Biology - Chemistry Frontier. Part 1. Roger Adams Award Lecture
Samuel Danishefsky, Memorial Sloan-Kettering Cancer Center
Biology - Chemistry Frontier. Part 1. Roger Adams Award Lecture
Samuel Danishefsky, Memorial Sloan-Kettering Cancer Center
Colleagues
Multivalent Antitumor Vaccine Jennifer Allen Stacy Keding Qian Wan Erythropoietin / Peptide Ligation Gong Chen Jiehao Chen Zihao Hua Cindy Kan Zhongping Tan John Trzupek Qian Wan David Warren Bin Wu Yu Yuan Grandisine A David Maloney Migrastatin Christoph Gaul Jon Njardarson Lucy Perez Isomigrastatin Isaac Krauss Mihir Mandal Epothilones Alexey Rivkin Young Shin Cho Fumihiko Yoshimura Cycloproparadicicol Xudong Geng Kana Yamamoto Zhi-Quang Yang
Colleagues
Multivalent Antitumor Vaccine Jennifer Allen Stacy Keding Qian Wan Erythropoietin / Peptide Ligation Gong Chen Jiehao Chen Zihao Hua Cindy Kan Zhongping Tan John Trzupek Qian Wan David Warren Bin Wu Yu Yuan Grandisine A David Maloney Migrastatin Christoph Gaul Jon Njardarson Lucy Perez Isomigrastatin Isaac Krauss Mihir Mandal Epothilones Alexey Rivkin Young Shin Cho Fumihiko Yoshimura Cycloproparadicicol Xudong Geng Kana Yamamoto Zhi-Quang Yang
Acknowledgement
I dedicate this Roger Adams Award Lecture to Sarah Danishefsky for a lifetime of creative support, encouragement and leadership.
Acknowledgement
I dedicate this Roger Adams Award Lecture to Sarah Danishefsky for a lifetime of creative support, encouragement and leadership.
Biology Intellectualization at the level of function Mechanisms: Identification of all components required to attain and maintain biological function → reconstitution! The power of bioreplicative synthesis
Chemistry Intellectualization at the level of structure Mechanisms: Pathways of chemical transformations usually involving breaking and making of covalent bonds → prediction of new reactions! The power of unencumbered synthesis
Biology - Chemistry Frontier
Biology Intellectualization at the level of function Mechanisms: Identification of all components required to attain and maintain biological function → reconstitution! The power of bioreplicative synthesis
Chemistry Intellectualization at the level of structure Mechanisms: Pathways of chemical transformations usually involving breaking and making of covalent bonds → prediction of new reactions! The power of unencumbered synthesis
Biology - Chemistry Frontier
On the Value of Natural Products in Pharma Discovery The de novo discovery of a new molecular agent of value in medicine is a daunting task. The risks are virtually incalculable. The study of small molecule natural products (SMNPs) may allow for entry into the discovery progression at a more advanced stage than the screening of medicinal chemistry sample collections, let alone standard diversity libraries. If properly exploited, this advantage may well compensate for the difficulties of exploration, collection maintenance, isolation, proof of structure and more complicated follow up synthesis studies which are often cited against the natural products based discovery route.
Highly Successful Small Molecule Natural Product Derived Blockbuster Drugs (Partial List) (a) Antibiotics: β-lactam, aminoglycosides, macrolides (b) Statins: Zocor, Pravastatin, Lipitor (c) Steroids: Population control, antiinflammatories, cardiotonics, antiestrogens, antiandrogens, dermatological applications, etc. (d) Alkaloids: Antidepressants, antitumor agents (Vinca, thecins) (e) Terpenoids: Taxol (f) Polyketies: Anthracyclines
Natural Products as Lead Candidates:
The Statin Class of Antilipidemic Drugs
HO HO O O H O O O O H HO O F N O
CO2H OH
O NHPh Lovastatin (Mevacor®) Natural Product (Isolated from A. terreus) Simvastatin (Zocor®) Natural Product-Derived Atorvastatin (Lipitor®) Natural Product-Inspired
Why Have Natural Products Been a Rich Source of Drug Discovery? (a) Wisdom of the ages: While the developmental justifications for the biosynthesis of secondary metabolites are often mysterious, in many instances there could well be some overall rationale which can be exploited. (b) The small molecule natural product has possibly been designed for its ability to bind to biomacromolecules. (c) Presumably the biosynthesis of the natural product was under enzymatic control. At its “launching,” the natural product had been contained and extruded from some protein pocket. (d) There is often a built in advantage in that the natural product has already been maintained in some living host, without unmanageable toxicity.
