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Versatile Conjugation of Octreotide to Dendrimers by Cycloaddition by nih35233


									                                                                                                                               Bioconjugate Chem. 2009, 20, 1323–1331                                            1323

                                                                             Versatile Conjugation of Octreotide to Dendrimers by Cycloaddition
                                                                             (“Click”) Chemistry to Yield High-Affinity Multivalent Cyclic Peptide
                                                                             Cheng-Bin Yim,†,‡ Otto C. Boerman,‡ Monique de Visser,§ Marion de Jong,§ Annemarie C. Dechesne,†
                                                                             Dirk T. S. Rijkers,† and Rob M. J. Liskamp*,†
                                                                             Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Department of Pharmaceutical
                                                                             Sciences, Faculty of Science, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, Department of Nuclear
                                                                             Medicine, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6525 GA Nijmegen, The Netherlands, and
                                                                             Department of Nuclear Medicine, Erasmus Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The
                                                                             Netherlands. Received February 5, 2009; Revised Manuscript Received May 13, 2009

                                                                                       The somatostatin analogue Tyr3-octreotide, which has a high binding affinity for the SSTR2 receptor (somatostatin
                                                                                       receptor subtype 2) expressed on tumor cells, is used clinically for the diagnosis and treatment of a variety of
                                                                                       neuroendocrine tumors and gastrointestinal disorders. There is growing interest in the development of multivalent
                                                                                       peptide systems, because they may have enhanced binding affinity compared to monovalent analogues. In this
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                                                                                       report, we describe the design and synthesis of a series of Tyr3-octreotide-containing monomeric, dimeric, and
                                                                                       tetrameric dendrimeric conjugates. These multivalent dendrimeric cyclic peptides were obtained using Cu(I)-
                                                                                       catalyzed 1,3-dipolar cycloaddition between peptidyl azides and dendrimeric alkynes. Their affinities for the SSTR2
        Publication Date (Web): June 18, 2009 | doi: 10.1021/bc900052n

                                                                                       receptor were determined by a competitive binding assay on rat brain sections.

                                                                             INTRODUCTION                                                            monomeric and dimeric analogues, presumably due to enhanced
                                                                                                                                                     statistical rebinding (20, 21). These findings have inspired us
                                                                                Somatostatin is a cyclic peptide hormone, which circulates
                                                                                                                                                     to prepare dendrimeric multivalent octreotide-based molecules.
                                                                             in the blood in two biologically active forms: somatostatin-14
                                                                                                                                                     On the basis of our experience with previously developed
                                                                             and its N-terminally extended form somatostatin-28, consisting
                                                                                                                                                     dendrimeric conjugates (22-26), these octreotide-based den-
                                                                             of 14 and 28 amino acids, respectively (1-3). Both hormones
                                                                                                                                                     drimers may have enhanced binding affinity for the SSTR2
                                                                             exert different biological effects in the gastrointestinal system
                                                                                                                                                     receptor. The aim of this study was to synthesize multivalent
                                                                             and in the nervous system. These effects are mediated by high-
                                                                                                                                                     peptide dendrimers of octreotide, and to compare their in vitro
                                                                             affinity G protein-coupled somatostatin receptors (SSTRs) and
                                                                                                                                                     binding characteristics with monovalent congeners.
                                                                             include modulation of hormone release and neurotransmitter
                                                                             release and inhibition of cell proliferation (4, 5). Five different        Especially challenging is the efficient conjugation of these
                                                                             SSTR subtypes have been identified and characterized (SSTR1-5)           biologically active peptides to the dendrimer. A fast coupling
                                                                             (6). As some of these receptors are overexpressed in several            reaction with high efficiency and chemoselectivity is desirable
                                                                             human tumors, especially neuroendocrine tumors and their                to allow the conjugation of unprotected peptides to dendrimers
                                                                             metastases, there is growing interest in the development of             to obtain well-defined multivalent peptide dendrimers with
                                                                             potent analogues to target these receptors. The octapeptide             preservation of receptor binding affinity. Several methodologies
                                                                             analogues octreotide and octreotate, having high binding affinity        have been investigated, and the Huisgen 1,3-dipolar cycload-
                                                                             for SSTR2-receptor-expressing tumor cells, are used clinically          dition (27) of an azide and an alkyne forming a triazole linkage
                                                                             for the diagnosis and treatment of a variety of neuroendocrine          is particularly suitable for chemoselective bioconjugation reac-
                                                                             tumors and gastrointestinal disorders (7-11).                           tions. The copper-catalyzed variant (28, 29), which regiospe-
                                                                                Much research has focused on the development of multivalent          cifically gives 1,4-disubstituted 1,2,3-triazoles has led to many
                                                                             dendrimeric molecules, because they show interesting properties         applications ranging from therapeutics to macromolecules (30-35).
                                                                             for enhancing the overall binding affinity compared to monova-           We recently showed that unprotected peptides could be ef-
                                                                             lent ligands (12-16). Several research groups have prepared             ficiently attached to an alkyne-derivatized version of our earlier
                                                                             multimers of cyclic Arg-Gly-Asp (RGD) peptides and evaluated            developed dendrimers (36-39) using the 1,3-dipolar cycload-
                                                                             theirbindingcharacteristicstowardtheRv 3 integrinreceptor(17-19).       dition reaction (21, 40). Thus, in this report we describe the
                                                                             Our studies have shown that tumor uptake of a tetrameric RGD            synthesis of multivalent dendrimeric octreotide peptides from
                                                                             dendrimer was significantly higher compared to that of the               dendrimeric alkynes and azido peptides. To evaluate the effect
                                                                                                                                                     of multivalency, the SSTR2 receptor binding characteristics of
                                                                                                                                                     the corresponding monovalent, divalent, and tetravalent oct-
                                                                               * To whom correspondence should be addressed. Rob M. J. Liskamp,      reotide peptide dendrimers were studied in comparison to
                                                                             Medicinal Chemistry and Chemical Biology, Utrecht Institute for
                                                                             Pharmaceutical Sciences, Department of Pharmaceutical Sciences,
                                                                             Faculty of Science, Utrecht University, PO Box 80082, 3508 TB
                                                                             Utrecht, The Netherlands. Phone: +31 30 253 7396. Fax: +31 30 253       EXPERIMENTAL PROCEDURES
                                                                             6655. E-mail:
                                                                                 Utrecht University.                                                   Materials and Instruments. Unless stated otherwise, chemi-
                                                                                 Radboud University Nijmegen Medical Centre.                         cals were obtained from commercial sources and used without
                                                                                 Erasmus Medical Center Rotterdam.                                   purification. Peptide-grade solvents were purchased from Bio-
                                                                                                                 10.1021/bc900052n CCC: $40.75  2009 American Chemical Society
                                                                                                                                    Published on Web 06/18/2009
                                                                             1324 Bioconjugate Chem., Vol. 20, No. 7, 2009                                                                             Yim et al.

