency of the
extraction of total chlorines. The authors found that only changes in tempera-
ture produced a significant variation in yield. They optimized the procedures
for both chlorines and alkenones. Equivalent results to repeated extractions
by ultrasonication were obtained from a single extraction step of 5 min using
10 mL of solvent at a temperature of 70 ◦ C. Microwave-assisted extraction was
found to be a more efficient, faster and less labour-intensive method than
ultrasonic extraction.
   Martinez et al. used a combination of organic extraction and ultra-
sonication for determination of antifouling pesticides and their degrada-
tion products in marine sediments [9]. The determination of these com-
pounds in sediment samples was performed by means of methanolic ul-
trasonic extraction then clean-up on an Isolute ENV+ solid phase extrac-
tion (SPE) cartridge. The resulting extract was then analyzed by reversed-
phase high-performance liquid chromatography coupled with atmospheric-
pressure chemical-ionization mass spectrometry in negative and positive ion
modes (HPLC-APCI-MS). Recovery was 54–109% for antifouling agents and
their degradation products. The determination limits for the different com-
pounds varied between 0.2 and 1.6 µg/kg dry sediment. Pino et al. have pub-
lished a paper concerning the extraction of polycyclic aromatic hydrocarbons
(PAHs) from marine sediments with a micellar medium of polyoxyethylene
10-lauryl ether by an ultrasound-assisted method [10]. They optimized the
Downstream Processing in Marine Biotechnology                               69

relationship of extraction time, surfactant concentration and surfactant vol-
ume to amount of sediment. The results suggest that surfactant concentration
is statistically the most significant factor.
    A possibility for cell lysis under very mild conditions is the use of hy-
drolysing enzymes. In addition enzymes offer selectivity during product re-
lease. Enzymes hydrolyse the walls of cells, and when sufficient wall has been
removed, the internal osmotic pressure bursts the periplasmic membrane al-
lowing the intracellular components to be released [3]. The effect of lytic
enzymes is specific to particular groups of cell types, which is attributed to
the differences in cell wall composition. For example, the most efficient lytic
enzyme for bacteria is lysozyme from hens’ egg. This enzyme is also used in
large scale processes for enzyme production [11]. The use of enzymes for cel-
lular disruption is nowadays discussed for the recovery of, e.g. astaxanthin
from Haematococcus spp. [12].
    Even more highly specialized procedures for sample disruption can be
applied in marine biotechnology. For example, high yields of intracellular
enzymes from yeast can be obtained by applying a series of electric field
pulses [13]. Using this technique up to 90% of the total activity can be re-
leased without any further or previous treatment of the cells. The method
is based on electro-induced changes in the cell envelope leading to a leak-
age of part of the intracellular proteins without the formation of debris and
permits the treatment of large volumes. The treatment of at least 20% wet
weight suspensions is possible. However, it must be mentioned that in our
post-genomic area, cell disruption and the problems related to the subsequent
downstream processing can often be avoided since various strategies have al-
ready been attempted at the genetic level to make the cells secrete the desired
product. These techniques will also become much more important in marine
biotechnology within the next few years.

2.2
Solid–Liquid Separations

Solid–liquid separation is a very important procedure during downstream
processing in marine biotechnology for cell separation, cell debris removal
and also for product recovery. The most important solid–liquid separation
techniques in use for are filtration and centrifugation. Filtration separates
solids from a liquid by forcing the liquid through a solid support or filter
medium. Two different designs of filtration can be used:
 • Dead end filtration
 • Cross flow filtration
In dead end filtration mode the total process fluid stream flows through the
membrane. The retained solids accumulate on the membrane and build up
a filter cake. The membrane has to be changed when the membrane pores are
70                                                           K. Muffler · R. Ulber

clogged by the solids. When the feed flow is directed parallel to the membrane
surface, the term cross-flow filtration is used. The tangential flow of liquid re-
moves any retained molecules or particles from the membrane surface, which
results in a stable flux for a longer time period. The capital investment for
dead end filtration is low relative to other techniques. However, costs for filter
aids can be very high depending on the filtration media. The pressure drop
and the shear stress increase with filtration time while the flow rate through
the filter decreases. By using cross-flow filtration, relatively low shear stress
is possible and filter aids are not required. In addition, scale-up is simple
and cell washing is possible in a single process step. In comparison to dead
end filtration the capital investment is high. One common problem in cross-
flow filtration is membrane fouling. Thus, frequent membrane replacement
is often necessary. The separation is based mainly on molecular size, but to
a lesser extent on shape and charge. During a membrane separation process
low-viscosity feed suspension is usually applied on one side of a membrane.
The stream that passes through the membrane under the influence of the
pressure force is termed the permeate (filtrate). After removal of the required
amount of permeates, the remaining material is termed retentate (concen-
trate). The extent of the concentration is characterized by the concentration
factor (f ), which is the ratio of feed volume to final volume.
   Membrane processes are easy to scale up and the possibility of using
the same materials and configurations in different sizes from laboratory to
process scale reduces the validation effort enormously. However, filtration
processes are limited with regard to selectivity. The fractionation of pro-
teins can only be achieved with large differences in the molecular weight of
the proteins and it is important to keep in mind that a certain difference
in the molecular weight of two proteins does not mean the same degree of
difference in molecular size. Proteins that differ in molecular weight by ten
times may differ in size by only three times when in globular or folded form.
The function of membranes has now been enhanced to more than their role
as a selective barrier for filtration of molecules. The selective adsorption of
molecules to the membranes for their separation, based on different chemical
behaviour, is being increasingly applied as an integrated downstream pro-
cessing operation. Over the last 30 years, a number of membrane processes
have been developed for molecular separation. These filtration techniques can
be divided into four major groups: reverse osmosis (hyperfiltration) (RO),
nanofiltration (NF), ultrafiltration (UF) and microfiltration (MF) [15]. The
dimensions of the components involved in these separations are given in
Fig. 3.
   One possible problem encountered when using membrane processes for
product recovery can be the slow retentate flux, which can result in the for-
mation of a thick secondary membrane. Another possibility is the strong
interaction of the sample with the membrane material. This often depends on
unspecific protein adsorption, which is effected by several factors [16]:
Downstream Processing in Marine Biotechnology                                       71




Fig. 3 Pressure-driven membrane processes and their separation characteristics (accord-
ing to: Datar and Rosén [14] and Lewis [15])


 • Proteins adhere to all wetted surfaces
 • The amount of adsorbed protein depends on:
    –   Surface tension of the surface
    –   Molecular interaction between product and surface
    –   Protein concentration
    –   pH and ionic strength
    –   Area of wetted surface (membranes!)
The most important problems resulting for downstream processes are be-
cause binding is:
 • Unspecific
 • Irreversible
72                                                          K. Muffler · R. Ulber

The adsorption is caused by hydrophobic interaction such as Van der Waals
forces and weak interactions. Thus, different membrane types have to be
screened to minimize the unspecific binding of the sample when a new fil-
tration procedure is to be developed. It should be easy to change the flow
conditions (e.g. flow rates, feed temperature) to minimize this unspecific
binding. Thus, at least in research, cross-flow filtration devices should have
a very flexible set-up. Lignot et al. [17] have developed a downstream pro-
cess for the recovery of chondroitin sulfate (CS), a fishery by-product. CS
is a glycosaminoglycan well known for its chondroprotective effect. After
an enzymatic extraction cross-flow filtration is used to concentrate and pu-
rify CS. In their article the authors compared the performances of UF and
MF membranes in terms of flux and selectivity. For CS purification, mem-
brane processes were chosen as they are safe and provide, as a concentration
mode, a low-energy, mild alternative to evaporation processes. They are also
well suited to the industrial development of continuous processes and can
be coupled with an enzymatic bioreactor. The article shows that CS solution
can be partially concentrated effectively by UF up to four times. If necessary,
further concentration can be performed by precipitation with ethanol. Such
a combination reduces solvent consumption and the related costs of hand-
ling and recycling. Moreover, partial purification takes place along with the
reduction in feed volume during UF, which saves high quality water needed
for subsequent desalting.
   Cross-flow filtration can also be used to isolate colloids (here defined as
particles or macromolecules between 1 kD and 0.2–1 µm) from sea water.
Laboratory and field studies were performed by Dai et al. [19] to evaluate two
1 kDa cross-flow UF membranes (a Millipore preparation-scale CFF mem-
brane constructed primarily from regenerated cellulose and an Amicon CFF
polysulfone membrane). In the paper three crucial aspects of cross-flow filtra-
tion were examined: retention characteristics, sorptive potential and ultrafil-
ter breakthrough. Laboratory results showed that both CFF systems retained
greater than or equal to 91% of a 3000 nominal molecular weight (NMW)
dextran standard, consistent with the manufacturer’s rated cutoff. Both mem-
branes showed higher losses of a protein standard (lactalbumin) added to sea
water. For bulk organic carbon (OC), both membranes usually had reason-
able recovery (100 ± 10%) as long as the membranes were preconditioned.
However, when using this technique for real samples problems may occur;
this is discussed for several other cross-flow systems by the same group of
authors [20]. They recommend that considerable care must be taken in quan-
tifying cross-flow filtration blanks and in assessing the cut-off for each system
prior to use in marine applications. Time-series sampling and the use of stan-
dard molecules in controlled experiments are encouraged in order to further
understanding of the behaviour of natural compound assemblages in cross-
flow filtration processing.
Downstream Processing in Marine Biotechnology                               73

   MF and UF techniques have also become suitable processes for the separa-
tion of microorganisms in a variety of biotechnical applications. For example,
eight commercial membranes were evaluated by Rossignol et al. [18] for
the harvesting of two marine microalgae: Haslea ostrearia and Skeletonema
costatum, both widely cultivated in western France. The effects of cross-flow
velocity, transmembrane pressure, concentration and the characteristics of
suspensions are discussed in the paper cited. The use of a UF membrane
(polyacrylonitrile, 40 kDa) proved to be the most efficient method under the
particular conditions of low pressure and low tangential velocity for long-
term operation. However, harvesting microalgae often means concentrating
the biomass from a concentration of < 1 g dry weight L–1 in the photobiore-
actor to as much as 250 g dry weight L–1 . Here centrifugation is the method
of choice [12]; this is only slightly more expensive than other techniques like
flocculation or filtration.

2.3
Membranes for Ion Exchange

Compared with conventional column chromatographic procedure the use of
membrane adsorption techniques offers some advantages, which lead to bet-
ter process performance such as:
 • Lower manufacturing costs
 • No diffusion-controlled exchange kinetics so that higher fluxes are pos-
   sible
 • Easier handling in various module forms
 • Easier upscaling [16].
The basic idea behind using modified microporous membranes as the station-
ary matrix in liquid chromatography is to raise the separation efficiency by
maximizing mass transfer. Membranes can be converted into efficient adsor-
bers by attaching functional groups to the inner surface of synthetic micro-
porous membranes. Affinity adsorption, ion exchange or immobilized metal
affinity chromatography can be carried out by these membranes. Membrane
ion exchangers of strong acidic (sulfonic acid), strongly basic (quarternary
ammonium), weakly acid (carboxylic acid), and weakly basic (diethylamine)
types are commercially available. A chelating membrane based on the imin-
odiacetate (IDA) group is applicable for IMAC. Membrane adsorber technol-
ogy has several major advantages compared to classical separation methods.
Due to the membrane structure the binding of proteins is not limited by diffu-
sional processes, therefore loading and elution can be performed at very high
fluxes resulting in very short cycle times. Compressibility of the membrane
under normal operation conditions can be neglected, channelling cannot
occur, and the pressure distribution inside the modules is designed to have
plug flow through the module, all of which lead to sharp breakthrough curves.
74                                                              K. Muffler · R. Ulber

Scale-up is very easy, materials and systems allow CIP (cleaning in place) and
the validation of the process is made easier due to standard products and the
validation service of suppliers.
   These membranes are available in products for laboratory and process
scale. For process applications the modules and systems can be adopted to the
special needs of the specific separation process to achieve optimal conditions.
For production and large-scale application the Sartobind Factor-Two Family
(Sartorius, Göttingen, Germany) of membrane adsorber modules has been
developed. The modules consist of a Sartobind membrane rolled up like a roll
of paper to form a cylindrical module sealed at both ends with POM (poly-
oxymethylene) caps. For scaling up, the modules have areas between 0.12 m2
and 8 m2 . Since the direction of flow is from the inside to the outside of the
membrane adsorber cylinder, a solid core of the appropriate size is inserted
into the module to keep hold-up volume as small as possible. The solid POM
cores are also available in lengths of 3, 6, 12, 25 and 50 cm and the thickness
varies with the number of membrane layers used. The module is inserted into
a specific housing, which consists of a top and base plate, the housing tube
and the solid core. To operate the system the unit is first filled with starting
buffer. The feed solution enters the unit at the top.
   The central cylindrical core distributes the fluid to the inside of the mod-
ule. The flow is directed from the inner channel radial through the module
to the outer channel. The permeate leaves the housing at the bottom plate.
For large-scale protein isolation, adsorber modules of different sizes can be
combined to achieve desired yield and productivity. The modules offer un-




Fig. 4 Scheme of Sartobind Factor-Two Family module (Sartorius, Göttingen, Germany);
the arrows indicate the flow direction of the feed [16]
Downstream Processing in Marine Biotechnology                                 75

precedentedly high flow rates and short cycle times of within a few minutes.
Typical applications for the membrane adsorber technology are the con-
centration of minor proteins [21] and monoclonal antibodies, removal of
contaminants (e.g. DNA, endotoxins) and reduction of virus content. How-
ever, this system can also be used to isolate marine enzymes such as sulfite
oxidase [22]. Sulfite oxidase (sulfite acceptor oxidoreductase) catalyses the
oxidation of sulfite to sulfate. The enzyme transfers electrons to oxygen, cy-
tochrome c and a variety of other electron acceptors. In mammalian tissues
the physiological importance of sulfite oxidase is its role as a terminal enzyme
in the degradation of sulfur-containing amino acids. Furthermore it is im-
portant in the detoxification of endogenous sulfite and sulfur dioxide, a sulfite
oxidase deficiency in the human organism leading to severe neurological dis-
orders. Sulfite oxidase has been located in several mammalian tissues, such as
the liver, but also in plants and in bacteria. Many phototrophic bacteria also
contain sulfite oxidases. Sulfite oxidase is commercially available (sulfite oxi-
dase from chicken liver, Sigma) and, apart from preparations containing only
the purified enzyme, preparations in which sulfite oxidase is the key com-
ponent in an enzyme-based analytical sulfite test kit are also available (Sulfite
Test Kit, r-biopharm, Germany). In the study reported Sulfitobacter pontia-
cus is used as the source of a new sulfite oxidase. Sulfitobacter pontiacus is
a Gram-negative bacterium that was isolated from water samples taken from
a depth of 100–140 m at the H2 S – O2 interface in the eastern part of the Black
Sea [23]. Sulfitobacter pontiacus is strictly heterotrophic and is unable to
grow autotrophically on H2 , thiosulfate or sulfite. The organism is strictly aer-
obic and requires NaCl (5–80 g/L, optimum 20–25 g/L). Temperature and pH
ranges are 4–35 ◦ C and pH 6.5–8.5, respectively. During metabolic studies of
the organism Sorokin et al. [24] found that in acetate-limited continuous cul-
ture, after an adaptation period, Sulfitobacter pontiacus tolerates extremely
high sulfite concentrations of up to 63 mmol/L. Furthermore, they discovered
that the oxidation of sulfite to sulfate by a highly active AMP-independent sol-
uble sulfite oxidase leads to an increase in biomass concentration, indicating
the ability of the organism to use sulfite as an additional source of energy.
In recent studies on the growth conditions of Sulfitobacter pontiacus in batch
culture the focus was to produce and purify sulfite oxidase from this organism
on a larger scale. The authors concentrated their investigations on the com-
mercially available cultivation medium Marine Broth 2216 and focussed on
the influence of:
 • Complexing agent EDTA
 • Concentration of the carbon source acetate
 • Concentration of the basic medium MB2216
 • Sodium thiosulfate
 • HEPES as a buffering agent
 • Temperature and oxygen supply
76                                                                  K. Muffler · R. Ulber

on the performance of the cultivations with respect to biomass concentration
and specific sulfite oxidase activity. To isolate sulfite oxidase the following
isolation steps were performed:
 • Centrifugation of the fermentation broth
 • Ultrasonification of the cell pellet
 • 2nd centrifugation
 • Cation exchange of the supernatant of the 2nd centrifugation
 • 30 kDa ultrafiltration
The main part of this downstream procedure is the cation exchange step.
In Fig. 5 a typical chromatogram of the isolation of sulfite oxidase from the
crude extract is shown. For the elution of the bound protein the following
gradient was used:
 • Buffer A containing 20 mM acetate at pH 4.6
 • Buffer B containing 200 mM NaCl, 20 mM acetate at pH 4.6
 • 0–5 min buffer A
 • 5–20 min 0–20% buffer B
 • 20–60 min 20–35% buffer B
 • 60–70 min 35–100% buffer B
 • 70–80 min buffer B
When using the membrane adsorber in the downstream procedure described
above, a highly purified sulfite oxidase can be isolated from the marine bac-
terium Sulfitobacter pontiacus. The specific activity of the enzyme in compar-




Fig. 5 Cation exchange chromatography of crude cell extract of Sulfitobacter pontiacus for
the isolation of sulfite oxidase using membrane adsorber (Sartorius, Germany) [22]
Downstream Processing in Marine Biotechnology                                         77




Fig. 6 Comparison of specific activities of sulfite oxidase during the downstream process
and in comparison to a commercially available enzyme (isolated from chicken liver) [22]

Table 1 Purification of sulfite oxidase from Sulfitobacter pontiacus

Fraction             Vol    Vol        Protein Total       Specific    Units   Yield
                            activity   content protein     activity
                     [mL]   [U/mL]     [µg/mL] [µg]        [U/mg]     [U]     [%]

Crude extract        10     3.17       141       1415       22.5      31.76   100
IEC-membrane         24     0.61         3.42      82.1    178.0      14.61    46
UF                   2.2    6.24        13.57      29.9     460       13.73    43


ison with a commercially available sulfite oxidase from chicken liver is shown
in Fig. 6. Table 1 gives an overview of the concentration factors and the yield
of each purification step.

2.4
Solvent Extraction

Solvent extraction is the most common method for the recovery of hy-
drophilic substances and, therefore, a method for separating well-soluble
metabolites from cultivation media or samples from seawater or sediment.
Classical extraction processes use organic solvents, which are often rarely
suitable for effective recovery of the solute. Recently, new extractions have
been developed which form specific adducts with the metabolite in ques-
tion and allow its recovery with high efficiency and selectivity [25]. Solvent
78                                                          K. Muffler · R. Ulber

extraction in biotechnology focuses on the recovery both of primary metabo-
lites (e.g. ethanol, acetic acid, citric acid and amino acids) and of secondary
metabolites (e.g. antibiotics or vitamins). The concentration of secondary
metabolites is usually much lower than that of primary metabolites. Since
most secondary metabolites from marine sources are for use as therapeu-
tics, the quality requirements of the products are high. Solvent extraction
can help to fulfil these requirements. In addition, the set-up of integrated
bioprocesses (production and down streaming) can be performed by solvent
extraction [26].
    Solvent extraction or microwave-assisted solvent extraction is commonly
used in analytical procedures for marine sediments. For example, a method
based on solvent extraction followed by chromatographic separation by
diode-array detection has been applied to determine polycyclic aromatic hy-
drocarbons (PAHs) in marine sediment samples [27]. The aim of a study by
Flotron et al. [28] was to develop a reliable and fast analytical procedure for
the determination of polycyclic aromatic hydrocarbons in sewage sludges,
using focussed microwave-assisted extraction. Optimization of the extrac-
tion conditions was performed on real matrices. The results showed that
extraction time was the only influential factor. The selected conditions (30 W,
10 min, 30 mL solvent) were used for real sludges and a certified marine
sediment, leading to recoveries of between 56% and 75%. Chung et al. [29]
reported the use of solvent extraction for the isolation of bromophenols from
brown algae.
    However, in the field of marine biotechnology at least small-scale down-
stream processes based on solvent extraction are described in the literature.
For example, chlorogentisylquinone, a new inhibitor of neutral sphingomyeli-
nase activity, was purified from the culture broth of a fungal strain FOM-
8108 isolated from a marine environment by solvent extraction, silica gel
chromatography and Sephadex LH-20 chromatography [30]. Mycosporine-
like amino acids (MAAs), produced by Heterocapasa sp. in indoor cul-
tures, were extracted along with water-soluble compounds in 25% aqueous
methanol [31]. Before extraction, the cells were washed once with 0.9% am-
monium formate for partial desalting. Extraction was performed at 45 ◦ C for
2.5 h. After centrifugation the extract was evaporated to dryness under vac-
uum at 30 ◦ C. The MAAs produced have great potential as UV absorbing
compounds in the λmax range of 310 nm to 360 nm.
    Gao et al. reported the application of supercritical fluid extraction (SFE)
for the isolation of halogenated monoterpenes from the marine red alga Plo-
camium cartilagineum. Most of the halogenated monoterpenes have been
found to exhibit varied biological activities, including antifungal, antimicro-
bial, and molluscicidal activity [32]. A supercritical fluid (SCF) is charac-
terized by physical and thermal properties that are between those of a pure
liquid and gas. The fluid density is a strong function of the temperature and
pressure. The diffusivity of SCF is much higher than for a liquid and SCF
Downstream Processing in Marine Biotechnology                                 79

readily penetrates porous and fibrous solids. P. cartilagineum samples were
extracted by SFE with carbon dioxide and modified carbon dioxide contain-
ing up to 10% methanol under different pressure and temperature conditions
to establish the optimum conditions for extraction. In marine biotechnology
the application of SFE also offers important advantages compared with other
solvent extraction methods. It is possible to work in an oxygen-free system,
which prevents oxidation. The low temperatures applied minimize thermal
degradation and microbes or their spores are not soluble. In addition, super-
critical fluids for extractions are inexpensive. The successful implementation
of this technique can lead to improved sample throughput, more efficient re-
covery of analytes, cleaner extracts, economic replacement of halogenated
solvents and a high level of automation, compared to conventional sample
preparation procedures [33]. SCF processes are being commercialized in the
polymer, pharmaceutical, specialty lubricants and fine chemicals industries.
SCFs are advantageously applied to increase product performance to levels
that cannot be achieved by traditional processing techniques.
   Macías-Sánchez et al. described the use of SFE for the extraction of dif-
ferent carotenoids [34]. Conventional methods of carotenoid extraction from
natural matrices are time-consuming since they require multiple extraction
steps and need large amounts of organic solvents, which are often expensive
and potentially harmful. Therefore, there is growing interest in the devel-
opment of simpler, faster and more efficient methods of carotenoid extrac-
tion from food and natural products. The aim of the study was to ascertain
the influence of pressure and temperature on carotenoid extraction with
a supercritical fluid from freeze-dried powder of the marine microalga Nan-
nochloropsis gaditana. Supercritical fluid extraction was performed using an
ISCO SFX 220 extractor with a 0.5 mL volume chamber, and a syringe pump
(model 260 DX) that supplies the supercritical carbon dioxide. The extraction
flow rate was controlled with a micrometric valve at the outlet of the extractor.
The highest extraction yield was obtained for β-carotene under all extraction
conditions. In particular, at 50 ◦ C and 300 bar, 60 ng pigment/mg dry weight
were extracted after 60 min, that value becoming a possibly asymptotic one.
A comparison of SFE with conventional solvent extraction is shown in Table 2.


Table 2 Comparison of extraction methods

                                     Yield [mg/mg dry weight]
                    Extraction             Extraction           Extraction
                    with methanol          with acetone         with CO2 SF

Beta-carotene       20.09 × 10–10          1.39 × 10–10         6.5 × 10–5
Canthaxanthin        4.03 × 10–7           2.82 × 10–7          1.34 × 10–5
Violaxanthin         4.5 × 10–6            2.14 × 10–6          2.11 × 10–5
80                                                           K. Muffler · R. Ulber

   Another application of SFE in marine biotechnology, the downstream pro-
cessing of PUFAs, is described below.

2.5
Affinity Adsorption (Chromatography)

While other separation techniques rely primarily on molecular size, charge
or solubility, affinity adsorption relies on highly specific binding interactions.
Chromatography was the first technique to be converted into an affinity-
based approach and this technique is now well-established in a large number
of separation processes [35, 36]. Affinity adsorption offers the advantages of
high purification factors (up to 1000-fold) and high recovery rates. It is based
on the formation of a reversible complex between the target and the ligand.
Often the ligand is used in an immobilized form (insoluble matrix). Some
affinity-based separation techniques are shown in Fig. 7.
   In downstream procedures for highly purified enzymes or antibodies of
marine origin, affinity adsorption steps are often included. For example,
Berteau et al. reported the purification and characterization of an α-L-
fucosidase (EC 3.2.1.51) [37]. The enzyme was purified by three chromato-
graphic steps, including an essential affinity chromatography based on the
glycosidase inhibitor analogue 6-amino-deoxymannojirimycin as the ligand.
More information on fucoidan and fucosidases can be found below. A new
affinity procedure for the isolation and further characterization of the blood
group B-specific lectin from the red marine alga Ptilota plumose is described
by Sampaio et al. [38]. They loaded the aqueous extract onto a Sephadex G-
200 column (1.6 × 18 cm), equilibrated and eluted with PBS containing 1 mM
CaCl2 at 10 mL h–1 until the column effluent showed absorbance at 280 nm of




Fig. 7 Affinity-based separation techniques [35]
Downstream Processing in Marine Biotechnology                                 81

less than 0.05. Adsorbed proteins were eluted with 50 mL 0.1 M D-glucose in
PBS containing 1 mM CaCl2 . Purification was 212-fold with the specific activ-
ity increasing from 218 U mg–1 in the crude extract to 46 282 U mg–1 in the
purified material. Another lectin from the marine green alga Caulerpa cupres-
soides was purified by α-lactose-agarose affinity chromatography, followed
by gel filtration on Bio-Gel P-100 [39]. A marine bacterial strain produc-
ing a particularly heat-labile alkaline phosphatase was selected by Hauksson
et al. from a total of 232 strains isolated from North Atlantic coastal wa-
ters [40]. The alkaline phosphatase was purified 151-fold with 54% yield from
the culture medium using a single-step affinity chromatography procedure
on agarose-linked L-histidyldiazobenzylphosphonic acid. Moreover other en-
zymes from marine organisms, such as serine proteases [41], thermostable
phosphatase [42], sialyltransferase [43] or exopolyphosphatases [44], were
isolated and purified by affinity adsorption techniques.


3
Examples

3.1
Isolation and Purification of Enzymes

To date there have been a very few reports that focus on the isolation of
enzymes from the marine environment such as acetylcholinesterase [45], ure-
thanase [46], L-asparaginase [47, 48] or β-1,3-xylanase [49]. However, the
marine environment is an excellent source of extremozymes. Since many in-
dustrial enzymes are required to function under extreme conditions (e.g.
heat, cold, salinity) there is also commercial pressure to discover stable
biocatalysts in modern biotechnology [50]. Turkiewicz et al. described the
purification of cold-adapted β-galactosidase [51]. They found that the ma-
rine, psychrotolerant, rod-shaped, Gram-negative bacterium 22b, classified
as Pseudoalteromonas sp. based on the 16S rRNA gene sequence, isolated
from the alimentary tract of Antarctic krill Thyssanoessa macrura, synthe-
sizes an intracellular cold-adapted β-galactosidase; this efficiently hydrolyzes
lactose at 0–20 ◦ C, as indicated by its specific activity of 21–67 U mg–1 of
protein (11–35% of maximum activity) in this temperature range. The max-
imum enzyme synthesis (lactose as a sufficient inducer) was observed at
6 ◦ C, thus below the optimum growth temperature of the bacterium (15 ◦ C).
The enzyme extracted from cells was purified to homogeneity (25% recov-
ery) by using a fast, three-step procedure, including affinity chromatography
on PABTG-Sepharose. They started their downstream process by producing
a cell-free extract in two different ways: first, a wet biomass extraction (4 ◦ C,
24 h) with 0.5% sodium cholate in 0.05 M potassium phosphate buffer, pH 7.6,
82                                                          K. Muffler · R. Ulber

enriched with 15 mM EDTA, 0.2 M Mg2+ , and 1 mM PMSF, 2 mL aliquots of
the buffered cholate solution being used per 1 g samples of wet biomass; sec-
ond, sonification of the wet biomass (twice for 2.5 min, 0 ◦ C, Vibrocell 72480,
Bioblock Scientific, USA) in the same buffer as above, enriched with 1 mM
PMSF and 2 mM EDTA. In both cases the residual insoluble cell debris was
discarded after centrifugation (10 000 × g, 4 ◦ C, 30 min). The further purifi-
cation consisted of different chromatographic steps. It started with a DEAE-
Sepharose column (elution with a linear NaCl gradient from 0 to 0.2 M;
flow rate 0.22 mL min–1 ), followed by affinity chromatography using agarose
coupled with p-aminobenzyl-1-thio-β-D-galactopyranoside (PABTG-agarose,
Sigma; elution with 0.1 M sodium borate, pH 10). After concentrating with
a 30 kDa filter the final product polishing was done by molecular sieving on
Sepharose Cl-6B column (0.6 × 80 cm, flow rate of 0.16 mL min–1 ), previously
equilibrated with 0.05 M potassium phosphate buffer, pH 7.6, and calibrated
with molecular mass standard proteins (67–450 kDa). The efficiency of all
purification steps is shown in Table 3.
   Laroche et al. [52] describe the purification and properties of a novel iron-
dependent L-serine dehydratase (EC 4.2.1.13) from Paracoccus seriniphila.
This first L-serine dehydratase of the genus Paracoccus is oxygen sensitive
and can be stabilized with Fe2+ and dithiothreitole. The enzyme catalyses
the irreversible non-oxidative deamination of L-serine to pyruvate and L-
threonine to 2-oxo-butyrate. The deamination process of the substrate starts
with intermediary dehydration. This β-elimination is followed by tautomer-
ization of the aminoacrylate and hydrolysis of the resulting imine. L-Serine
dehydratases have been purified from bacteria, yeast [53], other filamentous
fungi [54], mammal liver [55] and plants. Eukaryotic L-serine dehydratases
usually contain PLP as a cofactor and deaminate L-threonine to a certain
extent as a side reaction. On the other hand, PLP-dependant L-threonine de-
hydratases also deaminate a special amount of L-serine. Bacterial L-serine
dehydratases are independent of PLP and highly specific for L-serine. These
enzymes are very unstable during exposure to air and some of them can
be activated by iron, substrate or competitive inhibitors. Serine dehydratase
is usually composed of subunits which form monomers, dimers, tetramers
or octamers [56]. The subunits are linked to Fe–S clusters. During expo-
sure to oxygen these clusters are oxidized, become unstable and lose Fe2+ ,
so that the activity is lost. Some L-serine dehydratases can be reactivated by
Fe2+ and dithiothreitole. In nature L-serine dehydratase is important for the
metabolism of 2-hydroxyamino acids. In Clostridium propionicum L-serine
and L-threonine are similarly completely decomposed to carbon dioxide, am-
monia and propionate. As described in the cited paper Paracoccus seriniphila
is able to grow in a minimal medium containing L-serine as the sole car-
bon and nitrogen source. For the cultivation of the microorganism a com-
plex medium containing 5 g/L peptone, 5 g/L yeast extract, 34.3 g/L sea salts
(Sigma) was used. For inducing L-serine dehydratase activity 1 g/L of L-
Table 3 Purification of Pseudoalteromonas sp. 22b β-galactosidase [51]

Purification step           Volume            Protein            β-Galactosidase activity             Yield              Purification
                                                                Specific            Total                                (fold)
                           [mL]              [mg]               [U mg–1 ]          [U]               [%]

Sodium cholate extract     32                166.4                0.45             101.5             100                 –
                                                                                                                                      Downstream Processing in Marine Biotechnology




DEAE-Sepharose             20                  7.8               13.0              101.4             100                 29
Affinity chromatography      3                  0.65              75.0               48.8              48                167
Sepharose Cl-6B             9                  0.22             115.0               23.0              25                255

The enzyme was obtained from 16 g of fresh biomass, harvested from 1 L of culture medium after 8 days of agitated culture
                                                                                                                                      83
84                                                            K. Muffler · R. Ulber

serine (filter sterilized) was added after autoclaving. After cultivation the
cells were harvested by centrifugation at 3300 × g, for 15 min at 4 ◦ C and
washed by resuspending in 3% NaCl solution. The suspension (0.3 g of wet
cells in 2.7 mL of 100 mM KPP buffer, pH 7.6 containing 1 mM L-cysteine)
was cooled on ice/water and treated with ultrasonic sound for 3 min at 50 W
with an interval of 0.6 s/s for cooling. The resulting extract was centrifuged
for 15 min at 14 000 rpm at 4 ◦ C. The supernatant (crude extract) was used
for the further purification procedure. All purification steps were performed
under aerobic conditions at room temperature. The buffers and solutions (all
containing 1 mM L-cysteine) were sonicated for 10 min to remove oxygen.
The purification procedure consisted of three steps:
 • Step 1: Ammonium sulfate precipitation. The crude extract was saturated
   with ammonium sulfate to 60%. The precipitate was removed by cen-
   trifugation at 14 000 rpm for 20 min. Ammonium sulfate was added to the
   supernatant to 80% saturation. After centrifugation the precipitate was
   dissolved in 2 mL of 100 mM potassium phosphate buffer, pH 7.6.
 • Step 2: Hydroxyl apatite chromatography. The dissolved precipitate of step
   1 was applied to a 5 mL CHT II cartridge (Bio-Rad) equilibrated with
   1 mM potassium phosphate buffer, pH 7.6. The elution was performed by
   a linear gradient of a 400 mM potassium phosphate buffer, pH 6.8 (40 min,
   0.5 mL/min). Fractions of 1 mL were collected. Those showing maximum
   activity were pooled.
 • Step 3: Anion exchange chromatography. The pooled fractions of Step 2
   were applied to a 5 mL High Q cartridge (Bio-Rad) equilibrated with
   100 mM potassium phosphate buffer, pH 7.6. The elution was performed
   by a linear gradient of the equilibration buffer plus 1 M NaCl (40 min,
   2 mL/min). Fractions of 4 mL were collected. Those showing maximum
   activity were pooled and concentrated by UF (cut off: 30 kDa) using Vi-
   vaspin concentrators (Sartorius).
A summary of the purification procedure is presented in Table 4. The purity
of the fractions of the purification steps was controlled by gel electrophore-
sis (Fig. 8). The samples were treated with SDS to divide the enzyme into


Table 4 Purification of L-serine dehydratase

Purification step         Protein     Units    Activity   Purification     Yield
                         [mg]                 [U/mg]     (fold)          [%]

Cell-free extract        140         140       1.04       1              100
Ammonium sulfate          21          95       4.56       4               68
CHT II                     1.41       16      11.28      11               11
High Q                     1.00        0.94   75.24      72                1
Downstream Processing in Marine Biotechnology                                     85




Fig. 8 Analysis of L-serine dehydratase at different steps of purification by SDS-PAGE
(20% polyacryl amide). Lane 1 marker, lane 2 crude extract (1:20), lane 3 ammonium
sulfate precipitation, lane 4 CHT II, lane 5 High Q (after concentration 50 fold)


subunits. After the last purification step the gel showed only two bands at
14.5 kDa and 40 kDa.

3.2
Downstream Processing of Oligo- and Polysaccharides

In this chapter the downstream process of chitin/chitosan and fucoidans is
described. These compounds belong to the group of poly- and oligosaccha-
rides and play an important role in marine biotechnology efforts today. In
view of the limited space, this chapter cannot describe all investigations in the
downstream of oligo- and polysaccharides of marine origin and is therefore
focussed on these two topics.