Why Chemical Synthesis of Complex Molecules? 1. The sheer challenge! 2. A setting for the evaluation of the scope of current methodology. 3. A setting for the evaluation of “problem areas”. 4. Incitement to strategy level and new methodology centered creativity. 5. A medium for the discovery and development of new drug possibilities through diverted total synthesis. 6. To provide material for pre-clinical and clinical investigation. 7. Molecular editing and diverted total synthesis.
Diverted Total Synthesis
Allow for the exploration of chemical space not available directly from the natural product. Through the process of Diverted Total Synthesis, it is possible to manipulate chemical functionality that can not be altered through manipulation of the natural product itself.
DTS Starting Materials
Analogs
add complexity Chemical Synthesis Total Synthesis Natural Product reduce complexity Biosynthesis
Advanced Intermediate DTS
Analogs
DTS = DIVERTED TOTAL SYNTHESIS
Summary of the Epothilone Program: Molecular Editing through Total Synthesis
S N O O Me O OH O OH S N H O O OH S N H
Remove Epoxide Decrease toxicity
Me
Install Unsaturation Improve Potency and Biological Stability
Me
O
O OH
O OH
O OH
Epothilone B (EpoB)
dEpoB (KOS-862) Phase II
9,10-dehydro-dEpoB (KOS-1584) Phase I
S
O
N
Install Trifluoro Group Decrease Toxicity and Broaden Therapeutic Index
F3C
H
N O O OH
Modify Heterocyclic Sector Increase Efficacy and Stability
F3C
H
O
O OH
O OH
O OH
Fludelone (KOS-1591) Preclinical
Iso-Fludelone (KOS-1803) Preclinical
Iso-Fludelone: Therapy of Extra Large Tumors
Day 25 Day 39
Therapeutic effect of Iso-fludelone against extra-large MX-1 tumors (30 mg/kg, Q12Dx4, 6hr-infusion, N=4)
Day 53
The Radicicol Program
Radicicol and Cycloproparadicicol
Mode of Action: Potent inhibition of Hsp90. The first total synthesis of radicicol was completed in our laboratory in 2001. As reported, radicicol was confirmed to be a potent inhibitor of the molecular chaperone, Hsp90. Radicicol was largely inactive in mouse xenograft studies. We suspected that the epoxide moiety was responsible for the failure to translate protein inhibition to in vivo efficacy. Cycloproparadicicol was synthesized in our laboratory and shown to be an effective inhibitor of Hsp90. Importantly, this fully synthetic analog is active in in vivo settings.
O H O OH Cycloproparadicicol H O HO Cl O H O H
O HO
O
Cl OH
Radicicol Isolated from M. nordinii
Total Synthesis of Cycloproparadicicol
H O H Me OTBS + HO Me H 1) n-BuLi; CO2 2) PPh3, DIAD 47% (2 steps) OTBS O Me H Me Co2(CO)8 100%
TMSO H O O Me (CO)3Co (CO)3Co H OTBS Me 1) Grubbs' 2nd gen. cat. 57% 2) I2, 69% Ring-Closing Metathesis
H O HO O Me H OTBS OH
OH Cycloproparadicicol HO O O Me Cl H O
Me Me
H O O Me H OTMS 140єC, then silica gel 75% Diels-Alder Cycloaddition
H
OTBS
Me Me
The Radicicol Program
Radicicol and Cycloproparadicicol
Total Synthesis of the Tumor Cell Migration Inhibitor Migrastatin
O O NH O O
OMe O OTMS OMe
H2O O
O
O
Migrastatin
OH OMe
OMe
O O O HO O NH O
OTBS OMe OMe OH
Diverted Total Synthesis: Migrastatin
Wound Healing Assay
No Serum Serum
Serum + 200 nM Core
O O
Serum + 200 nM Migrastatin
O O O O NH O
OH OMe Migrastatin Core
OMe Migrastatin OH
Diverted Total Synthesis: Migrastatin
Inhibition of Metastasis in Mouse Breast Tumor (4T1) Model
O NH O
OH OMe Lactam OMe Ketone
OH
Tumor size on day 21
16 14 Diameter of tumor (mm) 12 Number of colonies 10 8 6 4 2 0
Ke to ne (1 0m g/ kg ) Ke to ne (2 0m g/ kg ) La ct am (1 0m g/ kg ) La ct am (2 0m g/ kg ) co nt ro l
Clonogenecity Assay
900 800 700 600 500 400 300 200 100 0 -100
Ke to ne (1 0m g/ kg ) Ke to ne (2 0m g/ kg ) La ct am (1 0m g/ kg ) La ct am (2 0m g/ kg ) co nt ro l
Anti-Metastatic Activity of Migrastatin Analogs (MDA-MB-231 cells)
O
3
O
O
Me
O
Me
O
Me
Serum
OH
OMe
HN
O
Me
OH
OMe
Me
Migrastatin
Pre-surgery:
2,3-dihydro-migrastatin ether (ME) Serum + 10µM ME ME (pre)
control group
No Serum
Post-surgery: control group
ME (post)
ME (pre+post)
Collaboration with Moore Lab at MSKCC
Migrastatin - Isomigrastatin Family
Isolated from S. Platensis
Me O O Me OH O OMe
Isomigrastatin
3
O NH O
O HN O
3
O O
Me O Me
OH OMe
Migrastatin
Isolation Isomigrastatin: Woo, E. J.; Starks, C. M.; Carney, J. R.; Arslanian, R.; Cadapan, L.;Zavala, S.; Licari, P. J. Antibiot. 2002, 55, 1411. Migrastatin: (a) Nakae, K.; Yoshimoto, Y.; Sawa, T.; Homma, Y.; Hamada, M.;Takeuchi, T.; Imoto, M. J. Antibiot. 2000, 53, 1130. (b) Nakae, K.;Yoshimoto, Y.; Ueda, M.; Sawa, T.; Takahashi, Y.; Naganawa, H.;Takeuchi, T.; Imoto, M. J. Antibiot. 2000, 53, 1228.