                                                                             solve and used directly except for dimethylformamide (DMF),        (3 × 10 mL, 2 min), DMF (3 × 10 mL, 2 min), and DCM (3
                                                                             N-methylpyrrolidone (NMP), and dichloromethane (DCM),              × 10 mL, 2 min). The loading of the resin, as calculated from
                                                                             which were dried on 4 Å molecular sieves prior to (solid phase)    an Fmoc determination, was 60% (0.66 mmol/g). The linear
                                                                             synthesis. 2-Chlorotrityl chloride resin was purchased from        peptide sequence was synthesized according to Fmoc/tBu solid-
                                                                             Tianjin Nankai Hecheng Science & Technology Company Ltd.           phase peptide synthesis protocols (32). In a typical 2-(1H-
                                                                             and NR-fluorenylmethyloxycarbonyl (Fmoc) amino acids were           benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro phos-
                                                                             purchased from Advanced ChemTech, Alexis, or Novabiochem.          phate(HBTU)/N-hydroxybenzotriazole(HOBt)-couplingprocedure,
                                                                             Solid-phase synthesis was carried out in plastic syringes with     the resin was treated for 3 h with a 4-fold excess of a selected
                                                                             PE frit (20 µm) obtained from Applied Separations Inc.             amino acid. After coupling of the N-terminal Fmoc-D-Phe-OH,
                                                                             Analytical HPLC runs were carried out on a Shimadzu HPLC           a portion of the resin (0.60 g, 0.23 mmol) was treated with 20%
                                                                             system, and preparative HPLC runs were performed on a Kratos       piperidine/NMP (3 × 5 mL, 10 min) to remove the N-terminal
                                                                             HPLC workstation. Analytical HPLC runs were performed on           Fmoc group, followed by washing with NMP and DCM. The
                                                                             an Alltech Alltima C8 column (250 × 4.6 mm, pore size 90 Å,        6-azido-hexanoic acid 1 (0.15 mg, 0.94 mmol) was dissolved
                                                                             particle size 5 µm) at a flow rate of 1.0 mL/min using a linear     in NMP (4 mL) in the presence of HBTU and HOBt (0.35 mg
                                                                             gradient of buffer B (0-100% in 20 min) in buffer A (buffer        and 0.14 mg, respectively, 0.94 mmol, 1 equiv), and to this
                                                                             A: 0.1% trifluoroacetic acid (TFA) in CH3CN/H2O 5:95 v/v,           solution DIPEA (0.31 mL, 1.88 mmol, 2 equiv) followed by
                                                                             buffer B: 0.1% TFA in CH3CN/H2O 95:5 v/v). Preparative             the peptide resin were added and the obtained slurry was gently
                                                                             HPLC runs were performed on an Alltech Adsorbosphere XL            swirled for 16 h at room temperature. The resin was subse-
                                                                             C8 column (250 × 22 mm, pore size 90 Å, particle size 10           quently washed (NMP, DCM), dried, suspended in TFA/H2O/
                                                                             µm), and semipreparative HPLC runs were performed on an            triisopropylsilane (TIS) (95:2.5:2.5 v/v/v; 10 mL) and stirred
                                                                             Alltech Adsorbosphere XL C18 column (250 × 10 mm, pore             for 4 h to cleave the peptide alcohol from the resin and to
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                                                                             size 90 Å, particle size: 10 µm) at a flow rate of 12.0 mL/min      remove the side chain protecting groups. The crude peptide was
                                                                             (semipreparative HPLC: 4.8 mL/min) using a linear gradient         isolated by precipitation with cold (-20 °C) methyl tert-butyl
                                                                                                                                                ether (MTBE)/hexane (1:1 v/v). After centrifugation, the pellet
        Publication Date (Web): June 18, 2009 | doi: 10.1021/bc900052n