3.2.1
Isolation and Purification of Chitin and Chitosan

Chitin is the most abundant nitrogen-bearing biopolymer found in nature. It
consists of linear β-1,4-linked N-acetylglucosamine residues (GlcNAc) and is
a common constituent of insect exoskeletons, fungal cell walls and shells of
crustaceans [57]. Due to availability only the latter is important for the extrac-
tion of chitin or chitosan. Chitosan is a copolymer of GlcNAc (approx. 20%)
and glucosamine (GlcN, 80%), which is a product of the de-N-acetylation in
the presence of hot alkali deacetylating enzymes. Thus chitosan is a collec-
tive name representing a whole family of deacetylated chitins, which differ
in their degree of deacetylation. Alternatively, chitosan can be obtained as
86                                                           K. Muffler · R. Ulber

a component of fungal cell walls [58]. For an application of chitosan the
degree of N-acetylation and the degree of polymerization are important pa-
rameters, because these parameters play significant roles in biochemical and
biopharmaceutical applications [59]. Chitosan has been used in several appli-
cations because it is biologically renewable, biodegradable, almost non-toxic.
Therefore a few important applications are explained more extensively be-
low. Today, several companies produce chitin and chitosan on a commercial
scale. The majority of them are located in Japan, which produces more than
100 billion tons each year from the shells of crabs and shrimps [60]. Chi-
tosan is widely used in the clarification of waste and effluent water [61].
Furthermore, chitosan and its derivatives carboxymethyl chitosan and cross-
linked chitosan have been successfully used in the removal of Pb2+ , Cu2+
and Cd2+ from drinking water [62, 63], owing to complex formation of the
amino group and heavy metal ions. In comparison with activated charcoal it
is more efficient in the removal of polychlorinated biphenyls from contami-
nated water [59]. In recent years chitosan has been increasingly investigated
for pharmaceutical applications [64]. Due to its cationic character it possesses
a unique property for controlled drug release techniques [65]. In wound heal-
ing it has been shown that chitosan and its derivatives can reduce scar tissue
by inhibiting fibrin formation and affecting macrophage activity [66]. Besides
pharmaceutical applications, chitosan has become a standard substance for
enzyme immobilization. Enzyme and whole cells can easily be immobilized in
ionotropic chitosan gels, which are formed by mixing chitosan solutions with
solutions of anionic polymers [67]. Furthermore, chiotosan can be applied as
a flocculating agent in separation processes [68].
   Concerning the poor availability of chitin of terrestial origin (e.g. in-
sects, fungi), that of marine origin (e.g. crab, shrimp and prawn) is much
better. However, as the raw material chitin is contaminated by other com-
pounds harsh treatments are necessary to remove compounds such as pro-
teins from the ground shell. Generally the chitin isolation consists of three
steps: demineralization, deproteinization and bleaching. The order of the
first two steps depends on further chitin applications and the recovery of
associated carotenoids and proteins [69]. The order cited should be used
when chitin is applied as an adsorbent or enzyme support. This removal of
salts assures chitin deacetylation under mild alkali conditions, with a high
deacetylation level of the polymer, whereas strong alkali conditions often pro-
duce fragments of chitin. For the purpose of carotinoid recovery from shells,
deproteinization is used as the first step [69]. Demineralization is usually
carried out by an extraction of 1–3 h with diluted hydrochloric acid [70],
but harsher conditions such as 90% formic acid, 22% HCl, 6 N HCl or 37%
HCl have also been applied [71, 72]. A gentle method to avoid cleavages of
the polymer and removal of mineral salts is digestion with ethylenediamine-
tetra-acetic acid [73]. Deproteinization can be implemented by treating the
raw material with either sodium hydroxide or potassium hydroxide. Concen-
Downstream Processing in Marine Biotechnology                                   87

trations of hydroxide solutions ranging from 1% to 10% (w/v) are applied
at temperatures of 65 to 100 ◦ C [72, 74]. The reaction times commonly range
from 0.5 to 6 h. These harsh conditions of alkaline digestion cause depoly-
merization and deacetylation of chitin. Therefore the number of enzymatic
applications by digestion with proteolytic enzymes such as papain, pepsin,
trypsin or pronase [75] increased. These methods avoid the cleavage of gly-
cosidic bonds. An enzymatic procedure which can be used as an alternative
to alkaline processing is outlined in Fig. 9. Such enzymatic deproteinization
methods cannot ensure the complete removal of proteins and their degrada-
tion products, contrary to the application of strong hydroxide [76].




Fig. 9 Flow sheet for production of chitin from crustacean shell waste by enzymatic
digestion [76]
88                                                           K. Muffler · R. Ulber

   The next step included the removal of pigments like melanins and caroti-
noids by solvent extraction (e.g. acetone, chloroform, ethanol) or oxidation
using 0.02% potassium permanganate at 60 ◦ C, or hydrogen peroxide and
sodium hypochlorite [77].
   Chitosan production is usually carried out by treating chitin with sodium
hydroxide or potassium hydroxide (30–60% w/v) at temperatures ranging
from 80 to 140 ◦ C to deesterify the N-acetyl linkages [77] (see Fig. 10). The
resulting molecular weight distribution and distribution of deacetylated units
along the polysaccharide chain depends on the applied alkali concentration,
temperature and time of the process [73]. The degree of deacetylation of the
polysaccharide can be increased by using high temperatures in the process,
but such harsh conditions also cause depolymerization and lead to a reduc-
tion in the size of the molecules [78].
   Chitosan can also be prepared at moderate concentration of the alkali,
relatively low temperature and therefore longer reaction times. However, such
conditions cause randomly distributed deacetylated residues. The deacety-
lation process is usually followed by a drying process to produce “flaked
chitosan”. To increase the purity of the chitosan obtained, in a first step it
can be dissolved in an acid (e.g. acetic acid) and filtered to remove unwanted
compounds. This step is followed by lyophilization to gain a water-soluble
chitosonium acid salt, or by precipitation with sodium hydroxide. The latter
step is followed by washing and drying to obtain the free amine form.
   To obtain more defined chitosans and to avoid polysaccharide degrada-
tion by oxygen it is also recommended to carry out the deacetylation pro-
cess under nitrogen [79], thiophenol or sodium borohydride addition as
scavengers of oxygen [80, 81]. Due to the harsh conditions, thermodynamic
degradation processes have a few disadvantages: high energy consumption,
generation of a large amount of concentrated alkaline solution waste and
generation of a wide range of varying molecular weights and heterogeneous
deacetylation. However, many biomedical applications recommend materials
with specific physical and chemical properties. To overcome these drawbacks
of thermodynamic degradation processes, chitin deacetylases deriving from
different organism sources can be used. The application of such enzymes in
the chitosan preparation process has been widely investigated, but these stud-
ies indicate poor deacetylation of crystalline and amorphous chitin [82–84].
Therefore chemical pre-treatment of such chitin substrates is necessary to
improve the accessibility of the acetyl groups to the chitin deacetylase and
to increase the yield of the deacetylation process. However, the potency of
chitin deacetylases can be used in deacetylation reactions of water-soluble
chitin oligomers. Whereas pretreated chitosans are deacetylated up to 97% by
a chitin deacetylase from Mucor rouxii [84], the yield of deacetylation of crys-
talline chitin and amorphous chitin is only 0.5% and 9.5% [85], respectively.
Due to the enzymatic process new polymers with defined different physical
and chemical characteristics can be produced.
Downstream Processing in Marine Biotechnology                               89




Fig. 10 Chitosan manufacturing process [59]


3.2.2
Isolation of Fucoidan and Fucoidanases

Sulfated water-soluble polysaccharides containing large amounts of L-fucose,
were first isolated from marine brown algae in 1913 and named fucoidin. For
a long time it seemed that marine brown algae are the only sources of fu-
coidin. In 1948 such sulfated polysaccharides were also obtained from marine
invertebrates. Sulfated polysaccharides consist mainly of L-fucose units but
can additionally contain other sugars such as galactose, mannose, xylose, or
uronic acid, and sometimes proteins [86]. The composition also depends on
the algal species, the extraction procedure [87], the season and place of har-
vesting [88]. Due to the range of their composition many names still exist for
this group of molecules. However, it is recommended to define “sulfated fu-
can” as a polysaccharide based mainly on sulfated L-fucose, with less than
10% other monosaccharides [86]. This term is commonly used for sulfated
fucans from marine invertebrates [89, 90], whereas the term “fucoidan” has
been used for fucans extracted from algae [91].
   As already mentioned, the structure of fucoidans differs when they are
extracted from different algal species; the structure of sulfated fucans of ma-
rine invertebrate origin is simpler in comparison with those of algae. They
commonly possess a clearly regular structure and each species has its own
sulfated fucan. As far as is known, no naturally occurring fucans without sul-
fate groups have been reported [86]. It is worth mentioning that it has not
90                                                               K. Muffler · R. Ulber

been investigated how the conformation of sulfated fucans determines their
biological properties [92, 93]. Such sulfated fucans exhibit a wide range of
biological activity. It has been shown that these compounds have anticoagu-
lant activity [94–97], venous antithrombic activity [98], are in vitro potential
inhibitors of native [99] and recombinant HIV reverse transcriptase activity
and, due to interference with molecular mechanisms of cell-to-cell recogni-
tion, they can be used to block cell invasion by different retroviruses such as
HIV, herpes, cytomegalovirus and African swine fever virus [100–102]. Due
to their heparin-like behaviour, fucoidan preparations have been proposed as
alternatives to the anticoagulant heparin, prepared from mammalian mucosa.
The application of such pharmaceutically useful compounds of vegetable
origin is less likely to entail infectious agents (e.g. viruses or prions) [86]. Fur-
thermore these macromolecules can act as anti-angiogenic agents [103], can
inhibit sperm binding to oviductal epithelium [104] and sperm-egg binding
in many species [105–107]. In addition, algal-sulfated fucoidans have antipro-
liferative and antitumoral properties [108].
    However, the biological activity of fucoidans results from their structure,
which depends on the fucoidan source. Due to the high molecular weight
of fucoidans they are too large for use in drug-application [109]. There-
fore research is focussed on substructures of these macromolecules, called
low molecular weight fucoidans (LMWF). These subunits are obtainable by
partial acid hydrolysis, enzymatic hydrolysis or radical depolymerization of
the polysaccharides. By using acid hydrolysis and radical depolymerization,
oligosaccharide subunits are gained [109], which also show biological activ-
ity. However, these methods can cause structural alterations, like debranch-
ing or desulfation, and possibly lead to modified biological activity, when
the fucoidan is compared with its subunits. An advantage is the use of en-
zymes [110], called fucoidanases, for partial degradation of these polysaccha-
rides. Thus, the glycosidic linkages are cleaved specifically without modifying
the structural units composing the original fucoidan.
    The dry weight of cell walls from brown algae can contain more than
40% of total dry weight [111]. Thus, in general sulfated L-fucose can eas-
ily be extracted from these cell walls by treatment with cold and hot mild
acid solution. Bakunina et al. treated ground wet or frozen seaweeds with
enthanol and acetone and extracted with 0.4% HCl at 20–25 ◦ C and with
hot water at 60–70 ◦ C [91]. The crude fucoidan extracted with hot wa-
ter is further purified by hydrophobic chromatography and precipitated
with ethanol. The crude extract can also be treated directly with alcohol
from a non-dialyzable fraction of water extract of the source material can
also be carried out. The precipitate is digested with water, filtered, dial-
ysed and freeze-dried [112]. Further purification is implemented by frac-
tional precipitation with cetyl trimethyl ammonium hydroxide (CTA-OH)
or cetylpyridinium chloride (CPC). This is followed by fractional solubi-
lization with acetic acid to release the polysaccharide from its precipitated
Downstream Processing in Marine Biotechnology                              91

salt, and subsequent precipitation with ethanol [113]. In contrast, Shan-
mugam et al. applied precipitation with KCl to fractionate polysaccharides
to obtain high active anticoagulant fractions from Codium spp. [112]. Fur-
thermore, sugar fractionation has also been implemented by using cop-
per salts or boric acid to form stable complexes [114]. Another useful ap-
plication for the precipitation of sulfated fucans is isolation with prote-
olytic enzymes. Vilela-Silva et al. extracted polysaccharides from jelly coat
by papain digestion and conducted partial purification by ethanol precip-
itation [115]. A similar method was also applied for fucoidan extraction
from brown algae, with subsequent precipitation with cetylpyridinium chlo-
ride [116].
    In general, the crude polysaccharide fractions obtained by precipitation
are further purified by chromatographic methods such as ion-exchange, gel
permeation or affinity chromatography [113], followed by molecular weight
determination with gel permeation methods. The fractionated and purified
fucoidans and sulfated fucans can be applied in biotransformation processes
to obtain low molecular weight poly- and oligomers for bioactivity tests. This
is commonly carried out by acid or enzymatic hydrolysis.

3.3
Downstream Processing of Polyunsaturated Fatty Acids

Polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA,
20:5n-3) and docosahexaenic acid (DHA, 22:6n-3) have become a focal point
of marine biotechnology processes due to their significance for human
health. These PUFAs are obtained from marine organisms and have cis, non-
conjugated double bonds. Counting starts from the terminal methyl group,
thus, the first double bond occurs at carbon three (n-3). Depending on their
double bonds, the application of PUFAs can prevent a tremendous number
of diseases like inflammation, hypotriglyceridemic effect, allergies, diabetes
and medical disorders of the heart and circulatory diseases [117–120]. Fur-
thermore, it has been shown that PUFAs can be used to treat cancer [121].
Therefore polyunsaturated fatty acids are widely used to enhance food prop-
erties [122, 123]. The most commonly used sources of PUFAs are fish oils, but
most of this oil is diverted for producing butter and margarine. Given the
limited availability and the increasing demand for fish, the search for alter-
native sources is becoming more important [124]. PUFAs are also available
from many terrestrial tissues such as ox liver, ox brain, hog brain or other
innards, and from marine organisms such as algae. However, due to the in-
creasing number of diseases caused by the consumption of prion-containing
contaminated innards the application of these tissues does not seem to be
applicable. In comparison to innards and fish oils, microalgae have many ad-
vantages. They show a less complex fatty acid profile and have no maladour.
In addition, concerning the food chain, phytoplankton is the source of PUFAs
92                                                            K. Muffler · R. Ulber

in fish. Due to their less complex fatty acid profile, PUFAs of microalgal origin
are well suitable for pharmaceutical applications.
   Purification steps of PUFAs must take into account the oxygen sensitivity
of non-conjugated methylene groups. Thus, hydroperoxides with conjugated
double bonds are produced, which causes a series of autocatalytic reactions
with the production of low molecular weight compounds. Polymers are also
produced as a side reaction. The production rate of hydroperoxides depends
on the degree of unsaturation of the fatty acid and is accelerated by heat, light,
moisture and the presence of metal ions. Therefore purified PUFAs must be
protected against oxygen, which is usually done by storage in ampoules with
minimum headspace or by an addition of antioxidants such as tertiary butyl-
hydroquinone, butylated hydroxytoluene and octyl gallate at concentrations
of 0.01–0.01% to reduce the oxidation rate [125].
   Usually, the cultivated microalgae are separated from the culture broth by
filtration or centrifugation, often followed by pre-treatment operations for
homogenization such as ultrasonication, freezing and grinding [126] or dis-
ruption in bead mills [127]. In general the lipids are directly extracted with
solvents in the wet state, because the solvents applied also induce breakage
of cells. The organic solvents applied should be inexpensive, volatile to allow
easy removal, and should be free of impurities. Concerning the removal of
non-lipids the solvent should be able to form a two-phase system with wa-
ter and have low solubility of unwanted compounds. In the following steps
the crude extract is purified using selective methods, such as chromatography
and precipitation, which are explained more extensively later.
   The type of solvent to apply depends strictly on the type of lipid (neu-
tral or polar) to be purified. It is worth mentioning that lipids can have
different linkages to other cell components. Lipids that are bound via hy-
drophobic or van der Waals interactions to these components can be sepa-
rated by non-polar organic solvents due to the low energy of this bond [128].
The application of polar solvents such as low chain alcohols and water is
useful for breaking existing hydrogen bonds of membrane-associated po-
lar lipids. Such alcohol-containing mixtures are also capable of inactivating
several lipid-degrading enzymes like phosphatidases and lipases. Other unde-
sirable compounds such as saccharides, amino acids, salts and pigments are
also extracted and must be removed in following purification steps.
   The extraction of stronger linked lipids via ionic bounds requires a drastic
shift in pH value. However, during these separation processes the disrup-
tion agents utilized must not alter the structure of the lipid to be purified.
Therefore, the solvents should be de-aerated by bubbling with an inert gas
such as nitrogen or argon to prevent oxidation of unsaturated lipids [129].
A gentle procedure for the extraction of lipids was described by Bligh and
Dyer [130], which is applicable to tissues of animal and plants, but also
to non-homogenized microorganisms. A mixture of chloroform-methanol-
water (1 : 2 : 0.8 by vol) is used as a single phase extractant, which is further
Downstream Processing in Marine Biotechnology                                         93

diluted with chloroform and water (2.2 : 1.8) to obtain a biphasic system. In
this system the lipid contents of the crude extract are enriched in the lower
non-polar phase, whereas unwanted non-lipids and salts are solved in the
upper methanol-water phase. Due to the simplicity of this method it is now
widely used in lipid extractions of microalgal origin. To overcome problems
in industrial applications, caused by the toxicity of chloroform and methanol,
nowadays other systems are also applied.
   The low-toxic systems hexane-isopropanol and butanol-ethanol are also
worth mentioning. The latter was compared by Nagle and Lemke [131]
with other extraction methods applied to diatoms and has showed a better
performance. Unlikely hexane-isopropanol extraction results in lower lipid
yields [132, 133]. In the following steps the lipidic extracts have to be saponi-
fied to obtain free fatty acids. The saponification can be done directly by
adding of alkali to the extract [133]. In contrast to the usual method applied,
which separates extraction and specifications, by omitting one step this pro-
cess is faster and more economical. A schematic flowsheet of the downstream
process of EPA is given in Fig. 11.
   By addition of urea to solvents containing organic compounds it is possible
to fractionate them. If the urea crystallizes from a saturated solution contain-
ing organic molecules it encloses these molecules in hexagonal crystals. The




Fig. 11 Schematic flowsheet for eicosapentaenoic acid purification from wet microalgal
biomass. 1 Culture from photobioreactor, 2 centrifuge, 3 freezer (this step may be omit-
ted if the biomass paste is used immediately after centrifuging), 4 stirred tank under
nitrogen or argon atmosphere, 5 filter press, 6 solvent extraction, 7 vacuum distillation,
8 crystallizer, 9 preparative HPLC [134]
94                                                          K. Muffler · R. Ulber

captured molecules are attached to urea via van der Waals forces, London dis-
persion forces or induced electrostatic attractions. These forces depend on
the shape, size and geometry of the organic compound, which co-precipitates
with urea. In general the urea-inclusion-compound method is used for the
separation of straight-chain compounds from branched or cyclic compounds.
If the method is applied to fatty acid solutions, the long and straight chain
saturated and monounsaturated fatty acids are included in the urea crystals,
whereas polyunsaturated fatty acids remain in the supernatant. As a rule,
the greater the tendency of fatty acids and esters to precipitate with urea,
the higher the unsaturation and the chain length. Therefore, this technique
is most commonly used to obtain concentrates of PUFAs containing ≥ four
double bonds.
    In general the precipitation is carried out in solvents such as methanol or
ethanol. The application of methanol can also lead to unwanted by-products
via methylation of fatty acids [135]. To obtain a reduced fatty acid profile
and concentrate the PUFAs it is important to use saturated urea concen-
trations, otherwise the yield of PUFAs recovered decreases [125]. Attention
must also be paid when the solvent itself is an adduct former and com-
petes with other fatty acids in complexing with urea. Therefore long chain
hydrocarbons and acetone should not be used. An important factor in urea
precipitation is the urea/fatty acid ratio (U/FA), which is strongly related
to temperature. If temperatures higher than – 12 ◦ C are used the concen-
tration of PUFAs increases with the urea/fatty acid ratio. The most appro-
priate is a U/FA of 4 : 1 [125, 136, 137]. Temperatures below – 12 ◦ C indicate
a decrease in PUFAs, if U/FA is increased, which is presumably caused by
precipitation of urea [138, 139]. However, the most appropriate temperature
for concentrating PUFAs such as DHA or stearidonic acid is approximately
4 ◦ C. At higher temperatures the tendency of these compounds to precipitate
with urea is much lower. But if EPA is the desired PUFA the urea inclusion-
compound method should be carried out at temperatures of 20–28 ◦ C [138,
139]. Therefore the temperature must be chosen carefully with respect to
the PUFA profile of the extracted organism to avoid loss of the PUFA com-
pounds desired. This method is simple to apply, more efficient and much
cheaper than other methods like fractional crystallization or selective sol-
vent extraction. Nevertheless, the disadvantage of this method is often the
low PUFA recovery due to the formation of co-precipitates with urea (e.g.
EPA) [140].
    A preparative HPLC separation method seems to be very suitable for the
recovery of PUFAs, although little work exists in this field to date. Despite
the fact that most fatty acids do not absorb UV radiation, conjugated unsat-
urated fatty acids can be detected. At 192 nm the PUFAs can be selectively
detected by an UV detector. The sensitivity is increased the higher the num-
ber of double bonds of the PUFA. But attention must be paid to possible
interference deriving from the mobile phase at this wavelength (e.g. chloro-
Downstream Processing in Marine Biotechnology                                        95

form, methanol). The isolation/separation of EPA and DHA was possible by
using 217 nm for the detection [138].
   The application of reverse-phase HPLC is also possible. In contrast to C18
materials, C8 phases allow faster separations but the separation of the for-
mer is sharper due to the unpolar interactions of the column and the fatty
acids. Free fatty acids are separated more rapidly than the more unpolar cor-
responding esters. Furthermore, the separation of cis and trans isomers is
also possible. The cis isomers of PUFAs are eluated faster due to steric ef-
fects on the eluent-double bond interaction. Commonly used mobile phases
are methanol/water and acetonitrile/water. The application of acetonitrile is
more selective than methanol and sharper separation peaks are obtained,
but many fatty acids are difficult to solve in acetonitrile and separation takes
longer. PUFAs used in clinical trials or in the food industry should be purified
with less toxic mobile phases. Therefore a biocompatible ethanol/water mix-
ture may be applied, but unlike methanol or acetonitrile the flow rate must
be lower concerning the higher viscosity of ethanol [125]. Analytical columns
have to be scaled-up for preparative purposes for commercialization, or for
physiological or nutritional research. Robles Medina et al. [138] scaled up
a reverse phase C18 column to obtain stearidonic acid, EPA and DHA frac-
tions of 90–95% purity from cod liver oil and the marine microalga Isochrysis
galbana.
   Supercritical fluid extraction (SFE) belongs to the green technology of sep-
aration and is relevant to food and pharmaceutical applications due to its
non-toxic and non-flammable behaviour. Furthermore, it is available in high
amounts, allows the handling of thermolabile compounds and simple removal
from the fatty acids (a process flow sheet is given in Fig. 12).
   The carbon dioxide atmosphere also protects the PUFAs from oxygen,
which causes auto-oxidation. Despite many such advantages, SFE is carried
out only in a few cases with marine algae or bacteria. From the green alga
Scenedesmus obliquus Choi et al. [142] extracted lipids from freeze-dried
samples to obtain neutral lipids. The residue was then re-extracted with




Fig. 12 Schematic diagram based on the supercritical fluid separation process [141]
96                                                          K. Muffler · R. Ulber

ethanol to increase the solubility of polar lipids. Extractions of Skeletonema
costatum and Ochromonas danica with supercritical CO2 , were carried out by
Polak et al. [143] at 17–31 MPa and 40 ◦ C on freeze-dried samples. Maximum
solubility was found at 24 MPa, without extraction of chlorophyll under these
conditions. Wang et al. [141] applied SFE for separation of DHA and EPA
from the crude fat of a PUFA-producing marine strain, which was isolated
from fish.

3.4
Downstream Processing of Bioactive Compounds With Low Molecular Weight

Investigations to find bioactive compounds from marine organisms were
first successful in the early 1950s, when Ross Nigrelli extracted a toxin
(holothurin) from the Cuverian organs of the Bahamian sea cucumber
(Actinopyga agassizi) [144]. A few years ago, Rosenfeld and ZoBell reported
on the bacteriostatic behaviour of sea water to some non-marine bacteria and
isolated nine microorganisms capable of producing antimicrobial bioactive
compounds [145]. However, interest in marine bacteria as sources of bioactive
compounds has been growing since Burkholder et al. and Lovell [146, 147]
determined the first novel structure of an antibiotic of marine origin. Due
to the tremendous amount of phyla found in the oceans, it seems that the
best sources of pharmacologically active metabolites are bacteria, fungi, cer-
tain groups of algae, sponges, soft corals, gorgonians, sea hares, nudibranchs,
bryozoans, and tunicates [148]. Bioactive metabolites deriving from these or-
ganisms show a whole range of new chemical structures, which are presently
not found in terrestrial organisms. These novel structures often serve as
leading structures in synthetic organic chemistry for pharmaceuticals. Unfor-
tunately, many marine producers of such bioactive compounds are difficult to
cultivate in the laboratory or are uncultivable.
   Concerning the sustainability of biological diversity, the traditional
method of harvesting vast amounts of material from invertebrates for the iso-
lation of secondary metabolites is not a good practice. When a chemical syn-
thesis of bioactive molecules is possible and economically viable, this should
be the method of choice. Otherwise it seems possible to apply mariculture
as a supply for harvesting. Mariculture has been used with considerable ef-
forts by CalBioMarine Technologies in the cultivation of Bugula neritina and
Ecteinascidia turbinata for the production of the anticancer drugs Bryostatin
1 and ET-743 [149]. Cultivation of the sponge Acanthella cavernosa is also
promising for the production of antiparasitic and anti-infective kalihinols in
culture tank systems, which can presumably be assigned for cultivation of
other sponges to overcome the supply problem [149]. In many cases marine
bacteria associated with such invertebrates are the real sources of the bioac-
tive compounds [150]. In general marine bacteria are isolated from seawater,
marine sediments and from the surface and tissues of higher organisms,
Downstream Processing in Marine Biotechnology                               97

well known as producers of secondary metabolites. The highest number of
structures has been isolated from the order Streptomyces, followed by uniden-
tified bacteria, Alteromonas, Vibrio, Agrobacteria, Bacillus, Pseudomonas and
Actinomyces. Secondary metabolites from other orders of bacteria have been
isolated in only a few cases [151]. However, it seems that many obligate ma-
rine symbionts bacteria cannot at present be cultured [148]. Therefore only
a small percentage of total marine bacteria are cultivatable using traditional
methods of cultivation. To overcome this problem many groups are devel-
oping new culture techniques to isolate slow-growing bacteria [152]. New
perspectives have been opened up by the possibility of transfer of the active
gene and subsequent splicing into another organism that is easier to cul-
ture [153]. For example, it has been proposed that Bugula neritina is not the
source of bryostatin, but that the compound is synthesized by a symbiotic
microorganism [154, 155].
    After cultivation of bacterial strains and centrifugation and harvesting of
macroorganisms, the biomass is extracted by an adequate solvent system
(e.g. methanol or acetone), followed by a gradient partition with polar and
non-polar organic solvents to separate compounds from the crude extract
(see Fig. 13). Such a procedure can be conducted by an appropriate assay to
identify bioactive fractions. The fractions obtained contain compounds de-
pending on their polarity. Water-soluble organic compounds (i.e. alkaloid
salts, amino acids, polyhydroxysteroids, saponins) were mainly found in the
fractions of high polarity, whereas peptides and depsipeptides were mainly
found in medium polarity fractions (i.e. CH2 Cl2 ). The low polarity fractions
(i.e. CCl4 or hexane) contain hydrocarbons, fatty acids, acetogenins, terpenes,
etc. [156].
    In general, the low and medium polar fractions of marine extracts are the
most investigated; therefore most bioactive compounds found are recovered
in this phase. This is due to the fact that lipophilic compounds are usually
easier and cheaper to isolate than hydrophilic compounds [156]. After ob-
taining the different polarity fractions, lipophilic compounds from the low
and/or medium polarity fractions can be further separated and purified by
standard or reverse phase column chromatography or MPLC, followed by
HPLC to obtain single molecules (see Fig. 13). In contrast, the purification
of high polarity fractions should begin by desalting to get rid of the mineral
salts and sodium chloride. This can be easily achieved by column chromatog-
raphy using a solid state material such as amberlite XAD and the organic
components retained on the non-ionic resin. Afterwards the reunited or-
ganic material can be further separated by size-exclusion chromatography on
Sephadex, followed by HPLC using appropriate column materials (i.e. C18,
amino, etc.).
98                                                                K. Muffler · R. Ulber




Fig. 13 Separation of the crude extract into fractions of low/medium and high polarity
and procedures for the isolation of bioactive compounds from these fractions [156]
Downstream Processing in Marine Biotechnology                                        99

4
Conclusion

As already mentioned at the beginning of this chapter, downstream process-
ing seems to be the neglected child of bioprocess engineering. Only a few
groups really focus on this very important part of the whole process. To be
able to use (also in terms of commercialization) the variety of biologically
active compounds from marine microorganisms big efforts must be made
in the downstream process. Only efficient and well-understood procedures
will enable us to produce sufficient material for application in food, feed, and
pharmaceuticals or as bulk chemicals. As described in this chapter, there are
no standard applications in downstream processing that are suitable for all
kind of products. However, wide varieties of downstream tools are available
and can be applied to marine biotechnology. In the future, more integrated
marine bioprocesses will be developed which will lead to a higher product
yield and better process performance.


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Adv Biochem Engin/Biotechnol (2005) 97: 105–131
DOI 10.1007/b135824
© Springer-Verlag Berlin Heidelberg 2005
Published online: 24 August 2005

Marine Pharmacology: Potentialities in the Treatment
of Infectious Diseases, Osteoporosis
and Alzheimer’s Disease
M.-L. Bourguet-Kondracki1 · J.-M. Kornprobst2 (u)
1 Muséum    National d’Histoire Naturelle, Laboratoire de Chimie – UMR 5154 CNRS,
  63 rue Buffon, 75005 Paris, France
  bourguet@cimrs1.mnhn.fr
2 Laboratoire de Chimie Marine, Groupe SMAB,

  Institut Substances et Organismes de la Mer (ISOMer), 2, rue de la Houssinière,
  44322 Nantes cedex 3, France
  jean-michel.kornprobst@isomer.univ-nantes.fr

1     Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   106

2     Antituberculosis Agents of Marine Origin . . . . . . . . . . . . . . . . . .         110

3     Marine Natural Products as Sources of New Antimalarial Agents . . . . .              117

4     Zoanthamine Family as Potent Antiosteoporosis Agents . . . . . . . . . .             120

5     A Promising Therapeutic Candidate for Alzheimer’s Disease . . . . . . . .            124

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   127

Abstract Several molecules isolated from various marine organisms (microorganisms, al-
gae, fungi, invertebrates, and vertebrates) are currently under study at an advanced stage
of clinical trials, either directly or in the form of analogues deduced from structure–
activity relationships. Some of them have already been marketed as drugs. The goal of
this article is not to present a complete panorama of marine pharmacology but to show
that new models and new mechanisms of action of marine substances bring new solutions
for tackling some of the major public health problems of the 21st century. These in-
clude: malaria, which assails mainly the southern hemisphere; tuberculosis, an infectious
disease once believed to be eliminated but alarmingly increasing, especially among HIV-
positive populations; and osteoporosis and Alzheimer’s disease, the extension of which
are correlated with ageing populations, especially in the developed countries.

Keywords Alzheimer’s disease · Malaria · Osteoporosis · Tuberculosis


Abbreviations
IC50 Inhibitory concentration 50%
ID50 Inhibitory dose 50%
SI   Selectivity index
MIC Minimal inhibitory concentration
106                                     M.-L. Bourguet-Kondracki · J.-M. Kornprobst

1
Introduction

New drugs are discovered via new models of the bioactive molecules that are
used as starting points for structure–activity relationships, fully implement-
ing many sophisticated techniques. These include total and combinatorial
synthesis, high-flow screening and pharmacological studies. Therefore, the
process of perfecting a marketable drug is a multidisciplinary task that im-
plies huge human and financial resources over many years. A new substance
can be marketed as a drug when an official agreement has been delivered by
the proper administrative authorities of the country concerned. These autho-
rizations are granted after several years of costly research and development
studies at the laboratory stage (in vitro and in vivo bioassays) and then at
the clinical stage in hospitals (involving four distinct phases). Thus, the de-
velopment of a new drug starting from a marine or terrestrial model requires
12–15 years of work and a total budget of about 800 million euros (or US $).
The entire research and development process is summarized in Table 1. In
this context, and with about ten molecules from marine sources already mar-
keted since the 1970s, the results obtained from marine natural molecules can
be considered as normal and even as very promising. A general panorama of
the current state of these results is presented in Table 2. The literature is abun-
dant in this field and only the most significant papers published during the
last 5 years have been selected [1–15].
   Basically, marine pharmacology consists of the search for new models
of bioactive compounds which, with the help of traditional or combinato-
rial chemical synthesis and of structure–activity relationships, generate new
drugs. This research approach was recently illustrated with KRB 7000, 1,
a synthetic α-galactosylceramide, which originated from the agelasphins such
as 2 isolated from the marine sponge Agelas mauritianus. Antitumoral by
immunostimulation, KRN 7000 is not toxic for mice, even at daily doses of
2.2 mg/kg for one month [16–18].
   Synthesis of new derivatives based on structure–activity relationships can
improve natural bioactive compounds. However, there are some examples for
which the use of the naturally occurring molecule remains necessary. This
poses the thorny problem of the supply of the concerned organisms while
waiting for the development of production by aquaculture, culture of symbi-
otic or associated microorganisms, or genetic engineering. In the best case,
we can cite bryostatin 1, 3, a potent anticancer (melanoma) compound that
cannot be easily synthesized and whose average yield in the organism is ex-
tremely low (about 10–6 –10–7 %). The bryozoan Bugula neritina has been
collected in hundreds of kilograms by specialized companies [19–21] but it is
now produced by aquaculture [22]. Thus, the supply of bryostatin 1 is time-
consuming and costly but not insurmountable.
Marine Pharmacology: Potentialities in the Treatment of Infectious Diseases             107

Table 1 Summary of research and development stages for marketing a new drug∗

Number of            Research and development         Minimum                 Part of
molecules            stages                           duration                costs∗∗
tested                                                (years)                 (%)

                                                                                ⎫
10 000               Fundamental research:                                     5⎪
                                                                                ⎪
                                                                                ⎪
                     in vitro screening                                         ⎪
                                                                                ⎪
                                                                                ⎪
                                                                                ⎬
    20      R        Characterization                  3–5
                     of a lead compound                                       10⎪ 30
                                                                                ⎪
                                                                                ⎪
                     Improvement of the                                         ⎪
                                                                                ⎪
                                                                                ⎪
                                                                                ⎭
                     lead compound                                            15⎫
    10               In vivo bioassays
                                                                              15⎪
                                                                                ⎪
                                                                                ⎪
                     on laboratory animals             2                        ⎪
                                                                                ⎪
                                                                                ⎪
                                                                                ⎪
     5               Clinical trials in humans:                                 ⎪
                                                                                ⎪
                                                                                ⎪
                                                                                ⎪
                     Phase I (tolerance)               2                        ⎬
            D        Clinical trials in humans:                               15⎪ 60
                     Phase II (therapeutic interest) 2                          ⎪
                                                                                ⎪
                                                                                ⎪
                                                                                ⎪
                     Clinical trials in humans:                                 ⎪
                                                                                ⎪
                                                                                ⎪
                                                                                ⎪
     1               Phase III (large scale bioassays) 3                        ⎪
                                                                                ⎪
                                                                                ⎭
                     Approval                          1                      30⎫
                     Post-marketing surveillance                               5⎪
                                                                                ⎬
Yield:      Phase IV Improvement                      Undetermined            10⎪ 20
0.01%                New indications                                            ⎭
                                                                               5

R research, D development
∗ Numbers are given as usual values and can fluctuate
∗∗ Data on financial aspects of drugs are available on internet web sites



   In the worst case, the production by total synthesis is not possible and the
biomass of the concerned organisms, even on a world level, is insufficient to
ensure clinical trials. This last case is illustrated by the series of halichon-
drins B, which are powerful antitumoral compounds isolated from a very rare
and deep sponge of the genus Lissodendoryx, which is currently found in one
very restricted area in the east of South Island in New Zealand. These sub-
stances, and especially isohomohalichondrin B 4, were found in some other
species but in quantities approximately ten times smaller than in Lissoden-
drodoryx sp., for which the average content is 1 mg/kg of sponge but whose
total biomass does not exceed 300 tons. Currently, the total sponge biomass
available is less than necessary to achieve clinical trials [23].
   Between these two extreme cases, there is that of ecteinascidin-743, 5
(YondelisTM ), a promising anticancer compound isolated from the ascidian
Ecteinascidia turbinata, which is currently in phase II/III of clinical trials.
This ascidian is currently produced by aquaculture in Florida [24–26].
   To find in a marine organism a bioactive compound that is easy to synthe-
size and efficient enough to yield a new drug without any chemical modifica-
                                                                                                                                           108


Table 2 Molecules of marine origin already marketed as drugs or currently in final phases of clinical trials

Name                             Stage R/D      Origin                          Activity                      Chemical class

  ınic
Ka¨ acid                         On market      Digenea simplex                 Anthelmintic                  Cyclic aminoacid
                                                (Rhodophyceae)
Cytarabine (Ara-C)               On market      Cryptotethya crypta             Antileukemic                  Nucleoside
                                                (Sponge)                                                      (Arabinose)
Cephalosporins                   On market      Cephalosporium acremonium       Antibiotics                   β-Lactames
                                                (Fungus)
Spongoadenoside (Ara-A)          On market      Cryptotethya crypta             Antiviral                     Nucleoside
                                                (Sponge)                        (Herpes)                      (arabinose)
Keyhole limpet hemocyanin        On market      Megathura crenulata             Anticancer                    Peptide
(KLH, Immucothel® )∗                            (Mollusc)                       (bladder)
Squalamine                       On market      Squalus acanthias               Antiangiogenic                Sulfated aminosterol
(Squalamax)∗∗                                   (Fish)                          (NSCLC)
Ziconotide (SNX-111)             Phase III      Conus magus                     Antipain                      Peptide
                                                (Mollusc)                                                     (ω-conotoxine)
Ecteinascidin-743 (ET-743)       Phase II/III   Ecteinascidia turbinata         Anticancer                    Alkaloid
(YondelisTM )                                   (Ascidian)                      (alkylating agent)            (tetrahydroisoquinolein)
Bryostatin-1                     Phase II       Bugula neritina                 Anticancer                    Macrolide
                                                (Bryozoan)                      (melanoma)
Dehydrodidemnine B               Phase II       Aplidium albicans               Anticancer                    Macrocyclic depsipeptide
(Aplidin® )                                     (Ascidian)                      (prostate, bladder)
Dolastatine-10                   Phase II       Dolabella auricularia           Antimitotic                   Peptide
                                                (Mollusc)                       (ovaries, colon)
GTS-21                           Phase II       Amphiporus lactifloreus          Alzheimer’s disease           Anabasein-derived alkaloid
                                                (Nemertina)
∗   Biosyn Arzneimittel, Fellbach, Germany, ∗∗ Nu-gen Nutrition, Wellington, FL, USA
                                                                                                                                           M.-L. Bourguet-Kondracki · J.-M. Kornprobst
Marine Pharmacology: Potentialities in the Treatment of Infectious Diseases   109

tion is, of course, the ideal case, and can be illustrated by squalamine 6 and
ziconotide 7 (Table 2 and Fig. 1).
   This article aims to present the most recent results in topical fields chosen
according to their current importance for public health:
 • Tuberculosis, due to Mycobacterium sp., was at one time stabilized in Eu-
   rope but has again become of concern, partly because of the weakening of
   immune defenses that is a consequence of AIDS.
 • Malaria, which remains the first cause of mortality in the world and for
   which new therapies are needed. This disease is the consequence of an in-
   festation by Plasmodium species introduced into the blood by mosquito
   bites.
 • Osteoporosis and Alzheimer’s disease, the extension of which seems to be
   one of the inescapable consequences of the ageing population in the de-
   veloped countries.