Instability of Isomigrastatin
OMe Me R O O O HO Me OMe HO Me O OH HO O OMe
H2O
SN2’
Me R
Dorrigocin A epi-Dorrigocin A
H2O H2O
Isomigrastatin
additionelimination
Me R O
HO Me OH HO
Dorrigocin B
O
R=
O
O NH
OMe
water-assisted 3,3rearrangement
HO Me O R O
Ju, J.; Shen, B. et. al. JACS 2005, 1622
Migrastatin
O Me
Unsuccessful Early Strategies to Isomigrastatin
O
O Me
R OP OMe
O
O Me
R OP OMe
(R)
(R)
trans-trans-dienolide
R O O Me R OP OMe O Me O OP OMe
2,3-cis-6,7-trans-dienolide
Total Synthesis of Isomigrastatin – Fragment Union
OMe O + OTMS OMe Me OMe Me O O O NH O Ph O NB O Me Me H Ph 1) Ph3P 2) H 2, Pd/C PPh3 Ph3P B O O O NH =R O O OMe Me OMe Me A O OH MOMO O O
BH3 O A + B Me O OMOM OMe R
R
LiMeCuCN
Me HO Me R OTBS OMOM OMe
OTBS OMOM OMe
Krauss, I. J., Mandal, M. , Danishefsky, S. J. Angew. Chem. Int. Ed. Engl. 2007, ASAP
Delayed Implementation of Dienolide Core – Completion of Synthesis
(±) O OH PhSe
3
Me HO Me TBSO OMOM OMe
Me O Me R OTBS OMOM OMe O PhSe O Me
Me R OH O OMe
O =R NH O
xs.
O PhSe
EDCI/DMAP
kinetic r esolution
>8:1 d.r. at C2
Me O PhSe Grubbs II O Me OH O OMe R mCPBA either C2 epimer O O Me
Me R OH O OMe cat. PMe3
Me O O Me OH OMe
R O
21 % trans 36 % cis (separable)
(+)-Isomigrastatin
Krauss, I. J., Mandal, M. , Danishefsky, S. J. Angew. Chem. Int. Ed. Engl. 2007, ASAP
Total Synthesis of Grandisine A (background)
H
• displays affinity for the human δ-opioid receptor (2.7 µM). • µ and κ agonist have been associated with adverse side effects when administered. • in animal models, δ-opioid agonists have been well tolerated.
O O H O
H H H
8
9
N
γ-pyridone
Grandisine A
Grandisine A 8-epi 9-epi
rel. energy 0 kcal/mol ~-6 kcal/mol ~-4 kcal/mol
J. Org. Chem., 2005, 70, 1889
Total Synthesis of the Grandisine A
Nuc.. E+
OH NCbz H NCbz
O H NCbz H3C
OTIPS H NCbz
+
O
O
O
OTIPS
BF3OEt2 -78 oC endo H
ax.
OTIPS NCbz
+
O NCbz
TBAF AcOH/THF
H H
ax.
NCbz
eq.
H
H H O H
O
H OTES
H
O
H
~5-10% from opposite face
H O H H H O H NCbz O H O H O H H N
O H
OSi(Et)3
HO
LHMDS
(+)-Grandisine A
unpublished results
Carbohydrate-Based Antitumor Vaccines: Rationale
• •
Cell surface (tumor) antigens are carbohydrate structures commonly expressed in glycoproteins, mucins, and glycosphingolipids. Antibodies that recognize these glycoconjugates can, on occasion, be found in human sera. It has been postulated that these antibodies are part of an immune response to the tumor state. Antibody formation is provoked by suitable glycoconjugates and not by the oligosaccharides alone. Having been elicited in this way, most antibodies are primarily sensitive to the structure of the carbohydrate domain. Vaccination with carbohydrate vaccines may provide immunological protection against micrometastases and circulating tumor cells. The possibility of an immune response to cancer could have an enormous impact on the in vivo diagnosis and therapy of cancer.