                                                                             of buffer B (0-100% in 120 min) in buffer A (buffer A: 0.1%
                                                                             TFA in CH3CN/H2O 5:95 v/v, buffer B: 0.1% TFA in CH3CN/            was dissolved in CH3CN/H2O (1:1 v/v), lyophilized and
                                                                             H2O 95:5 v/v). Electrospray ionization (ESI) mass spectrometry     subsequently purified by HPLC (C8), and fractions collected at
                                                                             was measured on a Shimadzu QP8000 single quadruple bench-          46% buffer B (44% CH3CN). The linear peptide 3 was obtained
                                                                             top mass spectrometer operating in a positive ionization mode.     in 29% (78 mg) yield. Rt: 20.17 min (C8). ESI-MS: m/z )
                                                                             MALDI-TOF analysis was performed on a Kratos Axima CFR             1176.30 [M+H]+, 1198.25 [M+Na]+, (C55H77N13O12S2: M )
                                                                             apparatus with human ACTH(18-39) (monoisotopic [M+H]+              1175.53).
                                                                             2465.198), bovine insulin oxidized B chain (monoisotopic              N3-Ahx-D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Thr-Cys)-Threol (4).
                                                                             [M+H]+ 3494.651), or bovine insulin (monoisotopic [M+H]+           Compound 4 was obtained by dimethylsulfoxide (DMSO)-
                                                                             5730.609) as external references and R-cyano-4-hydroxycin-         mediated oxidation of 3, according to a procedure of Tam
                                                                             namic acid as matrix. 1H NMR spectra were recorded on a            et al. (47). Peptide 3 (44 mg, 37.4 µmol) was dissolved in
                                                                             Varian G-300 (300 MHz) spectrometer, and chemical shifts are       aqueous 2.5% acetic acid (50 mL), and the pH was adjusted to
                                                                             given in ppm (δ) relative to TMS. 13C NMR spectra were             6.0 with aqueous ammonia (25%). To this solution, DMSO (12
                                                                             recorded on a Varian G-300 (75.5 MHz) spectrometer, and            mL) was added. After stirring overnight at room temperature,
                                                                             chemical shifts are given in ppm (δ) relative to CDCl3 (77.0       the solution was partially concentrated in vacuo and the
                                                                             ppm). The 13C NMR spectra were recorded using the attached         remaining DMSO was evaporated. The residue was dissolved
                                                                             proton test (APT) sequence. Solid-phase synthesis was              in CH3CN/H2O, filtered, and purified by HPLC (C8), and
                                                                             monitored with the Kaiser (41, 42) or the bromophenol blue         fractions of pure peptide were collected at 44% buffer B (42%
                                                                             (BPB) test (43). Where needed, the resin loading was               CH3CN). The cyclic peptide 4 was obtained in 79% (35 mg)
                                                                             determined by measuring the UV absorbance of the                   yield. Rt: 20.05 min (C8). ESI-MS: m/z ) 1174.00 [M+H]+,
                                                                             piperidine-dibenzofulvene adduct (λmax 300 nm) (44) using          1196.10 [M+Na]+, (C55H75N13O12S2: M ) 1173.51).
                                                                             a Thermo Electron Corporation UV/vis spectrophotometer.               MonoValent Tyr3-Octreotide Peptide Conjugate (11). The
                                                                             Solution-phase reactions were monitored by thin layer              synthesis of 11 was performed using activated copper-in-
                                                                             chromatography (TLC), and Rf values were determined on             charcoal (Cu/C) as the catalyst (48, 49). Alkyne 5 (0.5 mg, 2.9
                                                                             Merck precoated silica gel 60 F-254 (0.25 mm) plates. Spots        µmol) and azide 4 (3.7 mg, 3.2 µmol, 1.1 equiv) were dissolved
                                                                             were visualized with UV light or ninhydrin or by staining          in DMF (200 µL). To this solution, Cu/C (10 mg, excess) and
                                                                             with chlorine/N,N,N′,N′-tetramethyl-4,4′-diaminodiphenyl-          triethylamine (TEA) (5 µL) were added. The mixture was
                                                                             methane (TDM) (45). The syntheses of compounds 1 and 2             allowed to react under microwave conditions (100 °C) for 5-10
                                                                             are given in the Supporting Information. The syntheses of          min. The cycloaddition reaction was monitored by HPLC. A
                                                                             dendrimers 5-10 have been described previously (21, 40).           yellow-brownish solution was formed during the reaction.
                                                                                Chemistry. N3-Ahx-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Th-           Following microwave irradiation, the mixture was filtered over
                                                                             reol (3). The 2-chlorotrityl chloride resin (909 mg, 1.00 mmol)    Hyflo and concentrated in vacuo. The residue was dissolved in
                                                                             was treated with SOCl2/DCM (1:1 v/v; 2 × 10 mL, 10 min) to         CH3CN/H2O (1:1 v/v), lyophilized, and purified by semiprepara-
                                                                             convert it completely into the chloride form prior loading of      tive HPLC (C18). Yield: 1.8 mg (46%). Rt: 20.48 min (C8).
                                                                             the first amino acid derivative. To remove any residual SOCl2,      ESI-MS: m/z ) 1364.30 [M+H]+, 1386.65 [M+Na]+. MALDI-
                                                                             the resin was extensively washed with DCM (6 × 10 mL, 10           TOF: 1364.693 [M+H]+, 1386.749 [M+Na]+ (C66H85N13O15S2:
                                                                             min). Then, Fmoc-Thr(tBu)Ψ[CH2OH] 2 (1.53 g, 4.00 mmol)            M ) 1363.573).
                                                                             was dissolved in DCM (10 mL), and to this solution N,N-               DiValent Tyr3-Octreotide Peptide Dendrimer (12). Alkyne 6
                                                                             diisopropylethylamine (DIPEA) (1.42 mL, 8.00 mmol) followed        (0.7 mg, 2.8 µmol) and azide 4 (6.9 mg, 5.9 µmol, 2.1 equiv)
                                                                             by the 2-chlorotrityl chloride resin were added and the obtained   were dissolved in degassed DMF (200 µL). To this solution
                                                                             slurry was gently swirled for 2 h at room temperature. The resin   CuOAc (0.8 mg, 6.5 µmol, 2.2 equiv) was added, and the
                                                                             was subsequently washed with DCM/CH3OH/DIPEA (17:2:1               mixture was allowed to react under microwave conditions (100
                                                                             v/v/v; 3 × 10 mL, 10 min) to quench any remaining reactive         °C) for 20 min. Following microwave irradiation, the obtained
                                                                             2-chlorotrityl chloride, followed by a washing step with DCM       yellow mixture was concentrated in vacuo, and the residue was
                                                                             Synthesis of Multivalent Octreotide Dendrimers                                          Bioconjugate Chem., Vol. 20, No. 7, 2009 1325