Fig. 1 Structure of some lead compounds in marine pharmacology
110                                   M.-L. Bourguet-Kondracki · J.-M. Kornprobst

2
Antituberculosis Agents of Marine Origin

Although the majority of molecules from marine origin currently in clinical
trials come from anticancer programs, new trends and potencies have been
uncovered due to the need for more effective treatments in many diseases.
This is the case for infectious diseases such as tuberculosis, a new thera-
peutic challenge with the emergence of multiresistant strains. Tuberculosis,
once believed to be eliminated, is now the second leading cause of death
in the world [27]. More than 9 million people are infected by an active tu-
berculosis and each year 2 million of people die from it. This incidence is
strongly associated with the AIDS pandemic. According to WHO one-third
of the 41 million people who are HIV positive, are infected by tuberculosis.
Moreover, the increased mobility of the world population due to migration
and the development of air travel contributes to a devastating impact world-
wide. In order to find new drugs to cure tuberculosis without relapse, the
antimycobacterial activity of numerous marine molecules has been evaluated.
Various chemical classes including peptides, terpenes, steroids, and alkaloids
showed promise against M. tuberculosis and are presented herein. Some of
these molecules have been included in the excellent review reported by Brent
Copp on antimycobacterial natural products from both marine and terrestrial
sources, which covers the literature between 1990 and 2002 [28].
    The first reports concerning potent antituberculosis molecules from ma-
rine origin date from the years 1997–1999. In 1997, Andersen’s group isolated
two cyclic depsipeptides, massetolide A 8 and viscosin 9 from the cultures of
two Pseudomonas sp. isolated from a marine alga and a marine tube worm,
respectively. Both compounds exhibited in vitro antimicrobial activity against
M. tuberculosis and M. avium intracellulare with MIC values of 5–10 µg/mL
and 2.5–5 µg/mL for massetolide A 8, respectively, and of 10–20 µg/mL
against the two strains of Mycobacterium for viscosin 9 [29]. Following the
precursor-directed biosynthesis procedure, new antituberculosis analogues
were obtained by the incorporation of non-protein amino acids in the cul-
tures of Pseudomonas sp., as previously reported in several cases [30]. How-
ever, they were only obtained in inseparable mixtures of two derivatives and
in quantities too small to evaluate their antimycobacterial activity. Later,
further cyclodepsipeptides demonstrated their potential as antituberculosis
templates, as illustrated with kahalalide A 10 and kahalalide F 11 isolated
from the sacoglossan mollusc Elysia rufescens and its algal diet [31, 32]. Ka-
halalide A 10 exhibited an inhibitory activity of 83% on the growth of M.
tuberculosis H37 Rv at 12.5 µg/mL, while kahalalide F 11 was less active with
67% growth activity on the same strain at 12.5 µg/mL. In contrast to kaha-
lalide F 11, kahalalide A 10 is not toxic, which makes it a good candidate for
future investigations [33]. The marine cyanobacterium Lyngbya majuscula
Marine Pharmacology: Potentialities in the Treatment of Infectious Diseases   111

collected in Guam yielded the cyclodepsipeptides pitipeptolides A 12 and B
13 as antimycobacterial constituents [34]. These molecules also stimulated
elastase activity (Fig. 2).
   In 1999, Rodriguez et al. isolated from the West Indian gorgonian coral
Pseudopterogorgia elisabethae two diterpene alkaloids named pseudopterox-
azole 14 and seco-pseudopteroxazole 15 [35]. Both possess the rare benzox-
azole core and exhibited a significant antituberculosis activity against M. tu-
berculosis H37 Rv with 97% and 66% growth inhibition at a concentration
of 12.5 µg/mL, respectively. In 2001, Corey’s group proposed an enantiospe-
cific synthesis of these compounds, which led to their structural revision,
as presented [36]. Previous investigation of P. elisabethae also yielded two
moderate antituberculosis terpenes with novel skeletons: elisabethine C 16
and elisabanolide 17. Both inhibited the growth of M. tuberculosis H37 Rv
with 42% and 39%, respectively, at 12.5 µg/mL [37]. In 2000, Rodriguez et al.
reported the isolation of further novel terpenoids with a novel cagelike frame-
work from P. elisabethae. One of them, named elisapterosin B 18 displayed
79% inhibitory activity against M. tuberculosis H37 Rv at 12.5 µg/mL and no
significant cytotoxicity [38]. Again in 2001 and from the same extract, Ro-




Fig. 2 Chemical structures of antituberculosis marine natural products – 1
112                                    M.-L. Bourguet-Kondracki · J.-M. Kornprobst

driguez et al. reported the isolation of two additional antimycobacterial diter-
penes of the serrulatane type: erogorgiaene 19 and its hydroxylated analogue
7-hydroxyerogorgiaene 20. Erogorgiaene 19 and 7-hydroxyerogorgiaene 20
exhibited 96% growth inhibition at 12.5 µg/mL and 77% growth inhibition
at 6.25 µg/mL against M. tuberculosis H37 Rv, respectively [39]. These results
provide evidence that the benzoxazole core is not essential for the activity
and that the hydroxylation in position 7 does not decrease the activity. No
significant toxicity was detected for 7-hydroxyerogorgiaene 20 in the NCI tu-
mor 60 cell line panel. Erogorgiaene 19 could not be tested due to the low
available quantity of the compound. More recently, in 2003, the gorgonian
coral P. elisabethae yielded homopseudopteroxazole 21 as minor compound,
which exhibited 80% growth inhibition at 12.5 µg/mL against M. tuberculosis
H37 Rv [40], highlighting diterpene alkaloids of serrulatane type as very good
antituberculosis candidates.
    In 1999, the polyhalogenated monoterpene 22 isolated by König’s group
from the tropical marine red alga Plocamium hamatum, exhibited a mod-
erate antimycobacterial activity towards M. tuberculosis and M. avium with
MIC values of 32 and 64 µg/mL, respectively [41]. Antialgal and mod-
erate cytotoxic activities were also mentioned. From a sponge Agelas sp.
from the Philippines an additional monocyclic diterpene, associated to
a 9-methyladeninium chloride unit, agelasine F 23 revealed activity against
Mycobacterium strains including the isoniazid and ethambutol resistant
strains, with a MIC value of 3.13 µg/mL [42].
    In addition to peptides and terpenes, steroids and brominated spirocyclo-
hexadienylisoxazolin-type compounds are two further potent antituberculo-
sis structural classes highlighted by Hamann’s group from studies of forty-
eight marine natural or synthesis molecules. 19-Hydroxysteroids such as
litosterol 24 and nephalsterols B 25 and C 26 isolated from Nephthea sp. [43]
showed significant antimycobacterial activity with 90, 69 and 96% growth
inhibition against M. tuberculosis H37 Rv at 12.5 µg/mL, respectively [33].
These results indicated that the presence of a free hydroxyl group in pos-
ition 7 decreases the activity. However, the low solubility of these molecules
in the aqueous culture media limit their potential for the treatment of tuber-
culosis. More recently, the steroid saringosterol 27 originally isolated from
the brown alga Sargassum ringgoldianum [44], was reported as the strong
antituberculosis compound of the Chilean brown algae Lessonia nigrescens.
Saringosterol 27, isolated as a 1 : 1 mixture of 24S and 24R epimers, was
found to be remarkably effective and selective against M. tuberculosis with
a MIC value of 0.25 µg/mL. Saringosterol 27 exhibited no toxicity against
Vero cells. Its 24S and 24R epimers, separated by normal-phase HPLC, exhib-
ited MIC values of 1 and 0.125 µg/mL, respectively [45]. The aerothionines of
brominated spirocyclohexadienylisoxazolin structure form the second class
of potent antituberculosis inhibitors of the Hamann group studies. They were
isolated from sponges of the genus Aplysina (=Verongia). Compounds 28 and
Marine Pharmacology: Potentialities in the Treatment of Infectious Diseases   113

29 demonstrated inhibition of 70% and 60% on M. tuberculosis, respectively,
whereas compound 30 was completely inactive. These results suggest the im-
portance of hydroxylation in position 11 or 12 to enhance the antituberculosis
activity in this series (Fig. 3).
   Sesterterpenes of the scalarane type, illustrated with heteronemin 31, are
also interesting in the search for novel antimycobacterial molecules. Het-
eronemin 31 was initially isolated in 1976 from the Red Sea marine sponge
Heteronema erecta [46] then from several Hyrtios erecta sponges of various




Fig. 3 Chemical structures of antituberculosis marine natural products – 2
114                                    M.-L. Bourguet-Kondracki · J.-M. Kornprobst

locations [47–52]. In 1991, Eggleston’s group established the absolute config-
uration after X-ray crystallographic analysis [53]. More recently, the isolation
of heteronemin from the dorid nudibranch Glossodoris atromarginata col-
lected off Mandapan in India strengthened the hypothesis of an ecological
role of this sesterterpene as a chemical defense in opistobranch molluscs,
which specifically fed upon sponges of the genus Hyrtios [54]. Heterone-
min 31 exhibited a MIC value of 6.25 µg/mL on M. tuberculosis [33]. However,
the cytotoxic activity observed towards some tumour cell lines implies the
necessity of chemical modifications to reduce toxicity. The sesquiterpene
quinones such as puupehenone 32 and the hundreds of natural or synthetic
analogues [55] including 15-cyanopuupehenone 33 [56], proved to be a fertile
source for the development of antituberculosis drugs. Puupehenone 32 was
initially isolated in 1979 by Scheuer’s group from the Hawaiian Chondrosia
chucalla sponge [57] but its absolute configuration, and consequently that of
its many co-metabolites, was only determined in 1996 by Capon’s group [58].
These compounds displayed a wide range of biological activities such as anti-
fungal, antimicrobial, antimalarial, antiviral, cytotoxic, and immunodulatory
activity but have not, until now, progressed to more thorough development.
The best antimycobacterial results were obtained with puupehenone 32 and
15-cyanopuupehenone 33, which showed inhibitory activities of 99 and 90%
against M. tuberculosis at 12.5 µg/mL, respectively [33]. While the quinone
methide system is essential for activity, substitutions or additions in pos-
ition 15 preserve the activity and reduce toxicity except for oxidized and
methylated derivatives, which become inactive. Puupehenone 32 with a MIC
value of 12.5 µg/ml (i.e. 38 µM) against the development of M. tuberculosis,
is the most promising compound in this series despite a significant cytotox-
icity [33]. Recently, a new enantiospecific synthesis developed by Quideau’s
group [59], in addition to the two total synthesis previously reported [60],
allows consideration of the production of less toxic analogues.
    Also in 2000, the isonitrile group arose as a relevant group for further
antimicobacterial investigations after evaluation of the activity of 39 ma-
rine molecules against M. tuberculosis and M. avium by König’s team [61].
Of the 11 active compounds selected with a MIC value of 16 µg/ml or less,
only seven of them remained interesting thanks to a low cytotoxicity. Among
these molecules, three of them are structurally closely related and possess an
isonitrile group. The most active non- toxic antimycobacterial compound is
the axisonitrile-3, 34 with a MIC value of 2 µg/mL against M. tuberculosis.
Axisonitrile-3, 34 was previously isolated from the marine sponge Acanthella
klethra Pulitzer-Finali, collected off Australia. This compound had been se-
lected for its strong antimalarial activity [62].
    Additional interesting studies come from Stenger’s group, who are in-
volved in the search for novel therapeutic approaches to fight the multidrug-
resistant tuberculosis strains, and are focused on the study of CD1 molecules,
which play a role in the immunity to mycobacteria [63]. Investigations
Marine Pharmacology: Potentialities in the Treatment of Infectious Diseases   115

with α-galactosylceramide 1, a sponge-derived glycolipid well-known as
KRN 7000, 1, were prompted after the discovery of the implication of
α-galactosylceramide 1 in activation of NKT cells [64, 65]. α-Galactosylceram-
ide 1 is a synthetic analogue of galactosylceramides, previously isolated from
the marine sponge Agelas mauritianus [16]. It also demonstrated strong
immunomodulatory and antitumor activities [66] and is currently under-
going clinical trials as an antitumor agent. Results from Stenger’s group
demonstrated that α-galactosylceramide 1 can participate in human host
defense against the pathogen M. tuberculosis in activating CD1d-restricted
T cells [67]. Previously, α-galactosylceramide 1 showed a protective immunity
in a murine malaria model when combined with vaccination [68].
   Some new strategies have recently emerged in order to find useful com-
pounds with antituberculosis potential, as illustrated with mycothiol-S-conju-
gate amidase (MCA), a new MSH-dependent detoxification enzyme. MCA was
recently characterized by Newton et al. and proved to be a valid target for
the discovery of novel inhibitors of M. tuberculosis strains [69]. Mycothiol
(MSH) is the major low molecular mass thiol produced by most actino-
mycetes [70]. It appears to play an analogous role to glutathione in eukary-
otes and Gram-negative bacteria and is specifically cleaved by the amidase
mycothiol-S-conjugate. Mycothiol and mycothiol-S-conjugate amidase have
been shown to play an important role in the detoxification of alkylating agents
and in protection of actinomycetes against oxygen toxicity [69].
   In 2002, a series of 14 natural and synthetic bromotyrosine-derived com-
pounds related to psammaplin A were screened using a fluorescent assay
by Bewley’s group in order to determine their ability to inhibit the MCA
enzyme [71]. In this study, natural compounds exhibited greater activities
than synthetic ones with IC50 values ranging from 2 µM to 2.7 mM. Their
structures contained either a spirocyclic isoxazoline skeleton, or a reduced
bromophenyl oximinoamide moiety. The most active compounds were com-
pounds 35 and36. Compound 35, a spirocyclic isoxazoline derivative, dis-
played an IC50 value of 2 µM. Compound 36, which possesses a bromophenyl
oximino amide moiety, yielded an IC50 value of 2.8 µM. These compounds
were previously isolated from an Australian non-verongid sponge Oceanapia
sp. [72]. The unique common element in the series seems to be a central
amide group.
   In their ongoing biological investigations with the mycobacterial detoxi-
fication enzyme MCA, in 2003 Bewley’s group screened 1500 crude organic
extracts: 1200 from marine plants and invertebrates and 300 from terrestrial
fungal cultures [73]. Twenty extracts were selected for their ability to inhibit
MCA at concentrations less than 50 µg/ml. From the active extracts studied
so far, 13 compounds were isolated and identified, ten of which were from
marine sponges. In addition to the bromotyrosine-derived molecules pre-
viously reported [71], eight further compounds (five from marine sponges)
showed a significant inhibitory activity with micromolar IC50 values. These
116                                        M.-L. Bourguet-Kondracki · J.-M. Kornprobst

are oceanapiside 37 and oceanapiside aglycon 38, first isolated from the ma-
rine sponge Oceanapia sp. [74], which showed an IC50 value of 10 and 0.5 µM
and 100 and 50 µM on MCA of M. tuberculosis and M. smegmatis, respec-
tively. Suvanine 39 [75] and halisulfate 1, 40 [76], isolated from the marine
sponge Coscinoderma matthewsi, exhibited an IC50 value of 50 and 60 µM
on MCA of M. smegmatis, respectively. The greatest inhibitory activity was
observed with 1 ,3-pyridinium polymer 41, which yielded an IC50 value of
0.1 µM on MCA of both M. tuberculosis and M. smegmatis [77]. The var-
ied chemical structure of these compounds does not provide any valuable
structure–activity relationship (Fig. 4).
   In conclusion, the current assessment of potent marine antimycobacterial
compounds has revealed a series of interesting new leads among available
marine molecules. The exciting results obtained on new targets such as the




Fig. 4 Chemical structures of antituberculosis marine natural products – 3
Marine Pharmacology: Potentialities in the Treatment of Infectious Diseases   117

mycothiol-S-conjugate amidase enzyme should stimulate collaborations be-
tween natural product chemists and pharmacologists in the search for novel
antituberculosis molecules.


3
Marine Natural Products as Sources of New Antimalarial Agents

With an average of 2 million deaths per year, mainly in the Southern hemi-
sphere, and the appearance of chloroquine-resistant Plasmodium strains,
malaria appears as one of the greatest challenges for mankind in this new
century, although this disease was already known in antiquity (http://www.
malarianetwork.org). Four species of Apicomplexa parasites are responsible
for this alarming disease: the well-known and probably the most dangerous
Plasmodium falciparum but also P. malariae, P. ovale, and P. vivax, all trans-
mitted to humans by the bite of a female mosquito of the genus Anopheles.
These parasites feed on blood erythrocytes, taking proteins and leaving the
heme, which is toxic for Plasmodium. However, the parasite is able to poly-
merize the heme into the non-toxic β-hematine. Accordingly, one possibility
for finding antimalarial compounds is to obtain substances that can block
the polymerization of heme. Currently, two groups of natural compounds are
able to do so: quinoline derivatives discovered with quinine and structural
analogues such as mefloquine (LariamTM ), marketed many years ago. Over
two thousand years ago the traditional Chinese pharmacopeia used to rec-
ommend Artemisia annua leaves, the active principle of which is artemisinin,
an endoperoxide-containing sesquiterpene which is able to alkylate the heme,
thus preventing its polymerization [78, 79].
   Marine biodiversity has already yielded new models of antimalarial sub-
stances that have been reviewed up to 1996 [62, 80], and consequently we
shall only present results published after that date. Lyophilized extracts of
two Cyanobacteria strains of the genus Calothrix led, after bioassay-directed
fractionations, to calothrixins A 42 and B 43. These two pentacyclic alkaloids
possess the new and unusual indolo[3,2-j]phenanthridine ring system, both
structures being confirmed by X-ray diffraction analysis [81]. Calothrixins
A and B are active in vitro at nanomolar range against the chloroquine-
resistant strains of Plasmodium FAF6, with an IC50 value of 58 and 180 nM re-
spectively, showing the importance of the N-oxide group for the antimalarial
activity. The IC50 value of chloroquine is higher than 70 nM towards the same
strain (80). Calothrixins are closely related to cryptolepin 44, an alkaloid iso-
lated from roots and bark of the African plant Cryptolepis sanguinolenta. The
in vitro activity of cryptolepin (IC50 ) against chloroquine-resistant P. falci-
parum strains D6, K1 and W2 are 27, 33, and 41 ng/mL, respectively [82, 83].
Under the same experimental conditions chloroquine and artemisinin display
IC50 values of 2, 72, 68 and 2.5, 3.3, and 2.7 ng/mL, respectively.
118                                      M.-L. Bourguet-Kondracki · J.-M. Kornprobst

   From the culture medium of the marine fungus Halorosellinia oceanica
collected in Thailand (strain BCC5149) five compounds were isolated. Two
compounds, 5-carboxymellein 45 and halorosellinic acid 46 (a new sestert-
erpene with ophiobolane skeleton), display against a multiresistant strain
of P. falciparum noticeable antimalarial activity with IC50 values of 4 and
13 µg/mL, respectively. Under the same conditions artemisinin displays an
IC50 value of 1 ng/mL [84] (Fig. 5).
   Among marine organisms, sponges are still the greatest source of new po-
tential candidates for antimalarial drugs. From 1992 a series of sesqui- and
diterpenes bearing one or two isocyano, and/or isocyanate groups displayed
interesting activities against D6 and W2 strains of P. falciparum chloroquine-
resistant. This is especially the case for axisonitrile-3, 34 and for the three iso-
cyanoditerpenes 47–49, the latter three isolated from the sponge Cymbastela
hooperi. Their activities were similar to that of artemisinin [62, 85, 86]. These
isocyanoterpenes also display an interesting selectivity index (SI) which is
defined as the ratio of KB cell cytotoxicity over P. falciparum strains D6 or
W2 cytotoxicity (Table 3). Later, it was shown that isonitrile groups interact
with heme to form a coordination complex with the heme iron atom, then
inhibiting the detoxification process [87]. Another diterpene analogue, kali-
hinol A 50, isolated from an unidentified sponge of the genus Acanthella,
displayed an antimalarial activity (IC50 ∼ 400 ng/mL) with a selectivity index
(SI = 320) slightly lower than those of compounds 47–49 [88].
   Manzamine A 51 and 8-hydroxymanzamine A 52 represent a new model of
in vivo bioactive compounds. Manzamine A was discovered in 1986 from an




Fig. 5 Chemical structures of antimalarial marine natural products isolated from
sponges – 1
Marine Pharmacology: Potentialities in the Treatment of Infectious Diseases         119

Table 3 Antimalarial activity and selectivity index for diterpenes 47–49 (Fig. 5)

Diterpene           P. falciparum D6 strain            P. falciparum W2 strain
                    IC50 (ng/mL)          SI           IC50 (ng/mL)         SI

47                   3.2                 1340          2.5                  1710
48                   4.7                 1000          4.3                  1100
49                  14.1                  230          9.3                   340
Artemisinin          2.8               > 7100          2.1                > 9400
Mefloquine           11.5                  300          3.8                   920



unidentified sponge of the genus Haliclona [89]. 8-Hydroxymanzamine A was
characterized in 1994 from another sponge of the genus Pachychalina (Am-
phimedon) [90] and manzamine F was isolated from Xestospongia sp. [91].
These three genera belong to the same Order Haplosclerida [92]. Manza-
mine A 51 and 8-hydroxy-manzamine A 52 inhibit the growth of Plasmodium
berghei in infected mice. Without treatment all the infected mice die at the
end of 4 days. However, with a single i.p. injection of manzamine A at 50–
100 µM/kg the time survival of mice is enhanced to 10 days, and 40% of
the treated mice were still alive after 60 days. 8-Hydroxymanzamine A is
slightly less active than manzamine A. Manzamine F 53, which has a keto
group, is inactive against malaria [93]. The most recent compounds isolated
from sponges that display antimalarial activity are endoperoxides, especially
three nor-sesterterpenes isolated from Diacarnus erythraeanus collected in
the Red Sea. Two of them, sigmosceptrellin B 54 [94] and muqubilin 55 [95]
are slightly active against D6 and W2 strains with IC50 values of 1.2 and
3.4 µg/mL, respectively. Under the same conditions, muqubilone 56 displays
no activity [96].
    Structure–activity relationships showed that antimalarial activity in en-
doperoxides implies the presence of the three following structural elements:
a 1,2-dioxane ring, a conjugated diene-containing side chain, and a methyl
group in α position from a carboxymethyl group. Two of these conditions are
fulfilled for both diastereoisomers 57and 58 that display potent antimalarial
activity against cycloguanyl-resistant FCR3 strains. They are a hundred times
more active than compounds 54 and 55 [97–99] (Fig. 6).
    The phylum Cnidaria has also yielded antimalarial diterpenes with mod-
erate in vitro activities, as illustrated with briarellin P 59, polyanthellin A 60,
and its derivative 61, which display IC50 values of 14, 16, and 16 µg/mL, re-
spectively [100]. Other chemical changes have been performed on Cnidarian
diterpenes, such as the transformation of lactones into lactames, which made
it possible to convert the inactive sarcophine 62 into active N-substituted aza-
sarcophines such as 63 and 64 with an IC50 value of about 1.0 µg/mL against
P. falciparum D6 and W2 strains [101] (Fig. 7).
120                                        M.-L. Bourguet-Kondracki · J.-M. Kornprobst




Fig. 6 Chemical structures of antimalarial marine natural products isolated from
sponges – 2




Fig. 7 Chemical structures of antimalarial marine natural products isolated from Cnidaria



4
Zoanthamine Family as Potent Antiosteoporosis Agents

The zoanthamine family represent an unprecedented source of polycyclic al-
kaloids, which could be potent candidates for preventing osteoporosis. Zoan-
Marine Pharmacology: Potentialities in the Treatment of Infectious Diseases   121

thamine 65 was the first reported member of this new polycyclic alkaloids
class, isolated in 1984 by Faulkner’s group from an unidentified colonial
zoanthid Zoanthus sp. (Phylum Cnidaria, Class Anthozoa, Order Zoanthidea,
Family Zoanthidae) collected off the Visakhapatnam coast of India. Its struc-
ture was established by X-ray crystallographic analysis but only the relative
stereochemistry was determined [102]. Currently, 17 analogues have been so
far isolated from colonial Indian, Arabian, or Japanese zoanthids of the genus
Zoanthus.
    In 1985, two additional derivatives: zoanthenamine 66 and zoanthamide
67 were isolated during a search for antiinflammatory agents in Zoanthus
sp. All three compounds inhibited phorbol myristate acetate-induced inflam-
mation of the mouse ear [103]. In 1989, the structure of two new isomeric
alkaloids, 28-deoxyzoanthenamine 68 and 22-epi-28-deoxyzoanthamine 69,
were reported from a species of Zoanthus from the Bay of Bengal [104].
28-Deoxyzoanthenamine 68 exhibited potent antiinflammatory and analgesic
properties [104]. The same year, Atta-ur-Rahman’s group isolated zoan-
thaminone 70 from an unidentified Arabian zoanthid [105]. It was noted that
the biosynthetic origin still remains unknown, although an isoprenoid or
a polyketide origin could be suggested [105]. The complexity of the hepta-
cyclic structure of this family stimulated synthetic studies, as illustrated by
the stereocontrolled synthetic study reported in 1994 of the C-9 to C-22 por-
tion of zoanthamine [106].
    In 1995, five new derivatives: norzoanthamine 71, oxyzoanthamine 72, nor-
zoanthaminone 73, cyclozoanthamine 74, and epinorzoanthamine 75 were
isolated from a Japanese Zoanthus sp. These compounds exhibited cyto-
toxic activity towards murine leukemia cells P 388 with IC50 values of 24.0,
7.0, 1.0, 24.0 and 2.6 µg/ml, respectively [107]. In 1997, Uemura’s group de-
termined the absolute configuration of norzoanthamine 71 by a modified
Mosher’s method and proposed a polyketide biogenetic pathway for norzoan-
thamine 71 and previous isolated norzoanthamine-related alkaloids as shown
in Fig. 8 [108]. This possible polyketide pathway is strengthened by the iso-
lation of zooxanthellamine 82 from a symbiotic dinoflagellate Symbiodinium
sp. [109] (Fig. 9). The structural similarity of zooxanthellamine 82 with zoan-
thamine congeners suggests a microalgal origin of the series and supports
a polyketide biogenetic origin starting from a glycine unit, in line with other
marine polyketides [109].
   In 1998, Norte’s group reported the isolation of epioxyzoanthamine 76
from a zoanthid collected along the north coast of Tenerife. The occur-
rence in the animal of epioxyzoanthamine 76 and its epimer oxyzoan-
thamine 71 reinforced the hypothesis of a polyketide origin for the se-
ries [110]. In 1999, five additional zoanthamine analogues were reported
by Norte’s group from a Zoanthus sp. collected off Tenerife (Canary Is-
lands), named 3-hydroxynorzoanthamine 77, 30-hydroxy-norzoanthamine
78, 11-hydroxynorzoanthamine 79, 11-hydroxyzoanthamine 80, and zoan-
122                                      M.-L. Bourguet-Kondracki · J.-M. Kornprobst




Fig. 8 Proposed biogenetic pathway of norzoanthamine by Uemura’s group [108]

thenol 81 [111]. Although significant pharmacological activities have been
reported in this family, the most interesting activity concerns their abil-
ity to inhibit interleukin-6 production, known to stimulate the formation
of osteoclasts, which are involved in bone resorption. Thereby, inhibitors
of interleukin-6 could be potent candidates to prevent osteoporosis [112].
Norzoanthamine 71, first reported in 1995, strongly inhibited the produc-
tion of interleukin-6 in preosteoblastic MC3T3-El cells with an IC50 value of
13 µg/ml. However, the most pharmacologically promising candidate is its
hydrochloride derivative, norzoanthamine hydrochloride, which inhibited IL-
6 induction with an IC50 value of 4.7 µg/ml [113].
   Establishment of structure–activity relationships of antiosteoporosis activ-
ity in the family outlined the importance of the presence of the double bond
in position C15-C16 and of the lactone moiety [114]. Concerning norzoan-
thamine hydrochloride, the presence of an iminium structure, as suggested
by 13 C NMR data, was explained by an equilibrium between the lactone and
the iminium structures as shown in Fig. 10 [114]. These attractive results have
stimulated new synthesis investigations, illustrated by the recently reported
studies concerning formation of decalin ring systems [115] or construction of
the pentacyclic aminal core of zoanthamine/norzoanthamine alkaloids [116].
   The effects of norzoanthamine hydrochloride on bone weight and strength
were evaluated in ovariectomized mice, a postmenopausal osteoporosis
model, in comparison with 17-β-estradiol [114]. In this model, norzoan-
thamine hydrochloride, by oral administration, significantly suppressed the
decrease of bone weight at a daily dose of 0.08–2.0 mg/kg over a period of
4 weeks without affecting the uterine weight. This property is important for
a future therapeutic development. Furthermore, norzoanthamine hydrochlo-
ride at a daily dose of 0.4 mg/kg also suppressed the reduction in bone
strength caused by ovariectomy. In comparison, 17-β-estradiol at the daily
dose of 0.08 mg/kg by intraperitoneal administration also suppressed bone
weight loss but it significantly and dose-dependently increased the uterine
weight and lacked activity at a daily dose of 0.4 mg/kg. In terms of failure
load, 17-β-estradiol showed a suppressive effect by intraperitoneal adminis-
tration at the daily dose of 0.016–0.4 mg/kg. Norzoanthamine hydrochloride
Marine Pharmacology: Potentialities in the Treatment of Infectious Diseases   123




Fig. 9 Structures of zoanthamine alkaloids
124                                      M.-L. Bourguet-Kondracki · J.-M. Kornprobst




Fig. 10 Equilibrium of norzoanthamine hydrochloride reported by Uemura’s group [114]


also exhibited protective effects on both the trabecular and cortical bone in
the humerus. It may act as both a suppressor of bone resorption and as an
enhancer of bone formation [112]. Moreover, norzoanthamine hydrochloride
did not provide the side effects of estrogens on reproductive organs and may
act by a different mechanism of action. However, its target molecule remains
unclear because its direct inhibitory effect on IL-6 secretion has not been
confirmed in vivo.
   More recently, a new biological property was pointed out in the series
after their screening on the aggregation of human platelets. This homoge-
neous family, from a chemical point of view, displayed varied activities in this
assay [117]. 11-Hydroxyzoanthamine 80 and the semisynthetic analogue 83
appeared as strong inhibitors of the human platelet aggregation induced by
several stimulating agents. 11-Hydroxynorzoanthamine 79 showed a specific
inhibitory activity of collagen- and arachidonic acid-induced aggregation. In
contrast, zoanthaminone 70 behaved as a stimulating agent whereas, zoan-
thamine 65 lacked activity in this assay. These markedly different behaviors
suggest that the oxidized function and the methyl group in position 26 play
an important role in the modulation of the human platelet aggregation in the
series. These results could offer a new future for a biological development of
the zoanthamine-type alkaloids series.


5
A Promising Therapeutic Candidate for Alzheimer’s Disease

According to recent epidemiologic studies, Alzheimer’s disease (AD), a pro-
tein misfolding disease, is recognized as one of the major problems of public
health in industrialized countries. This progressive neurodegenerative disease
generally occurs after 65 years with a prevalence around 2–4% at age 70 and
somewhere between 30 and 50% by age 90 [118]. It begins with short-term
memory loss and continues with more widespread cognitive dysfunctions.
Marine Pharmacology: Potentialities in the Treatment of Infectious Diseases   125

   Currently, the main available drugs for treatment of AD are acetyl-
cholinesterase inhibitors and muscarinic M1 receptors agonists. Implication
of the nicotinic system in the disease has also stimulated new therapy ap-
proaches [119]. Especially, low nicotine affinity α7 subtype receptors have
become a new target for β-amyloid mediated neurotoxicity studies, in order
to find α7 receptors agonists that could stem the ravages of AD [120]. In
this context, anabaseine 84 (a paralyzing toxin isolated from the nemertine
worm Amphiporus lactifloreus [121] and also found in ants [122]), which is
structurally related to nicotine, was elicited for its agonist nicotinic receptor
activity, providing a good model for searching potential candidates. Start-
ing from the anabaseine structure, numerous analogues were synthesised,
which led to the discovery of 3-(2,4-dimethoxybenzylidene)-anabaseine, also
called GTS-21 or DMXBA 85 (Fig. 11). It was selected for its partial agonist
activity on the α7 nicotinic receptor in Xenopus oocytes, in a dose-dependant
manner [123–125]. At higher doses, DMXBA displays a moderate antagonist
activity on other central nicotinic receptors such as α4β2 receptors.
   DMXBA 85, which is much less toxic than nicotine, displays significant
improvement in cognitive and learning performance in rats, rabbits, and
monkeys [126–128]. DMXBA 85 also provides neuroprotective effects on cul-
tured neuronal cells exposed to β-amyloid [129]. Interestingly, Kem’s group
reported that DMXBA has the advantage of not affecting the nicotine to
cue [130].
   DMXBA 85 is metabolized extensively after oral administration. O-
demethylation of both ortho and para methoxy substituents occurred readily,
as described in Fig. 12, 4-OH-MBA 86 being the major metabolite of DMXBA
85 in Kem’s studies. In rats, both DMXBA and 4-OH-MBA 86 behave as α7
receptors agonists both in vitro and in vivo. In humans, DMXBA does not
exhibit any α7 agonist activity in vitro, while 4-OH-MBA shows an α7 ag-
onist activity both in vitro and in vivo, suggesting that DMXBA 85 in vivo
activity is due to its major metabolite 4-OH-MBA 86. From these behav-
ior studies, Kem et al. proposed that DMXBA 85 was probably a pro-drug




Fig. 11 Anabaseine and GTS-21
126                                        M.-L. Bourguet-Kondracki · J.-M. Kornprobst




Fig. 12 Biotransformation of DMXBA [129]


in humans [129]. Kem’s group also demonstrated in recent studies that the
two metabolites 87 and 88 are also α7 receptor partial agonists and that
all three metabolites display a higher efficacy than DMXBA for stimulating
rat and human α7 receptors [131]. The partial agonist activity of DMXBA
85 is more potent for α7 receptors in rats than in humans. In a separate
paper, Papke’s group reported the differences between α7 in humans and
rats in the agonist binding site [132]. Through the study of DMXBA 85, the
role and mode of activation of α7 receptors, especially in neuromodulation
and cytoprotection, were explored and analyzed [133]. Hence, recent stud-
ies demonstrated that the protective effects of DMXBA on EtOH-induced
neurotoxicity may be mediated through the activation of α7 nicotinic recep-
tors [134]. DMXBA provides increased understanding about the action of α7
subtype nicotinic acetylcholine receptors and reveals their great functional
heterogeneity [135].
   Activity of the DMXBA and its three hydroxy metabolites were also eval-
uated in Xenopus oocytes on the 5-hydroxytryptamine receptors (5-HT3A ),
which belong to the superfamily of ligand-gated ion channels including nico-
tinic acetylcholine receptors. In this model, DMXBA acts as an antagonist.
In contrast, all three hydroxy metabolites are partial agonists of the murine
5-HT3A receptor [136]. New behavior studies reveal that DMXBA 85 and its
metabolites 86–88 are antagonists of the human 5-HT3A receptor, also greatly
contributing to a better knowledge of the 5-HT receptor [131]. Currently, the
selective α7 nicotinic-acetylcholine receptor partial agonist DMXBA is under
Marine Pharmacology: Potentialities in the Treatment of Infectious Diseases        127

clinical development at Taiho Pharmaceutical Company as a promising ther-
apeutic approach for the cure of Alzheimer’s disease.