•
• •
Oligosaccharide Synthesis: How We Got Started
OMe R1 R3SiO R2 O R3
OMe
Lewis Acid
R1 R3SiO R2
O R3
R1 HO R2
O R3
HO O HO HO OH OH
HO O HO HO
Classic Approach
Glycal Assembly Approach
Oligosaccharide Synthesis: The Glycal Assembly Method
O O RO RO O O OH RO RO O O RO RO O O OH RO RO O O OH RO RO RO Oligosaccharide RO glycal RO RO O RO RO O RO P RO O P O RO O P HO O RO O O O O O OH RO O RO X O OH O O O O O OH XR
O O
RO
RO E glycosyl donor RO O OR'
E
E+
E+
RO in situ generated glycosyl donor O
+ O I
O I+ O PhSO2NH2 I O NHSO2Ph
SEt
I+ O
NHSO2Ph O HO
HO I O O
I O
O O
I
O O O
O O NSO2Ph O O
NHSO2Ph
Carbohydrate Cancer Vaccine Program
Angew. Chem. Int. Ed. 1996, 35, 1380
Breast Cancer-Associated Antigen: MBr1 Antigen
O
O O
OTIPS AcO O O O
OTIPS O SEt
OBn OBn HO O BnO O OBn O O BnO BnO OBn OBn O O O
NHSO2Ph
O OBn OBn
OBn
HO HO
OH O O O
HO O
OH O AcNH
HO O
OH O HO O HO OH O OH O HO OH O OH O
O C25H51 HN C13H27 OH
OH
OH HO
Globo-H
Clinical Trials with Carbohydrate-Based Antitumor Vaccines
HO HO OH O O O OH HO OH HO O OH O HO OH O HO O HO OH O OH O OH
HO HO
O NHAc
OH O OH
HO O
OH O AcHN O AcHN OH
HO HO
OH O OH H N
HO O O
OH O AcHN O N H Linker O KLH
O OH HO
O Linker
KLH
Globo-H SCLC (Phase I) Prostate (Phase I) Breast (Phase I)
OH O HO HO OH O O O OH HO
HO HO Me OH OH O O O OH OH OH O O AcHN O O OH HO HO NHAc HO
O O AcHN O HO O OH
HO HO
O OH
TF(c) Prostate (Phase I)
OH OH O HO OH O OH
OH O O
O NHAc
O Linker
KLH
OH
LewisY Ovarian (Phase I)
HO HO OH O AcHN O HO HO H N O AcHN O O OH O AcHN O N H Linker O KLH
O
OH O
OH HO2C
O OH HO
OH O OH
O Linker
KLH
AcHN
Fucosyl-GM1 SCLC (Phase I)
HO HO
O OH
Tn(c) Prostate (Phase I)
Flexible Accesses to Glycosyl-amino Acids
pentenyl glycosides
Protected carbohydrate
allyl and pentenyl glycosides
X
Protected carbohydrate
glycosyl donnors
Protected carbohydrate
O X = CH2 X=O CO2R
O n
X
X = Glycosyl Donor CO2Bn NHFmoc HO CO2Bn NHFmoc
O MeO P MeO
NHBoc
Horner -Emmons
Protected carbohydrate
Cross Metathesis
CO2R NHBoc
Protected carbohydrate
Glycosylation (L.A.)
CO2Bn NHFmoc
Protected carbohydrate
O
O
n
O
CO2Bn NHFmoc
H2 (S, S)-Et-DuPHOS-Rh
H2/Pt-C
H2/Pt-C
Protected carbohydrate
O
n
CO2Bn
NHFmoc n = 1 or 3
Unimolecular Multivalent Vaccine for Prostate Cancer: A Construct Containing Five Different Antigens
OH OH O HO O Me HO O OH OH OH OH OH O O O O NAc HO O H OH HO Globo-H HO HO OH AcN H OH OH O OH HO O HO HO O OH O N H O N H O CO2H STn O HO O O AcHNO HO HO OH O AcHN O Tn
HAcN HO HO O OH AcHN O O O HO2C OH HO O HO HO HO OH NHAc GM2 OH O O OH O OH HO HO O
H N
H N O
H N O
Linker
KLH
OH O O OH
AcHN
O TF
HO
O OH
A fully synthetic, unimolecular pentavalent vaccine incorporating five of the known antigens associated with breast cancer has been synthesized and evaluated in preclinical settings. This compound is scheduled to enter Phase I clinical trials at MSKCC in fall / winter 2007.
