Biomacromolecules 2001, 2, 557-561
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Fully Convergent Solid Phase Synthesis of Antifreeze Glycoprotein Analogues Adewale Eniade and Robert N. Ben* Department of Chemistry, State University of New York at Binghamton, Binghamton, New York 13902 Received January 15, 2001
The convergent solid phase synthesis of C-linked analogues of antifreeze glycoprotein (AFGP) has been achieved. In this approach, three to six carbohydrate residues are simultaneously coupled to a resin-bound polypeptide. Glycopeptides ranging from 1.6 to 3.0 kDa are easily prepared in 26-44% yield demonstrating the utility of this approach. Introduction Carbohydrates play fundamental roles in many different biological processes.1a,b In most organisms, the carbohydrate moiety exists as a glycoconjugate. While many different types of glycoconjugate are known, the glycoprotein and glycolipid are most common. In a glycoprotein, the carbohydrate is convalently attached to a polypeptide backbone through an oxygen, nitrogen or sulfur atom while in a glycolipid, the carbohydrate is attached through an oxygen atom. Several native glycoconjugates possessing desirable therapeutic applications are now in clinical use2a,b and some glycoprotein analogues have proven useful as biological “probes” to study carbohydrate-protein interactions.2c-g Despite these advances, the discovery of useful glycoconjugate ligands for relevant biological processes has been hindered by the difficult synthesis of these compounds.3 Consequently, the development of efficient preparative protocols for glycoprotein and glycoprotein analogues continues to be of interest. Prior to conventional solid phase synthesis (SPS), the preparation of glycoconjugates relied on solution-phase strategies. In practice, two different strategies are utilized: a stepwise or linear strategy and a convergent strategy. In the linear approach, the glycopeptide is assembled in a stepwise fashion using glycosylated amino acid derivatives. While this approach necessitates the judicious choice of protecting groups, it has been successfully used to prepare complex glycoconjugates.4a-k In the convergent approach, a preformed polypeptide backbone is glycosylated late in the synthesis using the appropriate saccharide. Unfortunately, this strategy has not been widely adopted since preliminary results suggest that it is less effective than a linear approach.4b,5a,b Further complications arise from the low solubility of large peptides in dichloromethane6 and the fact that the primary structure of the polypeptide backbone has been shown to influence glycosylation.7 Conventional solid-phase synthesis (SPS) has revolutionalized peptide synthesis.8a-e New coupling reagents, resins and protocols now facilitate the preparation of large peptides * Corresponding author. E-mail:
[email protected].
Figure 1. Typical antifreeze glyroprotein (AFGP).
that previously represented formidable synthetic challenges. Despite these advances, the preparation of glycoconjugates continues to center on the linear approach where orthogonally protected glycosyl-amino acid residues are assembled on an insoluble polymer support or resin.9a,b To date, only a handful of papers have appeared describing the convergent synthesis of O-linked,10a,b N-linked,10c-e or C-linked glyconjugates10f using conventional solid-phase synthesis. In each of these cases only a single carbohydrate or oligosaccharide is coupled to a polypeptide backbone prior to removal of the glycopeptide from the polymer support. During the course of preparing antifreeze glycoprotein analogues, we found that a convergent coupling of multiple C-linked saccharide derivatives to resin-bound peptides is very efficient and economical. This paper describes the convergent synthesis of antifreeze glycoprotein analogues where up to six individual carbohydrate residues are linked to a resin-bound polypeptide backbone in a single step and demonstrates the utility of a convergent approach. Results and Discussion Over the last several years, our laboratory has been interested in antifreeze glycoproteins (AFGPs).11a-c These are naturally occurring glycoconjugates composed of a repeating tripeptide unit (L-threonyl-L-alanyl-L-alanyl) where the L-threonyl side chain is glycosylated with the disaccharide β-D-galactosyl-(1,4)-R-D-N-acetyl galactosamine (Figure 1). Native AFGP is composed of 4-55 of these repeating tripeptide units and thus, the molecular weight of AFGP subtypes varies from 2.4 to 34 kDa. These glycoproteins have
10.1021/bm0155120 CCC: $20.00 © 2001 American Chemical Society Published on Web 03/23/2001
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Eniade and Ben
Figure 2. Linear solid-phase synthesis of a C-linked AFGP mimic.