                                                                             dissolved in CH3CN/H2O (1:1 v/v) and lyophilized. After            mented with 5 mM MgCl2, 1% bovine serum albumin (BSA),
                                                                             purification by semipreparative HPLC (C18), compound 12 was         and 40 µg/mL bacitracin. To generate the competitive inhibition
                                                                             obtained in 42% yield (3.1 mg). Rt: 27.33 min (C18). ESI-MS:       curves, the sections were incubated in the presence of an
                                                                             m/z ) 1296.90 [M+2H]2+. MALDI-TOF: m/z ) 2593.995                  increasing amount (10-10 to 10-5 M) of the nonradioactive
                                                                             [M+H]ave+, 2616.045 [M+Na]ave+, 2632.121 [M+K]ave+,                dendrimeric octreotide constructs. After incubation, the sections
                                                                             (C124H162N26O28S4: Mave ) 2593.074).                               were washed two times for 5 min each in 167 mM Tris (pH
                                                                                TetraValent Tyr3-Octreotide Peptide Dendrimer (13). Analo-      7.6), 5 mM MgCl2, 0.25% BSA, (4 °C), 5 min in 167 mM Tris
                                                                             gous to the preparation of 12, alkyne 7 (0.7 mg, 1.0 µmol),        (pH 7.6), 5 mM MgCl2 (4 °C), and finally rinsed in Milli-Q
                                                                             azide 4 (5.3 mg, 4.5 µmol, 4.5 equiv), and CuOAc (0.5 mg, 4.1      water (4 °C). The sections were then dried and exposed to
                                                                             µmol, 4.1 equiv) were used in DMF (200 µL) as the solvent.         phosphor imaging screens (Perkin-Elmer) for 48 h. The imaging
                                                                             After purification by semipreparative HPLC (C18), compound          screens were read using a Cyclone Storage Phosphor System
                                                                             13 was obtained in 24% yield (1.5 mg). Rt: 29.17 min (C18).        (Packard), and the autoradiograms were quantified using Op-
                                                                             MALDI-TOF: m/z ) 5375.938 [M+H]ave+, 5397.834                      tiquant Software (Packard). Fitting of IC50 curves and calculation
                                                                             [M+Na]ave+, 5414.927 [M+K]ave+, (C258H334N54O58S8: Mave )          of IC50 values were performed with GraphPad Prism 4.02
                                                                             5376.349).                                                         (GraphPad Software Inc.).
                                                                                MonoValent 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraace-
                                                                             tic acid (DOTA)-Conjugated Tyr3-Octreotide Construct (14).         RESULTS AND DISCUSSION
                                                                             Synthesis of 14 was performed using a combination of CuSO4            Syntheses of Cyclic Tyr3-Octreotide Azides and Dendrim-
                                                                             and copper powder as the catalyst (50). Alkyne 8 (4.2 mg, 5.4      eric Alkynes. Azido octreotide 4 was prepared on the solid phase
                                                                             µmol, 1.1 equiv) and azide 4 (5.5 mg, 4.7 µmol, 1.0 equiv)         via Fmoc/tBu chemistry protocols (46), as depicted in Scheme
                                                                             were dissolved in DMF/H2O (1:1 v/v; 0.2 mL). To this solution,
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                                                                                                                                                1. To enable click chemistry, an N-terminal azide functionality
                                                                             aqueous 0.1 M CuSO4 (50 µL, 5.0 µmol, 1.1 equiv) and copper        was introduced via 6-azidohexanoic acid 1, which was prepared
                                                                             powder (5 mg) were added, and the mixture was stirred for 18 h     from 6-bromohexanoic acid (52). The preparation of octreotide
        Publication Date (Web): June 18, 2009 | doi: 10.1021/bc900052n