Erythropoietin (EPO): A Multiply Glycosylated Protein
126 38 161
Cysteine pair
Cysteine pair
7 24 Sialic acid Galactos e annose M
33
83 29
Fucose N-Acetylglucosamine
166-Residue glycoprotein
N-Glycosylated at Asn24, Asn38, and Asn83 O-Glycosylated at Ser126 Two cysteine pairs: Cys7-161 and Cys29-33 Heterogeneous glycoprotein Treatment of anemia Various glycoforms have different biological properties
Hematopoietic growth factor
Erythropoietin (EPO): A Multiply Glycosylated Protein
EPO is a heterogeneous glycoprotein that may exist as a number of glycoforms. The amino acid sequence is highly conserved among isoforms. Current manufacturing processes yield inseparable mixtures of glycoforms, which can lead to complications at the regulatory level. Could we use total synthesis to gain access to single glycoforms of erythropoietin? We elected to synthesize the EPO isoform containing the consensus amino acid sequence and displaying highly branched and sialidated carbohydrate sectors. Incorporation of these types of carbohydrates is known to convey biostability. We will evaluate whether our fully synthetic erythropoietin is able to fold properly and whether it possesses EPO-like biological activity. The successful completion of this undertaking would require the development of some new synthetic methodologies to allow for the preparation and merging of very large and complex glycopeptide fragments.
Toward Erythropoietin (EPO): The Development of Novel Methods for Glycopeptide Ligation
Glycan O Peptide1 X H2N R Peptide2 O Glycan
The Challenge Glycopeptide Ligation
Glycan Peptide1 O R N H O
Glycan Peptide2
I. A Cysteine-Based Glycopeptide Ligation
Glycan O Peptide1 SEt S O StBu S H2N Peptide2 O Glycan Glycan O Peptide1 O Glycan O Peptide1 S HS H2N O Peptide2 Glycan HO H2N O HS Peptide1 O S Peptide2 Glycan Peptide1 Glycan Glycan O HS N H O Glycan Peptide2
Reduce S-S
Toward Erythropoietin (EPO): The Development of Novel Methods for Glycopeptide Ligation
II. A Cysteine-Free Ligation Model
Glycan
O
Peptide1
P O S O
H2N
Glycan R Peptide2 O
Glycan Glycan
O
Peptide1
P O S HN
R Peptide2 O
Removing P
Glycan Glycan
O
Peptide1 R
Glycan
Glycan
S
Peptide2 O
O
Peptide1
R
O O
HO
S HN
SH HN
O
Peptide2
S
Glycan
N
Glycan Glycan Glycan
O
Peptide1
R
O
Peptide2 O Peptide1
R
N SH
N H
Peptide2 O
HO
Dr. Gong Chen
Toward Erythropoietin (EPO): The Development of Novel Methods for Glycopeptide Ligation
III. A Cysteine-Free Glycopeptide Ligation
SEt Glycan O Glycan SP O H2N MeO OMe OMe MeO OMe OMe Glycan O Peptide1 SH N O R Peptide2 Glycan R Peptide2 O Peptide1 S O Glycan R
Philip Dawson
R'S
S
HN O
Peptide2
Glycan O Peptide1 S HN O R
Glycan Peptide2
MeO
OMe OMe Glycan O Peptide1 N H R Peptide2 O
MeO
OMe OMe
Glycan
Toward Erythropoietin (EPO): The Development of Novel Methods for Glycopeptide Ligation
IV. A Cysteine-Free Glycopeptide Condensation
SEt S O H2N O Glycan R Peptide2
Glycan O Peptide1
Saburo Aimoto AgCl
HOOBt, DIEA DMSO, RT
Gly / Pro
Glycan O Peptide1 N H R
Glycan Peptide2 O
Lys and Cys Protected
SEt S O H2N O Glycan R Peptide2 O S O O
Glycan O Peptide1
Gly / Pro
TCEP
HOOBt, DIEA DMSO, RT
Glycan O Peptide1 N H R
Glycan O Peptide2 O S O O
Lys and Cys Protected
Toward Erythropoietin (EPO): The Development of Novel Methods for Glycopeptide Ligation
V. Free Radical Desulfurization: Cys to Ala
Glycan O Peptide1 N H Peptide2 O SH Glycan
TCEP, tBuSH, VA-50 H2O, RT
Glycan O Peptide1 N H
Glycan Peptide2 O
S-H
Peptide
In
.
. S
Peptide
R P R R
S
Peptide
.
R P R R
.