Figure 3. Convergent approach for C-linked AFGP.
the unusual ability to prevent the growth of ice crystals in organisms inhabiting sub-zero environments, a condition that would otherwise be lethal. Despite the fact that these glycoproteins were discovered over 30 years ago, a molecular mechanism of action has not been generally accepted.12a-e This is unfortunate given the tremendous medical, industrial and commercial applications of such compounds.13 To realize these applications, a detailed understanding of how these compounds function at the molecular level must be attained. Consequently, new synthetic protocols affording AFGP analogues for structure-function studies are urgently required.14 As part of our continuing effort to further elucidate the molecular mechanism by which antifreeze glycoproteins function, we have utilized a linear solid phase strategy to prepare structurally diverse analogues of native AFGP with molecular weights of approximately 1.5 kDa.15 As illustrated in Figure 2, our efforts have focused on C-linked analogues because of their enhanced chemical and biological stability.16 This is an attractive property for many medical and industrial applications. Substitution of the L-threonine residue with the glycosylated-L-lysine in 3 serves as a structural analogue for L-arginine that is frequently found in the core tripeptide of native AFGP 7-8.11d-f Recent structure-function studies
with type I AFP analogues have shown that substitution of with L-lysine produces an analogue that exhibits antifreeze protein-specific activity.17 While this linear solid phase approach has proven effective, it necessitates a costly and lengthy synthesis of the glycosylated tripeptide building block (3). In addition, 3-4 equiv of 3 are utilized in each coupling step, and recovery of unreacted material is not cost-effective. These factors led us to consider a fully convergent approach to prepare C-linked AFGP analogues. The convergent approach necessitates construction of a polypeptide backbone and simultaneous removal of multiple tert-butylcarbamate protecting groups followed by coupling of the resulting -amino termini with multiple C-linked saccharide derivatives in a single step as illustrated in Figure 3. Preparation of the orthogonally protected tripeptide building block 7 is necessary to prepare the resin-bound polypeptide backbone (Scheme 1). Building block 7 was prepared by reaction of commercially available tert-Boc-glycine with 4 in the presence of 1,1′-carbonyldiimidazole (CDI), N,Ndiisopropylethylamine, and dichloromethane to furnish dipeptide 5 in 90% yield. Treatment of 5 with TFA/CH2Cl2 followed by another CDI-mediated coupling with comL-threonine
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Antifreeze Glycoprotein Analogues Scheme 1. Synthesis of Tripeptide Building Block
Scheme 2. Convergent Solid Phase Synthesis of C-Linked Mimics of AFGP
mercially available lysine derivative 6 produced tripeptide 7 in 87% overall yield. Debenzylation of 7 was accomplished using hydrogen in the presence Pd/C catalyst and resulted in the formation of 8 in nearly quantitative yield. The choice of resin is crucial for a convergent approach since it is necessary to selectively remove the tert-butyl and Fmoc-carbamate residues without cleaving the peptide from the resin. Therefore, a commercially available base labile HMBA resin was chosen. The resin was first loaded with Fmoc-Gly-OH using diisopropylcarbodiimide (DIC) as coupling agent and the loading verified using standard protocols. After removal of the Fmoc group, the polypeptide backbone