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Adv Biochem Engin/Biotechnol (2005) 97: 133–203
DOI 10.1007/b135825
© Springer-Verlag Berlin Heidelberg 2005
Published online: 25 August 2005

Asymmetric Total Synthesis
of Complex Marine Natural Products
Jorma Hassfeld1 · Markus Kalesse1 · Timo Stellfeld1 ·
Mathias Christmann2 (u)
1 Institut
        für Organische Chemie, Universität Hannover, Schneiderberg 1B,
 30167 Hannover, Germany
 Markus.Kalesse@oci.uni-hannover.de
2 Institut
        für Organische Chemie, RWTH Aachen, Professor-Pirlet-Strasse 1,
 52074 Aachen, Germany
 Christmann@oc.RWTH-Aachen.de

1       Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   136

2       Selected Syntheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   137
2.1     Leucascandrolide A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     137
2.1.1   Carreira’s Formal Total Synthesis . . . . . . . . . . . . . . . . . . . . . . . .    138
2.1.2   Introduction of the Side Chain (Leighton) . . . . . . . . . . . . . . . . . . .      139
2.1.3   Hetero Diels-Alder Reaction (Paterson) . . . . . . . . . . . . . . . . . . . .       140
2.1.4   Spontaneous Macrocyclization (Kozmin) . . . . . . . . . . . . . . . . . . .          141
2.1.5   Selectivity Through a Spiroketal (Crimmins) . . . . . . . . . . . . . . . . .        142
2.1.6   Asymmetric Allylation (Williams) . . . . . . . . . . . . . . . . . . . . . . .       143
2.1.7   Chiral Crotylsilane [4 + 2]-Annulation (Panek) . . . . . . . . . . . . . . . .       144
2.1.8   Rychnovsky’s Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . .      145
2.2     Bryostatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   146
2.2.1   Evans’ Total Synthesis of Bryostatin 2 . . . . . . . . . . . . . . . . . . . . .     147
2.2.2   Evans’ Asymmetric Synthesis of the A-ring Sulfone . . . . . . . . . . . . .          148
2.2.3   The B-ring Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     149
2.2.4   The C-ring Synthon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     150
2.2.5   Fragment Coupling and Macrolactonization . . . . . . . . . . . . . . . . .           151
2.2.6   Practical Synthesis of a Highly Potent Bryostatin Analog . . . . . . . . . .         154
2.2.7   Synthesis of the Southern Hemisphere . . . . . . . . . . . . . . . . . . . . .       154
2.2.8   Synthesis of the Simplified A/B-ring Spacer Domain . . . . . . . . . . . . .          156
2.2.9   Segment Coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     156
2.3     Halichlorine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   157
2.3.1   Radical Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     161
2.3.2   Nitrone-Olefin [3 + 2] Cycloaddition Approaches . . . . . . . . . . . . . . .         162
2.3.3   Imine- or Iminium-Based Approaches . . . . . . . . . . . . . . . . . . . . .         165
2.3.4   Michael-Initiated Ring Closure (MIRC)/Curtius Rearrangement Approach                 166
2.4     Apratoxin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .    167
2.4.1   The Forsyth Synthesis of Apratoxin A . . . . . . . . . . . . . . . . . . . . .       168
2.4.2   The Ma Synthesis of an Oxazoline Analog of Apratoxin A . . . . . . . . . .           171
2.5     Tetrodotoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   174
2.5.1   Syntheses and Application . . . . . . . . . . . . . . . . . . . . . . . . . . .      174
2.6     Ciguatoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   181
2.6.1   The Hirama Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     182
134                                                                            J. Hassfeld et al.

2.6.2 Synthetic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .      184
2.6.3 A Synthesis-Based Immunoassay . . . . . . . . . . . . . . . . . . . . . . . .          188
2.7 Cephalostatin Analogs – Synthesis and Biological Activity . . . . . . . . . .            190

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .     197

Abstract Among nature’s ecosystems, the marine environment has been an extremely rich
source of structurally complex and biologically active molecules. This review aims to
cover the recent developments in the synthesis of marine natural products, also reflect-
ing the trend of their increased use to address biological questions. The examples chosen
should be viewed as representative of the different structural motifs on the one hand and
the strategies and stimuli for their synthesis on the other.

Keywords Asymmetric synthesis · Total synthesis · Marine natural products


Abbreviations
9-BBN      9-borabicyclo[3.3.1]nonane
Ac         acetyl
AIBN       2, 2 -azobis-isobutylonitrile
BEP        2-bromo-1-ethyl-pyridinium tetrafluoroborate
BINAP      2, 2 -bis(diphenylphosphino)-1,1 -binaphthalene
BINOL      1, 1 -bi(2-naphthol)
Bn         benzyl
Boc        tert-butyloxycarbonyl
BOM        benzyloxymethyl
Bz         benzoyl
CAN        ceric ammonium nitrate
Cbz        benzyloxycarbonyl
Cp         cyclopentadienyl
CSA        10-camphorsulfonic acid
Cy         cyclohexyl
DAST       (diethylamino)sulphur trifluoride
dba        trans, trans-dibenzylideneacetone
DBU        1,8-diazabicyclo[5.4.0]undec-7-en
DDQ        2,3-dichloro-5,6-dicyano-p-benzoquinone
de         diastereomeric excess
DEAD       diethyl azodicarboxylate
DHQD       dehydroquinidine
DIAD       di-iso-propyl azodicarboxylate
DIBALH di-iso-butylaluminum hydride
DIC        N, N -diisopropylcarbodiimide
DIP        diisopinocampheylborane
DIPEA      N-ethyldi-iso-propylamine
DIPS       di-iso-propylphenylsilyl
DMAP       N, N-(dimethylamino)pyridine
DMDO 2,2-dimethyldioxirane
DMF        dimethylformamide
DMP        dimethoxypropane
DMS        dimethylsufide
Asymmetric Total Synthesis of Complex Marine Natural Products                  135

DMSO      dimethyl sulfoxide
DPPA      diphenylphosphoryl azide
dppf      diphenylphosphinoferrocene
dr        diasteremeric ratio
EDCI      N-ethyl-N -(3-dimethylaminopropyl) carbodiimide hydrochloride
ee        enantiomeric excess
Fmoc      9-fluorenylmethoxycarbonyl
HATU      O-(7-azabenzotriazol-1-yl)-N, N, N , N -tetramethyluronium hexafluorophos-
          phate
HAD       di-iso-propylamine
HMPA      hexamethylphosphoric acid triamide
HOBT      1-hydroxybenzotriazole
IBX       o-iodoxybenzoic acid
imid      imidazole
Ipc       isopinocampheyl
KHMDS     potassium bis(trimethylsilyl)amide
LDA       lithium di-iso-propylamide
LiHMDS    lithium bis(trimethylsilyl)amide
m-CPBA    meta-chloroperbenzoic acid
MMPP      magnesium monoperoxyphthalate
MMTr      p-methoxyphenyldiphenylmethyl
MOM       methoxy methyl
Ms        methanesulfonyl
MS        molecular sieves
NaHMDS    sodium bis(trimethylsilyl)amide
NBS       N-bromosuccinimide
NMM       4-N-methylmorpholine
NMO       N-methylmorpholin-N-oxide
NMR       nuclear magnetic resonance
PCC       pyridinium chlorochromate
PMB       p-methoxybenzyl
PMBM      p-methoxybenzyloxymethyl
PMP       p-methoxyphenyl
PPTS      pyridinium p-toluenesulfonate
PVP       polyvinylpyridine
PyAOP     7-azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorphosphate
PyBroP®   bromotripyrrolidinophosphonium hexafluorophosphate
pyr       pyridine
RCM       ring closing metathesis
r.t.      room temperature
TBAF      tetra-n-butylammonium fluoride
TBDPS     tert-butyldiphenylsilyl chloride
TBHP      tert-butyl hydroperoxide
TBS       tert-butyldimethyl silyl
TEMPO     2,2,6,6-tetramethylpiperidine 1-oxyl
TES       triethylsilyl
Tf        trifluoromethanesulfonate
TFA       trifluoroacetic acid
THF       tetrahydrofuran
THP       tetrahydropyranyl
136                                                             J. Hassfeld et al.

TIPS     tri-iso-propylsilyl
TMEDA    N, N, N , N -tetramethylethylenediamine
TMS      trimethyl silyl
TMSSPh   phenylthiotrimethylsilane
tol      tolyl
TPAP     tetrapropylammonium perruthenate
Ts       toluene sulfonyl




1
Introduction

The synthesis of natural products is one of the most important research top-
ics in organic chemistry [1, 2]. Apart from the more classical fields such as
isolation and structure elucidation, a special focus is placed on efficient and
selective synthesis of biologically important naturally occurring compounds.
On the other hand, the synthesis of complex natural products, which is judged
by the highest synthetic standards, can no longer be the sole reason for the
initiation of research programs. The variety of different techniques and new
methods in organic chemistry allows for synthesizing even the most complex
natural products. In this context, the evaluation of biological mechanisms
and targets connects synthetic organic chemistry with cell biology. Only the
combination of both allows for broadly addressing biological questions such
as cell cycle regulation or transport through membranes. In this context, we
will focus on marine natural products that offer an extension to the struc-
tural diversity of known terrestrial natural products. Furthermore, we will
emphasize elegant techniques that allow for the rapid assembly of complex
structures in addition to their application in simplifying and deconvoluting
structure-activity relationships as a starting point for more detailed biologi-
cal investigations.
   All natural products covered herein have unique biological activities and
synthetically challenging frameworks. At the beginning of each chapter, we
will introduce the reader to their chemical and biological properties and pro-
vide a short overview on the status quo. Additionally, the focus on particular
transformations allows readers who are not accustomed to synthetic prob-
lems to appreciate the covered achievements and to put the retrosynthetic
analyses into perspective with the biological properties.
   The selection of marine natural products covered herein provides ex-
amples where synthesis had to overcome shortages of supply from natural
sources (leucascandrolide). Modified natural products are the essential tools
for target identification (bryostatin), and simplified structures provide a prac-
tical access to structures relevant for pharmacology (bryostatin, ciguatoxin).
Asymmetric Total Synthesis of Complex Marine Natural Products               137

2
Selected Syntheses

2.1
Leucascandrolide A

In 1996, Pietra and coworkers reported the isolation and characterization
of leucascandrolide A (1), an 18-membered macrolide from the calcareous
sponges Leucascandra caveolata Borojevic and Klautau along the northeast-
ern coast of New Caledonia in the Coral Sea east of Australia (Fig. 1) [3].
    Its relative and absolute stereochemistry was assigned using 2D NMR tech-
niques and Mosher ester analysis, respectively. By far more powerful than
leucascandrolide B (2), a second structurally unrelated bioactive compound
isolated from these calcareous sponges and reported 3 years later [4], 1 shows
remarkable cytotoxicity against human KB throat epithelial cancer (IC50 of
0.05 µg/mL) and P388 murine leukemia cells (IC50 of 0.25 µg/mL) in biolog-
ical assays. Leucascandrolide A (1) also displays antifungal activity for which
the side chain was accounted, and, following structure-activity relationship
studies, with the macrocyclic core being responsible for the observed cytotox-
icity.
    The isolation crew suggests a microbial origin of the leucascandrolides.
Samples of L. caveolata collected in 1994, 5 years after the initial sampling at
an unrelated site, contained no trace of these compounds. Isolation of 1 and 2
in high abundance from the earlier sampling could be explained by the pres-
ence of extensive dead, and thus possibly extensively colonized portions of
the sponges collected. A mixed assembly of opportunistic rather than symbi-
otic microbes would best explain these findings and the structural differences
in the two macrolides.
    Structually, (1) possesses two trisubstituted tetrahydropyran rings embed-
ded in the 18-membered macrolactone. The side chain bears an oxazole and




Fig. 1 Structures of leucascandrolide A (1) and B (2)
138                                                                           J. Hassfeld et al.

a carbamate moiety as well as two Z-configured double bonds. These struc-
turally challenging features in combination with the lack of availability and its
biological profile have made 1 a challenging target for the synthetic commu-
nity in recent years [5–20].

2.1.1
Carreira’s Formal Total Synthesis

Among the different approaches, Carreira’s synthesis of the macrocyclic core
of 1 is one of the most convergent strategies [5, 6]. For the synthesis of the first
key intermediate, the catalytic enantioselective addition of dienolate 4 to cro-
tonaldehyde (3) was employed (Scheme 1). The modest yield of this reaction
compared to saturated aldehydes is compensated by possible multigram scale
reactions affording the aldol product 5 in excellent selectivity (91% ee) using




Scheme 1 Carreira. a (R)-Tol-BINAP (2.1 mol %), Cu(OTf)2 (2 mol %), n-Bu4 NPh3 SiF2
(4 mol %), THF, – 78 ◦ C, then 4 and 3, 4 h, then TFA, 44% (91% ee). b 7, Zn(OTf)2 , (-)-N-
methyl ephedrine, Et3 N, PhMe, then 8, r.t., 48 h, 75% (dr 94 : 6). c sodium triacetoxy borohy-
dride, AcOH, MeCN, – 40 ◦ C, 70 h, 97% (dr > 95 : 5). d K2 CO3 , MeOH, r.t., 40 h, 92%. e 2,4,6-
triisoproylphenylselenyl bromide, 2,6-di-tert-butyl-4-methylpyridine, CH2 Cl2 , – 78 ◦ C, 11,
4 h, 74% (dr 88 : 12)
Asymmetric Total Synthesis of Complex Marine Natural Products                        139

2 mol % of a (R)-Tol-BINAP-based copper fluoride catalyst. Further elabora-
tion to methyl ketone 6 provided the eastern segment of the natural product.
   The western hemisphere is constructed employing an asymmetric zinc
acetylide addition to (R)-isopropylidene glyceraldehyde (8) with a stoichio-
metric amount of (-)-N-methyl ephedrine as chiral ligand. Although this
aldehyde is known to cause problems due to polymerization [21], propar-
gylic alcohol 8 was obtained in high yield and selectivity for the E-selective
reduction of the triple bond, and elaboration to the C11 aldehyde set the
stage for fragment coupling with methyl ketone 6. The use of a boron eno-
late aldol reaction employing either n-Bu2 BOTf or chiral (-)-DIPCl furnished
the desired anti-Felkin aldol product through 1,5-stereoinduction in good
yield and excellent selectivity. After antiselective reduction of the ketone and
selenium-mediated cyclization to the second terahydropyran ring, Yamaguchi
macrolactonization [22] concluded Carreira’s formal total synthesis of the
macrocyclic core of leucascandrolide A (1). The side chain could be intro-
duced to 13 by the two-step sequence described by Leighton and coworkers
(Sect. 2.1.2).

2.1.2
Introduction of the Side Chain (Leighton)

As described above, the Leighton group was also involved in the synthetic ef-
forts to synthesize 1, resulting in the completion of the first total synthesis (20
linear steps). Their approach to constructing and incorporating the side chain
will be discussed in detail (Scheme 2).




Scheme 2 Leighton’s side chain synthesis. a n-BuLi, CO2 , THF, – 78 ◦ C to 0 ◦ C. b Lind-
lar’s catalyst, quinoline, H2 , EtOAc, 73% (2 steps). c i-BuOCOCl, N-Me-morpholine,
Ser-OMe·HCl, THF, 75%. d DAST, CH2 Cl2 , – 20 ◦ C, BrCCl3, DBU, 0 ◦ C, 64%. e DIBALH,
THF, 0 ◦ C, 86%. f CBr4 , Ph3 P, 2,6-lutidine, MeCN, 83%. g n-Bu3 SnCH = CH2 , Pd2 dba3 ,
tri(2-furyl)phosphine, THF, reflux, 82%. h 9-BBN, THF, H2 O2 . i (COCl)2, DMSO, Et3 N,
CH2 Cl2 , – 78 ◦ C to 0 ◦ C, 71% (2 steps)
140                                                                 J. Hassfeld et al.




Scheme 3 Leighton’s acylative introduction of the side chain. a (CF3 CH2 O)2 P(O)CH2
CO2 H, EDCI·HCl, HOBT·H2 O, CH2 Cl2 . b KHMDS, 18-crown-6·MeCN, 19, THF, – 100 ◦ C,
55% (2 steps)


   The synthesis began with the known carbamate 14, derived from propar-
gyl amine and methyl chloroformate. Addition of CO2 and Lindlar reduction
to Z-enoic acid 15 were followed by peptide coupling to L-serine methyl ester
through carboxyl activation. Amide 16 was converted to oxazole 17 in good
yield by employing Wipf ’s one-pot method [23]. DIBALH reduction of es-
ter 17 and subsequent Appel reaction gave bromide 18. Stille coupling with
vinyltributyltin introduced an olefinic C2 fragment that was subjected to hy-
droboration and Swern oxidation of the resultant primary alcohol to afford
aldehyde 19 in 10 steps and 14% overall yield from propargyl amine.
   For the introduction of the side chain to macrolide 13, the C5 hydroxyl
group was acylated with bis(2,2,2-trifluoroethyl)phosphonoacetic acid to give
phosphonoacetate 20 (Scheme 3). Deprotonation of 20 with KHMDS and
treatment with aldehyde 19 at – 100 ◦ C gave synthetic leucascandrolide A (1)
in good yield as a 7 : 1 mixture of the Z- and E-olefins.

2.1.3
Hetero Diels-Alder Reaction (Paterson)

For the construction of the eastern tetrahydropyran ring, a hetero Diels-
Alder approach using Jacobsen’s trivalent chromium catalyst 23 [24] was
chosen by the Paterson group (Scheme 4) [8, 9]. In this reaction, 2-siloxydiene
22 was reacted with the α-oxygenated aldehyde 21 and then hydrolyzed
to afford the required cis-tetrahydropyran 24 in good yield without the
need of a solvent. The C5 ketone obtained from this reaction could then
be selectively reduced to the axial alcohol using L-Selectride or NaBH4
to give the required equatorial alcohol for the envisioned invertive side
chain introduction. Followed by TIPS protection of the C5 hydroxyl group,
the primary TBS alcohol was elaborated to methyl ketone 26 and then
coupled to aldehyde 25 through a boron enolate aldol reaction with 1,5-anti-
stereoinduction to provide the anti-Felkin product 27 in almost quanti-
tative yield. Through conversion of 27 to the anomeric acetylated lac-
tol 28, the stage was set for the addition of silyl enol ether 29 in the pres-
Asymmetric Total Synthesis of Complex Marine Natural Products                             141




                                                ˚
Scheme 4 Paterson’s HDA approach. a 4 A MS, r.t., 20 h, then acidified CHCl3 , 4 h,
80%, (dr > 20 : 1, > 95% ee). b c-Hex2 BCl, Et3 N, Et2 O, 0 ◦ C, 25, – 78 ◦ C to – 30 ◦ C, 99%
(dr 17 : 1). c ZnBr2 , CH2 Cl2 , 29, 0 ◦ C, 81% (dr 50 : 1)


ence of ZnBr2 to give the desired trans-substituted western tetrahydropyran
in 30, presumably through axial attack on the intermediate oxycarbenium
ion.
   The C17 ketone was then syn-selective reduced, and macrocycle 31 could
be established via Mitsunobu macrolactonization and deprotection. The Mit-
sunobu protocol under inversion of the C5 equatorial hydroxyl group was
also employed for the attachment of the side chain 32 to overcome steric hin-
drance preventing direct acylation of the axial C5 alcohol (Scheme 5). Lindlar
hydrogenation of both alkynes afforded the synthetic natural product 1 in 23
steps and 6% overall yield under essentially complete stereocontrol.

2.1.4
Spontaneous Macrocyclization (Kozmin)

In addition to the efforts of other groups to affect macrolactonization,
Kozmin and coworkers discovered a spontaneous intramolecular macroac-
142                                                                 J. Hassfeld et al.




Scheme 5 Paterson’s invertive introduction of the side chain. a DEAD, Ph3 P, THF/PhH
(3 : 2), 0 ◦ C to r.t., 90%. b H2 , Lindlar’s catalyst, quinoline, EtOAc, 92%




Scheme 6 Spontaneous acetalization in the Kozmin synthesis. a Pb(OAc)4 , EtOAc, 92%.
b PCC, CH2 Cl2 , 85%. c DDQ, CH2 Cl2 /pH7 buffer, 99%


etalization [10]. Upon treatment of diol 33 with lead(IV)acetate for diol cleav-
age, the resulting aldehyde underwent ring closure to lactol 34 as a single
diastereomer due to the rigid molecular framework (Scheme 6).
   Subsequent oxidation to the lactone and cleavage of the benzyl protecting
group afforded macrolactone 31, which was then coupled with the fully func-
tionalized side chain using the Mitsunobu protocol in analogy to the Paterson
synthesis under inversion of the C5 stereocenter.

2.1.5
Selectivity Through a Spiroketal (Crimmins)

In the Crimmins synthesis of a C1-C13 fragment (40) of leucascandrolide A,
pyrone 36 was deprotonated and added to α-alkoxy aldehyde 35 to afford
a 1 : 1 mixture of diastereomers (37, Scheme 7) [12]. The C9 hydroxyl was pro-
tected as TBS ether followed by removal of the PMB group and then cyclized
to give a thermodynamic 1 : 1 mixture of spiroketal 38 and starting pyrone
37. The material was separated by chromatography and resubjected to the re-
action condition to afford spiroketal 38 in 80% yield after 3 cycles. For the
following reactions, this spiroketal serves as a rigid template, allowing for ex-
Asymmetric Total Synthesis of Complex Marine Natural Products                         143




Scheme 7 Crimmins’ pyrone approach. a 36, LiHMDS, THF, – 78 ◦ C, 35, 84% (dr 1 : 1).
b TBSOTf, 2,6-lutidine, CH2 Cl2 , 0 ◦ C, 90%. c DDQ, CH2 Cl2 /pH7 buffer, 78%. d TFA, PhH,
80% (after 3 cycles). e Et3 SiH, AlCl3, CH2 Cl2 , – 78 ◦ C, 73%

cellent stereocontrol in the forthcoming ketone reductions of the C5 and C9
hydroxyl groups. The required cis-2,6-disubstituted tetrahydropyran 40 was
obtained through bidentate coordination of AlCl3 to the C13 benzyl and the
C11 spiroketal oxygen of 39 to the metal center, which allowed for selective
activation of the C11 oxygen-anomeric carbon bond. Subsequent reduction of
the resulting oxocarbenium ion resulted in axial approach of the hydride to
give tetrahydropyran 40 as single diastereomer.

2.1.6
Asymmetric Allylation (Williams)

In the course of their total synthesis, Williams and coworkers demonstrated
the application of reagent-based asymmetric allylation methodology [14, 15].
Starting from the known epoxide 41 [25, 26], copper-catalyzed addition of
allyltrimethylsilane and TBS protection of the resulting hydroxyl group af-
forded dithiane 42 (Scheme 8).
   Transformation of the allyl silane to the corresponding stannane through
intermediate bromination using NBS gave the precursor for selective allyla-
tion of aldehyde 45. The C3 stereocenter of 46 was set in good selectivity
using Corey’s diazoborolidine 44 (Scheme 9) [27]. Tosylation of the C3 alco-
hol, deprotection of the TBS group at C7, and treatment with NaH furnished
2,6-cis-tetrahydropyranyl aldehyde 47 by invertive displacement at C3 and re-
moval of the dithiane moiety.
   Asymmetric allylation methodology was also employed for the introduction
stannane 48 using the enantiomer of diazoborolidine 49, giving alcohol 50 in
quantitative yield in good selectivity. Elaboration to the acetyl protected lactol
51 gave the precursor for the attachment of the western side chain by addition
of TMS enol ether 29 to the oxycarbenium ion as described in the Paterson syn-
144                                                                             J. Hassfeld et al.




Scheme 8 Williams’ asymmetric allylation. a Mg, THF, (2-bromoallyl)trimethylsilane,
then 41, CuI, – 50 ◦ C to – 10 ◦ C, 2 h, 79%. b TBSCl, imid, DMF, 100%. c NBS, propylene
oxide, CH2 Cl2 /DMF (2 : 3), – 78 ◦ C. d n-Bu3 SnLi, CuBr·DMS, THF, – 78 ◦ C to – 40 ◦ C, 77%
(2 steps). e (S, S)-44, 43, r.t., 10 h, then 45, – 78 ◦ C, 1.5 h, 100% (dr 11 : 1). f TsCl, Et3 N,
DMAP, CH2 Cl2 , 100%. g HF·pyr, MeCN, 99%. h NaH, PhH, 90 ◦ C, 75%. i MeI, CaCO3 ,
MeCN/H2 O (9 : 1), 16 h, 100%




Scheme 9 Fragment coupling through allylation. a (R, R)-44, CH2 Cl2 , 48, r.t., 10 h, then
– 78 ◦ C, 47, 2 h, 100% (dr 91 : 9)


thesis (see above), followed by cyclization to the macrocyclic core. This formal
total synthesis was achieved in 21 linear steps and 7% overall yield.

2.1.7
Chiral Crotylsilane [4 + 2]-Annulation (Panek)

Recently, Panek et al. described the synthesis of a C1-C22 fragment of 1
through [4 + 2]-annulation of chiral crotylsilanes [16, 17]. Using this method-
ology, reaction of crotylsilane 52 with aldehyde 53 catalyzed by TMSOTf at
– 50 ◦ C gave the desired dihydropyran 54 in excellent yield and sets the three
stereocenters diastereoselctively (Scheme 10). Hydrogenation of the double
bond was followed by Hg(OAc)2 -mediated oxidation of the silyl group to give
alcohol 55.
   Following further elaboration of 55 to aldehyde 56, fragment coupling with
the eastern hemisphere was accomplished with a Mukaiyama aldol reaction
Asymmetric Total Synthesis of Complex Marine Natural Products                             145




Scheme 10 Panek’s chiral crotylsilane annulation. a TMSOTf, CH2 Cl2 , – 50 ◦ C, 4 h, 95%,
dr > 20 : 1). b H2 , Pd/C, EtOAc, r.t., 99%. c Hg(OAc)2 , CH3 CO3 H, r.t., 76%. d BF3 ·OEt2 ,
CH2 Cl2 , – 78 ◦ C, 81% (dr > 15 : 1). e Me3 OBF4 , proton sponge, 4 ˚ MS, CH2 Cl2 , r.t., 99%
                                                                     A


of silyl enol ether 57. Treating a mixture of both fragments with BF3·OEt2 at
– 78 ◦ C, the coupling product 58 was obtained in 81% yield under 1,3-anti-
stereoinduction of the aldehyde [28–30]. Methylation of the C9 hydroxyl
group concluded the fragment synthesis, setting the basis for further syn-
thetic studies toward the natural product.

2.1.8
Rychnovsky’s Approach

In their formal total synthesis of 1, Kopecky and Rychnovsky employed
a Mukaiyama aldol/prins cyclization cascade reaction for fragment coupling,
building up the eastern tetrahydropyran ring and most of the macrolide back-
bone in the cyclization step [18]. Using BF3 ·OEt2 as the Lewis acid, enol ether
60 was coupled to aldehyde 59 (Scheme 11). The intermediate oxonium ion




Scheme 11 Rychnovsky’s Mukaiyama aldol/prins cyclization approach. a BF3 ·Et2O, 2,6-
di-tert-butylpyridine, CH2 Cl2 , – 78 ◦ C, then NaBH4 , EtOH, 78% (dr 5.5 : 1 at C9).
b Me3 OBF4 , proton sponge, 4 ˚ MS, CH2 Cl2 , 94% (79% and 15% separated at this stage)
                              A
146                                                                      J. Hassfeld et al.

was directly trapped by intramolecular attack of the allyl silane, exclusively
affecting cyclization to the desired 2,6-cis-tetrahydropyran and under good
1,3-anti-stereocontrol [28] in the aldol reaction. The C9 hydroxyl group was
subsequently methylated for easier separation of the diastereomeric mixture.
Cleavage of the C5 olefin followed by selective reduction and further elabora-
tion to the macrocycle through stannane addition of the western side chain
to a C17 aldehyde and Yamaguchi macrolactonization to complete the formal
total synthesis of leucascandrolide A (1).

2.2
Bryostatin

In the course of their search for new anticancer drugs from the bryozoan
invertebrates Burgula neritina Linnaeus and Amathia convulata, Pettit et al.
isolated a structurally novel family of marine natural products that they
called the bryostatins (Fig. 2) [31–45]. Since 1982, 18 related structures have
been isolated from these two organisms that can be found in the Gulfs of
Mexico and Sagami off Japan. These compounds exhibit potent biological
activities in stimulating immune system responses and regulating apoptotic
function. Additionally, they reverse multidrug resistance and might therefore
act synergistically with other cytostatic agents. Consequently, bryostatin 1
(62) is currently in phase I and II clinical trials for several malignancies.
    The exact mode of action is not known to date, but it has been shown
to bind with high affinity to protein kinase C isozymes [46]. Unfortunately,
the natural abundance of the bryostatin group is very low, which complicates
clinical trials, studies on its mode of action, and establishment of structure-
activity relationships leading to superior clinical candidates.
    Due to their limited availability, difficult isolation, and structural complex-
ity, the bryostatins have attracted the interest of the synthetic community




Fig. 2 Bryostatins 1 (62) and 2 (63) and Wender’s novel structural analog (64)
Asymmetric Total Synthesis of Complex Marine Natural Products               147

over the past 20 years, culminating in 3 total syntheses so far [48–50]. (See
also [47] and references therein.) These synthetic efforts, although impressive
in content, are still not suitable for providing the large quantities needed for
clinical studies or the preparation of superior derivatives.
   The encouraging clinical trials and natural scarcity have ignited substan-
tial interest in the synthesis of analogous structures. The identification of
a simplified analog remains a primary goal of many research groups from
which only the Wender group has made significant progress.
   In this chapter, we will discuss Evans’ synthetic efforts toward bryostatin 2
(63) as a representative example of this class of compounds, followed by
Wender’s rational design and practical synthesis of a potentially superior
bryostatin analog (64).

2.2.1
Evans’ Total Synthesis of Bryostatin 2

For more than 10 years, the Evans group was involved in synthetic studies to-
ward the bryostatins [51–54]. In 1999, a concise total synthesis was published,
using state-of-the-art methodology to establish the complex polyketide struc-
ture of bryostatin 2 (63). In their retrosynthetic analysis, the macrocycle was
constructed from three fragments 67, 68, and 69, which were successively




Fig. 3 Evans’ retrosynthetic plan for the synthesis of 63
148                                                                         J. Hassfeld et al.

coupled (Fig. 3). The subsequent macrolactonization step was followed by
several functional group modifications.

2.2.2
Evans’ Asymmetric Synthesis of the A-ring Sulfone

The Evans synthesis of the A-ring fragment starts from aldehyde 74 that can
be synthesized in five steps according to a sequence developed by Julia and
Zimmerman (Scheme 12) [55, 56]. Evans aldol reaction, using an α-chloro
functionalized acetate 75 that is needed for good stereocontrol, afforded
amide 76.
   Removal of the halide and reduction of the amide furnished diol 77 under
recovery of the oxazolidinone auxiliary. Appropiate functionalization of 77
was achieved through acetalization, reductive cleavage of the acetal, and sub-
sequent oxidation to aldehyde 78.
   For building up the C5 stereocenter in combination with the introduction
of a diacetate fragment, Evans chose to employ a titanium-mediated dienolate
aldol reaction that proceeds in excellent 1,3-anti-selectivity through chelation
control (Scheme 13).
   The β-hydroxy ketone 80 was selectively reduced to the antidiol 81, and
lactonization allowed the differentiation of the hydroxyl groups (Scheme 14).
Silylation of the remaining C3 hydroxyl group and Lewis acid-mediated lac-
tone opening with aniline provided amide 82. Upon treatment with ozone,
a mixture of the lactol diastereomers 83 was obtained. Selective acetalization




Scheme 12 a PhOH, 76%. b KOt-Bu, DMSO, 100 ◦ C. c N2 CH2 CO2 Et, Cu bronze. d PhLi.
e NaHSO4 , acetone/H2 O, 80%. f 75, Bu2 BOTf, i-Pr2 NEt, CH2 Cl2 , then 74, – 78 ◦ C to 0 ◦ C
(dr 9 : 1). g Zn, THF/AcOH. h LiBH4 , MeOH, THF, 0 ◦ C, 67% (3 steps). i PMPCH(OMe)2 ,
PPTS, CH2 Cl2 . k DIBALH, CH2 Cl2 , 0 ◦ C, 94% (2 steps). l (COCl)2, DMSO, Et3 N, CH2 Cl2 ,
– 78 ◦ C, 96%
Asymmetric Total Synthesis of Complex Marine Natural Products                         149




Scheme 13 a TiCl2(OiPr)2 , PhMe, – 78 ◦ C, 83% (dr 94 : 6)




Scheme 14 a Me4 NHB(OAc)3 AcOH/MeCN, – 35 ◦ C, 92%. b PPTS, PhMe, 80 ◦ C. c TBSOTf,
2,6-lutidine, CH2 Cl2, – 10 ◦ C, 64% (2 steps). d AlMe3 , H3 NPhCl (better PhNH2 ·HCl),
CH2 Cl2 , 0 ◦ C, 75%. e O3 , CH2 Cl2 /MeOH (10 : 1), – 78 ◦ C, then Me2 S, 72% (dr 3 : 2).
f Ac2 O, pyr 48 h. g PhSTMS, ZnI2 , n-Bu4 NI, CH2 Cl2 (dr 97 : 3). h m-CPBA, NaHCO3 ,
EtOAc, 76% (3 steps)


and conversion to the α-sulfide was achieved in very good selectivity and was
followed by m-CPBA oxidation to provide the A-ring fragment, sulfone 68.

2.2.3
The B-ring Synthesis

Starting from commercially available benzyloxyacetaldehyde 85, Evans em-
ployed his chiral copper pybox 86 catalyst to affect the second dienolate aldol
reaction with almost perfect selectivity (Scheme 15). Again, the keto group
was selectively reduced to the antidiol using tetramethylammonium triace-
toxyborane, followed by lactonization and TES protection to 88. The remain-
ing side chain was introduced by the addition of an oxygen-functionalized
organolithium species to form a mixture of hemiacetals. Upon ionic reduc-
tion, good stereoselection for diol 89 was observed. Hydroxyl protection,
hydrogenation of the benzyl group, and Swern oxidation completed the syn-
thesis of B-ring aldehyde 67.
150                                                                           J. Hassfeld et al.