CH2
Peptide
H-S CH3
Peptide
R S P R R P(CH2CH2COOH)3
TCEP
O FmocN S Me S Acm S S
O O
HN N N H2N
NH HCl NH2
tolerated
VA-50
Progress Toward Erythropoietin (EPO): Synthesis of Glycan
BnO
OBn O
OBn F
BnO
OBn O OBn O O O BnO PhSO2NH OTBS
BnO O AcO BnO PhSO2NH
SEt
TIPSO HO BnO
O
BnO O AcO BnO PhSO2NH
HO O BnO
O
BnO O HO BnO PhSO2NH
Ph
O O PMBO
OBn O S
BnO BnO BnO Ph BnO OBn O OBn BnO O O O BnO PhSO2NH OBn O O O BnO PhSO2NH OTBS
OBz O
O
2x
SEt
OH BnO BnO BnO O O BnO BnO BnO BnO O BnO OBn O OBn BnO O O O BnO PhSO2NH OBn O O O BnO PhSO2NH OTBS
HO BnO HO
O OH
Progress Toward Erythropoietin (EPO): Synthesis of Glycan
O
O O
OBn O + HO BnO
OBn O
O
O O
OBn O BnO O BnO
OBn O HO HO OBn O BnO OBn O SEt O BnO OBn O SEt
O O
TMSESO2NH2 EtSH
AcO OAc O AcO PivO O
OBn + HO BnO O SEt
CCl3 NH
NPht
AcO OAc O O AcO BnO PivO
NPht
NPht
AcO OBn O BnO O SEt
OAc OAc O
OP(OBn)2 CO2Me
AcO
HO HO
OBn O BnO
AcHN AcO
CO2Me AcO OBn O O O AcHN O BnO AcO OBn OAc OAc
OBn O SEt
NPht
NPht
Progress Toward Erythropoietin (EPO): Synthesis of Glycan
OH CO2Me AcO OAc OBn AcO OBn OAc O O O O AcHN O BnO AcO OBn PhtN BnO BnO BnO O O SEt BnO OBn O OBn BnO O O O BnO PhSO2NH OBn O O O BnO PhSO2NH OTBS
+
BnO BnO BnO
BnO
O
2x
O OH
AcO
CO2Me OBn AcO OBn O O O O AcHN O BnO AcO OBn PhtN BnO BnO BnO OAc OAc
O BnO
O O BnO O
OBn O OBn
AcO
CO2Me AcO OBn BnO O O O O AcHN O BnO AcO OBn PhtN OAc OAc
BnO BnO BnO
OBn BnO O O O BnO PhSO2NH
O
1. O O 2. O BnO PhSO2NH OTBS
Global Deprotection Kochetkov Amination
O
Progress Toward Erythropoietin (EPO): Synthesis of Glycan
HO
CO2H OH OH OH O O O O AcHN O HO HO OH AcHN HO HO HO OH OH
O OH OH O OH O HO O HO O AcHN O OH O AcHN NH2 O O HO
O O HO O
HO
CO2H OH OH HO O O O O AcHN O HO HO OH AcHN OH OH
HO HO HO
O
Progress Toward Erythropoietin (EPO): Synthesis of Glycan
HO
CO2H OH OH OH O O O O AcHN O HO HO OH AcHN OH OH HO HO HO
O
O O OH O
HO
CO2H OH OH OH O O O O AcHN O HO HO OH AcHN OH OH HO OH OH
HO HO O HO
O
HO O HO
O AcHN
HO O HO
O AcHN
NH2
O
CO2H OH OH HO O O O O AcHN O HO HO OH AcHN
O
Progress Toward Erythropoietin (EPO): Synthesis of Glycan
OAc OAc AcHN AcO OAc OAc AcHN AcO AcO AcO OAc OAc AcHN AcO O O O O OAc HO N3 AcO OAc OAc AcHN AcO AcO OAc OAc AcHN AcO FmocHN O OBn O O O O OAc OAc O O NHAc O OAc O O COOMe O FmocHN O O OBn OAc O SEt O O O O OAc OAc AcHN AcO OAc OAc AcHN O AcO OAc O COOMe O O OP(OBn)2 COOMe O O N3 O O OTIPS O O O N3 OH O COOBn NHFmoc OTIPS O O CCl3 NH
AcO
OP(OBn)2 COOMe HO
OH
OTIPS O O
O
OTIPS O
AcO
AcO
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
HO CO2H OH OH OH O O O O AcHN O HO HO OH AcHN HO HO HO OH OH
O O O HO O OH O OH OH O HO O HO O AcHN O O OH OH O AcHN NH2 O O HO
HO
HO CO2H HO OH OH HO HO O O O O AcHN O HO HO OH AcHN OH OH
+
O Ala Glu Asp Ile
Dmab
O Thr Thr Gly O SSEt
Aspartylation
HO
CO2H OH OH OH O O O O AcHN O HO HO OH AcHN HO HO HO OH OH
O O O HO O OH O OH OH O HO O HO O AcHN O Ala Glu Asp Ile O
Dmab
OH O AcHN O Thr Thr Gly O SSEt H2N Cys Ala Glu His Cys Ser Leu Asn Glu H N O HS
O O HO
CO2H HO OH OH OH OH HO O O O O AcHN O HO HO OH AcHN
HO HO HO
NCL
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
HO
CO2H OH OH OH O O O O AcHN O HO HO OH AcHN HO HO HO OH OH
O O O HO O OH O OH OH O HO O HO O AcHN O Ala Glu Asp Ile O
Dmab
OH O AcHN Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu H N
O O HO
O
HO
CO2H OH OH HO O O O O AcHN O HO HO OH AcHN OH OH
HO HO HO
EPO 22-37
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