was assembled using tripeptide 7 and O-(7-azabenzotriazol1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) as coupling agent (Scheme 2). A 20% solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in N,N-dimethylformamide was used for successive removal of the Nterminal Fmoc protecting group. This coupling sequence was repeated three to six times and furnished polypeptide backbones of 12 or 21 amino acid residues after the addition of two glycine residues to the N-terminus. Each polypeptide contained three or six L-lysine residues. Treatment of the resin-bound peptide with a 20% solution of TFA in dichloromethane for 30 min removed all
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tert-butylcarbamate protecting groups on the -amino termini. The requisite C-linked galactose tetraacetate derivative (1) was prepared as previously described by our laboratory.15 Each resin-bound polypeptide was then treated with 1 (either 4 or 8 equiv), HATU and N,N-diisopropylethylamine in DMF for 2-3 h at room temperature after which time resin tests (TNBS and Kaiser) indicated that the couplings were complete. Cleavage of the glycopeptides from the resin using hydrazine and concomitant removal of the acetate protecting groups yielded 9 (approximately 1.6 kDa) and 10 (approximately 3.0 kDa) in 44% and 26% yields, respectively, after purification by reversed-phase HPLC. In summary, we have demonstrated that complex C-linked glycoconjugates ranging in molecular weight from 1.6 to 3.0 kDa can be easily prepared using a truly convergent solid phase synthesis. In this approach, multiple (three or six) saccharide residues are covalently attached to a resin-bound polypeptide backbone. These glycopeptides are structurally diverse C-linked analogues of native AFGP 7-8 and are currently being assessed for antifreeze protein-specific activity in our laboratory. The results of these studies will be presented in a future publication. Experimental Section General Data. All glycopeptides were prepared on HMBA-MBHA resin (Novabiochem) using standard SPS protocols.18 Anhydrous DMF was purchased from Sigma/ Aldrich and was used without further purification. A ninhydrin assay (Kaiser test) and trinitrobenzenesulfonic acid (TNBS test) were used to monitor each coupling reaction. All couplings were carried out using HBTU as a coupling reagent in the presence of DIPEA and DMF. Tripeptide 7, dipeptide 5, and saccharide derivative 1 have been previously prepared and characterized.15 Compound 9. MBHA-HMBA resin (150 mg, 0.7 mmol/g loading) was swollen in dry DMF (15 mL) for 30 min. The DMF was drained and the resin washed three times (DMF, 3 × 10 mL). FmocGlyOH (187 mg, 0.63 mmol), HOBt (80 mg, 0.63 mmol), 1,3-diisopropylcarbodiimide (80 mg, 0.63 mmol), and DMAP (catalytic) were dissolved in DMF (15 mL). This solution was then added to the resin and agitated at room temperature for 12 h. The DMF solution was drained away and the resin washed with 3 × 15 mL of dry DMF. The resin loading was determined using UV spectrophotometry. A 20% solution (v/v) of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF (15 mL) was then added and the resin was agitated for 30 min. The resin was washed with DMF (3 × 10 mL) and Kaiser and TNBS tests confirmed removal of the N-terminal Fmoc carbamate. FmocLys(Boc)GlyGlyOH (7, 200 mg, 0.34 mmol), Obenzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, 120 mg, 0.32 mmol), and N,N-diisopropylethylamine (40 mg, 0.31 mmol) were dissolved in DMF (15 mL) and this solution was added to the resin. After 4 h, both Kaiser and TNBS tests confirmed that the coupling was complete. The DMF solution was drained and the resin washed with 3 × 10 mL of DMF followed by treatment with a 20% (v/v) DBU solution in dry DMF (15 mL) for 30 min.