                                                                             at room temperature. Then, the copper powder was removed           required a C-terminal threoninol derivative. For this purpose,
                                                                             by filtration, the solvent was evaporated under reduced pressure,   Fmoc-protected threonine was reduced to its corresponding
                                                                             and the residue was taken up in CH3CN/H2O (1:1 v/v; 2 mL)          alcohol 2 (53). Following coupling of the protected threoninol,
                                                                             and lyophilized. The crude product was purified by semi-            peptide 3 was prepared on the solid phase, and 6-azidohexanoic
                                                                             preparative HPLC (C8, 5f95% CH3CN in 90 min) to yield              acid 1 was coupled to the N-terminal D-phenylalanine before
                                                                             tert-butyl-protected 14 (lyophilized: 8.0 mg, 87%). The tert-      cleavage from the resin. After cleavage and concomitant removal
                                                                             butyl esters of the DOTA moiety were cleaved by treatment          of the side chain protection groups, the unprotected peptide 3
                                                                             with TFA/TIS/H2O (95:2.5:2.5 v/v/v; 2 mL) for 3 h. The solvent     was obtained in an isolated yield of 29%. Despite the fact that
                                                                             was removed under reduced pressure, and the residue was            the azide is susceptible to reduction in the presence of free thiols
                                                                             dissolved in CH3CN/H2O (1:1 v/v; 2 mL) and lyophilized. After      as are present in cysteine-2 and cysteine-7, the synthesis and
                                                                             semipreparative HPLC (C8, 5f95% CH3CN in 90 min) and               subsequent ring closure by a DMSO-mediated disulfide forma-
                                                                             subsequent lyophilization, 14 (3.4 mg, 1.9 µmol) was isolated      tion of the peptide proceeded without noticeable side reactions
                                                                             in overall yield of 40%. Rt: 18.10 min (C8). MALDI-TOF: m/z        and the azide remained stable throughout this procedure. Thus,
                                                                             ) 1780.234 [M+H]ave+, 1801.672 [M+Na]ave+, 1843.146                linear peptide 3 was oxidized to the cyclic disulfide 4 by
                                                                             [M+Cu]ave+, (C83H115N19O21S2: Mave ) 1779.05).                     treatment with DMSO (47). After HPLC purification, the cyclic
                                                                                DiValent DOTA-Conjugated Tyr3-Octreotide Dendrimer              azido octreotide 4 was obtained in an overall yield of 23%.
                                                                             (15). Alkyne 9 (2.2 mg, 2.7 µmol, 1.0 equiv), azide 4 (6.8            Several studies have reported on the convergent synthesis of
                                                                             mg, 5.8 µmol, 2.1 equiv), aqueous 0.1 M CuSO4 (55 µL, 5.5          amino acid based dendrimers with peripheral propargyl groups
                                                                             µmol, 2.0 equiv), and copper powder (5 mg) were added to           to enable a 1,3-dipolar cycloaddition (“click”) reaction with
                                                                             DMF/H2O (1:1 v/v; 0.2 mL), and the mixture was stirred for         peptide derived azides (21, 40). A similar methodology was
                                                                             18 h at room temperature. The workup, TFA treatment, and           used here to prepare alkyne-derivatized dendrimers 5-7 and
                                                                             isolation of 15 were similar to that described for 14. After       their corresponding DOTA-conjugated derivatives 8-10 (Figure
                                                                             semipreparative HPLC and subsequent lyophilization, 15 (0.9        1) as a scaffold for the multimerization of octreotide.
                                                                             mg, 0.3 µmol) was isolated in an overall yield of 12%. Rt:            Conjugation of Octreotide to Dendrimers by the Click
                                                                             18.48 min (C8). MALDI-TOF: m/z ) 3008.674 [M+H]ave+,               Cycloaddition Reaction Procedure. After obtaining azide-
                                                                             3070.520 [M+Cu]ave+, (C141H192N32O34S4: Mave ) 3007.49).           functionalized octreotide 4 and alkynes 5-10, the stage was
                                                                                TetraValent DOTA-Conjugated Tyr3-Octreotide Dendrimer           set for the preparation of dendrimeric peptide systems using
                                                                             (16). Alkyne 10 (1.8 mg, 1.5 µmol, 1.0 equiv), azide 4 (7.6        chemoselective “click” chemistry, as shown in Scheme 2. To
                                                                             mg, 6.5 µmol, 4.3 equiv), aqueous 0.1 M CuSO4 (58 µL, 5.8          prove the concept, the first experiments were conducted with
                                                                             µmol, 3.9 equiv), and copper powder (10 mg) were added to          alkynes 5-7 according to the literature procedure for microwave-
                                                                             DMF/H2O (1:1 v/v; 0.2 mL), and the mixture was stirred for         assisted cycloaddition chemistry. Azide 4 was reacted with
                                                                             18 h at room temperature. The workup, TFA treatment, and           acetylene 5 in the presence of CuSO4/Na-ascorbate in DMF/
                                                                             isolation of 16 were similar to that described for 14. After       H2O (9:1 v/v) for 5-15 min under microwave irradiation. Since
                                                                             semipreparative HPLC and subsequent lyophilization, 16 (0.9        the Rf values of the starting material and product were
                                                                             mg, 0.16 µmol) was isolated in overall yield of 11%. Rt: 18.93     comparable on TLC, HPLC analysis was used to monitor the
                                                                             min (C8). MALDI-TOF: m/z ) 5790.326 [M+H]ave+, 5851.068            progress of the click cycloaddition reaction. After 5 min at 100
                                                                             [M+Cu]ave+, (C275H364N60O64S8: Mave ) 5790.72).                    °C, the monovalent cycloaddition conjugate 11 was formed.
                                                                                Receptor Autoradiography. The receptor binding affinity          Different sources of copper catalyst, including CuOAc and
                                                                             of the peptide dendrimers was determined in a competitive          activated copper-in-charcoal (Cu/C) (48, 49), were used for
                                                                             binding assay on rat brain cryostat sections (10 µm) using         comparison to find the best procedure and to assess the
                                                                             methods previously described with minor modifications (51).         versatility of this approach. Microwave-assisted cycloaddition
                                                                             The sections were incubated for 1 h at room temperature with       with any of the three Cu(I)-sources afforded monovalent
                                                                             0.1 nM [111In-DOTA0,Tyr3]octreotate (135 MBq/nmol) in 167          cycloadducts in yields ranging from 40% to 70%, with Cu/C
                                                                             mM tris(hydroxymethyl)aminoethane (Tris) (pH 7.6), supple-         giving the highest yield. However, the use of CuOAc showed
                                                                             1326 Bioconjugate Chem., Vol. 20, No. 7, 2009                                                                                 Yim et al.