Scheme 15 a 86 (0.05 eq), CH2 Cl2 , – 90 ◦ C, 85% (99% ee). b Me4 NHB(OAc)3 , AcOH/MeCN,
– 35 ◦ C, 84% (dr 94 : 6). c F3 CCO2 H, CH2 Cl2 . d TESCl, imid, CH2 Cl2 , 0 ◦ C, 77% (2 steps).
e PMBOCH2Li, THF, – 78 ◦ C to – 50 ◦ C. f BF3 ·OEt2 , Et3 SiH, CH2 Cl2 , – 20 ◦ C, 64% (2 steps,
dr 94 : 6). g TBSCl, imid, DMAP, CH2 Cl2 . h H2 , 10% Pd/C, cat. AcOH, EtOAc. i (COCl)2,
DMSO, Et3 N, CH2 Cl2 , – 78 ◦ C to – 50 ◦ C, 66% (3 steps)


2.2.4
The C-ring Synthon

The synthesis of the C-ring was started from selective tosylation, nucleophilic
displacement, and double oxidation of the commercially available diol 90 to
afford aldehyde 91 in 76% yield over four steps (Scheme 16). Reaction of 91
with Grignard reagent 92 was followed by Swern oxidation of 93 and oxidative
cleavage of the double bond using a dihydroxylation/diol cleavage sequence
to keto aldehyde 94.
   The methyl ketone 97 that is required for further elaboration of the back-
bone was synthesized through kinetic resolution of allyl alcohol 95 using
the Sharpless epoxidation protocol. PMB protection and ozonolysis led to
97, which was coupled to aldehyde 94 via its chiral ((-)-Ipc)2 -boron enolate.
The hydroxyl ketone 98 was reduced to the antidiol employing p-nitrobenzyl
aldehyde under Tishchenko oxidation conditions, which selectively gave the
monoprotected compound 99 in excellent yield and regio- and diastereose-
lectivity. The remaining hydroxyl group was TBS protected and the benzoate
was cleaved. Upon treatment with CSA, dihydropyran 69 was formed through
dehydration of the intermediate glycal. Dihydropyran 69 was then used for
fragment coupling since further functionalization on this stage to the exo-
cyclic α,β-unsaturated ester led to instability under the basic conditions in
the forthcoming Julia olefination reaction.
Asymmetric Total Synthesis of Complex Marine Natural Products                              151




Scheme 16 a TsCl (0.2 eq), pyr, CH2 Cl2 , 0 ◦ C to r.t.. b NaH, PhSH, DMF, 80 ◦ C. c m-
CPBA, CH2 Cl2 , 0 ◦ C to r.t. d (COCl)2, DMSO, Et3 N, CH2 Cl2 , – 78 ◦ C, 76% (4 steps).
e 92, Et2 O/CH2 Cl2 (1 : 1), 0 ◦ C to r.t. f (COCl)2, DMSO, Et3 N, CH2 Cl2 , – 78 ◦ C. g i : 1:
K2 OsO4 (OH)2 (0.02 eq), quinuclidine (0.02 eq), K3 Fe(CN)6 , K2 CO3 , t-BuOH/H2 O 1 : 1;
ii : 2-NaIO4 , NaHCO3 , t-BuOH/H2 O/THF 2 : 2 : 1, 78% (4 steps). h L-(+)-DIPT (0.15 eq),
Ti(OiPr)4 (0.1 eq), TBHP (0.7 eq), CH2 Cl2 , – 20 ◦ C. j NaH, PMBBr, cat. n-Bu4 NI, THF, 0 ◦ C
to r.t. k O3 , CH2 Cl2 /MeOH, – 78 ◦ C, then Me2 S, 25–30% (3 steps). l 97, (-)-DIPCl, Et3 N,
CH2 Cl2 , – 78 ◦ C, then 94, – 70 ◦ C, 87% (dr 93 : 7). m SmI2 (0.2 eq), p-NO2 PhCHO, THF,
0 ◦ C, 76%. n TBSOTf, 2,6-lutidine, CH2 Cl2, – 15 ◦ C. o LiOH, THF/MeOH/H2 O 2 : 2 : 1,
88% (2 steps). p CSA (0.05 eq), PhH, 80 ◦ C 90%


2.2.5
Fragment Coupling and Macrolactonization

Having the three segments in hand, the stage was set for coupling studies. As
a result of extensive optimization, a Julia olefination reaction was chosen to
first join the B- and C-ring segments (Scheme 17). This three-step procedure
exclusively gave the desired E-olefin 100 through acetylation and mercuric-
chloride-mediated cleavage of the intermediate hydroxysulfone to form 100
in good yield. For the introduction of the third ring, the primary TBS group
was selectively deprotected under basic conditions and converted to the cor-
responding triflate 101.
   The lithium dianion of A-ring sulfonylamide 68 was treated with the com-
bined A/B-ring segment 101 to form the ABC lactol 102 in excellent yield
(Scheme 18). This intermediate was reported to be synthesized in multigram
scale. Having tricycle 102 in hand, the appropriate functionalization to the
macrolactonization precursor had to be accomplished.
   Therefore, lactol 102 was opened by TES protection of the open chain
equilibrium derivative followed by amide conversion to the benzyl ester 103.
152                                                                          J. Hassfeld et al.




Scheme 17 a 69, n-BuLi, THF, – 78 ◦ C, then 67, – 78 ◦ C to – 50 ◦ C. b Ac2 O, DMAP, CH2 Cl2 .
c Mg, HgCl2 (0.2 eq), EtOH, 64% (3 steps). d TBAF, THF, – 15 ◦ C. e Tf 2 O, 2,6-lutidine,
CH2 Cl2 , – 10 ◦ C, 71% (2 steps)




Scheme 18 a 68, n-BuLi (2 eq), THF, – 78 ◦ C, then HMPA, then 101, SiO2 , 87%. b TESCl,
imid, MeCN, 85%. c Boc2 O, DMAP, MeCN. d BnOLi, THF/MeOH 1 : 1, – 30 ◦ C, 75%
(2 steps). e m-CPBA, MeOH, – 20 ◦ C. f ClCH2 CO2 H, MeOH, 0 ◦ C. g DMP, CH2 Cl2 , pyr,
79% (3 steps). h HF·pyr, THF/MeOH/pyr 4 : 4 : 1, 80%. j TESCl, DMAP, CH2 Cl2 , 10 ◦ C,
65%. k 1,4-cyclohexadiene, 10% Pd/C (0.5 eq), EtOAc, 62% (2 steps)



Simple methanolysis failed due to reversal of the reaction after purification
possibly due to the acidity of the anilide proton. The enol double bond was
then epoxidized and in situ subjected to methanolysis followed by oxidation
to form the oxoketal 104 as a single diastereomer. Complete removal of the
silyl protecting groups led to recyclization of the A-ring and was followed by
selective TES protection of the triol, retaining the free hydroxyl group at C26.
Hydrogenolysis of the benzyl ester led to seco-acid 66 as macrocyclization
precursor.
Asymmetric Total Synthesis of Complex Marine Natural Products                         153




Scheme 19 a 2,4,6-trichlorobenzoyl chloride, i-Pr2 NEt, PhH, then DMAP, 81%. b PPTS
(0.2 eq), (MeO)3 CH/MeOH 1 : 2, CH2 Cl2 , – 30 ◦ C. c DMP, CH2 Cl2 , pyr, 66% (2 steps).
d 107, NaHMDS, THF, – 78 ◦ C to – 15 ◦ C, 93% (> 12 : 1 E:Z). e KHMDS, THF, – 78 ◦ C, then
OHCCO2 Me. f Et3 NSO2 NCO2 Me, PhH, 54% (2 steps, dr 12 : 1). g 109, BH3 ·SMe2 , CH2 Cl2 ,
then MeOH, not clear (CH3 OCH2 CO)O2 , pyr, DMAP (89%, dr 91 : 9). h PPTS, THF/H2 O.
j Na2 CO3 , MeOH. k p-TsOH, MeCN/H2 O 4 : 1, 80% (3 steps). l 111, DIC, DMAP, CH2 Cl2 ,
62%. m DDQ, CH2 Cl2 /H2 O 10 : 1, pH7 phosphate buffer, 57% i) step missing


    Employing the Yamaguchi conditions, the hydroxyl acid was smoothly
converted to the macrocycle. Selective removal of the C13 TES group under
acidic conditions and Dess-Martin oxidation furnished ketone 106, which
was converted to the corresponding α,β-unsaturated ester 108 by the add-
ition of the chiral Horner-Wadsworth-Emmons reagent 107, giving 108 in
excellent yield and sufficient selectivity. The exocyclic ester moiety of the
C-ring was then incorporated by a two-step aldol reaction/dehydration se-
quence followed by asymmetric CBS reduction of the C20 ketone under in situ
protection as the methoxy acetate. The chiral catalyst 109 was needed since
various simple reducing agents gave only moderate selectivity and yield.
Treatment with PPTS hydrolyzes the C19 acetal under removal of the silyl
protecting groups and deprotection of the hydroxyl group under basic con-
ditions gave 110. Treatment with p-TsOH led to hydrolysis and equilibration
of the A-ring acetal to form the thermodynamically favored natural configu-
ration. The addition of the DCC-activated C20 carboxylic acid 111 furnished
the monoacetylated triol, which was deprotected to the natural product 62
154                                                              J. Hassfeld et al.

through DDQ oxidation of the remaining PMB protecting groups under
buffered conditions.
   This synthetic approach to the synthesis of bryostatin 2 (62) consists of 40
linear steps and demonstrates the efficacy of convergent fragment coupling.
The flexibility of late stage functionalization makes it suitable for structure-
activity studies and biological assays, but the synthesis is too complex for
scaling up to larger quantities for clinical development

2.2.6
Practical Synthesis of a Highly Potent Bryostatin Analog

As indicated before, none of the current total syntheses of the bryostatins
is able to solve the supply problem for in-depth clinical studies. The Wen-
der group has addressed this issue by providing analogs that are up to 100
times more potent than natural bryostatin and can be produced in sufficient
quantities through total synthesis (see [57] and references therein).
   Wender et al. suggest the C1-carbonyl and the C19/C26-hydroxyl groups
to be responsible for selective binding to PKC isozymes, imitating the func-
tional group geometry of its natural ligand 1,2-diacylgylcerol, which is known
to activate PKC. Based on this model, the northern part of the molecule is
only responsible for the appropriate conformation of the molecule that affects
the orientation of the pharmacophoric groups and can be simplified without
loss in binding activity and selectivity, while being optimized for facile syn-
thesis. In recent publications [57, 58], Wender et al. successfully demonstrated
this concept in practical synthesis of a simplified C-ring fragment in com-
bination with an esterification/macroacetalization protocol employing their
second-generation A/B-ring analog spacer domain.

2.2.7
Synthesis of the Southern Hemisphere

Wender et al.’s second-generation synthesis of the southern hemisphere
started with diol 112, which was monoprotected as TBS ether and oxidized
to generate aldehyde 113 (Scheme 20). The addition of a Grignard reagent
to elongate the chain and Swern oxidation was followed by highly selective
asymmetric Keck allylation to form homoallylic alcohol 114. Acid-catalyzed
hemiacetal formation proceeded under dehydration, and the so-formed dou-
ble bond was epoxidized and elaborated to ketone 115 through methanolysis
and oxidation of the major diastereomer.
   The introduction of the α,β-unsaturated ester 116 was accomplished by
treatment of ketone 115 with K2 CO3 in methanol with methyl glyoxylate. Sub-
sequent reduction of the keto group and esterification afforded ester 116 in
excellent diastereoselectivity.
Asymmetric Total Synthesis of Complex Marine Natural Products                             155




Scheme 20 a NaH, TBSCl, THF, r.t. b SO3 ·pyr, Et3 N, DMSO, CH2 Cl2 , r.t. c (i) 4-chloro-1-
butanol, MeMgCl, THF, – 78 ◦ C to r.t.; (ii) Mg, reflux; (iii) 113, – 78 ◦ C. d (COCl)2, DMSO,
CH2 Cl2 , Et3 N, – 78 ◦ C, 54% from 112. e 10 mol % R-BINOL, 4 ˚ MS, 5 mol % Ti(OiPr)4 ,
                                                                    A
B(OMe)3 , allyl-SnBu3 , CH2 Cl2, r.t., 77% (92% ee). f cat. p-TsOH·H2 O, 4 ˚ MS, PhMe, r.t.,
                                                                              A
85%. g MMPP, NaHCO3 , CH2 Cl2 /MeOH (2 : 1), 0 ◦ C, 78% (dr 4 : 1). h 10 mol % TPAP,
NMO, 4 ˚ MS, CH2 Cl2 :MeCN (6 : 1), 0 ◦ C, r.t., 78%. i K2 CO3 , OHCCO2 Me, MeOH, r.t.,
          A
72%. j NaBH4 , CeCl3·7H2 O, MeOH, – 30 ◦ C. k C7 H15 CO2 H, DIC, DMAP, CH2 Cl2 , r.t., 93%
from 116


   Deprotection of the primary TBS group and oxidation was followed by
the addition of a vinyl zincate species that was reported to be one of
few nucleophiles that were successful in the homologation to aldehyde 119
(Scheme 21). The terminal double bond was asymmetrically dihydroxylated
using Sharpless’ protocol. After separation of the diastereomers, selective
protection of the primary hydroxyl group generated the fully elaborated
recognition domain 120 in 17 steps and 3% yield.




Scheme 21 a HF·Et3 N, THF, r.t. b DMP, NaHCO3 , CH2 Cl2 , 0 ◦ C f r.t., 87% (2 steps).
c (i) (Z)-1-bromo-2-ethoxyethene, t-BuLi, Me2 Zn, then 119, Et2 O, – 78 ◦ C, (ii) 1 M
HCl, – 78 ◦ C, r.t., 90%. d (DHQD)2 PYR, K2 OsO2 (OH)4 , K3 Fe(CN)6 , K2 CO3 , t-BuOH:H2 O
(1 : 1), 0 ◦ C, 71% (d.r 5 : 2). e p-TsOH·H2 O, MeCN, H2 O. f TBSCl, imid, CH2 Cl2 , r.t., 46%
(2 steps)
156                                                                        J. Hassfeld et al.

2.2.8
Synthesis of the Simplified A/B-ring Spacer Domain

Very recently, the Wender group published a second-generation synthesis of
the northern hemisphere of the analog 64 that relies on three commercially
available building blocks [58]. The first two segments are coupled through
methyl ketone addition to acid chloride 121 (Scheme 22).




Scheme 22 a LDA, 4-benzyloxy-2-butanone, – 78 ◦ C, 68%. b Ru-(S)-BINAPCl2 , MeOH, H2 ,
(95 atm), 30 ◦ C, 92%. c silica, PhMe, reflux, 95%. d TBDPSCl, imid, DMF, 85%. e ethylace-
toacetate, LDA (2 eq), – 78 ◦ C. f Et3 SiH, TFA, – 30 ◦ C, 70% (2 steps). g Ru-(R)-BINAPCl2,
EtOH, H2 (78 atm), 91%. h H2 , Pd(OH)2 , Et2 O, then LiBH4 , 96%. i 2,2-dimethoxypropane,
p-TsOH, DMF, then silica, CH2 Cl2 , 93%. j TEMPO, NaOCl, NaClO2 , MeCN, 50 ◦ C, 92%.


   Employing the Noyori protocol, asymmetric hydrogenation of diketone
122 proceeded in excellent yield and diastereoselectivity. Differentiation of
the diol was accomplished by selective lactonization and protection of the re-
maining hydroxyl group, followed by introduction of the remaining backbone
atoms through dienolate addition to lactone 123. Reduction of lactol 124 to
the tetrahydropyran and subsequent Noyori hydrogenation selectively set the
remaining stereocenters. Liberation of the primary hydroxyl group through
Pd/C hydrogenation and in situ reduction to the corresponding triol was fol-
lowed by acetalization. A one-step oxidation to carboxylic acid afforded the
desired A/B-ring spacer domain 126 in 10 steps and 25% yield.

2.2.9
Segment Coupling

With the 2 segments in hand, coupling to the lactone was accomplished by
activation of the carboxylic acid of the northern hemisphere 126 and esterifi-
cation with the southern part 120 to form the precursor for transacetalization
(Scheme 23). Upon treatment with HF·pyridine, both silyl protecting groups
were removed while the acidic conditions mediated the macroacetalization,
Asymmetric Total Synthesis of Complex Marine Natural Products                      157




Scheme 23 a PyBroP®, i-Pr2 NEt, 120, DMAP, CH2 Cl2 , 70%. b 70% HF·pyr, THF, r.t., 90%


setting the C15 stereocenter under thermodynamic control. All these trans-
formations could be readily adapted to large scale, thus allowing the 19 step
synthesis (longest linear sequence) for supplying the macrocyclic product 64
in the quantities needed for clinical studies.

2.3
Halichlorine

In 1996, Uemura and coworkers isolated a novel marine alkaloid from the
sponge Halichondria okadai near the city of Kadota. Halichlorine (127) was
shown to inhibit the induced expression of VCAM-1 (vascular cell adhesion
molecule-1) at IC50 7 µg mL–1 (Fig. 4). VCAM-1 regulates the transport of
leucocytes, which makes it a potential target for the treatment of arterioscle-
rosis, inflammatory diseases, and cancer [59, 60]. Interestingly, closely related
pinnaic acid (128) [61] displays inhibitory activity against cytosolic phospho-
lipase A2 . The challenging azaspirocyclic core and its limited availability from
biological sources (3.5 × 10–7 % from wet sponge), along with a promising bi-
ological profile, make halichlorine an ideal candidate for a synthetic venture.




Fig. 4 Halichlorine (127) and pinnaic acid (128)
158                                                                        J. Hassfeld et al.

   Not surprisingly, several groups have reported their efforts toward the
azaspirocyclic core. In 1999, the Danishefsky group at Columbia University
published an elegant total synthesis of halichlorine [62–64]. In this chapter
we will discuss their total synthesis, followed by an overview of some recent
approaches.

The Danishefsky Synthesis

Starting with the Meyers lactam 129 [65–68], the crucial quaternary stere-
ocenter at C9 was introduced using a Sakurai reaction (Scheme 24). After
replacement of the phenylglycinol moiety with a Boc protecting group, a se-
lective methylation from the convex face of the molecule afforded the bicyclic
lactam 132. The latter compound was converted to the TBDPS ether 133 and
subjected to a B-alkyl-Suzuki coupling [69] with the (Z)-iodo acrylate 134.
Concomitant cleavage of the Boc protecting group and stereoselective aza-
Michael reaction furnished the azaspirocyclic scaffold.
   Ester 136 was then subjected to Claisen condenzation with tert-butyl ac-
etate followed by a Mannich reaction with formaldehyde (Scheme 25). Con-
version of the β-keto ester to the corresponding α,β-unsaturated ester 137
was achieved using a procedure developed by Ganem et al. [70].
   The TBPDS ether was removed and the resulting primary alcohol oxidized
with tetra-n-propylammonium perruthenate (TPAP) and excess N-methyl-
morpholine N-oxide (NMO) (Scheme 26) [71]. Homologation of the aldehyde
intermediate with the Gilbert-reagent [72–74] afforded alkyne 138, which was
subjected to hydrozirconation followed by transmetallation with dimethyl




Scheme 24 a allyl trimethylsilane, TiCl4 , CH2 Cl2 , – 78 ◦ C → r.t. (99%). b Na, NH3 , THF,
EtOH, – 78 ◦ C (92%). c Boc2 O, DMAP, THF (96%). d 1. LiHMDS, THF, – 40 ◦ C, 2. MeI, –
78 → 0 ◦ C (90%). e LiOH, THF, H2 O (89%). f 1. ClCOOEt, Et3 N, THF, 2. NaBH4 , MeOH
(82%). g TBDPSCl, Et3 N, DMAP, CH2 Cl2 (95%). h 1. 9-BBN, THF, 2. 134, [Pd(dppf)Cl2 ],
AsPh3 , Cs2 CO3 , DMF, H2 O
Asymmetric Total Synthesis of Complex Marine Natural Products                              159




Scheme 25 a 1. TFA, CH2 Cl2, 2. H2 O, K2 CO3 (77% from 133). b t-BuOAc, LiHMDS, THF,
– 50 ◦ C → r.t. (86%) c H2 CO, EtOH (73%). d 1. LiHMDS, THF, 0 ◦ C, 2. [Cp2 Zr(H)Cl], r.t.
(91%)




Scheme 26 a HF/pyr, pyr, THF (94%). b TPAP, NMO, MeCN, r.t., N2 CHP(O) (OMe)2 ,
KOtBu, THF, – 78 ◦ C (47% from 136). c 1. [Cp2 Zr(H)Cl], CH2 Cl2 , 2. Zn2 Me, heptane,
65 ◦ C, 3. 141 (10%), – 65 → – 30 ◦ C, 4. 140, 30 ◦ C → r.t. (67% yield, 4 : 1-mixture favoring
142). d TBSOTf, 2,6-lutidine, CH2 Cl2 , – 78 ◦ C → r.t. e NH4 F, MeOH, H2 O (66% from 141).
f EDCI, DMAP, DMAP·HCl, CHCl3 , THF, reflux, (54%). g HF/pyr, pyr, THF (95%)

zinc. This species was added to the aldehyde 140 [75] in the presence of chiral
amino alcohol 141 [76–78] to yield the desired (17R)-isomer 142 in a 4 : 1 di-
astereomeric ratio. Treatment of 142 with TBSOTf led to the protection of the
C17-hydroxy group and the conversion of the tert-butyl ester to the silyl ester.
Treatment with ammonium fluoride in aqueous methanol [79, 80] resulted
in the cleavage of the silyl ester and the selective liberation of the primary
alcohol. A macrolactonization under Keck conditions [81, 82], followed by re-
moval of the remaining TBS protecting group with HF/pyridine completed
the first total synthesis of halichlorine.
160                                                                       J. Hassfeld et al.

The Acyclnitroso-Ene Aproach

In 2003 Kibayashi and coworkers published a highly stereoselective ap-
proach for the construction of the azaspirocyclic core of the halichlorins [83].
The readily available ester 143 was converted to the corresponding hydrox-
amic acid 144 by treatment with hydroxylamine under basic conditions
(Scheme 27). Oxidation using tetrapropylammonium periodate afforded the
acylnitroso intermediate which participated in an intramolecular ene reac-
tion [84–86] with the cyclopentene moiety. It is noteworthy that the MOM
group was effective in shielding the α-face, thereby allowing the spirocyclic
lactam 145 to be isolated as a single diastereoisomer.
   The C5 side chain was introduced via the addition of lithium acetylide
followed by reduction of the resulting iminium ion with NaBH3 CN. Interest-
ingly, it was absolutely crucial to reduce the C10-C11 double bond prior to




Scheme 27 a NH2 OH·HCl, KOH, MeOH, 0 ◦ C, 82%; b Pr4 NIO4 , CHCl3 , 0 ◦ C, 82%.




Scheme 28 a H2 (5 atm), Pd – C, MeOH, 99%. b BnBr, NaH, Bu4 NIO4 , DMF, r.t., 98%.
c HC ≡ CLi,H2 NCH2 CH2 – NH2 , THF, 5 ◦ C, then NaBH3 CN, AcOH, MeOH, r.t., 67–74%.
d disiamylborane, Et2 O, r.t., then H2 O2 , NaOH, r.t., 96%. e NaBH4 , i-PrOH, 0 ◦ C, 91%.
f LiBF4 , MeCN – H2 O, 72 ◦ C, 83%. g TBSCl, Et3 N, DMAP, CH2 Cl2, r.t., 97%. h Dess-Martin
periodinane, CH2 Cl2 , r.t., 75%
Asymmetric Total Synthesis of Complex Marine Natural Products              161

the addition (Scheme 28). The alkyne 148 was converted to compound 149,
which could serve as an intermediate in Kibayashi’s projected synthesis of
halichlorine.

2.3.1
Radical Approaches

Radical cascade reactions have a long-standing tradition in the efficient con-
struction of complex molecular frameworks. Takasu and Ihara have reported
on the synthesis of the azaspirocyclic core of the halichlorins using a rad-
ical translocation/cyclization process [87]. In their strategy, an aryl radical
(153) dissociated a C – H bond by [1, 5]-radical translocation to generate an
α-aminyl radical (154), which further participated in a cyclization with an
intramolecular olefin (Scheme 29). In fact, when 152 was treated with 2 equiv-
alents of tributyltin hydride and 0.5 equivalents of AIBN in refluxing PhH,
the desired spirocyclic compound 155 was isolated in 78% yield as a 91 : 9
mixture of diastereoisomers favoring the title compound.
   This outcome was rationalized with a transition state where the olefinic
β-proton was in the pseudoaxial position in order to minimize interaction
with the piperidine ring. The conversion to funtionalized spirosystem is
outlined in Scheme 30. The lactam 155 was transformed to thiolactam 156
using Lawesson’s reagent. Treatment with 2-bromoacetoacetate followed by
deacetylation afforded the vinylogous carbamate 157. The stereoselective hy-
drogenation, which was established by Shishido and coworkers, furnished
the desired piperidine 158 as a single isomer [95]. The tert-butyl ester was
cleaved by treatment with TFA and converted to the corresponding lactam.
The ethyl ester was then reduced with LiEt3 BH to the alcohol, which was
protected as a triethylsilyl ether. As predicted by computer modeling, the




Scheme 29 a 2.0 eq Bu3 SnH, 0.5 eq AIBN, PhH, reflux, 78%
162                                                                               J. Hassfeld et al.




Scheme 30 a cat. Pd(OH)2 , H2 , cat. HCl (conc), t-BuOH, reflux. b Lawesson’s reagent,
PhMe, reflux. c (i) ethyl 2-bromoacetylacetate, NaHCO3 , CH2 Cl2 , r.t., (ii) NaOEt, EtOH,
40 ◦ C. d PtO2 , H2 , EtOH, r.t. e (i) TFA, CH2 Cl2 , r.t., (ii) EDCl, CH2 Cl2 , r.t. f (i) LiEt3 BH,
THF, 0 ◦ C, (ii) TESCl, Et3 N, cat. DMAP, CH2 Cl2 , r.t. g LDA, THF, – 78 ◦ C; then MeI,
– 78 ◦ C. h Li(NH2 )BH3 , THF, 40 ◦ C




Scheme 31 a Bu3 SnH, AIBN, PhMe, 75 ◦ C, 57%. b Na(Hg), MeOH, Na2 HPO4 , 75%


methylation of the lactam enolate occurred from the less bulky β-face to
give 160 as single diastereomer. Finally, the lactam ring was cleaved with
LI(NH2 )BH3 to give 161 in 59% yield. In conclusion, a highly stereoselec-
tive route to the spirocyclic nucleus of the halichlorine family was developed.
The powerful radical translocation/cyclization strategy should find further
application in the synthesis of polycyclic molecules.
   Clive and Yeh [88] reported on a 5-exo radical cyclization of an enamine
sulfone. When 162 was treated with Bu3 SnH and AIBN in PhH, the desired
spirocycle 163 was isolated in 57% yield. Desulfonylation with Na/Hg gave
the 6-azaspiro [4, 5] decane core (164) of halichlorine (Scheme 31).

2.3.2
Nitrone-Olefin [3 + 2] Cycloaddition Approaches

The [3 + 2] nitrone-olefin cycloaddition is an efficient method for the syn-
thesis of isoxazolidines. Since the N – O bond can be cleaved reductively, this
provides entry to the synthesis of γ -amino alcohols. There have been sev-
eral approaches to the azaspirocyclic core of the using a [3 + 2] nitrone-olefin
cycloaddition as the key step. The required nitrones were generated by inter-
Asymmetric Total Synthesis of Complex Marine Natural Products                     163

molecular, intramolecular, and transannular aza-Michael additions of oximes
to α, β-unsaturated esters or lactones, respectively.
   In 1999, Zhao and Lee published a synthesis of the halichlorine core using
a [3 + 2] nitrone/olefin-cycloaddition [89]. In fact, when oxime 165 was
heated with benzyl acrylate in xylene at 140 ◦ C, nitrone 166 was generated
(Scheme 32). Once formed, this species takes part in an intramolecular cy-
cloaddition with the olefinic double bond to yield the bicyclic compound 167
as a single diastereoisomer. The [3 + 2] cycloaddition is a stereospecific pro-
cess; therefore the Z-double bond geometry accounts for the observed selec-
tivity. Acidolysis of the tetrahydropyranyl group and Swern oxidation of the
resulting alcohol was followed by a Wittig olefination with the stabilized ylide
Ph3 P = CHCO2 Me. The N – O bond was then cleaved reductively with zinc in
aqueous acetic acid. When 169 was heated in refluxing 1,2-dichlorobenzene
for 24 h, an intramolecular Michael addition afforded intermediate 171, which
eliminated benzyl acrylate to furnish 172. In a retro-Michael/Michael equi-




Scheme 32 a CH2 = CHCO2 Bn, xylene, 140 ◦ C, 92%. b p-TsOH, 93%. c Swern oxidation,
97%. d Ph3 P = CHCO2 Me, 93%. e Zn, AcOH/H2 O, 55 ◦ C, 94%. f 1,2-dichlorobenzene, re-
flux, 84%
164                                                                     J. Hassfeld et al.

libration process, intermediate 172 was isomerized to 170, which molecular
mechanics predicted to be favored by 3.8 kcal mol–1 .
   In 1984, Grigg and coworkers reported on an intramolecular aza-Michael/
[3 + 2] cycloaddition reaction cascade to furnish the azaspiro[4.5] decan
system 175 [90–93]. Applying their methodology, Shishido and Zhao de-
veloped efficient one-pot protocols for this useful transformation [94, 95]
(Scheme 33). Unfortunately, the C5 stereocenter is epimeric to the natural
product and must be inverted. Following the reductive cleavage of N – O
bond, the Zhao group took advantage of a thermal equilibration process
(172 → 170, see above).
   Shishido et al. anticipated that the hydrogenation of a C4-C5 α, β-un-
saturated ester would give the desired isomer [95]. A bromination/dehy-
drobromination sequence provided the desired unsaturation at C4-C5 (176).
Concomitant with the cleavage of the N – O bond (Zn, AcOH, H2 O) an iso-
merization of the C – C double was observed, which might be rationalized
by hydrogen bond stabilization in the Z-configuration. With the primary
hydroxyl group protected as the TBDPS ether, a catalytic hydrogenation af-
forded the desired spirocyclic compound 178 as a single isomer. In a few more
steps, 178 was converted to the tricyclic core of halichlorine.
   An inherent problem of these intramolecular approaches is the undesired
stereochemistry at C5. The White group [96] envisioned that this problem
could be overcome using a transannular nitrone cycloaddition (Scheme 34).
In fact, azide 179 was subjected to a Staudinger reaction followed by an aza-
Wittig reaction with p-anisaldehyde. The resulting imine was oxidized with
m-chloroperbenzoic acid to yield the oxaziridine 180. When 180 was treated
with p-toluenesulfonic acid in aqueous methanol, simultaneous hydrolysis
of the dioxolane and the oxaziridine occurred. Intramolecular condensa-
tion of the resulting keto hydroxylamine moiety (181) afforded nitrone 182,




Scheme 33 a NH2 OH·HCl, NaOAc, EtOH, reflux, 4 h, 90% (Shishido); NH2 OH·HCl,
NaOAc, xylene/H2 O 10 : 1, reflux, 92% (Zhao). b Zn, AcOH/H2 O, 94%. c LDA, Br(CF2 )2 Br,
90%. d DBU, PhH, reflux, 3 d, 79%. e Zn, AcOH/H2 O, 50 ◦ C, 100%. f TBDPSCl, imid,
DMAP, CH2 Cl2 , 100%. g H2 , PtO2 , 100%
Asymmetric Total Synthesis of Complex Marine Natural Products                       165




Scheme 34 a Ph3 P, THF. b anisaldehyde, ∆. c m-CPBA, CH2 Cl2 , – 78 ◦ C → r.t., 79% from
179. d p-TsOH·H2 O, MeOH – H2 O (5 : 1), ∆, 70%. e PhMe, ∆, 64%. f K2 CO3 , MeOH, ∆,
88%. g SmI2 , THF, r.t., 64%


which upon heating in toluene took part in a transannular cycloaddition. In-
terestingly, the stereocenter at C5 induced the selective formation of three
contiguous stereocenters in 183; because of steric reasons the nitrone oxy-
gen cannot pass through the ring. As a consequence, the olefinic double was
approached from the bottom, leading to 183 as a single isomer.

2.3.3
Imine- or Iminium-Based Approaches

In 1999, Forsyth and Koviach described their efforts toward the spirobi-
cyclic core of halichlorine using an in situ iminium formation-allylation ap-
proach [97]. Aldehyde 185 was generated by oxidation using TPAP/NMO,
which was followed by treatment with trifluoroacetic acid. This not only
cleaved the Cbz protecting group but also induced the formation of a highly
reactive iminium ion. In fact, upon addition of allyl trimethyl silane the
iminium species was attacked stereoselectively to yield the desired spirobi-
cyclic compound 188 as a single isomer. The stereochemical outcome of this
reaction can be rationalized by minimization of steric interaction as indicated
in Scheme 35.
   Dake and coworkers [98] employed an α-hydroxyiminium ion semipinacol
rearrangement, a strategy pioneered by Paquette [99, 100] in the hydroxyox-
onium case (Scheme 36). When 189 was treated with hydrochloric acid in
dichloromethane, a cyclic iminium species (190) was formed, which readily
participated in semipinacol rearrangement to yield 191. The formation of the
major isomer could be explained by a chair conformation predominating over
a boat conformation in the transition state.
166                                                                   J. Hassfeld et al.




Scheme 35 a TFA, 5 min, then allyltrimethyl silane, – 42 ◦ C, 70%




Scheme 36 a HCl, CH2 Cl2 , 0 ◦ C, 93%, dr = 14 : 1




Scheme 37 a PhH, 60 ◦ C, azeotropic removal of solvent, 96%. b allylmagnesium bromide,
THF, 76%. c Grubbs II catalyst (10 mol %), p-TsOH, 56 h, 83%


   The Wright group’s approach takes advantage of the progress made in
field of ring closing metathesis (RCM) [101]. Allylation of an allyl imine
was followed by RCM of the resulting diene. Gratifyingly, the treatment of
193 with the more robust Grubbs second-generation catalyst in the pres-
ence of p-TsOH yielded the desired spirobicyclic compound 194 in 83% yield
(Scheme 37).

2.3.4
Michael-Initiated Ring Closure (MIRC)/Curtius Rearrangement Approach

In 1999, Arimoto and coworkers [102] reported their approach to the spiro-
cyclic core of pinnaic acid using an asymmetric MIRC (Michael-initiated ring
closure) reaction developed by the Enders group [103] (Scheme 38). Con-
jugate addition of the hydrazone 195 followed by nucleophilic displacement
of the iodo group afforded the five-membered ring 197. It is important to
note that this reaction established three contiguous stereocenters. Prenyla-
tion of the ester enolate of 197 occurred in a selective fashion since the
α-face was effectively shielded by the C13-side chain. 197 was transformed to
the carboxylic acid 198, which was treated with DPPA, to initiate a Curtius
rearrangement. Addition of benzyl alcohol to the resulting isocyanate pro-
vided the Cbz-protected amine 199. The latter was subjected to ozonolysis
followed by a Horner-Wadsworth-Emmons olefination to yield 200. Catalytic
Asymmetric Total Synthesis of Complex Marine Natural Products                              167




Scheme 38 a (i) LDA, THF, 0 ◦ C, 4 h, (ii) TMEDA, – 78 ◦ C, 0.5 h, (iii) 196, – 78 ◦ C → r.t.,
18 h, 89%. b (i) O3 /CH2 Cl2 , – 78 ◦ C, 0.5 h; (ii) DMS, 50%. c NaBH4 , MeOH, 0 ◦ C, 1 h, 89%.
d PMP – OH, Ph3 P, DEAD, THF, 80 ◦ C, 2 h, 93%. e (i) LDA, THF, – 78 ◦ C, 2 h, (ii) prenyl
bromide, – 78 ◦ C → r.t., 20 h, 69%. f 2 M aq KOH – DMSO (1 : 1), 120 ◦ C, 7.5 h, 89%.
g DPPA, Et3 N, PhH, reflux, 2 h. h BnOH, i-Pr2 NEt, PhH, reflux, 15 h, 86% (2 steps). i O3 ,
MeOH, – 78 ◦ C, 5 min. j LiCl, Et3 N, THF, 0 ◦ C → r.t., 15 h, 95%. k Pd(OH)H2 , AcOH (cat.),
EtOH, 93%


hydrogenation in the presence of acetic acid led to 201 in 93% yield by hydro-
genation of the double bond, removal of the Cbz-group, and reduction of an
intermediate imine.