HO CO2H OH OH OH O O O O AcHN O HO HO OH AcHN HO HO HO OH OH O O O HO HO HO CO2H HO OH OH HO HO O O O O AcHN O HO HO OH AcHN OH OH O OH O HO O HO OH OH O O AcHN OH O AcHN NH2 O O HO
+
OH O O Thr Thr Gly S O O
O O
Aspartylation Removing Fmoc
FmocHN
Lys Glu Ala Glu Asp Ile
Allyl ivDde Allyl
HO
CO2H OH OH OH O O O O AcHN O HO HO OH AcHN HO HO HO OH OH
O O O HO O OH O HO O HO OH OH O O AcHN OH O AcHN H N O O HO
O O O S O
HO CO2H HO OH HO OH OH HO OH HO O O O O AcHN O HO HO OH AcHN
O O
H2N
Lys Glu Ala Glu Asp Ile
Allyl ivDde Allyl
Thr Thr Gly
TCEP Fragment Condensation
+
O O SSEt
Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Glu Ala
Acm
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
HO
CO2H OH OH OH O O O O AcHN O HO HO OH AcHN HO HO HO OH OH
O O O HO O OH O OH OH O HO O HO O AcHN O O OH O AcHN H N O O O HO
HO
CO2H OH OH HO O O O O AcHN O HO HO OH AcHN OH OH
HO HO HO
?
Thr Thr Gly
Allyl ivDde Allyl
O S
O O
Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Glu Ala Lys Glu Ala Glu Asp Ile
Acm
EPO 1-28
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
AcO
OAc OAc O AcO OAc OAc O
COOMe O
AcHN AcO OAc OAc O AcO O O O OAc
O O AcNH O Ala Ala Asp Pro Pro Ser Ile Ser Ala SSEt O O SH HN Ala Pro Leu Arg Thr Ile ivDde Ala Glu Lys Gln Ala NHFmoc ivDde
AcHN
Auxiliary Ligation
Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn
+
MeO OMe OMe
Arg Asp Gly Thr Arg Cys Ala Glu Gly Thr Tyr Leu Lys leu Lys Gly Arg Leu Phe Acm ivDde ivDde
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
AcO
OAc OAc O AcO OAc OAc O
COOMe O
AcHN AcO OAc OAc O AcO O O O OAc
O O AcNH O Ala Ala Asp Pro Pro Ser Ile Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Arg Asp Gly Thr Arg Cys Ala Glu Gly Thr Tyr Leu Lys leu Lys Gly Arg Leu Phe Acm ivDde ivDde Ala Glu Lys Gln Ala NHFmoc ivDde
AcHN
ivDde
EPO 114-166
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
AcO
OAc OAc O AcO OAc OAc O
COOMe O
AcHN AcO OAc OAc O AcO O O O OAc
O O AcNH O Ala Ala Asp Pro Pro Ser Ile Ser Ala Ala Pro O SSEt O H2N-- Leu Arg Thr Ile ivDde Ala Glu Lys Gln Ala NHFmoc ivDde
AcHN
TCEP Fragment Condensation
Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn
Arg Asp Gly Thr Arg Cys Ala Glu Gly Thr Tyr Leu Lys leu Lys Gly Arg Leu Phe Acm ivDde ivDde
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
AcO
OAc OAc O AcO OAc
COOMe O
AcHN AcO OAc OAc O AcO O O O OAc OAc O
O O AcNH O Ala Ala Asp Pro Pro Ser Ile Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Arg Asp Gly Thr Arg Cys Ala Glu Gly Thr Tyr Leu Lys leu Lys Gly Arg Leu Phe Acm ivDde ivDde Ala Glu Lys Gln Ala NHFmoc ivDde
AcHN
ivDde
EPO 114-166
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
HO CO2H OH OH OH O O O O AcHN O HO HO OH AcHN HO HO HO OH OH O O O HO HO HO CO2H HO OH OH HO HO O O O O AcHN O HO HO OH AcHN OH OH O OH O OH OH O HO O HO O AcHN O SSEt O O O OH O AcHN O O HO OH O
+
NH2
Ser Ser Asp Val Leu Leu Ala Gln NHFmoc Gln Pro
HO
Aspartylation
CO2H OH OH OH O O O O AcHN O HO HO OH AcHN HO HO HO OH OH
O O O HO O OH O OH OH O HO O HO O AcHN O Ser Ser Asp Val Leu Leu Ala Gln NHFmoc O Gln SSEt O O Pro OH O AcHN H N O O HO
O
HO
CO2H OH OH HO O O O O AcHN O HO HO OH AcHN OH OH
HO HO HO
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
HO
CO2H OH OH OH O O O O AcHN O HO HO OH AcHN HO HO HO OH OH
O OH OH O OH O HO O HO O AcHN O Ser Ser Asp Val Leu Leu Ala Gln NHFmoc O SSEt O O Gln Pro OH O AcHN H N O O HO