                                                                             Scheme 1. Synthesis of the Cyclic N-ω-Azido Hexanoyl Octreotide Peptide 4
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        Publication Date (Web): June 18, 2009 | doi: 10.1021/bc900052n

                                                                             improved results in attaching peptidyl azide 4 to the divalent        It turned out that using an equimolar amount of CuOAc gave
                                                                             alkyne 6 giving divalent octreotide conjugate 12. Therefore,          the best result for all cycloaddition reactions. When using more
                                                                             CuOAc was chosen as the catalyst for the subsequent click             equivalents of copper catalyst, increasing amounts of impurities
                                                                             conjugation reaction with the tetravalent dendrimeric alkyne 7.       were detected by HPLC. The identity of the cycloadducts 11-13
                                                                             Considerable amount of side products were observed during the         was confirmed by MALDI-TOF and the spectrum of cyclic
                                                                             reactions toward divalent and especially tetravalent peptide          octreotide tetramer 13 is shown in Figure 2 as a representative
                                                                             dendrimers, and LC-MS analyses showed most of these to be             example.
                                                                             incomplete cycloadducts. These incomplete conjugates ham-                Scheme 3 shows the synthetic route toward DOTA-conjugated
                                                                             pered purification by HPLC and resulted into a low isolated            octreotide dendrimers 14, 15, and 16. In contrast to the synthesis
                                                                             yield of the desired product. The formation of 11 and 13 was          of conjugated peptide dendrimers 11-13, the DOTA moiety
                                                                             complete after 5 to 10 min microwave irradiation at 100 °C,
                                                                                                                                                   of dendrimers 8-10 required a different copper catalyst, since
                                                                             whereas the reaction time for 12 had to be extended to 20 min.
                                                                                                                                                   none of the above-mentioned Cu(I)-sources resulted in the
                                                                                                                                                   desired products. Fortunately, using a combination of CuSO4
                                                                                                                                                   and copper powder as described by Kessler and co-workers (50)
                                                                                                                                                   gave the desired conjugates in acceptable yields. Figure 3 shows
                                                                                                                                                   the analytical HPLC trace of azido octreotide 4 and alkyne 8
                                                                                                                                                   (A) and the resulting DOTA-conjugated tBu-protected precursor
                                                                                                                                                   of 14 after the click reaction (B), respectively. Conjugate 14
                                                                                                                                                   was obtained in 40% yield after purification. The isolated yields
                                                                                                                                                   for dimer 15 and tetramer 16 were 12% and 11%, respectively.
                                                                                                                                                   These relative low values were mainly due to the presence of
                                                                                                                                                   incomplete coupling products and concomitantly difficult pu-
                                                                                                                                                   rification to obtain the pure compound.
                                                                                                                                                      Since a relatively high amount of copper had to be used
                                                                                                                                                   during the synthesis of 14, 15, and 16, Cu-DOTA complexes
                                                                                                                                                   were formed in addition to the desired copper-free products, as
                                                                                                                                                   was inferred from the mass spectra. To remove residual copper
                                                                                                                                                   from the DOTA moieties, the product was treated with Na2S
                                                                                                                                                   as described in the literature (50). This treatment, however,
                                                                                                                                                   reduced the disulfide bridges in octreotide, which had to be
                                                                                                                                                   restored by a reoxidation step in aqueous DMSO. Figure 4
                                                                             Figure 1. Structures of the mono-, di-, and tetravalent dendrimeric   shows the mass spectrum of conjugate 14 with no indication of
                                                                             alkynes 5, 6, and 7, and their corresponding DOTA conjugates 8, 9,    Cu-DOTA complexes. However, mass analysis of either dimer
                                                                             and 10.                                                               15 or tetramer 16 still clearly showed copper-bound adducts
                                                                             Synthesis of Multivalent Octreotide Dendrimers                                            Bioconjugate Chem., Vol. 20, No. 7, 2009 1327