2.4
Apratoxin A

Cyclodepsipeptides are widespread in nature, and many of them pos-
sess interesting biological properties such as anticancer, antiinfective, anti-
inflammatory, and anticlotting activities [104]. Recently, Moore, Paul and co-
workers reported the characterization of apratoxins A–C (202–4, Fig. 5) [105,
106]. These metabolites of mixed biogenetic origin were isolated from the
cyanobacterium Lyngba majuscula collected in Guam and Palau. From
a structural point of view, the apratoxins consist of polyketide and par-
tially methylated polypeptide moieties. A remarkable feature is the α, β-
unsaturated thiazoline, which is extremely prone to acid-induced dehydration
to the corresponding (E)-34,35-dehydroapratoxin A (205). The synthetic
interest in the apratoxins was further stimulated by their high levels of cyto-
toxicity displayed against KB and LoVo cancer cells, with IC50 values of 0.52
and 0.32 nM, respectively. So far, the mode of action of these compounds is
not known, although they do not induce microtubule stablization or interfere
with topoisomerase I. First structure activity relationships from the naturally
occurring apratoxins 202–205 and the dehydration product 205 reveal that
168                                                             J. Hassfeld et al.




Fig. 5 Structure of the apratoxins


203 and 205 are 1 to 2 orders of magnitude less potent, suggesting that the bi-
ological activity is very sensitive to changes in the conformation. In addition
to more detailed structure activity studies, an efficient synthesis would also
provide sufficient material for further biological evaluations.

2.4.1
The Forsyth Synthesis of Apratoxin A

In 2003, Forsyth and Chen from the University of Minnesota disclosed
the first total synthesis of apratoxin A [107]. In their retrosynthetic analy-
sis, the formation of the sensitive thiazoline moiety was deferred to a late
stage in the synthesis (Scheme 39). They used an intramolecular Staudinger




Scheme 39 Retrosynthetic analysis of apratoxin A
Asymmetric Total Synthesis of Complex Marine Natural Products                             169

reduction/aza-Wittig process with the α-azido thiolester 206, which was de-
rived from tripeptide 207 and the polyketide ester 208.
    The synthesis of the polyketide portion of the molecule commenced with
the acylation [108] of known 209 [109] with acrylic acid, which gave diene
210 in 71% yield (Scheme 40). Ring-closing metathesis [110] furnished an α,
β-unsaturated lactone, which was subjected to a conjugate addition reaction
with methyl cuprate [111] to afford 211 as a single isomer. Reduction with
lithium aluminum hydride gave the corresponding diol, whose primary hy-
droxyl group was silylated with TBSCl. The secondary hydroxyl group of 212
was esterified with N-Boc-proline under Yamaguchi conditions [112]. Con-
comitantly, the primary hydroxyl group was liberated with TBAF and the
resulting primary alcohol 214 was oxidized with TPAP/NMO. Aldehyde 215
was subjected to an antiselective Paterson-aldol reaction with 216 [113] to
furnish hydroxyketone 217. The hydroxyl group was protected as TBS ether
(218), after which the α-benzyloxyketone moiety was degraded to the desired
carboxylic acid 208.
    The synthesis of the peptide moiety of apratoxin commenced with N-Boc,
N-methyl isoleucine methyl ester 219 (Scheme 41). In situ deblocking was
followed by PyAOP-mediated coupling [114] with N-Boc-N-methylalanine to
yield 220. In a similar fashion, an N-methyltyrosine unit was added. The de-




Scheme 40 a 2-chloro-N-methylpyridinium iodide, Et3 N, acrylic acid, CH2 Cl2 , reflux,
71%. b Grubbs’ catalyst I, CH2 Cl2 , reflux, 97%. c Me2 Cu(CN)Li2 , Et2 O, – 78 ◦ C, 86%.
d LiAlH4 , Et2 O, 0 ◦ C, 83%. e TBSCl, imid, CH2 Cl2 , 98%. f N-Boc-Pro-OH, Yamaguchi es-
terification, 91%. g TBAF, THF, 88%. h TPAP, NMO, 4 ˚ MS, CH2 Cl2 , 89%. i Me2 NEt,
                                                            A
c-Hex2 BCl, Et2 O, – 78 → – 20 ◦ C. j TBSOTf, 2,6-lutidine, CH2 Cl2 , – 50 ◦ C, 74% (2 steps).
k 1. K2 CO3 , MeOH; 2. NaIO4 , MeOH, buffer, 75% (2 steps)
170                                                                       J. Hassfeld et al.




Scheme 41 a TFA, CH2 Cl2. b N-Boc-N-Me-Ala-OH, PyAOP, DIPEA, CH2 Cl2 , 79%
(2 steps). c TFA, CH2 Cl2 . d Boc-OMe-Tyr-OH, PyAOP, DIPEA, CH2 Cl2 , 95% (2 steps).
e TFA, CH2 Cl2 . f 222, PyAOP, Et3 N, DMF, 76% (2 steps). g HF·pyr, THF, 98%. h DIAD,
Ph3 P, AcSH, 85%. i K2 CO3 , MeOH, 0 ◦ C. j 208, DPPA, Et3 N, CH2 Cl2 , 80% (2 steps).
k DDQ, CH2 Cl2, H2 O, 0 ◦ C → r.t., 93%. l DPPA, Ph3 P, DIAD, THF, 97%. m 1. HF·pyr,
THF; 2. TESOTf, 2,6-lutidine, CH2 Cl2 , – 78 ◦ C, 86% (2 steps). n Ph3 P, THF, 50 ◦ C, 63%.
o TBSOTf, CH2 Cl2 , 2,6-lutidine. p TBAF, THF, 0 ◦ C, 86% (2 steps). q LiOH, t-BuOH, THF,
H2 O. r PyAOP, CH2 Cl2 , DIPEA, 73% (2 steps). s HF·pyr, THF, 65%
Asymmetric Total Synthesis of Complex Marine Natural Products              171

rived diamide 221 was coupled with the cysteine surrogate 222. After the
tert-butyldimethylsilyl protecting group was removed with HF in pyridine,
the resulting hydroxyl group was converted to the corresponding thiol in
a Mitsunobu-type process [115]. The thiolester formation was mediated by
DPPA [116]. Oxidative removal of the PMB group was followed by introduc-
tion of the azide moiety with DPPA under Mitsunobu conditions [117]. At
a later stage it turned out that the C35-TBS protecting group could not be
removed without degradation of other parts of the molecule. Therefore, this
group was exchanged with the more labile TES group. Now the stage was set
for the crucial intramolecular aza-Wittig reaction.
   The accounted mechanism is depicted in Scheme 42. Azide 234 was treated
with triphenylphosphine to give a phosphinimine-intermediate 235, which
was formed via Staudinger reduction. Intramolecular attack of the nitrogen
at the carbonyl carbon led to the desired thiazoline (236) after elimination of
triphenylphosphine oxide. A key feature of this process was that the acid la-
bile thiazoline moiety could be installed under neutral reaction conditions.
In fact, when a solution of 5 in anhydrous tetrahydrofuran was treated with
triphenylphosphine, the desired thiazoline 229 was formed in 63% yield.




Scheme 42 Proposed mechanism for the thiazoline formation


   In the endgame, the N-Boc group was converted to the silyl carbamate
using TBSOTf [118]. Following the removal of the TBS group with TBAF, the
methyl ester was saponified and the resulting amino acid was subjected to
a PyAOP-mediated macrolactamization. Finally, the TES group was removed
with HF·pyridine in THF to afford 202, whose spectroscopic and chromato-
graphic properties matched those of an authentic sample.

2.4.2
The Ma Synthesis of an Oxazoline Analog of Apratoxin A

Shortly after the disclosure of the Forsyth synthesis, the Ma group from
Shanghai reported the synthesis of an oxazoline analog of Apratoxin A [119].
   Their synthesis of the polyketide domain of the apratoxins started with
a (R)-proline catalyzed aldol reaction [120, 121] between pivalaldehyde and
acetone, which gave the desired product 237 in 99% ee (Scheme 43). The
carbonyl group was reduced, and the resulting alcohol was converted to a me-
sylate and subjected to an elimination reaction with KOt-Bu. Ozonolysis gave
172                                                                          J. Hassfeld et al.




Scheme 43 a TBSCl, imid, DMF, r.t. b NaBH4 , MeOH, 0 ◦ C. c MsCl, Et3 N, CH2 Cl2, r.t.
d t-BuOK, PhMe, reflux, 71% (4 steps). e O3 /Me2 S. f LiCH2 CO2 Et, THF, – 78 ◦ C. g 40%
HF, MeCN, r.t. h MsCl, Et3 N, CH2 Cl2 , 0 ◦ C to r.t., 45% (4 steps). i Me2 (CuCN)Li2, Et2 O,
– 78 ◦ C, 94%. j LiAlH4 , THF, reflux. k AcCl, pyr, CH2 Cl2 , 0 ◦ C, then K2 CO3 , MeOH. l Dess-
Martin oxidation, 69% (3 steps). m N-propionylsultam, Et2 BOTf, DIPEA, CH2 Cl2 , – 15 ◦ C,
then 240, TiCl4, – 78 ◦ C, 90%. n LiAlH4 , THF, reflux. o DMP, PPTS, 67% (2 steps). p N-
Fmoc-L-proline, 2,4,6-trichlorobenzoyl chloride, DIPEA, PhH then 242, DMAP, r.t., 90%.
q TsOH, MeOH, r.t. r TEMPO, NaClO, aq. NaHCO3 , CH2 Cl2 , 0 ◦ C. s NaH2 PO4 , NaClO2,
t-BuOH, H2 O, 2-methylbutene, 81% (3 steps). t 245, TFA, H2 O, then 244, HATU, DIPEA,
CH2 Cl2 , r.t., 90%. u DAST, CH2 Cl2, – 78 ◦ C. v Pd(PPh3 )4 , N-methylaniline, THF, r.t., 70%
(2 steps)


the desired aldehyde, which was reacted with LiCH2 CO2 Et to give the α,
β-unsaturated lactone 238 after cyclization and elimination. A stereoselec-
tive methylcupration introduced the C37-methyl group in a manner similar
to the previous synthesis. Lactone 211 was converted to 240 in a straightfor-
ward fashion and subjected to an antiselective aldol reaction using Oppolzer’s
sultam methodology [122]. After reductive removal of the auxiliary and the
acetate protecting group, the 1,3-diol was protected as an acetonide (242).
Asymmetric Total Synthesis of Complex Marine Natural Products                          173

   Next, the polyketide moiety was connected to N-Fmoc-(S)-proline using
the Yamaguchi protocol, after which the diol was liberated using p-TsOH in
methanol. The primary alcohol was oxidized selectively using TEMPO/NaClO
followed by an oxidation with NaClO2 to give the carboxylic acid 244 [123].
Compound 244 was coupled with α, β-unsaturated ester 245, which can
be easily obtained from the commercially available Garner aldehyde. After
DAST-mediated oxazoline formation the allyl ester was cleaved using
Pd(PPh3 )4 with the help of N-methylaniline.
   The synthesis of the tripeptide part started with the BEP-mediated (BEP
= 2-bromo-1-ethyl pyridinium tetrafluoroborate) [124] coupling between
N-Fmoc-O-methyl-(S)-tyrosine 249 and 248 (Scheme 44). Hydrogenolysis of
the benzyl ester afforded the carboxylic acid 250. After removal of the Fmoc
group, BEP-mediated peptide coupling gave tripeptide 252. Treatment of 252
with diethylamine (to remove the Fmoc group) was followed by coupling with
the oxazoline-containing acid 247 to yield the macrocyclization precursor
253. Gratifyingly, after one-pot removal of the protecting group with TBAF,
the resulting amino acid could be cyclized with HATU/DIPEA in a dilute
methylene chloride solution to give the target molecule 254.




Scheme 44 a Et2 NH, MeCN, r.t. b 249, BEP, DIPEA, CH2 Cl2 , r.t. c Pd – C, H2 , EtOAc, 55%.
d 251, Et2 NH, MeCN, then 250, BEP, DIPEA, CH2 Cl2 , r.t.; 60% e Et2 NH, MeCN, then 247,
HATU, DIPEA; 59%. f TBAF, THF. g HATU, DIPEA, CH2 Cl2 , (0.002 M), r.t. 45%
174                                                             J. Hassfeld et al.

2.5
Tetrodotoxin

Tetrodotoxin (TTX, 255, Fig. 6) is one of the most famous and important
marine natural products. Its unique chemical architecture and potent bio-
logical activity evoked extensive efforts to elucidate the structure as well as
develop a synthesis of this fascinating molecule. Tetrodotoxin (255) was first
isolated from the ovaries of the puffer fish (Spheroides rubripes) in 1909 and
named after its family Tetraodontidae [125, 126]. Its structure was indepen-
dently elucidated by Hirata-Goto (1965) [127], Tsuda [128], and Woodward
(1964) [129]. The first racemic total synthesis was achieved in 1972 by Kishi
and coworkers [130, 131], and 31 years later, in 2003, Isobe reported the first
asymmetric synthesis of 255.




Fig. 6 Structures of tetrodotoxin 255 and analogs

   In the 1960s, the toxicity of tetrodotoxin (255) was revealed to be caused
by a specific blockage of sodium channels; due to its biological properties, 255
is widely used as a biochemical tool for neurophysiological studies [132, 133].
Further studies resulted in the identification and isolation of the ion-channel
protein and determination of its amino acid sequence [134]. Moreover, sev-
eral medicinal products based on tetrodotoxin 255 are currently in clinical
testing, including Tectin (pain management), Tetrodin (opiate withdrawal),
and Tocudin (anesthetic) [135].

2.5.1
Syntheses and Application

The Kishi Synthesis

In 1972, Kishi and coworkers reported the first racemic synthesis of tetro-
dotoxin (255), which represented a milestone in organic chemistry [130, 131].
It was the only known synthesis of this outstanding structure for over
30 years. The synthesis started with the benzoquinone derivative 261, which
Asymmetric Total Synthesis of Complex Marine Natural Products                         175




Scheme 45 Diels-Alder reaction and derivatization of quinone-derivative 251. a SnCl2 ,
MeCN, 83%. b 1. CH3 SO2 Cl, Et3 N, 2. H2 O, 100 ◦ C, 61% (2 steps). c NaBH4 , MeOH, 96%.
d m-CPBA, CSA, 75%. e CrO3 , pyr/H2 O, 90%. f ethylene glycol, BF3 ·Et2 O, CH2 Cl2 , 100%.
g 1. Al(Oi – Pr)3 , 2. Ac2 O, pyr, 95% (2 steps). h) SeO2 , xylene, 180 ◦ C, 100%


was subjected to a chemoselective Diels-Alder reaction with butadiene to
afford, after subsequent Beckmann rearrangement, diastereospecifically the
bicyclic product 262 (Scheme 45). The authors elegantly took advantage of the
cagelike conformation of this intermediate, which is responsible for the stere-
oselectivity in the following steps. Reduction of the less restricted ketone was
followed by the epoxidation of the electron rich C10, C11-double bond [136].
The reagents used in these steps approached the substrate from the convex
face, leading almost exclusively to the desired diastereomers. The epoxide in-
termediate 253 directly engaged in an epoxide opening/ring closure, forming
the tetrahydrofuran moiety. The resulting C10 alcohol was oxidized and the
corresponding ketone protected as a cyclic acetal.
   After reduction of the α, β-unsaturated ketone and acetylation of the so-
derived alcohol the vinylic methyl group could be oxidized with selenium
dioxide to obtain aldehyde 265 quantitatively. In a 12-step sequence, the car-
boxyl group at C10 was introduced and the lactone moiety of tetrodotoxin
generated (Scheme 46). First, the aldehyde was reduced, and the allylic al-
cohol could be selectively epoxidized in the same manner as before under
substrate control. After protection of the alcohol as acetate the cyclic ketal
at C10 was transformed into the diethyl ketal 266, elimination of which was
followed by epoxidation of the enol ether to give the α-acetoxy-substituted
ketone 267 after epoxide opening with acetic acid. Baeyer-Villiger oxidation
furnished the seven-membered lactone 268, which rearranged under treat-
ment with potassium acetate in acetic acid to provide the complete carbon
backbone of tetrodotoxin (255).
176                                                                       J. Hassfeld et al.




Scheme 46 a NaBH4 , MeOH, 100%. b m-CPBA, 4,4’-thiobis(6-tert-butyl-3-methylphe-
nol) (as radical inhibitor), 1,2-dichloroethane, 90 ◦ C, 95%. c Ac2 O, pyr, 100%. d 1. TFA,
H2 O, 2. Ac2 O, pyr, 80% (2 steps). e 1. HC(OCH2 CH3 )3 , CSA, 2. Ac2 O, pyr. f o-dichloro-
benzene, reflux. g m-CPBA, CH2 Cl2. h AcOH, 70% (5 steps). i m-CPBA, CH2 Cl2 , 100%.
j KOAc, AcOH, 90 ◦ C, 100%. k Ac2 O, CSA, 100 ◦ C, 100%. l high vacuum, 290–300 ◦ C, 80%




Scheme 47 a Et4 NBF4 , NaCO3 , CH2 Cl2 , 92%. b NaIO4 , THF/H2 O; c) NH4 OH, MeOH/H2 O,
5% (from the acetylated tetrodamine 271)

   The completion of the synthesis included the sophisticated guanidy-
lation [131] of the dihydrofuranamine 272 and the subsequent oxidative
cleavage of the dihydrofuran double bond (Scheme 47). The free nitro-
Asymmetric Total Synthesis of Complex Marine Natural Products             177

gen of the monoacetylguanidine engaged with the aldehyde to afford the
hemiaminal, and a deacetylation with ammonium hydroxide in aqueous
methanol consummated the synthesis of racemic tetrodotoxin (rac-255).
Since then, numerous synthetic endeavors have been reported by various re-
search groups [137–141], and finally, in 2003, Isobe and coworkers published
the first asymmetric synthesis of tetrodotoxin (255) after having previously
synthesized three different deoxy-analogs (256, 257, and 258) [142–145].

The Isobe Syntheses

Isobe and coworkers concentrated their attention on the synthesis of tetrodo-
toxin (255) and analogs after they had identified this issue to be most reward-
ing due to the biological potential of the naturally occurring tetrodotoxins.
Since extended bioorganic studies had been limited by the difficult derivati-
zation of the natural product [146–148], the chemical synthesis could provide
analogs such as labeled tetrodotoxins to approach relevant unsolved prob-
lems.
   The syntheses of three analogs of tetrodotoxin (255) started from the same
trichloroacetamide intermediate 276 (Scheme 48), which can be obtained in
a nine-step synthesis from levoglucosenone 278 [143]. The Diels-Alder re-
action of bromolevoglucosenone 279 with isoprene 277 and an Overman
rearrangement in order to introduce the amine function and the exocyclic
double bond are the key steps of this sequence. In an impressive diver-
gent manner 11-deoxytetrodotoxin (256), 8,11-dideoxytetrodotoxin (257),
and 5,11-dideoxytetrodotoxin (258) were synthesized by means of varying the
oxygenation state at C5 and C8, respectively (Scheme 49).
   Dexterous manipulation of the dibromide 280 furnishes either the bicyclic
iminoether 281 or the oxazolin 284, respectively. Hydrolysis of 281, followed
by epoxidation, inversion of the C – 7 alcohol via an oxidation/reduction se-
quence, and epoxidation of the cyclohexene double bond furnishes interme-
diate 283, in which the desired functionalities for 8,11-dideoxytetrodotoxin
(257) can be observed. Starting from oxazoline 284, key intermediates for
the syntheses of 5,11-dideoxytetrodotoxin 285 as well as the key intermedi-
ate for the synthesis of 11-deoxytetrodotoxin 287 are available as depicted in




Scheme 48 Retrosynthesis of the common intermediate 276
178                                                                      J. Hassfeld et al.




Scheme 49 Key steps in Isobes analog syntheses. a PyH·Br3 , K2 CO3 , CH2 Cl2 , 87%.
b K2 CO3 , MeOH, 90%. c AcOH, THF, H2 O. d CCl3 COCl, pyr. e K2 CO3 , MeOH, 82%
(3 steps). f m-CPBA, Na2 HPO4 , CH2 Cl2 , 85%. g PCC, 4 ˚ MS, CH2 Cl2 . h NaBH4 , MeOH,
                                                         A
89% (2 steps). i DBU, DMF, 86%. j p-TsOH, pyr, H2 O, 83%. k PCC, 4 ˚ MS, CH2 Cl2 .
                                                                         A
l NaBH4 , CeCl3(H2 O)7 , EtOH, CH2 Cl2 , 75% (2 steps). m m-CPBA, CH2 Cl2 , 86%. n Ti(Oi-
Pr)4 , 1,2-dichloroethane, reflux, 90%



Scheme 49. For the synthesis of 256, the inversion of the two hydroxyl groups
present in 287 and epoxidation were required. The general strategy for the
completion of the syntheses is exemplified in Scheme 50.
   Ozonolysis of the exocyclic double bond and stereoselective addition of
TMS-protected acetylene is followed by desilylation and protection of the
propargylic alcohol to achieve 289. The triple bond was cleaved through treat-
ment with ruthenium(IV)oxide, and the intermediate carboxylic acid 290
underwent an lactonization/epoxide opening reaction to provide bicyclic lac-
Asymmetric Total Synthesis of Complex Marine Natural Products                         179




Scheme 50 Synthesis of 5,11-Dideoxytetrodotoxin 258. a NaH, BnBr, THF, DMF, 95%.
b O3 , MeOH, – 78 ◦ C, Me2 S, 96%. c Me3 SiCCMgBr, THF. d n-Bu4 NF, THF. e Ac2 O, pyr,
86% (3 steps). f RuO2 (H2 O)n , NaIO4 , CCl4, MeCN, H2 O, 75%. g H5 IO6 , AcOEt. h CSA,
CH(OMe)3 , MeOH. i Ac2 O, pyr, DMAP, 78% (3 steps). j BnNH2 , Na2 CO3 , DMF, 140 ◦ C.
k KCN, EtOH. l CSA, acetone, 70% (3 steps). m Ph3 P, CBr4 , Et3 N, CH2 Cl2 . n BnNH2 ·HCl,
pyr, reflux. o Ac2 O, pyr, Et3 N, 85% (3 steps). p H2 (1 atm), Pd(OH)2 /C, Ac2 O, 81%.
q NH3 , MeOH, H2 O. r TFA, H2 O, 81% (2 steps)



tone 291. The final steps included the introduction of the guanidine part.
Therefore, the acetonide in 291 was transformed into the dimethyl acetal 292,
which gave the mixed cyclic acetal 293 during the guanidine synthesis. This
artifice prevented the racemization of the sensitive C9-acetoxy group. From
the fully acetylated precursor 294, the synthesis of 5,11-dideoxytetrodotoxin
(258) was achieved within two deprotection steps.
   The asymmetric synthesis of tetrodotoxin (255) turned out to be more
complex than the analog syntheses described above. Although the previously
developed chemistry could be applied, the synthesis of the key intermedi-
ate 303 (Scheme 52) was different from the former Diels-Alder approach.
The synthesis started with the known 2-acetoxy-tri-O-acetyl-d-glucal-derived
allylic alcohol 295 (Scheme 51). The chiral information of the precursor
was transferred into cyclohexenone 299 via a Claisen rearrangement of al-
lyl vinylether 296 and an intramolecular Mukaiyama aldol condensation of
TBS-enol ether 298. Reductive opening of the acetal and reprotection of the
1,2-diol as acetonide furnished allylic alcohol 300 (Scheme 52).
180                                                                    J. Hassfeld et al.




Scheme 51 a K2 CO3 , o-dichlorobenzene, 150 ◦ C, 94%. b TBAF, THF, H2 O. c Cl3 CCO Cl,
DMAP, pyr, 74% (2 steps)




Scheme 52 a t-BuOK, THF, 90%. b LiBH4 , THF. c MMTr – Cl, pyr, 98% (2 steps). d Boc2 O,
Et3 N, DMAP, THF. e LiOH, MeOH, 1,2-dichloroethane, H2 O, 84% (2 steps). f m-CPBA,
Na2 HPO4 , 1,2-dichloroethane. g BzCl, Et3 N, CH2 Cl2 , 93% (2 steps)

   Introduction of the nitrogen functionality mediated by the C5-alcohol pro-
vides the key intermediate 303 after epoxidation and benzoylation of the
primary alcohol, which corresponds to the intermediates 283, 285, and 287
in the analog syntheses. At that stage, only the formation of the lactone and
the introduction of the guanidine remained to accomplish the synthesis. Lac-
tonization was achieved by an epoxide opening with aldehyde enolate derived
from 304 via deprotonation with DBU (Scheme 53).
Asymmetric Total Synthesis of Complex Marine Natural Products                     181




Scheme 53 a DBU, o-dichlorobenzene, 130 ◦ C. b OsO4 , NMO, acetone, H2 O. c IBX, DMSO.
d NaBH4 , MeOH, 68% (4 steps). e HgCl2 , Et3 N, DMF, 308. f NaIO4 , MeOH, H2 O. g TFA,
MeOH, 90% (2 steps)


   The resulting enolether 305 was transformed into the α-ketolactone, which
could be selectively reduced to obtain α-hydroxylactone 306. After protecting
group manipulation, the guanidine moiety could be obtained using the well-
established thiourea derivative 308, and the synthesis was completed after
oxidative cleavage of the 1,2-diol and sequential deprotection.

2.6
Ciguatoxin

Ciguatera is one of the most important types of human poisoning caused by
the consumption of seafood. More than 20,000 people annually suffer from
the serious effects of the intoxication, mainly in tropical and subtropical
Pacific and Indian Ocean regions and the tropical Caribbean. The symp-
toms are represented by gastrointestinal, neurological, and cardiovascular
disturbances, which can, in severe cases, lead to paralysis, coma, or death.
Although ciguatera fish poisoning has been reported for centuries [149], the
causative principle remained unknown until Scheuer and coworkers reported
the isolation of a group of toxins from the moray eel Gymnothorax javani-
cus [150–153] designated as ciguatoxins. In 1980, ciguatoxin (CTX1B (310),
182                                                               J. Hassfeld et al.




Fig. 7 Structures of selected Ciguatoxins


Fig. 7) was characterized as a polycyclic ether, and further efforts resulted in
the revelation of the relative [154–157] and absolute structure of 310.
    Ciguatoxin (310) is highly cytotoxic; the lethal potency is 0.35 µg/kg
(i.p.) [155]. The toxicity is exerted through the activation of voltage-sensitive
sodium channels (VSSC) [158, 159]. For the elucidation of its structure only
0.35 mg of 310 could be isolated from 4 t of G. javanicus [155]. Due to the
extremely low availability, biological studies are limited. Such studies are dir-
ected toward the development of immunochemical methods for detecting
ciguatoxins prior to consumption [160–163] as well as the detailed exam-
ination of the mechanism of action. Therefore, a synthetic access to 310 is
exquisitely desirable. In what follows, strategies and the employment of dif-
ferent synthetic methods in ciguatoxin syntheses will be discussed.

2.6.1
The Hirama Synthesis

The main challenges in the synthesis of the ciguatoxins are the size and com-
plexity of these molecules, which demand reliable synthetic tools as well
as sophisticated strategies. The most important achievement in this con-
text is the first and, so far, only total synthesis of a ciguatoxin congener,
videlicet CTX3C (311), by Hirama and coworkers in 2001 [164]. The key
steps in their synthesis are the coupling of two multicyclic fragments 313
and 314 (Scheme 54) [165–167] followed by a radical cyclization/ring-closing
metathesis approach for the formation of the F- and G-rings.
   The fragments were connected by the formation of cyclic acetal 315, which
was opened to give the O, S-acetal 316. The subsequent steps set the stage
for the radical cyclization reaction described in Scheme 55. Treatment of 317
with n-Bu3 SnH and AIBN led to the formation of the C-27 radical, which
attacked the unsaturated ester to furnish the G-ring. The following trans-
Asymmetric Total Synthesis of Complex Marine Natural Products                     183




Scheme 54 Connection of the fragments 313 and 314. a Sc(OTf)3 , PhH, 57%.
b 1. TMSSPh, TMSOTf, CH2 Cl2 , 2. K2 CO3 , MeOH, 61%. c PMBMCl, i-Pr2 NEt, Bu4 NBr,
(CH2 Cl)2 , 70%. d TBAF, THF, 92%. e methyl propiolate, NMM, CH2 Cl2 , 92%




Scheme 55 Formation of the F and G rings. a n-Bu3 SnH, AIBN, PhMe. b DIBALH, CH2 Cl2 .
c Ph3 PCH3 Br, NaHMDS, THF, 61% (3 steps). d TMSBr, CH2 Cl2 , 93%.
e SO3 · pyr, Et3 N, DMSO, CH2 Cl2 . f Ph3 PCH3 Br, NaHMDS, THF. g Grubbs I catalyst
(20 mol %), CH2 Cl2 , 60% (3 steps)
184                                                                  J. Hassfeld et al.

formations established two terminal double bonds in 319, and a ring-closing
metathesis reaction provided the F-ring. Global debenzylation of 320 com-
pleted the synthesis of 311.

2.6.2
Synthetic Studies

In recent decades several research groups engaged in the synthesis of cigua-
toxins and miscellaneous approaches have published studies [168, 169]. Some
selected examples will be discussed below. In 2000, Takeda and cowork-
ers described a method for the intramolecular olefination of esters bearing
a thioacetal moiety (322, Scheme 56) [170]. Treatment of 322 with a low valent
titanium species (321) gave the reactive metal carbene 324, which attacked
the ester carbonyl group and furnished enolether 326 after elimination of
titanocene oxide.
    This method was employed by Hirama for the synthesis of the HIJKLM
fragment of CTX3C (311) [167] (Scheme 57). Starting from thioacetal 327, the
titanium-mediated olefination afforded cyclic enolether 328 and, after 6 more
steps, the pentacyclic intermediate 329. From that point, 20 more steps were
needed to build up the H-ring to yield the key fragment 314.
    An improvement of their synthesis was reported by Hirama and Inoue
in 2003 [171]. In the key transformation for the construction of the H-ring,
they used the oxiranyl anion derived from 332 as a versatile building block
(Scheme 58). In this case the H-ring was generated prior to the JKLM part,
which could be synthesized as shown in Scheme 57. Alkylation of the oxiranyl
anion with alkyl triflate 331 gave adduct 333, and the H-ring was formed via
an epoxide opening/ring closure under acidic conditions.
    From intermediate 335 it took only 11 steps to yield a differently protected
analog of 314. Another strategy was used by Sasaki for synthetic studies
on CTX1B (310) utilizing the B-alkyl Suzuki coupling reaction [172, 173].
This method turned out to be most effective in coupling reactions of cyclic




Scheme 56 Intramolecular carbonyl olefination with low valent titanium complex 321
Asymmetric Total Synthesis of Complex Marine Natural Products                      185




Scheme 57 The intramolecular olefination as a key step in the synthesis of the HIJKLM
fragment of CTX3C (311)




Scheme 58 The oxiranyl anion strategy. a Tf 2 O, 2,6-lutidine, 4 ˚ MS, CH2 Cl2, then
                                                                 A
TESOTf. b 332, n-BuLi, HMPA, THF. c TsOH·H2 O, CH2 Cl2 , (MeO)2 CH(p-MeOPh), 46%
(3 steps)




Scheme 59 Sasaki’s B-alkyl Suzuki coupling approach in the synthesis of an advanced in-
termediate of CTX1B (310); formation of the GHI ring fragment: a 336, 9-BBN, THF, then
aqueous NaHCO3 , 337, [Pd(PPh3 )4 ], DMF, 85% (based on 337)
186                                                                      J. Hassfeld et al.




Scheme 60 Synthesis of a F ring building block for ciguatoxins: a allyl bromide, THF, 1 N
NaOH. b TMSCl, imid, THF, 46% (2 steps). c H2 CCHCH2 ZnBr, THF, 94%. d Grubbs I,
60 ◦ C, 70%. e yellow HgO/I2 , PhH, hν, reflux. f NaCNBH3 , glacial acetic acid, 43%
(2 steps). g TMSCl, imidazole, THF, 99%. h L-Selectride®, THF, 63%




Scheme 61 The acetylene cobalt complex based strategy for the synthesis of the E’FGH
ring fragment of CTX1B (310). a 353 (1.5 equiv), n-BuLi, THF, then 354, 73% (recovered
354 16%). b K2 CO3 , MeOH, 95%. c Ac2 O, pyr, DMAP, CH2 Cl2, 100%. d PPTS, MeOH, 96%.
e Co2 (CO)8 , CH2 Cl2 , 95%. f TsOH·H2 O, CH2 Cl2 , 86%. g H2 100 kg/cm2 , hexane, 46%
(conjugated enone 7%, diene 12%). h K2 CO3 , MeOH, 97%. i BF3 ·Et2 O, Et3 SiH, MeCN,
72%
Asymmetric Total Synthesis of Complex Marine Natural Products                        187




Scheme 62 Synthesis of the GHIJKLM ring system of CTX1B (310). a 339, 9-BBN, THF,
then 3 M aqueous Cs2 CO3 , 340, [Pd(PPh3 )4 ], DMF, 71% (based on 340). b BH3 ·THF, THF,
then 3 M NaOH, H2 O2 , 81%. c ethyl vinyl ether, CSA, CH2 Cl2 . d TBAF, THF. e TPAP, NMO,
4 ˚ MS, CH2 Cl2. f EtSH, Zn(OTf)2 , CH2 Cl2 . g Ph3 SnH, AIBN, PhMe, 56% (5 steps)
  A


alkylboranes with cyclic enol phosphates or vinyl triflates, respectively. Fur-
thermore, a highly convergent synthesis was developed using the same trans-
formation at different stages, i.e., for substrates with varying complexity. The
crucial steps in the synthesis of the GHIJKLM fragment of CTX1B (310) are
depicted in Scheme 59 and Scheme 62.
   The components 336 and 337 were coupled under Suzuki conditions to
furnish the GHI-ring fragment 339 through 19 additional steps. Tricyclic
intermediate 339 exhibited an excyclic olefin that provided the possibility
for the following palladium-mediated coupling reaction. After hydroboration
with 9-BBN the so-formed alkylborane directly engaged in the Suzuki coup-
188                                                               J. Hassfeld et al.

ling with vinyl triflate 340 (Scheme 62). The construction of the J-ring was
achieved by radical reduction of the O, S-acetal 344 to furnish key interme-
diate 345, representing the “right hemisphere” of CTX1B (310). The mixed
acetal 344 was available from coupling product 341 via the hydroboration of
the K-ring olefin, protection of the resulting alcohol as ethoxyethyl ether, and
formation of the ketone 343. Treatment of 343 with Zn(OTf)2 in the presence
of ethane thiol gives the thioketal that is reduced under radical conditions to
afford the polycyclic target 345.
   Recently, a new access to an F-ring building block for ciguatoxins was
reported by Perlmutter and Bond [174]. This approach starts from ascor-
bic acid and utilizes a ring expansion reaction as a key step. Derivatization
of cyclohexylidene-protected ascorbic acid 346 provided dienol 348 in three
steps. Ring closing metathesis furnished the cyclohexene derivative 349, and
the mono TMS-protected diol underwent oxidative cleavage with HgO/I2 to
yield tricarbonyl compound 350. Progressive reduction and protection af-
forded the F-ring building block 352.
   In step (a), bis C-allyled compound reflux in toluene to promote complete
Claisen rearrangement should be indicated as well as the fact that 347 is a 4 : 1
mixture of epimeric C-allyled compound
   An acetylene-cobalt-complex-based method for the construction of
medium-size ether rings was reported by Isobe et al. [175]. In their syn-
thesis of the E’FGH-ring fragment 358 of CTX1B (310), this method was
used for the formation of the FG region (Scheme 61) [176]. The coupling
of acetylene compound 353 and aldehyde 354 furnished the corresponding
propargyl alcohol, which was transformed into acetylene cobalt complex 355
after protecting group manipulation. The cyclization proceeded under acidic
conditions by displacement of the propargylic acetoxy group. The resulting
complex 356 was hydrogenated to give ketone 357. Treatment of 357 with
potassium carbonate in methanol led to the deacetylation and formation
of a cyclic hemiacetal, which was reduced with Et3 SiH in the presence of
BF3 ·Et2 O to provide 358.