O O HO O
TCEP Fragment Condensation
O
HO
CO2H OH OH HO O O O O AcHN O HO HO OH AcHN OH OH
HO HO HO
+
H2N Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser O O S O Thr Gly Leu Ala Arg Leu Leu Thr
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
HO
CO2H OH OH OH O O O O AcHN O HO HO OH AcHN HO HO HO OH OH
O HO
O O HO O OH O
OH O OH O AcHN Ser Ser Asp Val Leu Leu Ala Gln NHFmoc H N O O HO
HO O HO
O AcHN
O
HO
CO2H OH OH HO O O O O AcHN O HO HO OH AcHN OH OH
HO HO HO
O
O
Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser O O S O Thr Gly Leu Ala Arg Leu Leu Thr
EPO 78-113
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
O O S O
EPO 78-113 Gly
HO CO2H OH OH OH E O O O O AcHN O HO HO OH AcHN HO HO HO OH OH
NH2- Ala
EPO 114-166
O OH OH O OH O HO O HO O AcHN O OH O AcHN H N O O HO
O O HO O
AgCl Fragment Condensation
O
Saburo Aimoto
HO
CO2H OH OH HO O O O O AcHN O HO HO OH AcHN OH OH
HO HO HO
O
Ser Ser Asp Val Leu Leu Ala Gln NHFmoc Gln
AcO
OAc OAc O AcO OAc
Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg COOMe O Thr Ala Ala Asp Pro Pro Ser Ile O Ser Ala Glu Lys Gln Ala Gly Leu Ala Arg Leu Leu Thr ivDde Ser
AcHN AcO OAc OAc O AcO O O O OAc OAc O
AcHN
O AcNH O
ivDde
Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Arg Asp Gly Thr Arg Cys Ala Glu Gly Thr Tyr Leu Lys leu Lys Gly Arg Leu Phe Acm ivDde ivDde
EPO 78-166
Progress Toward Erythropoietin (EPO): Assembly of the Glycopeptide Building Blocks
Lys leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg Gly Arg Leu Ala Pro Pro Arg Leu Ile Phe Asn Ser Tyr Asp Val Arg Phe Leu Lys Arg Phe Thr Asp Ala Thr Ile Thr Arg Leu Pro Ala Ala Ser Ala Ala Asp Pro Pro Ser Ile Ala Glu Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Asp Val His Leu Gln Leu Pro Glu Trp Pro Gln Ser Ser Asn Val Leu Leu Ala Gln Gly Arg Leu Lys Val Ser Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Trp Gln Gly leu Ala leu leu Ser Glu Ala Val Lys Thr Asp Pro Val Thr Ile Asn Glu Asn Leu Ser Cys His Glu Ala Cys Gly Thr Thr Ile
EPO 1-28
Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Glu Ala Lys Glu Ala Glu Asn
EPO 29-77
EPO 78-166
Summary
We close this review with some thoughts concerning life on the chemistry– biology frontier. In this account, we have shown by historical progression how our laboratory, starting with fascinating problems in the field of “small molecule” natural products, has become involved in issues of tumor expression and tumor immunology. One of the singular contributions which we and like-oriented research groups bring to such coalitions is a sensitivity for precisely defined structures. When collaborating with biologists in identifying bioactive compounds and charting their functions, the chemist insists that the compounds in question be demonstrably pure and that the structural assignments, down to each stereogenic center, be corroborated. But chemistry’s contribution to the enterprise is certainly more than restraints arising from insistence on thoroughness and intellectual exactitude. Methodological building upon the principles of our science leads to the magic of synthesis – with its unique capability to prepare molecules of virtually any shape and juxtaposition of functional groups. Creative synthesis is the indispensable talent that the chemist will bring to the many exciting struggles and opportunities in the future.
Angew. Chem. Int. Ed. 1996, 35, 1380
Thank you!