                                                                             Scheme 2. Synthesis of Mono-, Di-, and Tetravalent Octreotide Dendrimeric Constructs 11, 12, and 13
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                                                                             after Na2S treatment (Supporting Information Figures SI 8,9,         synthesis of 111In-labeled cyclic RGD-DTPA conjugates.
                                                                             respectively).                                                       Alternative copper-free “click” approaches to obtain DOTA-
                                                                                To avoid copper contamination of the desired end-product,         conjugated multimeric octreotide dendrimers are currently under
                                                                             since it is cytotoxic toward mammalian cells and also hampers        investigation in our laboratory.
                                                                             efficient loading of the DOTA moiety with radioactive metals,            Receptor Binding Assay. Binding affinities toward the
                                                                             approaches toward metal-free azide-alkyne cycloadditions are         somatostatin receptor of the mono-, di-, and tetravalent DOTA
                                                                             particularly relevant (54). In a recent paper by Rutjes and co-      conjugates of octreotide 14, 15, and 16, respectively, were
                                                                             workers (55), it was shown that a copper-free tandem 1,3-dipolar     determined by a competitive binding assay on rat SSTR2-positive
                                                                             cycloaddition retro-Diels-Alder ligation enabled the efficient        brain cryostat sections, using [111In-DOTA0,Tyr3]octreotate as a
                                                                                                                                                  radiotracer. Figure 5 represents a typical example of an autorad-
                                                                                                                                                  iogram, in which [111In-DOTA0,Tyr3]octreotate was displaced by
                                                                                                                                                  tetravalent octreotide dendrimer 13 (first and second column) and
                                                                                                                                                  azido octreotide 4 (third and fourth column) in a concentration
                                                                                                                                                  range from 10-10 to 10-5 M (from top to bottom) in two
                                                                                                                                                  independent experiments. The autoradiograms were quantified, and
                                                                                                                                                  the IC50 values were calculated from fitted sigmoidal displacement
                                                                                                                                                  curves as shown in Figure 6 and summarized in Table 1. On the
                                                                                                                                                  basis of these data, it became clear that the IC50 values of conjugate
                                                                                                                                                  14, dimer 15, and tetramer 16 were all within the low nanomolar
                                                                                                                                                  range. However, no significant increase in receptor affinity for the
                                                                                                                                                  tetramer 16 compared to either dimer 15 or conjugate 14 could be
                                                                                                                                                  measured. A possible explanation for the absence of the multiva-
                                                                                                                                                  lency effect may be found in the nonoptimized spacer-arm length
                                                                                                                                                  either between the DOTA chelator and the dendrimeric core or
                                                                                                                                                  between the dendrimer core and the peptide. However, it is known
                                                                             Figure 2. MALDI-TOF spectrum of tetravalent cyclic octreotide        from our earlier work that elongating the spacer-arm length,
                                                                             dendrimer 13.                                                        between the dendrimer core and the carbohydrate moiety of a
                                                                             1328 Bioconjugate Chem., Vol. 20, No. 7, 2009                                                                                        Yim et al.

                                                                             Scheme 3. Synthesis of Mono-, Di-, and Tetravalent DOTA-Conjugated Octreotide Dendrimeric Constructs 14, 15, and 16
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                                                                             divalent galabiose derivative, resulted in a 2-fold increase in IC50      experiments using conjugates 11-13 without the DOTA-moiety
                                                                             value for the inhibition of Streptococcus suis bacteria (23),             (Supporting Information Table SI 1 and Figure SI 10). The resulting
                                                                             compared to the analogue without a spacer. Therefore, the present         IC50 values found for conjugate 11 and dimer 12 were higher than
                                                                             dendrimeric systems of the octreotide cyclic peptide may not be           those for their corresponding DOTA conjugates 14 and 15;
                                                                             ideal for augmenting receptor binding. Furthermore, the importance        probably, a poor aqueous solubility of tetramer 13 prevented even
                                                                             of the linker or other solubilizing moieties connected directly to        the calculation of a reliable IC50 value. Incorporating the DOTA
                                                                             the dendrimer scaffold was also apparent from similar binding             moiety, which contains three hydrophilic carboxylic acid func-