2.6.3
A Synthesis-Based Immunoassay

In order to prevent human intoxination by the ingestion of reef fish, the ac-
curate detection of ciguatoxins is most desirable. Among several methods for
this issue [177–181], an antibody-based immunoassay is probably most ap-
propriate. Hokama et al. used the natural toxins to prepare anticiguatoxin
antibodies, which exhibited cross reactivity to another marine toxin, okadaic
acid (361, Fig. 8) [182, 183]. The further development of these antibodies to
avoid this disadvantage has been hampered by the scarcity of the naturally
occurring toxins.
Asymmetric Total Synthesis of Complex Marine Natural Products                  189




Fig. 8 Structures of synthetic haptens 359 and 360 and okadaic acid




Fig. 9 Direct sandwich ELISA for CTX3C (311) (schematic): specific antibody mAb 10C9
is immobilized; mAb 3D11 is conjugated with horseradish peroxidase (HRP)



   Based on the total synthesis of CTX3C (311), Hirama and Fuji developed
an approach for a direct sandwich immunoassay for the specific detection of
311 [184]. Using haptens 359 and 360 (Fig. 8), monoclonal antibodies (mAbs)
against the left and right wings of 311 were prepared by immunizing with
protein conjugates of the synthetic fragments. In combination, mAb 10C9 for
the left wing of 311 and mAb 3D11 for the right wing of 311 led to the highly
sensitive detection of 311 utilizing a conventional sandwich ELISA protocol
with o-phenylenediamine (OPD) as a colorimetric substrate (Fig. 9) [184].
   Following this protocol, the detection of ppb levels (detection limits:
5 ng mL) of 311 was achieved, without exhibiting cross reactivity neither to
other marine toxins nor to the synthetic fragments 359 and 360. The so-
developed method not only provides the possibility to detect a discrete toxin
but offers a strategy for the development of immunoassays for all ciguatoxin
congeners. Moreover, it emphasizes the role that organic synthesis can play in
this context.
190                                                              J. Hassfeld et al.

2.7
Cephalostatin Analogs – Synthesis and Biological Activity

The cephalostatins and ritteracins represent an unprecedented class of bio-
logically active marine natural products (Fig. 10). They were first described
by Pettit et al. and Fusetani in 1988 and 1994, respectively. Pettit et al.
presented the structure together with the remarkable biological activity of
cephalostatin 1 (362). Since several reviews have already covered the activities
regarding isolation, structure elucidation, biological activities, and synthetic
efforts [185, 186], here we will focus on the direct synthesis of unsymmetrical
bissteroidal pyrazines as synthetic probes used to deconvolute the structure-
activity relationship.
   The starting point for the identification of these marine natural products
was when extracts derived from the marine worm Cephalodiscus gilchristi,
collected from the Indian Ocean along the South African coast, showed
in vivo tumor-inhibiting properties in the P388 model of murine lymphozytic
leukemia [187]. Pettit’s group then further reported the identification, isola-
tion, and structure elucidation of cephalostatin 1 (362) in 1988 [187]. This
compound was the major carrier of activity in the C. gilchristi extracts. It
turned out that cephalostatin 1 (362) with an ED50 value of 0.1 pM for the
in vitro P388 murine leukemia cell assay was one of the most potent tumor




Fig. 10 Cephalostatin 1 and ritterazine B
Asymmetric Total Synthesis of Complex Marine Natural Products               191

cell growth inhibitors tested at the NCI. It was shown to be about 25 times
more active than taxol in this assay system.1
   In the aftermath of the isolation, Pettit and his group were able to add
cephalostatins 2 to 19 as additional members to this class of compounds. It
is noteworthy that none of these additional cephalostatins could reach the
extraordinary high potency of cephalostatin 1 [188–194].
   The group of Fusetani added the ritterazines (363) to the new class of bis-
steroidal pyrazines [195]. The ritterazines were isolated from the tunicate
Ritterella tokioka from the coastline of Japan. Interestingly, both subgroups
showed strikingly similar frameworks and growth-inhibiting properties. The
identification of several members of the cephalostatin group was paralleld
by the identification of 26 ritterazines by the Fusetani group [195–200]. De-
spite several subtle differences, some members, such as cephalostatin 7 and
cephalostatin 16 on the one hand and ritterazine J, K, L, and M on the other,
share the same steroidal moiety (South 7, the upper right part is referred to
as the North moiety, the lower left part is referred to as the South moiety).
The known cephalostatins and ritterazines are combinations of six common
motifs and two “lone” motifs that only exist in one single natural product
(Fig. 11).
   Despite the achievements by the groups of Pettit and Fusetani it was appar-
ent that obtaining substantial amounts of cephalostatins from natural sources
for advanced pharmacological characterization or even clinical trials would
be difficult. In fact, 166 kg of the C. gilchristi worms yielded only 139 mg of
cephalostatin 1 (362) as the major cephalostatin. Similarly, collecting ritter-
azines yielded only in 13.4 mg of ritterazine B (363) from 5.5 kg of tunicate
material.
   The hurdles associated with the total synthesis of these compounds be-
come apparent if one analyzes the molecular architecture. They contain up to
a total of 13 rings annelated to the pyrazine. The attached highly oxygenated
spiroketals represent a challenging structural feature on their own. From the
synthetic standpoint, they are probably the most challenging segments.
   Finally, analysis of the biological activity of the bissteroidal pyrazines
reveals that active compounds require the selective assembly of two noniden-
tical steroid segments.
   A Gutknecht-type [201] pyrazine synthesis (Scheme 63) provides valu-
able dimeric products, but, unfortunately, starting from structurally different
α-aminoketones VII and VIII, the reaction also provides the two symmetrical
pyrazines IX and XI. Considering the synthetic efforts required to establish
the spiroacetal segments the Gutknecht-type pyrazine syntheses must there-
fore be ruled out as a practical route for the direct synthesis of unsymmetrical
pyrazines.


1   See http://dtp.nci.nih.gov/docs/dtp_search.html
192                                                                      J. Hassfeld et al.




Fig. 11 North 1, South 1, South 6, South 7, North A, North G (and the “lone” motives are:
South 9 and South V)




Scheme 63 Gutknecht-type pyrazine synthesis


   On the other hand, the condensation of a diketone XII with diamine XIII
will certainly be chemoselective with XII exclusively acting as the acceptor
and XIII being the donor molecule (Scheme 64).
   Unfortunately, this reaction also lacks regioselectivity and formation of
a mixture of isomers is observed [202].
Asymmetric Total Synthesis of Complex Marine Natural Products           193




Scheme 64 Pyrazine condensation using a diketone and a diamine


   A combination of both methods was reported in 1994 from the Heathcock
group. They performed the coupling of steroidal α-amino oxime ether 364
with 2-acetoxy-3-ketones 365 (Scheme 65) [202].
   The coupling precursor ketone 365 was prepared from readily available
androstanone 367 through enolization and subsequent epoxidation. Ring-
opening followed by epimerization provided 365 in 4 steps (Scheme 66).
   The α-amino oxime ether 364 was obtained in two steps starting from
known azido ketone 371. Treatment with methyl hydroxylamine hydrochlo-
ride followed by Staudinger reduction of the azido group gave α-amino oxime
ether 364 in high yield (Scheme 67).
   The coupling of α-acetoxy ketone 365 with α-amino oxime ether 364 finally
gave the unsymmetrical bissteroidal pyrazine 366 in 43% yield (Scheme 65).
   In order to obtain bissteroidal pyrazines that more closely resemble the
natural products, Heathcock also synthesized an acetoxy ketone bearing
a spiroketal moiety 372 (Fig. 12). Unsymmetrical coupling of this compound
with α-amino oxime ether 364 gave another bissteroidal pyrazine in 29%
yield.
   In 1996 Fuchs et al. reported a variation of Heathcock’s protocol. They
exchanged the acetoxy ketones 365 with α-azido ketone 373 and used ei-
ther polyvinylpyridine (PVP) or Nafion-H to initiate the condensation. Using
this modified protocol the protected unsymmetrical dihydrocephalostatin 1




Scheme 65 a (i) PhMe, 90 ◦ C, 24 h; (ii) 145 ◦ C, 24 h, 43%
194                                                                        J. Hassfeld et al.




Scheme 66 a Ac2 O, HClO4 , EtOAc, r.t., 78%. b DMDO, acetone, 0 ◦ C to r.t., 78%. c PhMe,
10% pyr, reflux, 83%. d HBr (cat.), AcOH, r.t., 59%




Scheme 67 a MeONH2 ·HCl, pyr, 0 ◦ C, 100%. b Ph3 P, H2 O, THF, r.t., 89%




Fig. 12 Heatchcock’s analog


(375) was obtained in good yield (75% based on recovered starting material)
(Scheme 68).
   They also reported the first cephalostatin-ritterazine hybrids (375) and
(376) under similar coupling conditions as described above.
   Compound 375 fulfilled the high expectations. While under evaluation at
the National Cancer Institute’s human cancer cell panel it displayed a mean
of GI50 < 7.4 (60 of 60 cell lines affected), which resembles only a tenfold de-
crease in activity compared to cephalostatin 1 (362) (GI50 < 8.5 nM). Hybrid
375 displayed a higher activity than cephalostatin 7 in all cell lines tested. The
Asymmetric Total Synthesis of Complex Marine Natural Products             195




Fig. 13 Cephalostatin-ritterazine hybrids




Scheme 68 a PVP, 10 mol % Bu2 SnCl2 , PhH, 51%


biological activity of 376, on the other hand, dropped significantly below the
activity of cephalostatin 1 (362), displaying a GI50 of > 6.1 nM. The missing
17-α-hydroxylation was given as a probable reason for this loss in activity.
   The Winterfeldt group developed a new unsymmetrical approach for the
coupling of two different steroids [203, 204]. Based on the observation that
aminoketone 377 did not dimerize under a variety of reaction conditions,
196                                                              J. Hassfeld et al.

they envisioned azirines as a suitable counterpart for an unsymmetrical
pyrazine synthesis. Due to the inherent reactivity of azirines fused to six-
membered rings, in situ formation of the reactive intermediates was applied.
The group used steroidal ∆2,3 -3-vinyl azides as the ideal precursors. Ther-
mally or photochemically generated azirines were attacked by the enam-
ino functionality of enamino ketone XIX, this being the driving force for
the regioselectivity observed in this coupling reaction. The so-formed aziri-
dine was ring-opened, followed by condensation with the resulting amino
group to the ketone and subsequent isomerization. Complete substrate speci-
ficity was observed since neither enamino ketone XIX nor vinyl azide XVIII
was observed to be capable of forming homodimers under these reaction
conditions.
   Using these conditions bissteroidal pyrazine 379 was formed as the first
cephalostatin analog via this strategy (Scheme 70).
   So far the following structure activity relationship (SAR) trends can be
directly derived from the 45 natural cephalostatins and ritterazines.




Scheme 69 The Winterfeldt synthesis of unsymmetrical pyrazines




Scheme 70 a PPTS (cat.), MS 3 ˚, dioxane, 100 ◦ C, 51%
                              A
Asymmetric Total Synthesis of Complex Marine Natural Products                  197

1. Additional methoxylations or hydroxylations in the steroidal A-ring core
   structure (1-position) are slightly reducing the activity.
2. Additional hydroxylations in the B-ring (7- and 9-position) do not have
   a strong effect.
3. Regarding 12-functionalization, it is apparent that all biologically active
   cephalostatins and ritterazines posses either a free hydroxy or a keto func-
   tion at this position. However, it is not apparent whether a 12, 12 -diol or
   a 12-keto-12 -ol is favored.
4. At least one 14,15-double bond is part of all highly active cephalostatins/
   ritterazines. All ritterazines lacking this feature display only low potency.
   However, the 14,15-double bond may be necessary only for stereochem-
   ical reasons creating a specific curvature of the molecule by bending the
   D-ring down. Also in line with the “curvature theory” is the fact that rit-
   terazine B (363) (14-β-hydrogen) is even more potent than ritterazine G
   (14,15-double bond).
5. At least one 17-hydroxy group is part of all highly active cephalostatins
   and ritterazines.
6. All highly active cephalostatins and ritterazines are substantially asym-
   metric.
7. In addition to the basic requirement of overall substantial asymmetry for
   high activity, it appears that a “polarity match” between both steroidal
   units (33) is required – as one must be substantially more polar (high
   hydroxylation grade) than the other (e.g., cephalostatin 1 (362).
8. Four core moieties are privileged, meaning all highly active ritterazines/
   cephalostatins are constructed out of them. These are North 1, South 1,
   South 7, and North G.


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Adv Biochem Engin/Biotechnol (2005) 97: 205–235
DOI 10.1007/b135827
© Springer-Verlag Berlin Heidelberg 2005
Published online: 25 August 2005

Seafood Allergy: Lessons from Clinical Symptoms,
Immunological Mechanisms and Molecular Biology
Ka Hou Chu1 · Chi Yan Tang1 · Adrian Wu2 · Patrick S. C. Leung3 (u)
1 Department   of Biology, The Chinese University of Hong Kong, Hong Kong, China
2 Department   of Medicine, The Chinese University of Hong Kong, Hong Kong, China
3 Divisionof Rheumatology/Allergy and Clinical Immunology, School of Medicine,
 University of California, Davis, CA 95616, USA
 psleung@ucdavis.edu

1     Food Allergy and Society . . . . . . . . . . . . . . . . . . . . . . . . . . . .                                                206

2     Clinical Symptoms of Food Allergies . . . . . . .                       .   .   .   .   .   .   .   .   .   .   .   .   .   .   207
2.1   Gastrointestinal Manifestations of Food Allergy .                       .   .   .   .   .   .   .   .   .   .   .   .   .   .   207
2.2   Dermatological Manifestations of Food Allergy . .                       .   .   .   .   .   .   .   .   .   .   .   .   .   .   207
2.3   Respiratory Tract Manifestations of Food Allergy .                      .   .   .   .   .   .   .   .   .   .   .   .   .   .   208
2.4   Systemic Manifestations of Food Allergy . . . . .                       .   .   .   .   .   .   .   .   .   .   .   .   .   .   208

3     Diagnosis of Food Allergies . . . . . . . . . . . . . . . . . . . . . . . . . .                                                 208

4     Immunological Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . .                                                    209

5     Molecular Biology of Seafood Allergens . . . . . . . . .                                .   .   .   .   .   .   .   .   .   .   214
5.1   History . . . . . . . . . . . . . . . . . . . . . . . . . . . .                         .   .   .   .   .   .   .   .   .   .   214
5.2   Studies of Fish Allergens . . . . . . . . . . . . . . . . . .                           .   .   .   .   .   .   .   .   .   .   214
5.3   Studies on Crustacean Allergens . . . . . . . . . . . . . .                             .   .   .   .   .   .   .   .   .   .   216
5.4   Studies of Mollusk Allergens . . . . . . . . . . . . . . . .                            .   .   .   .   .   .   .   .   .   .   218
5.5   Cross-reactivity Among Different Seafood . . . . . . . .                                .   .   .   .   .   .   .   .   .   .   219
5.6   Tropomyosins as Allergens in other Invertebrate Groups                                  .   .   .   .   .   .   .   .   .   .   221
5.7   Epitopes of Tropomyosin Allergens . . . . . . . . . . . .                               .   .   .   .   .   .   .   .   .   .   222

6     Applications and Future Directions      .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   226
6.1   Diagnosis and Profiling . . . . . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   226
6.2   Immunotherapy . . . . . . . . . . .     .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   227
6.3   Non-allergenic Seafood . . . . . . .    .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   .   229

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .                                              229

Abstract Food allergy consists of a wide range of disorders that result from adverse im-
mune responses to dietary antigens. Manifestations of allergic response includes acute,
potentially fatal anaphylactic reactions and a variety of chronic diseases that mainly af-
fect the gastrointestinal tract, skin, and respiratory tract. Tools for clinical diagnosis and
management, which have not changed much in the past two decades, include the clin-
ical history, tests for specific IgE antibody to suspected foods, elimination diets, oral
food challenges, and provision of medications such as epinephrine for emergency treat-
ment. On the other hand, recent immunological and molecular biological research have
206                                                                       K.H. Chu et al.

enhanced our understanding of the mechanisms of these disorders and revealed the iden-
tities of many food allergens. Here, we will discuss seafood allergies with respect to
the clinical manifestations, diagnosis, immunological mechanisms, and molecular biol-
ogy of seafood allergens. Furthermore, potential applications and future directions in the
clinical management of seafood allergies are discussed.

Keywords Hypersensitivity · IgE · Tropomyosin · Epitopes · Allergen · Seafood



1
Food Allergy and Society

Allergies are the sixth leading cause of chronic diseases with approximately
9–16% of people suffering from allergies in the United States [1]. Treatment
of allergies has a great economic impact in the United States, amounting to
$18 billion annually in health care costs [2]. Some of the different types of
allergies are allergic drug reactions, which make up about 5–10% of all nega-
tive drug reactions [2], and food allergies, which occur in children less than 6
years old and approximately 1–2% of adults [3, 4]. Hughes and Mills [6] stated
that around 33% of anaphylactic reactions are caused by food. About 4–8% of
children suffer from some forms of food allergy [7, 8]. The death count from
food allergies adds up to about 100 Americans per year, with most of them
being children [5]. Gastrointestinal manifestations of food allergies include
nausea, emesis, diarrhea, and abdominal cramping. Dermatological reactions
(flushing, urticaria, angioedema and atopic dermatitis) [9, 10], respiratory re-
actions (wheezing and rhinitis) and ocular reactions (conjunctivitis) [11, 12]
are frequent in patients with food allergies.
   Food is an intrinsic part of human civilization. In recent years, seafood has
been considered a healthier component of the human diet due to an increas-
ing concern about dietary fat and cholesterol. In 2001, seafood consumption
reached 14.8 lb per capita in the United States. Shrimp and tuna with an aver-
age per capita consumption of 3.4 and 2.9 lb, respectively, were the first and
second most popular seafood items. Physiologically, food is being catabolized
to provide energy and nutrients. Although the intake and breakdown of food
are taken very much for granted in human life, food is frequently implicated
in a variety of maladies, including food allergies. Currently, the only therapy
for food allergy is strict avoidance of the food that causes allergy. Chemi-
cals including cromoglycate histamine receptor antagonists and ketotifen are
not recommended as propholytic treatment of food allergy because of the
conflict of their efficacy [13, 14]. Occupational hypersensitivity affects a wide
spectrum of people ranging from fishermen, processing workers, fish meal
factory workers, oyster shuckers, caterers, and cooks [15–18]. The growing
demand for seafood and the increasing risk of getting seafood allergy make it
important to elucidate the clinical symptoms, diagnosis, and immunological
Seafood Allergy                                                               207

mechanisms of seafood allergy and to identify the seafood allergen(s) at the
molecular level.


2
Clinical Symptoms of Food Allergies

2.1
Gastrointestinal Manifestations of Food Allergy

Since the gastrointestinal tract is the first site to come into contact with food
allergens, gastrointestinal symptoms are frequent in patients with food al-
lergy. IgE-mediated reactions that cause nausea, vomiting, abdominal pain,
and diarrhea may occur within minutes of ingestion. Repeated ingestion of
allergenic foods may lead to poor appetite, intermittent abdominal pain, and
malabsorption. The direct contact of allergenic food with the oral mucosa
that causes itching and swelling of the lips, tongue, palate, and throat is called
the oral allergy syndrome. Aside from IgE-mediated reactions, food allergies
may cause other forms of gastrointestinal pathologies, including eosinophilic
oesophagitis or gastroenteritis. Symptoms include gastroesophageal reflux,
vomiting, abdominal pain, failure to thrive, low serum albumin, blood vom-
iting, and intestinal obstruction [19]. Elimination of the offending foods will
lead to resolution of symptoms in 3–8 weeks.

2.2
Dermatological Manifestations of Food Allergy

IgE-mediated reactions to food often present as urticaria (hives) and an-
gioedema (swelling). The onset of symptoms is rapid, sometimes within min-
utes of ingestion, and usually lasts for less than 24 h. However, patients are
usually able to identify and avoid offending foods that cause the symptoms.
Direct skin contact can also elicit a reaction, and this is sometimes seen in
fishmongers and kitchen workers. It is now clear that food allergy is an im-
portant factor in the development of eczema in young children. In a recent
study, 37% of children with moderate to severe eczema were found to be al-
lergic to food by serum IgE tests and food challenge [20]. Although one-third
of children with eczema and food allergies “outgrow” their allergies within
1–3 years [21], shellfish allergies tend to be an exception. It is therefore per-
tinent that all children with moderate to severe eczema should be tested for
food allergies and, if present, dietary advice should be given. A 2–3 week
elimination diet followed by gradual reintroduction of suspected foods one by
one while keeping a symptom diary is often sufficient for diagnosis.
208                                                                 K.H. Chu et al.

2.3
Respiratory Tract Manifestations of Food Allergy

Respiratory symptoms are common in patients who develop food-induced
anaphylaxis, and are invariably present in fatal or near-fatal cases. Patients
with a history of asthma or a history of previous severe food allergic reactions
are especially susceptible to severe anaphylaxis. Food allergy is rarely a factor
in allergic rhinitis; a survey of 323 patients with chronic rhinitis revealed that
only two patients had nasal symptoms during blinded food challenges [22].
A study of 88 children with eczema and asthma revealed that 15% wheezed
during food challenge, and 8% demonstrated a greater than 20% drop in
lung function [23]. However, food-induced respiratory symptoms appear to
be rare in patients without eczema.

2.4
Systemic Manifestations of Food Allergy

Systemic anaphylaxis is the most dangerous of all allergic reactions, and food
allergy is the most common cause of anaphylaxis [24]. Any food protein can
theoretically cause anaphylaxis but shellfish is especially likely. In our series
of 84 shellfish allergic patients in Hong Kong, one-third gave a history of ana-
phylaxis. Reactions invariably occur within 60 min and may include urticaria,
angioedema, respiratory difficulties, low blood pressure, throat swelling, ab-
dominal pain, vomiting, and diarrhea. Severe reactions will lead to asphyxia,
vascular collapse, irregular heartbeat, or myocardial infraction. The quantity
of food required to induce a reaction is dependent on patient sensitivity, po-
tency of the food allergen, and other unknown factors. Shellfish allergens may
invoke a fatal reaction in microgram amounts. The sensitivity of individual
patients may change with time. Some patients may have tolerated a certain
amount of a particular food without significant ill effects in the past, but
nevertheless developed a severe reaction when they ingest a similar amount
a second time. One interesting form of anaphylaxis is the exercise-induced,
food-specific anaphylaxis. These patients develop anaphylaxis if they exercise
within 2–4 h of ingesting a food that they are allergic to. However, ingesting
the same food without exercise will not elicit a reaction. Shellfish has been
found to cause 8% of exercise-induced anaphylaxis [25].


3
Diagnosis of Food Allergies

The history is the most important tool in the diagnosis of food allergy. How-
ever, it is frequently inaccurate and only about 40% of such histories can be
verified by food challenges [26]. Important points to note in the history in-
Seafood Allergy                                                             209

clude the nature of the reaction, the foods ingested at the time, the timing
of the reaction relative to food ingestion, repeatability and frequency of the
reaction, and other exacerbating factors such as exercise. Knowledge of food
allergy patterns found in a community is also helpful in discerning the likely
culprit. One must also bear in mind the “hidden allergens”, especially in pro-
cessed food. For example, shellfish is often used in Chinese dumplings and
shells are sometimes added to soup for flavor. Cross-contamination of cook-
ing utensils can also cause problems. A food diary is often a helpful tool in
revealing these hidden allergens. If an IgE-mediated food allergy is suspected,
skin prick tests (SPT) are useful in screening for likely culprits. One must
bear in mind that SPT have a low positive predictive value (hence high false-
positive rate) but a high negative predictive value. Treatment based solely on
positive SPT results would subject patients to unnecessary food avoidance.
For patients who are on antihistamines or have significant skin pathologies
that preclude the use of skin tests, measurement of serum food-specific IgE
by the radioallergosorbent test (RAST) or the Pharmacia CAP test is war-
ranted. When a list of possible candidate food allergens can be established,
a diagnostic elimination diet can support the diagnosis. If the offending aller-
gens are eliminated, there should be significant improvement in symptoms.
After two weeks of allergen elimination, a food challenge should be carried
out. In cases where there is a risk of severe anaphylaxis, or if the history is
strongly supported by SPT results, then food challenge can be omitted. Food
challenges can be done open, single-blind or double-blind. Double-blind,
placebo-controlled food challenge is the gold standard for the diagnosis of
food allergies and should be used when patient perception might significantly
influence symptom assessment.


4
Immunological Mechanisms

Ingested food is first processed through the gastrointestinal tract, which
forms an extensive barrier to the outside environment. Although the mucosal
immune system associated with the intestinal tract (gut-associated lymphoid
tissue) is proficient in inhibiting immune responses to non-dangerous anti-
gens and mounting a rapid response to pathogens, about 2% of ingested food
is absorbed and transported throughout the body in an immunologically ac-
tive form [4, 27]. Food antigens, are typically poor immunogens and induce
a state of unresponsiveness (oral tolerance). Thus intact food antigens do not
typically cause clinical symptoms because most individuals acquire tolerance.
For example, unresponsiveness of T cells to ingested food proteins is believed
to be due to T cell anergy. The regulation of T-cell responses in the intestinal
mucosa by intestinal epithelial cells as antigen-presenting cells, the secretion
of IL-10 and IL-4 by dendritic cells residing with the non-inflammatory en-
210                                                                  K.H. Chu et al.

vironment of the Peyer’s patches, and the production of transforming growth
factor beta by T regulatory cells in the mucosal lymphoid tissue in response
to low dose antigen are believed to contribute to oral tolerance [28–30].
   Despite the phenomenon of oral tolerance, one may be sensitized to food
allergens in the gastrointestinal tract after ingestion of food, which is clas-
sified as class 1 food allergy or after inhalation of an airborne allergen
that crossreacts with a specific food, which is considered a class 2 food al-
lergy [31]. IgE-mediated reactions develop when food-specific IgE antibodies
residing on mast cells and basophils recognize and bind to circulating in-
gested food allergens and thus activate the cells to release a number of potent
mediators and cytokines.
   A typical sequence of events in the development of a hypersensitive food re-
action consists of exposure to the antigen, activation of Th2 cells specific for the
antigen, production of IgE antibodies specific for the antigen, binding of IgE
to Fc receptors of mast cells, releasing of mediators upon exposure to the anti-
gen, and subsequent pathology reaction. The development of an IgE-mediated
response to an allergen is the result of a series of molecular and cellular inter-
actions, involving antigen-presenting cells (APCs), T cells, and B cells (Fig. 1).
APCs present small peptides in the context of MHC class II molecules to T cells.
The binding of allergen/MHC class II complex to the complementary T cell
receptor signals the T cells to proliferate and generate signals to promote an
IgE response [32, 33]. Recent studies have substantiated the understanding of
the roles of T cells in allergic inflammatory responses. A combination of inter-
leukin 4 (IL-4), IL-5, IL-10, IL-13 produced by a subset of CD4+ T cells (termed
Th2 cells) are found in increased abundance in allergic individuals [35]. CD4+
T cells play a role in the enhancement, recruitment, growth, and differentiation
of other cell types in allergic inflammatory responses through cytokine secre-
tion [34]. CD4+ T cells secrete IL-4 and IL-13, which enhance the induction
of IgE synthesis in B cells, mast cell growth, and recruitment of lymphocytes,
mast cells, and basophils to sites of inflammation [35]. In addition, CD4+ T cells
produce IL-5, which enhances the growth and differentiation of eosinophils
and B cells, and IL-10, which enhances the growth differentiation of mast cells
and inhibits the production of IFN-γ . This may partly explain why non-allergic
individuals are asymptomatic because they develop a Th1 response, which in-
hibits IgE synthesis and mast cell and eosinophil differentiation. A recent study
by Watanabe et al. [36] suggested that food antigen might negatively select
Th cells for IgE response to the food antigen by preferential deletion of Th1
cells in the liver. A novel mechanism of peripheral T cell response in atopic
disease was described by Akdis et al. [37]. The Th1 compartment of activated
memory/effector T cells selectively undergoes activation-induced cell death
and thus skews the immune response towards surviving Th2 cells. The apop-
tosis of circulating memory/effector T cells was confined to atopic individuals
whereas healthy controls showed no evidence for enhanced T cell apoptosis
in vivo. It is interesting to note that IL-25, a recently reported cytokine, has
Seafood Allergy                                                                         211




Fig. 1 Seafood allergens are first processed and presented by the antigen-presenting cells
(APC) through the major histocompatibility complex class II (MHC II). CD4+ helper
T cells (Th2) then recognize the allergens via the interaction between the T cell receptor
(TCR) and MHC II, while IL-4 and IL-13 are secreted to promote the formation of plasma
cell from B cell. Specific IgE are released by plasma cells and bind to the IgE receptors
(FcεRI) on the surface of mast cells or basophils. Upon subsequent exposure to the same
allergen, the cross-linking of the IgE receptors induce the degranulation of mast cells or
basophils. A potent Th2 response inducer IL-25, is also secreted by mast cells. IL-10 is se-
creted by Th cells as positive feedback to mast cells. Chemical mediators released by the
degranulation include histamine, prostaglandins, proteoglycans, leucotrienes, platelet-
activating factors and cytokines, which are responsible for the hypersensitivity reaction.
The secretion of IL-4 and IL-13 by mast cells as well as IL-5 secreted by Th2 cells lead to
late phase allergic reactions


been suggested to play a significant role in allergic diseases via the produc-
tion of IL-4, IL-5 and IL-13, and eotaxin [38], perhaps by mast cells [39]. IL-25
might play a significant role in the development of food hypersensitivity reac-
tions. Although it is generally agreed that allergy is caused by the development
of allergen-specific Th2 cells in allergic individuals, this is a much simplified
picture. Furthermore, the recent discovery of a different subset of CD4+ T
regulatory (Tr) cells and the manifestation of cytokine- (IL-10 and TGF-β) de-
pendent suppression of inflammatory response by CD4+ CD25– (Tr) T cells and
cytokine-independent suppression by CD4+ CD25– T cells substantiate the hy-
pothesis that Tr cells have a critical role in maintaining immune homeostasis
in vivo [40, 41]. The exact mechanism of the Th1/Th2 paradigm is not fully
understood and is likely to be influenced by a number of genetic and environ-
mental factors [42].
212                                                               K.H. Chu et al.

   The mast cell IgE and IgE receptor network plays an important role in
regulating an allergic reaction [43, 44]. IgE antibodies are bound to a high
affinity Fc receptor specific for ε heavy chains (FcεRI) that are expressed on
mast cells, basophils, and eosinophils. Each FcεRI molecule is composed of
one α chain that mediates IgE binding, and a β and two γ chains that are re-
sponsible for signaling. The amino terminal extracellular portion of the FcεRI
α chain consists of two Ig-like domains and constitutes the IgE binding site.
The β chain of the FcεRI chains consists of a single immunoreceptor tyrosine-
based activation motif (ITAM) in the carboxyl terminus and the two identical
γ chains, each containing one ITAM [45, 46].
   In an individual who is allergic to a particular antigen, a large proportion
of the IgE bound to mast cells is specific for that antigen. In contrast, in non-
atopic individuals, the mast cell’s associated IgE are highly heterologous in
antigen specificity. Thus, exposure to the allergen will cross-link sufficient IgE
molecules in an allergic individual but will be less likely to produce the same
effect in non-atopic individuals. Upon IgE cross-linking of the FcεRI, the Lyn
tyrosine kinase phosphorylates the ITAM of the β and γ chains of the FcεRI.
Syk tyrosine kinase is then recruited to the ITAMs of the γ chain, and acti-
vated, thus phosphorylating other proteins in the signaling cascade [47]. Sev-
eral key signaling players are involved in the IgE-mediated immune response:
(a) Phosphorylation of the γ isoform of phosphatidylinositol-specific phos-
pholipase (PLCγ ), which catalyses the breakdown of phosphatidylinositol
bisphosphate to inositol triphosphate (IP3) and diacylglycerol (DAG); (b) ac-
tivation of cytosolic phospholipase A2 (PLA2 ), which subsequently hydrolyzes
membrane phospholipase eventually leading to the release of mediators in-
cluding arachidonic leukotrienes especially LTC4 and prostagladin D2; and
(c) nuclear translocation of transcription factors including nuclear activator
of activated T cells (NFAT) and NF-kB, which are important in the stimulation
of transcription of cytokines [48, 49]. Mast cell activation is also negatively
regulated by cyclic adenosine monophosphate (cAMP). Activated cAMP ac-
tivates protein kinase A, which in turn inhibits mast cell degranulation. In
addition, the FcεRI activation pathway is regulated by the inhibitory Fc recep-
tor (Fcγ RIIb), which contains a immunoreceptor tyrosine-based inhibitory
motif (ITIM). Phosphorylation of ITIM leads to the recruitment of the tyro-
sine phosphatase Sh2 domain-containing inositol 5-phosphatase (SHIP) and
inhibition of IgE-mediated FcεRI signaling [50, 51].
   The effector functions of mast cells are mediated by soluble molecules re-
leased upon activation. The key mediators include: (a) histamines; (b) granule
proteins such as tryptase, chymase, carboxypeptidase A, cathepsin G, and
proteoglycans such as heparin [52]; (c) lipid mediators such as prostaglandin
D2 and leukotrienes that have a variety of effects on blood vessels, bronchial
smooth muscle, and leukocytes; and (d) cytokines including TNF, IL-1, IL-4,
IL-5, IL-6, IL-13, MIP-1α, MIP-1β [53, 54], and a potent Th2 response inducer
IL-25 [39]. In addition to mast cells, FcεRI are also expressed on basophils
Seafood Allergy                                                                       213

Table 1 Mediators produced by mast cells, basophils and eosinophils and their physiolog-
ical functions

Cell type         Mediators                        Physiological functions

Mast cells and basophils

Preformed         Histamines                       Increase vascular permeability,
  cytoplasmic                                      stimulate smooth muscle
  granules                                         contraction

                  Neutral proteases (tryptase,    Tissue damage/remodelling
                  chymase), acid hydrolyases,
                  cathepsin G, carboxypeptidase A

Lipid             Prostaglandin D2                 Vasodilation, bronchoconstriction,
  mediators                                        neutrophil chemotaxis

                  Leukotriene C4, D4, E4           Prolong bronchoconstriction, mucus
                                                   secretion, increase vascular
                                                   permeability

                  Platelet activating factor       Bronchoconstriction,
                                                   increase vascular permeability,
                                                   chemotaxis of leukocytes

Cytokines         IL-3                             Promotes mast cell proliferation

                  TNF-α, MIP-1α,                   Inflammation

                  IL-4, IL-13                      Th2 differentiation

                  Il-5                             Promotes eosinophil production
                                                   and activation

Eosinophils

Preformed         Peroxidase, lysosomal            Tissue damage/remodelling
  cytoplasmic     hydrolases
  granules

Lipid             Leukotriene C4, D4, E4           Prolong bronchoconstriction,
  mediators                                        mucus secretion,
                                                   increase vascular permeability

                  Lipoxins                         Inflammation

Cytokines         IL-3, IL-5 , GM-CSF              Promotes eosinophil production
                                                   and activation

                  IL-8, IL-10, MIP-1α              Leukocytes chemotaxis
214                                                               K.H. Chu et al.

and eosinophils. The ability of eosinophils to release granules is enhanced by
IL-5 [55]. Activated eosinophils produce and release lipid mediators such as
PAF, prostaglandins, and leukotrienes [56–59] (Table 1) in a similar fashion
to mast cells and basophils.


5
Molecular Biology of Seafood Allergens

5.1
History

Prausnitz and Küstner [60] performed a classical experiment by injecting
serum from a fish-allergic patient into the skin of a non-atopic individual.
The injection site was then challenged with fish extract and the allergic re-
sponses observed. This study represents an early study on seafood allergy.
The fish allergen, known as Allergen M (later designated as Gad m 1 or
Gad c 1), isolated from the Atlantic cod (Gadus morhua = G. callarias) in 1969
is among the first food allergens identified [61]. Crustaceans such as shrimp,
lobster, and crab have long been known to be the most common allergic
food items among seafood [62, 63]. Some allergic patients not only developed
allergic reactions to ingested shrimp, but also developed urticaria after hand-
ling shrimp directly, or wrapping containing shrimp or other crustaceans
such as lobster or crab [64]. Shrimp water or vapor from cooked shrimp
was shown to be a source of allergen in susceptible individuals [65]. Such
contacts present a threat of occupational allergy among seafood-processing
workers [18]. Some patients even developed adverse reactions towards crus-
taceans in the absence of previous contact [11]. Although isolation of shrimp
allergens by biochemical methods was reported in the 1980s [64, 66], the
identity of shellfish allergens remained elusive till the 1990s, despite the fact
that a number of allergens had been identified from common food items such
as lactoglobulin, lactalbumin, and casein from cow’s milk, and ovalbumin and
ovomucoid from hen’s egg in 1970–1980.