Eniade and Ben
Kaiser and TMBS tests confirmed removal of the N-terminal Fmoc-carbamate. The above coupling sequence was repeated two times. After removal the N-terminal Fmoc-carbamate a solution of FmocGlyOH (187 mg, 0.63 mmol), HBTU (80 mg, 0.62 mmol), and N,N-diisopropylethylamine (80 mg, 0.63 mmol) in DMF (15 mL) was added to the resin. Agitation continued for 2 h after which time Kaiser and TNBS test confirmed the coupling was complete. The N-terminal Fmoc-carbamate was removed (20% DBU in 15 mL of DMF) and the final FmocGlyOH residue was added. The resin was washed with DMF (3 × 10 mL) and dichloromethane (2 × 10 mL) and a 20% (v/v) solution of trifluoroacetic acid in 15 mL of dry DMF was added to the resin and the mixture was agitated for 30 min. A Kaiser test confirmed removal of the tert-Boc-carbamate protecting groups on the -amino termini. The resin was washed with 3 × 10 mL of DMF, and a solution of 1 (200 mg, 0.51 mmol), HBTU (200 mg, 0.53 mmol), and N,N-diisopropylethylamine (100 mg, 0.77 mmol) in DMF (15 mL) was added and the mixture agitated for 4 h after which time Kaiser and TNBS tests confirmed the reaction was complete. The resin was washed (3 × 10 mL DMF) and a solution of 20% DBU (v/v) in DMF (15 mL) was added for 30 min. Acetic anhydride (300 mg, 2.9 mmol) and pyridine (250 mg, 3.1 mmol) in dry DMF were added to the resin and agitated for 20 min. The solution was drained, and the resin was washed with 3 × 10 mL of DMF and then suspended in a 1:1 and dichloromethane/methanol solution (15 mL). Anhydrous hydrazine (80 mg, 2.5 mmol) was added and the mixture agitated for 4 h. The solution was drained and the filtrate was placed into dialysis tubing (500 MW cutoff) and dialyzed in a 1:1 methanol/water solution overnight. Concentration of the solution from the dialysis tubing and purification by reversed-phase HPLC produced 37 mg of 9 (44% yield) as a white solid. 1H NMR (300 MHz, D2O), δ: 4.48 (2H, m), 4.32 (2H, d, J ) 5.0 Hz), 4.00 (18H, m), 3.863.67 (12H, m), 3.34 (4H, s), 3.19 (6H, br s), 2.59 (6H, m), 2.05 (2H, d, J ) 4.3 Hz), 1.92 (4H, d, J ) 10.6 Hz), 1.68 (6H, br s), 1.52 (6H, m), 1.40 (6H, br s). 13C NMR (75 MHz, D2O), δ: 174.6, 173.1, 171.6, 72.6, 72.1, 69.3, 68.4, 67.3, 60.5, 53.6, 42.0, 40.9, 38.8, 32.0, 31.9, 30.0, 27.5, 22.1. LRMS (electrospray, methanol, positive ion mode, m/z): calculated for C62H105N17O31 (M+ + H), 1584.7; found, 1584.9. Compound 10. The requisite peptide backbone was prepared using the same protocol outlined for the preparation of 9 except that six successive coupling/deprotection sequences using building block 7 were employed. After coupling of two additional FmocGlyOH residues to the N-terminus of the resin-bound peptide, the resin was treated with a 20% (v/v) solution of trifluoroacetic acid in 15 mL of dry DMF, and the mixture was agitated for 30 min. A Kaiser test confirmed removal of the tert-Boc-carbamate protecting groups on the -amino termini. The resin was washed with 3 × 10 mL of DMF, and a solution of 1 (300 mg, 0.79 mmol), HBTU (300 mg, 0.79 mmol), and N,Ndiisopropylethylamine (150 mg, 1.16 mmol) in DMF (15 mL) was added and the mixture agitated for 4 h after which time Kaiser and TNBS tests confirmed the reaction was complete.