                                                                             Figure 3. (A) Analytical C8 HPLC trace (UV 220 nm) of peptidyl azide 4 (Rt: 19.6 min) and alkyne 8 (Rt: 22.9 min). (B) Analytical C8 HPLC trace
                                                                             (UV 220 nm) of crude tBu-protected precursor of 14 (Rt: 21.8 min) obtained after the click reaction.
                                                                             Synthesis of Multivalent Octreotide Dendrimers                                             Bioconjugate Chem., Vol. 20, No. 7, 2009 1329
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                                                                             Figure 4. MALDI-TOF spectrum of monovalent DOTA-conjugated cyclic octreotide construct 14.
        Publication Date (Web): June 18, 2009 | doi: 10.1021/bc900052n

                                                                                                                                                    Figure 6. Competitive binding assay of mono-, di-, and tetravalent
                                                                                                                                                    DOTA-conjugated systems of octreotide 14 (9), 15 (2), and 16 (1)
                                                                                                                                                    displacing [111In-DOTA0,Tyr3]octreotate in rat SSTR2-positive brain
                                                                                                                                                    sections (n ) 2 for each data point). [DOTA0,Tyr3]-conjugated
                                                                                                                                                    octreotide 17 (•) was used as reference compound. The error bars
                                                                                                                                                    indicate the range of the 95% confidence interval.

                                                                                                                                                    Table 1. IC50 Values for Competitive Binding Assay of Mono-, Di-,
                                                                                                                                                    and Tetravalent DOTA-Conjugated Systems of Octreotide
                                                                                                                                                    Displacing [111In-DOTA0,Tyr3]octreotate in Rat SSTR2-Positive
                                                                                                                                                    Brain Sections (n ) 2 for each data point)
                                                                                                                                                         competing ligand           IC50 (nM)         95% CI (nM)
                                                                                                                                                       DOTA-conjugate 14               2.86             1.51-5.41
                                                                                                                                                       DOTA-dimer 15                  17.6              2.61-119
                                                                                                                                                       DOTA-tetramer 16                8.34             1.55-44.7
                                                                                                                                                       DOTA-octreotide 17             20.0              10.8-37.1
                                                                             Figure 5. Autoradiograms of tetravalent octreotide peptide dendrimer   tion (“click”) reaction leading to the successful synthesis of
                                                                             13 (first two columns) and azido octreotide 4 (last two columns).       multivalent octreotide-based dendrimers, either in the absence
                                                                             Concentrations used (from top to bottom): 10 µM, 1 µM, 100 nM, 50
                                                                             nM, 10 nM, 5 nM, 1 nM, 0.5 nM, and 0.1 nM, respectively.
                                                                                                                                                    or in the presence of the strong metal chelating DOTA
                                                                                                                                                    moiety. This conjugation method represents a chemoselective
                                                                                                                                                    approach for the preparation of these dendrimeric peptides
                                                                             tionalities, improved the overall solubility, and an IC50 value for    from unprotected cyclic peptidyl azides and dendrimeric
                                                                             tetramer 16 could be determined.                                       alkynes. We have tested the hypothesis regarding whether
                                                                                                                                                    functionalization of our DOTA-conjugated dendrimeric alkynes
                                                                                                                                                    8-10 with octreotide 4 will augment the binding affinity
                                                                             CONCLUSIONS                                                            between receptor and multivalent ligand. Competitive binding
                                                                                We have reported on the versatile conjugation of octreotide         assays showed no significant increase in receptor affinity for
                                                                             to dendrimers by the Cu(I)-catalyzed 1,3-dipolar cycloaddi-            the DOTA-conjugated tetramer 16 or dimer 15 compared to
                                                                             1330 Bioconjugate Chem., Vol. 20, No. 7, 2009                                                                                      Yim et al.

                                                                             the monomer 14. The role of the spacer between the scaffold             (16) Wester, H.-J., and Kessler, H. (2005) Molecular targeting with
                                                                             and the cyclic peptide could play a major role in improving               peptides or peptide-polymer conjugates: Just a question of size.
                                                                             binding affinity of the dimeric and tetrameric conjugates, and             J. Nucl. Med. 46, 1940–5.
                                                                             this aspect is currently under investigation. Furthermore, to           (17) Thumshirn, G., Hersel, U., Goodman, S. L., and Kessler, H.
                                                                             ensure an efficient radiolabeling, alternative copper-free                 (2003) Multimeric cyclic RGD peptides as potential tools for
                                                                             “click” approaches to obtain DOTA-conjugated multimeric                   tumor targeting: solid-phase peptide synthesis and chemoselective
                                                                             octreotide dendrimers are currently being developed in our                oxime ligation. Chem.sEur. J. 9, 2717–25.
                                                                             laboratory.                                                             (18) Janssen, M., Frielink, C., Dijkgraaf, I., Oyen, W. J. G.,
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                                                                             dohexanoic acid 1, Fmoc-Thr(tBu)[CH2OH] 2, DOTA-conju-                    of radiolabeled RGD peptides using rapid dose fractionation.
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