5.2
Studies of Fish Allergens

The allergen in fish was first identified in the Atlantic cod [61, 67–70]. The
allergen (Gad c 1) consists of 113 amino acids and a glucose molecule, with
a molecular weight of about 12 kDa. The allergen belongs to a family of par-
valbumins, a group of calcium-binding proteins. Parvalbumins are present
in high amounts in white muscles of lower invertebrates [71] and in lower
amounts in fast twitch muscles of higher vertebrates [72]. They function in
calcium buffering and are involved in the relaxation process of muscles [73].
Seafood Allergy                                                              215

Analysis of amino acid sequences indicates that the parvalbumin protein
family could be subdivided into an α group and a β group [74]. Lindstrom
et al. [75] purified parvalbumin in the Atlantic salmon (Salmo salar) and con-
firmed its allergenicity. The allergen was designated as Sal s 1. A probe based
on the amino acid sequences of Sal s 1 was used to screen a salmon muscle
cDNA library to identify two distinct cDNA clones representing two differ-
ent parvalbumin genes. The deduced amino acid sequences of both clones
belong to the β-lineage of parvalbumins. This study represents the first mo-
lecular cloning study of a fish allergen. Van Do et al. [76] further confirmed
this finding.
   Das Dores et al. [77, 78] determined the DNA sequence of parvalbumin
allergen (Gad m 1) in the Atlantic cod. This recombinant allergen of a mo-
lecular weight of 11.5 kDa had 62.3% identity with Gad c 1 and surprisingly,
75% with Sal s 1. The parvalbumin gene of the Atlantic cod was also cloned
by Van Do et al. [79]. Using RT-PCR, two distinct cDNA clones, named T1 and
T2, were identified in the white muscle. Both belong to parvalbumin β group
with high sequence similarity to other members of the family Gadidae. When
we compare the amino acid sequences among Gad c 1 [70], Gad m 1 [77, 78],
T1, and T2 [79], the identity of Gad m 1 and T2 is 94.3%, suggesting these
two allergens represent very similar, if not identical isoforms. Their identi-
ties to the other cod allergens are less than 72%, suggesting that the allergens
represent distinct isoforms of parvalbumin. Parvalbumin has also been char-
acterized as the allergen in horse mackerel [80] and carp [81, 82]. A list of
known fish allergens is shown in Table 2. It should be noted that while parval-
bumin represents the major cross-reactive fish allergen, other fish allergens
have been reported in a number of fish species [83–86].
   The cod allergen is the most intensively investigated allergen and serves as
a model for epitope mapping of fish allergen [87]. As parvalbumin, the struc-
ture of Gad c 1 consists of three domains, AB, CD, and EF. A series of studies
using tryptic and synthetic peptides as inhibitors of RAST showed that Gad
c 1 contains at least five IgE-binding sites distributed along its polypeptide
chain at residues 13–32, 33–44, 49–64, 65–74, and 88–96 [88–96]. Peptide
13–32 is found in the AB domain. Peptide 33–44 and 65–74 are found on
the axis joining AB and CD domains and the axis joining the CD and EF
domains, respectively. A high amino acid homology in these two regions
suggests the presence of repeated allergenic determinants in Gad c 1. Pep-
tide 49–64 of the Ca2+ -coordinating CD domain consists of two repetitive
sequences (D-E-D-K) and (D-E-L-K) that are important for antibody bind-
ing. This peptide is also cross-reactive with birch pollen allergen [97]. Peptide
88–96 is found in the Ca2+ -coordinating domain EF. Interestingly Ca2+ deple-
tion reduces specific IgE binding to Gad c 1 [98]. This observation has also
been confirmed in the study on carp parvalbumins (Cyp c 1.01 and Cyp c
1.02) [82]. The reduction in IgE binding is possibly due to unfolding of the
protein or a change in conformational epitopes. Other Ca2+ -binding proteins
216                                                               K.H. Chu et al.

have been identified as allergens in plants and autoallergens in man [99].
Protein-bound Ca2+ is required for IgE binding to Ca2+ -binding plant aller-
gens [100, 101].

5.3
Studies on Crustacean Allergens

The molecular identity of the shrimp allergen was elucidated in the early
1990s by a number of research groups. Shanti et al. [102] reported that there
is high (86%) amino acid sequence homology between allergen Sa-II (also
referred as Pen i 1) from the shrimp Penaeus indicus [66] and the muscle
protein tropomyosin of the fruit fly Drosophila melanogaster. Tropomyosins
are a family of actin filament-binding proteins with distinct isoforms found
in muscles, brain, and various non-muscle cells. In association with the tro-
ponin complex, tropomyosins function in the regulation of calcium-sensitive
interaction of actin and myosin. Leung et al. [103] first reported the cloning of
the major shrimp allergen from Metapenaeus ensis, called Met e 1, by screen-
ing a cDNA library of shrimp muscle with sera from patients with shellfish
allergy. The cDNA of Met e 1 exhibited an open reading frame of 281 amino
acids, coding for a 34-kDa protein. This molecule had high homology to
Drosophila tropomyosin. Another shrimp allergen, Pen a 1, was also identified
in the same year from the shrimp Penaeus aztecus [104]. The identity of Pen
a 1 as tropomyosin has subsequently been confirmed by molecular cloning
and nucleotide sequence analysis [105]. Comparsion of the amino acid se-
quences of antigens Sa-II, Pen a 1 and Met e 1 showed that they are similar
or identical allergens.
   The identity of tropomyosin as an allergen has subsequently been con-
firmed in other crustaceans. Leung et al. [106, 107] identified the allergen
Pan s 1 from the spiny lobster Panulirus stimpsoni, Hom a 1 from the Amer-
ican lobster Homarus americanus, and Cha f 1 from the crab Charybdis
feriatus. All three have similar molecular weight (34 kDa) and their deduced
amino acid sequences are highly homologous to tropomyosin. While Cha f 1
appears to be slow muscle tropomyosin, the other crustacean allergens iden-
tified (Met e 1, Pen a 1, Hom a 1, and Pan s 1) are possibly fast muscle
tropomyosins.
   While tropomyosin is the major allergen among crustaceans, there have
been reports on the presence of multiple allergens [108, 109]. Yu et al. [110]
recently identified a novel allergen designated as Pen m 2 from the shrimp
Penaeus monodon by two-dimensional immunoblotting using sera from pa-
tients with shrimp allergy. The allergen was cloned and the cDNA con-
tained 1071 bp of open reading frame encoding a 356 amino acid protein
with a theoretical molecular weight of 39.9 kDa. The sequence of this pro-
tein showed similarity (60%) to arginine kinase of crustaceans (Penaeus
japonicus). Pen m 2 exhibited arginine kinase activity and reacted with IgE
Seafood Allergy                                                                       217

Table 2 Summary of seafood allergens identified

Source of allergen                Nomen-       MW      Identity       cDNA Reference
                                  clature      (kDa)                  cloned

Fish
Gadus morhua                      Gad m 1      12      Parvalbumin    –     61, 67
= (Gadus callarias)
(Atlantic cod)
Salmo salar (salmon)              Sal s 1      12      Parvalbumin    +     75
Horse mackerel                    Nil          12      parvalbumin    –     80
Cyprinus carpio                   Cyp c 1.01   11.4    Parvalbumin    +     81, 82
(common carp)                     Cyp c 1.02   11.4    Parvalbumin    –
Crustacea
Metapenaeus ensis                 Met e 1      34      Tropomyosin    +     103
(Greasy back shrimp)
Penaeus aztecus                   Pen a 1      36      Tropomyosin    +     104
(Northern brown shrimp)
Penaeus indicus                   Pen i 1      38      Tropomyosin    –     66, 102
(Indian white shrimp)
Penaeus monodon                   Pen m 2      39.9    Arginine kinase +    110
(Giant tiger shrimp)
Homarus americanus                Hom a 1      34      Tropomyosin    +     107
(American lobster)
Panulirus stimpsoni               Pan s 1      34      Tropomyosin    +     107
(spiny lobster)
Charybdis feriatus(crab)          Cha f 1      34      Tropomyosin    +     106
Mollusca
Haliotis midae (abalone)          Hal m 1      49      Nil            –     119
Haliotis diversicolor (abalone)   Hal d 1      38      Tropomyosin    +     128
Turbo cornutus (turban shell)     Tur c 1      35      Tropomyosin    –     125
Helix aspersa                     Hel as1      36      Tropomyosin    +     120
(brown garden snail)
Perna viridis (mussel)            Per v 1      38      Tropomyosin    +     128
Placopecten magellanicus          Nil          30      Tropomyosin    +     127
(sea scallop)
Chlamys nobilis (scallop)         Chl n 1      38      Tropomyosin    +     128
Crassostrea gigas                 Cra g 1.01   35      Tropomyosin    –     117, 123,
(Pacific oyster)                   Cra g 1.02   35      Tropomyosin    –     124, 126
                                  Cra g 1.03   31      Tropomyosin    +
Todarodes pacificus (squid)        Tod p 1      38      Tropomyosin    –     129
Octopus vulgaris (octopus)        Oct v 1      31–34   Tropomyosin    –     130



from shrimp-allergic patients. Strong reactivity of purified arginine kinase
from shrimp (Metapenaeus ensis), lobster (Homarus gammarus), crawfish
(Metanephrops thomsoni), and crab (Scylla serrata) with anti-Pen m 2 anti-
218                                                               K.H. Chu et al.

body and sera from shrimp-sensitive patients indicates that arginine kinase
as a common allergen among crustaceans, in addition to tropomyosin. Argi-
nine kinase has also been designated as an allergen in the moth (Plodia
interpunctella) [111]. A list of known crustacean allergens is included in
Table 2.

5.4
Studies of Mollusk Allergens

The mollusks commonly consumed include members of class Gastropoda
(e.g. limpet and abalone), class Bivalvia (e.g. scallop, clam, mussel, and oys-
ter) and class Cephalopoda (cuttlefish, squid, and octopus). There have been
a number of reports on allergies to mollusks including allergies to snail [112],
abalone [113], limpet [114, 115], cuttlefish [116], and squid [117]. Leung
et al. [118] showed that sera from patients with shrimp allergies reacted spe-
cifically to a 38-kDa protein in a wide variety of mollusks from the three
classes. The IgE-binding reactivity of the sera to mollusk extract could be
inhibited by preabsorption with recombinant shrimp tropomyosin Met e 1,
suggesting that tropomyosin is a common allergen among crustaceans and
mollusks. The identity of tropomyosin as the allergens in mollusks has sub-
sequently been confirmed in the three groups of mollusks.
   For gastropods, Lopata et al. [119] demonstrated the IgE reactivities of
abalone (Haliotis midae) sensitive subjects to two major allergens with mo-
lecular weights of 38 kDa and 49 kDa. While the 38-kDa protein was believed
to be tropomyosin, the identity of the 49-kDa allergen, named Hal m 1, re-
mains to be identified. Ishikawa et al. [124, 125] purified a 35-kDa allergen
(Tur c 1) in the turban shell Turbo cornutus. The amino acid composition and
partial amino acid sequences of Tur c 1 imply that it is tropomyosin. Asturias
et al. [120] isolated and cloned tropomyosin from Helix aspersa (brown gar-
den snail) and found that the tropomyosin (Hel as 1) was a 36-kDa protein
and shared 84% of its amino acid sequence identity with abalone (Haliotis di-
versicolor), 70% with mussel (Mytilus edulis), and 72% with scallop (Chlamys
nobilis) tropomyosins. Western blot inhibition of different mollusk and crus-
tacean extracts showed cross-reactivity between Helix aspersa, cuttlefish, oc-
topus, sea snail, and shrimp. The IgE-binding capacity of the recombinant
Hel as 1 was found to be weaker than its natural counterpart, possibly due to
the absence of some epitopes or improper folding of the recombinant protein.
These studies demonstrated that tropomyosin is indeed a major allergen in
various gastropod mollusks.
   Among bivalves, it is well known that oysters can cause hypersensitive
reactions upon ingestion, as well as occupational reactions in sensitized
workers [16, 121]. Oyster and crustacean allergens possibly share common
antigen epitopes, i.e., the common primitive allergenic structure shared by
the two taxa is conserved in evolution [122]. Ishikawa et al. [123] purified
Seafood Allergy                                                               219

two biochemically similar allergens (Cra g 1 and 2) from the Pacific oyster
Crassostrea gigas by gel permeation chromatography, ion exchange FPLC, and
reverse-phase HPLC. Their molecular weights and amino acid composition
strongly suggest that they are isoforms of tropomyosins [123–125]. Leung and
Chu [126] indicated that Cra g 1 and 2 represent the same or similar isoforms
of oyster tropomyosins and they should be designated as Cra g 1.01 and Cra g
1.02. In the same study, tropomyosin (Cra g 1.03) was confirmed to be the oys-
ter allergen using recombinant DNA technology. Patwary et al. [127] isolated
and characterized cDNA clones that encode tropomyosin from adductor mus-
cle of sea scallop (Placopecten magellanicus). The cDNAs encoded an open
reading frame of 284 amino acids and this protein had a molecular weight
of approximately 30 kDa. The amino acid sequence is about 70% identical
to tropomyosins from other mollusks. Using reverse transcriptase-PCR, Chu
et al. [128] amplified tropomyosin cDNA from abalone (Haliotis diversicolor),
scallop (Chlamys nobilis), and mussel (Perna viridis). The cDNAs were cloned
and expressed and the IgE reactivity of the recombinant proteins was demon-
strated. A comparison of amino acid sequences of tropomyosins of the three
mollusks to those of a wide variety of mollusks available from the GenBank
database shows that they are highly conserved (identity > 68%).
   Allergens have also been identified as tropomyosins in cephalopods. Miya-
zawa et al. [129] isolated a 38-kD heat-stable allergen (Tod p 1) from the
squid Todarodes pacificus by column chromatography. The peptide sequences
of Tod p 1 exhibited significantly high homology to the snail tropomyosin.
Ishikawa et al. [130] identified a major allergen, Oct v 1, in muscle of octopus
(Octopus vulgaris) by gel filtration and liquid chromatography. Its molecular
weight, amino acid composition, and partial amino acid sequence indicate
that this allergen is tropomyosin. Competitive ELISA inhibition experiments
suggested cross-reactivity between Oct v 1 and Tur c 1 (turban shell Turbo
cornutus allergen). A list of known mollusk allergens is shown in Table 2.

5.5
Cross-reactivity Among Different Seafood

Food allergens are able to sensitize and elicit IgE reactions after oral expo-
sure. Related food items are believed to trigger the same responses, leading to
cross-reactivity. For example, Ayuso et al. [131] demonstrated cross-reactivity
between meats. IgE reactivities to beef and lamb were most frequent, followed
by venison, pork, and chicken. The possible reason of cross-reactivity would
be the presence of shared or common epitopes on antigens and the confor-
mational similarity of epitopes to which the antibody would bind with similar
affinity.
   In fish, Hansen et al. [132] studied the reactions of clinically cod-sensitive
patients to other fish species by skin prick test, specific IgE tests, histamine re-
lease test, and immunoblotting. IgE-binding ability to cod, mackerel, herring,
220                                                                  K.H. Chu et al.

and plaice were analyzed using sera from eight cod-allergic patients. RAST
inhibition assay revealed cross-reactivity between cod and other fish species,
but not between cod and shrimp nor milk. Similar studies confirm that par-
valbumin exhibits cross-reactivity among different fish species [82, 98]. For
instance, the loss of most of the IgE reactivity to cod, tuna, and salmon total
fish extract in sera from fish-allergic patients preincubated with recombi-
nant carp parvalbumin (Cyp c 1.01) suggests common epitopes are present
in fish [82]. These findings are consistent with numerous reports on in vivo
cross-reactivity among fish species [133–139].
    The cross-reactivity among crustaceans has long been documented
[117, 122, 140, 141]. Daul et al. [142] demonstrated that monoclonal antibod-
ies against shrimp Pen a 1 exhibited similar reactivity patterns to crayfish,
crab, and lobster antigens. Leung et al. [106, 107, 118] demonstrated that
sera from shellfish-allergic patients lost their IgE reactivity to crude crus-
tacean extracts when they were preincubated in recombinant proteins Met
e 1, Pan s 1 or Hom a 1. These studies represent the first evidence of cross-
reactivity at the molecular level between different crustacean species that
accounts for previous results on the allergic cross-reactivity between crus-
taceans [117, 122, 140, 141]. It suggests that crustacean tropomyosins share
common allergic epitopes.
    Cross-reactivity among mollusks has been reported. Lopata et al. [119]
demonstrated cross-reactivity in a variety of mollusks including abalone
(Haliotis midae), snail (Helix aspersa), white mussel (Donax serra), black
mussel (Mytilus galloprovinicialis), oyster (Crassostrea gigas), and squid
(Loligo vulgaris) by binding with abalone-sensitive sera. Chu et al. [128]
demonstrated that sera of shellfish-allergic subjects lost their IgE reactivities
to mollusk extract after preincubation in recombinant tropomyosins of mol-
lusks or crustaceans. The results confirmed that tropomyosin is the major
cross-reactive mollusk allergen.
    Cross-reactivity between crustaceans and mollusks has been widely re-
ported [114, 115, 117, 122]. For example, Goetz & Whisman [17] reported a case
of a seafood handler who was sensitive to both shrimp and scallop. Cross-
reactivity between the crustaceans and mollusks at the molecular level was
demonstrated by Leung et al. [118]. Immunoblotting of sera from subjects
sensitive to shrimp showed positive results against muscle extracts from bi-
valves (mussel, fan shell, clam, oyster, and scallops), gastropods (abalone and
whelk) and cephalopods (cuttlefish, squid, and octopus). Thus tropomyosin
is the major cross-reactive allergen among crustaceans and mollusks. Leung
et al. [118] also showed that sera from subjects with crustacean allergy demon-
strated IgE reactivities against insects, suggesting that allergic cross-reactivity
occurs among all arthropods (see next section). Thus tropomyosin serves as
a good molecular model for investigating the relationship between protein
structure and allergenicity. It should also be noted that the IgE against shellfish
tropomyosins does not cross-react with vertebrate tropomyosins.
Seafood Allergy                                                              221

5.6
Tropomyosins as Allergens in other Invertebrate Groups

Leung et al. [118] showed that sera from subjects with crustacean allergy
demonstrated IgE reactivities against insects, suggesting that allergic cross-
reactivity occurs among arthropods. Crespo et al. [143] demonstrated cross-
reactivity between shrimp (Pandalus borealis) and German cockroach (Blat-
tella germanica). Immunoblotting using sera of patients with shrimp allergy
showed the strongest IgE binding to both shrimp and cockroach extracts, at
30 and 34 kDa, respectively. Inhibition studies by immunoblotting showed
that the IgE-binding capacity of German cockroach was totally abolished by
the shrimp extract, while the binding capacity of shrimp was partially af-
fected by German cockroach extract. Witteman et al. [141] also identified
tropomyosin as a cross-reactive allergen among the arthropod groups Crus-
tacea, Arachnida (such as mites), and Insecta.
    The allergic nature of tropomyosin has been well documented in mites. For
example, Asturias et al. [144] cloned and expressed the tropomyosin allergen
(Der p 10) from Dermatophagoides pteronyssinus. Tropomyosin allergen from
dust mite Lepidoglyphus destructor (Lep d 10) has also been identified [145].
The recombinant allergen Blo t 10 from the mite Blomia tropicalis shared
94–98% protein sequence homology with other mite allergens including Lep
d 10, Der p 10, and Der f 10 (from Dermatophagoides farinae) [146]. Skin
prick tests using patients sensitive to mite allergens showed exclusive reactiv-
ity to Blo t 10 and Der p 10, indicating the presence of shared epitopes in the
two allergens. Positive skin reactivity of seafood allergy patients to Blo t 10
suggested cross-reactivity between Blo t 10 and seafood. It should be noted
that other allergens have been reported in mites. For instance, group I aller-
gens in mites, such as Pso o 1 from sheep scab mite Psoroptes ovis [147] are
papain-superfamily of cysteine proteases.
    Tropomyosin has also been identified as an insect allergen. For instance,
Asturias et al. [148] and Santos et al. [149] cloned and expressed the Amer-
ican cockroach (Periplaneta americana) tropomyosin (Per a 7). Tropomyosin
expressed in both studies showed high sequence homology to tropomyosin of
other invertebrates. The DNA sequences of Per a 7 from both studies differ in
several nucleotides, suggesting that they are tropomyosin isoforms. Thus the
cockroach tropomyosin allergens identified by Asturias et al. [148] and Santos
et al. [149] should be designated as Per a 7.01 and Per a 7.02, respectively. The
tropomyosin of the silverfish (Lepisma saccharina) was also characterized to
be allergic [150]. The allergen from this primitive insect can be recognized by
sera from patients with household insect allergy.
    Anaphylactic reactions can be caused by Anisakis simplex, a parasitic ne-
matode of fish, after ingestion of parasitized fish [151, 152]. The nematode
was shown to induce specific IgE-mediated reactions [153] and is an im-
portant etiologic factor in acute urticaria after ingestion of fish [154]. Leung
222                                                             K.H. Chu et al.

& Chu [126] suggested that tropomyosin is the allergen in the nematode,
based on its high sequence homology with arthropod tropomyosins. Johans-
son et al. [155] reported cross-reactivity between this nematode and insects.

5.7
Epitopes of Tropomyosin Allergens

Tropomyosin is the cross-reactive allergen among crustaceans, mollusks,
mites, and insects, yet there is no cross-reactivity between tropomyosins
from these invertebrates and the vertebrates [118]. Likewise, extracts of dif-
ferent fish species did not significantly inhibit shrimp RAST [119], indicat-
ing the presence of unique IgE-binding epitopes in tropomyosins from the
various groups of invertebrates. Moreover, the cross-reactivity between var-
ious tropomyosin allergens suggests the presence of common IgE-binding
epitopes. To characterize IgE epitopes of Pen a 1, a peptide library was con-
structed to express 10–30 amino acid peptides of this allergen, which were
screened with sera from allergic patients [105, 156, 157]. Reese et al. [105]
identified four IgE-binding peptides, namely peptide E2 (residues 167–179),
E3 (136–148), E4 (262–282) and E6 (157–169). Peptide E6 partially over-
laps with an IgE-reactive peptide (residues 153–161) of Pen i 1 [98]. Reese
et al. [157] showed that the center and C terminus of Pen a 1 contain most of
the IgE-binding sites. A 21-mer peptide (residues 264–284) at the C terminus
that overlaps most of peptide E4 was suggested as a putative T-cell epitope,
which could reduce the IgE reactivity in a mouse model [158].
   In the most recent study, Ayuso et al. [156] analyzed eight IgE-binding
epitopes in five regions of Pen a 1 based on the reactivity of sera from
shrimp-allergic subjects with synthetic peptides having different amino acid
substitutions present in the lobster Homarus americanus, cockroach Peri-
planeta americana, and house dust mites Dermatophagoides pteronyssinus
and D. farinae. The cross-reactive epitopes are: residues 43–55 (epitope 1),
residues 87–101 (epitope 2), residues 137–141 (epitope 3a), residues 144–151
(epitope 3b), residues 187–197 (epitope 4), residues 249–259 (epitope 5a),
residues 266–273 (epitope 5b), and residues 273–281 (epitope 5c). IgE re-
activity of shrimp-allergic sera to peptides with homologous amino acid
sequence of the other invertebrates is the basis of cross-reactivity between
these species.
   By comparing amino acid sequences of these epitopes with corresponding
sequences in other tropomyosin allergens (Fig. 2), we could classify the eight
epitopes into three different types depending on the degree of identity be-
tween different taxa. First, epitope 5a is almost identical among all species.
This may represent the common epitope among crustaceans, insects, mites,
and mollusks. In the second type, including epitopes 2, 3a, 3b, and 4, the
amino acid residues are highly conserved in arthropods (crustaceans, insects,
and mites), but are variable when compared with mollusks. These may repre-
Seafood Allergy                                                                       223




Fig. 2 Amino acid sequence comparison of tropomyosins identified as allergens in vari-
ous taxonomic groups. Crustaceans: Met e 1 from Metapenaeus ensis (shrimp), Pen a 1
from Penaeus aztecus (shrimp), Hom a 1 from Homarus americanus (American lobster),
Pan s 1 from Panulirus stimpsoni (spiny lobster), and Cha f 1 from Charybdis feriatus
(crab). Insects: Per a 7.01 from Periplaneta americana (American cockroach), Per a 7.02
from P. americana, Bla g 1 from Blattella germanica (German cockroach), and Lep s from
silverfish (Lepisma saccharina). Mites: Der f 10 from Dermatophagoides farinae (house
dust mite), Der p 10 from D. pteronyssinus (house dust mite), Lep d 10 from Lepidogly-
phus destructor (dust mite), and Blo t 10 from Blomia tropicalis. Mollusks: Hal d 1 from
Haliotis diversicolor (abalone), Hel as 1 from Helix aspersa (brown garden snail), Per v 1
from Perna viridis (green mussel), Chl n 1 from Chlamys nobilis (scallop), and Cra g 1.03
from Crassostrea gigas (Pacific oyster). IgE binding epitopes of Pen a 1 [156] are shaded
224                                                             K.H. Chu et al.




Fig. 2 (continued)


sent epitopes that are common to arthropods, but distinct from mollusks. In
the third type, including epitopes 1, 5b, and 5c, the amino acid residues are
similar within each of the above four taxa but vary considerably between the
taxa. These may represent epitopes that are specific only to crustaceans.
   The presence of these different types of epitopes may account for the find-
ing by Leung and Chu [128] that pre-absorption of sera from oyster allergy
subjects with recombinant crustacean allergen was able to remove most but
Seafood Allergy                                                             225




Fig. 2 (continued)


not all of the IgE reactivity to either recombinant oyster allergen or extract.
Generally speaking, there appears to be cross-reactive and distinct IgE epi-
topes in the tropomyosin allergens from different taxonomic groups.
   Epitopes in mollusk tropomyosins have been reported. Ishikawa et al. [124,
125] proposed a 14-mer peptide (IQLLEEDMERSEER, residues 92–105) as
the IgE-binding epitope in the oyster allergen Cra g 1.01 and 1.02. Yet the epi-
tope in Tur c 1 (from the turban shell Turbo cornutus) is different and resides
at the C-terminus (residues 245–284) of the tropomyosin molecule [125]. In
the allergen Oct v 1 from the octopus Octopus vulgaris, two IgE-binding epi-
topes were identified at the central region (residues 77–112 and 148–160)
and one at C-terminal region (residues 269–281) [124]. Residues 269–281 is
nested within the epitope of Tur c 1 and its sequence is highly conserved
among mollusks. This may represent the common epitope among mollusks.
Peptide 77–112 of Oct v 1, which is the most reactive site for IgE bind-
ing, include the sequence of the epitope (residues 92–105) in Cra g 1. While
residues 92–105 are rather conserved among different mollusks, the other re-
gions in the residues 77–112 are highly variable. Interestingly, the amino acid
residues 92–105 in the oyster epitope are almost identical to the correspond-
ing residues in most arthropod tropomyosins, although the residues differ
significantly between arthropods and some mollusks. It should also be noted
that this oyster epitope overlaps with epitope 2 in Pen a 1. Clearly more in-
tensive studies are necessary to clarify the common and distinct epitopes in
tropomyosin allergens.
226                                                               K.H. Chu et al.

6
Applications and Future Directions

The molecular identification of seafood allergens, mapping of epitopes, expres-
sion of recombinant allergens, and the elucidation of the immunological mech-
anisms of allergies set the cornerstones and thus provide significant knowledge
in the potential applications in the diagnosis and design of therapeutic modal-
ities in seafood allergies. In addition, the recent development of gene transfer
technology in lower invertebrates, including shrimp, may allow the future de-
velopment of non-allergic transgenic shrimps for human comsumption.

6.1
Diagnosis and Profiling

The enormous immune repertoire and specific recognition of antigens by
antibodies are among the most fascinating phenomena of the human body.
Interestingly, the specificity of antibody/antigen reactions has formed the
molecular principle of immunodiagnosis. Early immunoassays capable of
multiplex analysis include ELISA, fluorescent-based immunoassays, and ra-
dioimmunoassays performed in microtiter plates and arrays of peptides on
pins, western blot analysis, and bacterial colonies/phage plaque-based as-
says [156, 160]. However, these methods are limited by requirement for rela-
tively large quantities of clinical samples. In the late 1980s, several groups
proposed the use of miniaturized as well as addressable immunoassays and
photolithography-generated arrays [161, 162]. This idea was further advanced
by robotic printing devices in the generation of DNA microarrays [163] and
was subsequently applied in the fabrication of protein arrays on microscope
glass slides [164, 165]. Recent advances in microarrays have focused on the
capability of high throughput detection of antigens using submicroliter quan-
tities of biological fluids [166].
    In vitro diagnosis of allergy is based on the detection of allergen-specific
IgE antibodies in the sera of atopic patients. Current available forms of diag-
nosis rely on allergen extracts prepared from allergen-containing biological
materials, which are a variety of allergenic and non-allergenic components
and are difficult to standardize [167–170]. With the molecular identifica-
tion of allergens and availability of recombinant proteins, protein micror-
rays have been developed to profile allergen-specific antibodies from human
sera [171–173]. Allergen protein array thus allows high throughput determin-
ation and monitoring of IgE reactivity profiles to a large number of allergens
by using minute amounts of serum with sensitivity between 0.16–2 µg/L of
allergens [172]. Since small bioactive molecules such as synthetic peptides
conjugated to macromolecules can be applied in microarray technologies,
with the growing knowledge of IgE epitopes of allergens, peptide epitopes
may also be used in allergen microarrays.
Seafood Allergy                                                            227

6.2
Immunotherapy

One of the most exciting recent developments in the treatment of food allergy
is novel immunotherapeutic strategies designed to alter the immune system’s
response to food allergens. These strategies are now being examined in an-
imal models as potential treatment modalities. They include plasmid DNA
immunotherapy, immunostimulatory sequence-modulated immunotherapy,
allergen-peptide immunotherapy, and IgE binding site modified protein im-
munotherapy. All of these methods strive to elicit a Th1-type response or
tolerance from the immune system in response to a specific food allergen.
   In the search for a novel immunotherapy with a low risk-to-benefit ratio,
immunostimulatory CpG motif DNA sequences have recently been shown
to provide an excellent tool for designing safer and more efficient forms
of allergen immunotherapy. These DNA-based immunotherapeutics include
allergen gene vaccines, immunization with allergen-DNA conjugates, and im-
munomodulation with immunostimulatory oligodeoxynucleotides. All three
DNA-based immunotherapeutics have been shown to be very effective in an-
imal models of allergic diseases and, at present, allergen-DNA conjugates
are being tested for their safety and efficacy in allergic patients [174]. Plas-
mid DNA immunization entails the introduction of a plasmid DNA encod-
ing a specific allergenic protein. The plasmid DNA is taken up by antigen-
presenting cells and the expressed allergen is presumably presented on the
surface of APC in the context of MHC. The presentation of endogenous
proteins is believed to induce Th1 response with the upregulation of IFN-
γ and suppression of IgE production. A recent study showed that genetic
immunization with bovine β-lactoglobulin cDNA induces a preventive and
persistent inhibition of specific anti-BLG IgE response in mice, but the win-
dow of response appears to be highly restricted [175]. Oral immunization
with chitosan DNA particles has been shown to be effective in reducing
allergen-induced anaphylaxis in an animal model of peanut allergy [176]. The
effect of immunostimulatory sequence-modulated immunotherapy in food
allergies has also been under active investigation [174, 177]. In general, pri-
mary gene and protein/immunostimulatory sequence oligodeoxynucleotide
vaccination of Th2-sensitized mice significantly reduced the risk of death
after anaphylactic challenge. In addition, gene and protein/ISS-ODN vaccina-
tion reduced post-challenge plasma histamine levels. Analysis of the immune
profiles of mice receiving DNA-based vaccines showed that both gene and
protein/ISS-ODN vaccination effectively prevented the development of Th2
biased immune profiles after sensitization. In contrast, vaccination with pro-
tein alone, the experimental equivalent of the traditional protein-based im-
munotherapy reagents used in clinical practice, provided no protection from
anaphylaxis, and did not prevent the development of a Th2 biased immune
profile after allergen sensitization [178–180]. These studies support the po-
228                                                               K.H. Chu et al.

tential application of DNA-based vaccination for the desensitization of food
allergic individuals .
   Specific immunotherapy (SIT), whereby an individual is repeatedly in-
jected with increasing doses of allergen extract is perhaps the only effective
therapeutic intervention of allergy. Over time, the individual becomes less
reactive to the allergen, perhaps due to CD4+ T cell tolerance. However,
traditional injection immunotherapy for food allergy is not recommended
due to high rate of adverse systemic reactions, including anaphylaxis and
death [181]. On the other hand, peptide immunotherapy reduces the allergen
to its component CD4+ T cell epitopes. This method relies on HLA-binding
peptides and thus it is important to verify the population-based immun-
odominant peptides for each ethnic group under consideration. Peptides are
administered in order to induce antigen specific CD4+ T cell tolerance. Be-
cause the peptides cannot crosslink IgE, they are considered a safer therapy
for SIT than intact allergens. This method is effective in animal models,
and is thus potentially promising in human immunotherapy [182, 183]. Re-
combinant allergens can be genetically modified to reduce IgE binding, but
keeping the T cell epitopes intact. This approach has been shown to be effect-
ive in inducing tolerance and offers a safe immunotherapeutic treatment of
allergy [184–187].
   It should be noted that the use of IgE binding site modified proteins or
peptide epitopes to induce allergen non-responsiveness requires the iden-
tification of individual allergens to be included in a therapy regimen on
a per patient basis. Moreover, knowledge on T cell epitopes in the allergen
of interest is necessary so that the T cell epitopes can be retained in the con-
struction of IgE binding modified proteins or the design of peptides. Since
most individuals with allergies are responsive to many allergens, a cocktail
approach may be sought in either peptide or modified IgE binding protein
SIT. Although allergen-peptide immunotherapy and IgE binding site modi-
fied protein immunotherapy for food allergy have not reached the stage of
clinical trials, their potential for food allegy therapy is evident.
   There is currently no treatment available for seafood allergy. Therefore,
patients must be taught to recognize and avoid seafood allergens. Those pa-
tients at risk of anaphylaxis must carry injectable epinephrine. The use of
a humanized anti-IgE (Omalizumab) might reduce the risk of serious re-
actions. This product inhibits the binding of IgE to its receptors, and has
been approved for the treatment of asthma. Data from peanut-allergic sub-
jects indicates that it can significantly increase the amount of peanut aller-
gen tolerated, thus protecting the patients from inadvertent exposure [188].
However, there is no data on seafood-allergic subjects and long-term effi-
cacy is unknown. Another approach is the use of allergen immunotherapy
to induce immunologic tolerance. This approach is being used successfully
in the treatment of allergic rhinitis and asthma, but the risk is unaccept-
ably high in subjects with food allergy [181]. Nevertheless, there have been
Seafood Allergy                                                                   229

anecdotal reports of successful oral desensitization in food allergic sub-
jects [189].

6.3
Non-allergenic Seafood

With the advent of molecular biological technology together with the in-
creasing knowledge on the immunological mechanisms and epitopes of the
seafood allergens, the generation of non-allergenic seafood may be at hand.
Gene transfer technique is a powerful approach for changing the heritable
traits of domesticated animals. This technique has been successfully applied
in many fish species, with the production of stable lines suitable for com-
mercial production [190]. Using DNA transformation technology, allergic
structures in seafood can be modified by changing the amino acid sequences
in epitopes to avoid allergy. Technological advances in genetic transformation
in marine invertebrates including shrimp were unsatisfactory until recent
years. In the case of transgenic shrimp, Preston et al. [191] first demonstrated
microinjection as a reliable technique for delivering DNA to early embryos.
Kau et al. [192] have recently developed an in vivo gene transfer technique
for producing transgenic shrimp, in which gene constructs were injected into
spermatophores and electroporation was used to assist the entry of constructs
into oocytes. These studies provide significant groundwork for the future
development of genetic transformation in marine organisms, including non-
allergic transgenic seafood.

Acknowledgements We thank M.C. Fung and C.K. Wong for their comments on the
manuscript. Our work on seafood allergy was supported by a grant from the Research
Grants Council of the Hong Kong Special Administrative Region, China (Project No.
CUHK4256/02M) and a grant from the International Life Science Institute.



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