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The resin was washed (3 × 10 mL DMF) and a solution of 20% DBU (v/v) in DMF (15 mL) was added for 30 min. Acetic anhydride (300 mg, 2.9 mmol) and pyridine (250 mg, 3.1 mmol) in dry DMF was added to the resin and agitated for 20 min. The solution was drained, and the resin was washed with 3 × 10 mL of DMF and then suspended in a 1:1 and dichloromethane/methanol solution (15 mL). Anhydrous hydrazine (80 mg, 2.5 mmol) was added and the mixture agitated for 4 h. The solution was drained, and the filtrate was placed into dialysis tubing (500 MW cutoff) and dialyzed in a 1:1 methanol/water solution overnight. Concentration of the solution from the dialysis tubing and purification by reversed-phase HPLC produced 43 mg of 10 (26% yield) as a white solid. 1H NMR (300 MHz, D2O), δ: 4.48 (2H, m), 4.24 (2H, br s), 4.20-3.68 (45H, m), 3.19 (9H, br s), 2.93 (s, 8H), 2.60 (m, 8H), 2.12 (s, 6H), 1.97 (6H, m), 1.75 (12H, br s), 1.50 (12H, br s), 1.33 (12H, br s). 13C NMR (75 MHz, D O), δ: 174.6, 174.5, 173.5, 173.1, 2 171.6, 171. 4, 169.8, 169.5, 72.6, 72.1, 69.4, 68.4, 67.3, 62.5, 60.5, 53.6, 52.9, 46.8, 44.1, 42.9, 42.2, 42.1, 40.9, 38.8, 38.6, 32.1, 32.0, 31.1, 30.7, 30.1, 28.2, 27.6, 27.4, 22.0; LRMS (electrospray, methanol, positive ion, m/z): calculated for C116H195N29O58 (M+ + H), 2922.8; found, 2922.7. Acknowledgment. The authors wish to thank the donors of the Petroleum Research Fund (ACS-PRF 35280-G1), administered by the American Chemical Society, and AF Protein Inc. for funding, the National High Magnetic Field Laboratory (NHMFL) in Tallahassee, FL for access to highfield NMR, and Dr. Jim Kerwin (Cornell University) for IT-ES and MALDI analysis. References and Notes (1) (a) Dwek, R. A. Chem. ReV. 1996, 96, 683. (b) Varki, A. Glycobiology 1993, 3, 97. (2) (a) Unverzagt, U. Carbohydr. Res. 1998, 305, 423. (b) Seitz, O.; Wong, C.-H. J. Am. Chem. Soc. 1997, 119, 8766. (c) Lundquist, J. J.; Debenham, S. D.; Toone, E. J. J. Org. Chem. 2000, 65, 8245. (d) Bertozzi, C. R.; Cook D. A.; Kobertz, W. R.; Gonzalez-Scarano, F.; Bednarski, M. D. J. Am. Chem. Soc. 1992, 114, 10639. (e) Toyokuni, T.; Dean, B.; Cai, Shaopei, C.; Boivin, D.; Hakomori, S.-I.; Singhal, A. K. J. Am. Chem. Soc. 1994, 116, 395. (f) Mortel, K. H.; Weatherman, R. V.; Kiessling L. L. J. Am. Chem. Soc. 2000, 118, 2297. (g) Wong, C.-H.; Moris-Varas, F.; Hung, S.-C.; Marron. T. G.; Lin, C.-C.; Gong, K. W.; Weitz-Schmidt, G. J. Am. Chem. Soc. 1997, 119, 8152. (3) Kunz, H. Angew. Chem., Int. Ed. Engl. 1987, 26, 294 and references therein. (4) (a) Hoogerjout, P.; Guis, C. P.; Erkelens, C.; Blowmoff, W.; Kerling, K. E. T.; van Boom, J. H. Recl. TraV. Chim. Pays-Bas 1985 104, 54 and references therein. (b) Anisuzzaman, A. K. M.; Anderson, L.; Navia, J. L. Carbohydr. Res. 1988 174, 265. (c) Tsuda, T.; Nishimura, S. I. Chem Commun. 1996, 2779. (d) Lacombe, J. M.; Pavia, A. A. J. Org. Chem. 1983, 48, 2557. (e) Paulsen, H.; Schultz, M.; Klamann, J. D.; Waller, B.; Pall, M. Liebigs Ann. Chem. 1985, 2028. (f)
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Lavielle, S.; Ling, N. C.; Saltman, R.; Guillemin, R. C. Carbohydr. Res. 1981 89, 229. (g) Garg, H. G.; Hasenkamp, T.; Paulsen, H. Carbohydr. Res. 1986 151, 225. (h) Ferrari, B.; Pavia, A. A. Int. J. Peptide Protein Res. 1983 22, 549. (i) Kim. J. M.; Roy, R. Tetrahedron Lett. 1997 38, 3487. (j) Bencomo, V. V.; Sinay, P. Glycoconjugate, J. 1984 1, 5. (k) Gobbo, M.; Biondi, L.; Filiar, F.; Rocchi, R.; Lucchini, V. Tetrahedron 1988, 44, 887. (a) Garg, H. G.; Jeanloz, R. W. Carbohydr. Res. 1979 76, 85. (b) Lancombe, J. M.; Pavia, A. A. J. Org. Chem. 1983, 48, 2557. Paulsen, H.; Schultz, M.; Klamann, J.-D.; Waller, B.; Paal, M. Liebigs Ann. Chem. 1985, 2028. Maeji, N. J.; Inoue, Y.; Chujo. R. Carbohydr. Res. 1986, 146, 174. a) Carboni, B.; Carreaux, F.; Pilard, J. F. Actual. Chim. 2000, 9. (b) Andres, C. J.; Denhart, D. J.; Deshpande, M. S.; Gillman, K. W. Comb. Chem. High Throughput Screening 1999, 2, 191. (c) Gordon, K.; Balasubramanian, S. J. Chem. Technol. Biotechnol. 1999, 74, 835. (d) Kingsbury, C. L.; Mehrman, S. J.; Takacs, J. M. Curr. Org. Chem. 1999, 3, 497. (e) Sucholeiki, I. Mol. DiVersity 1998, 4, 25. (a) Lavielle, S.; Ling, N. C.; Guillemin, R. C. Carbohydr. Res. 1981 89, 221. (b) Chan. T. Y.; Chen, A.; Allanson, N.; Chen, R.; Liu, D.; Sofia, M. J. Tetrahedron Lett. 1996 37, 8097. (a) Hollosi, M.; Kollat, E.; Laczko, I.; Medzihradszky, K. F.; Thurin, J.; Otvos, L. Tetrahedron Lett. 1991, 32, 1531. (b) Valentijin, A. R. P. M.; van der Marel; Sliedregt, L. A. J. M.; van Berkel, T. J. C.; Biessen, E. A. L.; Van Boom, J. H. Tetrahedron 1997 53, 759. (c) Vetter, D.; Tumelty, D.; Singh, S. K.; Gallop, M. A. Angew. Chem., Int. Ed. Engl. 1995, 34, 60. (d) Cohen-Anisfeld, S.; Lansbury, P. T., Jr. J. Am. Chem. Soc. 1993, 115, 10531. (e) For a convergent approach where an oligosaccharide is attached to a resin, see: Roberge, J. Y.; Beebe, X.; Danishefsky, S. J. J. Am. Chem. Soc. 1998, 120, 3915. (f) Arya, P.; Kristina, M. K. K.; Barnes, M. L. J. Comb. Chem. 1999, 1, 28. (a) Yeh, Y.; Feeney, R. E. Chem. ReV. 1996, 96, 601. (b) Davies, P. L.; Sykes, B. D. Curr. Opin. Struct. Biol. 1997, 7, 824. (c) Ewart, K. V.; Lin, Q.; Hew, C. L. Cell. Mol. Life Sci. 1999, 55, 271. (d) Morris, H. R.; Thompson, M. R.; Osuga, D. T..; Ahmed, A. I.; Chan, S. M.; Vandenheede, J. R.; Feeney, R. E. J. Biol. Chem. 1978 253, 5155. (e) Hew, C. L.; Slaughter, D.; Fletcher, G.; Shashikant, J. B. Can. J. Zool. 1981, 59, 2186. (f) Raymond, J. A.; Lin, Y.; DeVries, A. L. J. Exp. Zool. 1975, 193, 125. (a) Knight, C. A. Nature 2000, 406, 249. (b) Ananthanarayanan, V. S. Life Chem. Rep. 1989, 7, 1. (c) Wilson, P. Cryo-Lett. 1993, 14, 31. (d) Chao, H.; Houston, M. E., Jr.; Hodges, R. S.; Kay, C. M.; Sykes, B. D.; Loewen, M. C.; Davies, P. L.; Sonnichsen, F. D. Biochemistry 1997, 36, 6, 14652. (e) Haymet, A. D. T.; Ward, L. G.; Harding, M. M. J. Am. Chem. Soc. 1999, 121, 941. (a) Tablin, F.; Oliver, A. E.; Walker, N. J.; Crowe, L. M.; Crowe, J. H. J. Cell. Physiol. 1996, 168, 305. (b) Hays, L. M.; Feeney, R. E.; Crowe, J. H.; Oliver, A. E. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6835. (c) Pham, L.; Dajiya, R.; Rubinsky, B. Cryobiology 1999, 38, 1. (d) Koushafar, H.; Pham, L.; Lee, C.; Rubinsky, B. J. Oncology 1997, 66, 114. (e) Griffith, M.; Ewart, K. V. Biotechnol. AdV. 1995, 13, 375. Jiaang, J. W.; Hsiao, K. F.; Chen, S. T.; Wang, K. T. Synthesis 1999, 9, 1687. Ben, R. N.; Eniade, A.; Hauer, L. Org. Lett. 1999, 1, 1759. Elofsson, M.; Salvador, L. A.; Kihlberg, J. Tetrahedron Lett. 1997, 53, 369 and references therein. Wierzbicki, A.; Knight, C. A.; Rutland, T. J.; Muccio, D. D.; Pybus, B. S.; Sikes, C. S. Biomacromolecules 2000, 1, 268. 1999 NoVabiochem Catalog and Peptide Synthesis Handbook; Calbiochem-Novabiochem AG: Laufelfingen, Switzerland, 1999.
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