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The Use of Carbohydrate Protein Conjugates of Proteases [CPC(Proteases)] for the Catalytic Formation of Peptide Bonds

Published online by Cambridge University Press:  15 February 2011

Peng Wang ,
Tara G. Hill ,
Mark D. Bednarski  and
Matthew R. Callstrom
Peng Wang
Affiliation:
University of California at Berkeley, Department of Chemistry, Berkeley, CA 94720 Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley, CA 94720
Tara G. Hill
Affiliation:
The Ohio State University, Department of Chemistry, Columbus, OH 43210
Mark D. Bednarski
Affiliation:
University of California at Berkeley, Department of Chemistry, Berkeley, CA 94720 Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley, CA 94720
Matthew R. Callstrom
Affiliation:
The Ohio State University, Department of Chemistry, Columbus, OH 43210 Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley, CA 94720
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The discovery of catalysts that can selectively couple unprotected peptide fragments would revolutionize protein chemistry by allowing convergent polypeptide synthesis. Proteolytic enzymes have the capability to perform this chemistry because the protein can specifically recognize and bind to C-terminal and N-terminal peptide sequences, activate the C-terminal peptide sequence by forming an acyl-enzyme intermediate, and couple the two peptide fragments together. However, barriers that limit the use of proteases as catalysts for convergent peptide synthesis include (i) the stability of proteolytic enzymes in organic solvent systems; (ii) a simple and effective C-erminal and N-terminal protecting group strategy; and (iii) the isolation of the polypeptide product from the reaction mixture. In the previous paper we reported the stabilization of enzymes by the covalent attachment of proteins through their ο-lysine residues to a series of carbohydrate-based macromolecules. In this paper we report the use of carbohydrate protein conjugates of proteases [CPC(proteases)] as catalysts for peptide bond synthesis and a general strategy for convergent oligopeptide synthesis.

Type
Research Article
Information
MRS Online Proceedings Library (OPL) , Volume 218: Symposium V – Materials Synthesis Based on Biological Processes , 1990 , 23
Copyright
Copyright © Materials Research Society 1991

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References

References and Footnotes

1. Nakarsuka, T.; Sasaki, T.; Kaiser, E. T. J. Am. Chem. Soc. 1987, 109, 3808. Kaiser, E. T.; Mihara, I-L; Laforet, G. A.; Kelly, J. W.; Waiters. L.; Findeis, L.A.; Sasaki, T. Science 1989, 243, 187.CrossRefGoogle Scholar
2. Barbas, C. F.; Matos, L. R.; West, J. B.; Wong, C. -H. J. Am. Chem. Soc. 1988, 110, 5162.CrossRefGoogle Scholar
3. Kltagucli, I-L; Klibanov, A. M. J. Am. Chem. Soc. 1989, 111. 9272.Google Scholar
4. Kemp, D. In The Pepridesr Underfriend, S.; Meienhofer, J. Eds.; Academic Press: San Diego: 1979; Vo1. 1.Google Scholar
5. Bergmann, M.; Fraenkel-Conrat, I. J. Bio. Chem. 1937, 119, 707. Bergmann, M.; Fraenkei-Conrr, J. Ibid. 1938, 124, 1.CrossRefGoogle Scholar
6. Kullman, W. Enzymatic Peptide Synthesis; CRC Press: Boca Raton: 1987. Jakubke, H-D. In The Peptides, Underfriend, S.; Meienhofer, J. Eds.; Academic Press: San Diego; 1987; Vol. 9, Chapter 3.Google Scholar
7. Jaknbke, H. D.; Kuhl, P.; Konnecke, A. Angew. Chem., Int. Ed. Engl. 1985, 24, 85.Google Scholar
8. Sakina, K.; Kawazurn, K.; Morihara, K.; Yajima, H. Chem. Pharmn. Bull. 1988, 36, 3915. Sakina. K.; Kawazura. K.; Moraara. K., Yajima, H. Ibid. 1988. 36, 4345. Morihara. K. Trends Biotechnol. 1987, 5, 164. Morihara, K.; Oka. T.Biochem. J. 1977, 163, 531.CrossRefGoogle Scholar
9. For examples, see: (a) Cabarer, D.; Maillot, S.; Wakselman, M. Terrahedron Lett. 1990, 31, 2131. (b) Wong, C. -H.; Chen, S.-T.; Hennen. W. J.; Bibbs, J. A.; Wang, Y.-F.; Lui. J. L.-C.; Pantoliano, M. W.; Whitlow, M.; Bryan, P. N. J. Am. Chem. Soc. 1990, 112, 945. (c) Gaermer, H.; Puigsever, A. Eur. J. Biochem. 1989, 181, 207. (d) Ricca. J. IIA; Crout. D. H. G. J. Chem. Soc., Perkin Trans. I 1989, 2126. (e) Cassells. J. M.; Hailing, P. I. Biotechnol. Bioeng. 1989, 33, 1489. (f) Scholten, J. Stolowich. N, J.; Hogg, I. L., Scott, A. I.; Wong, C. -H. J. Am. Chem. Soc. 1988, 110, 3709. (g) Barbas, C. F., III; Matos, L R.; Wong, C. -H. J. Am. Chem. Soc. 1988, 110, 5162. (h) Cassells, J. M.; Halling, P. J. Enzyme Microb. Technol. 1988, 10, 486. (i) Ferjancic, A.; Puigserver. A.; Gaermer, H. Biorechnol. Lett. 1988, 10, 101. (j) Cheng. a.; Dc Miranda, M. T. M..; Tominaga, M. Int. J. Peptide Protein Res. 1988, 31, 116. (k) Kamibira, M.; Taniguchi, M.; Kobayashi, T. Agric. Biol. Chem. 1987, 51, 3427. (j) Barbas, C. F., III; Wong, C. -H.; J. Chem. Soc., Chem. Commun. 1987, 533. West, J. B.; Wong, C.-H. Terrahedron Lett. 1987, 28, 1629. (l) Margolin, A. L.; Tai, D. F.; Klibanov, A. M. J. Am. Chem. Soc. 1987, 109, 7885. (m) Margolin, A. L.; Klibanov, A. M. J. Am. Chem. Soc. 1987, 109, 3802. (n) De Miranda. M. T. M.; Cheng, E.; Muradian, J.; Seidel, W. F.; Tominaga, M. Bioorg. Chem. 1986, 14, 182. (o) Riechman. L.; Kasche. V. Biochim. Biophys. Acta 1986, 872, 269. (p) Nakanishi, K.. Marsuno. R. Eur. J. Biochem. 1986, 161. 533. (q) Nakanishi, K.: Kamikubo, T.; Matsuno, R. BiolTechnoiogy 1985, 3, 459. (r) Ooshima. H.; Mori, H.: Harano. Y. Biorechnol. Lett. 1985, 7, 789. (s) West, J. B.; Luthi, P.; Luisi, P. L J. Am. Chem. Soc. 1984, 106, 7285. (t) Oyama, K.; Nishimura, S.; Nonakca. Y.; Kihara. K.; Hashimoto, T. J. Org. Chem. 1981, 46, 5241. (u) Oyama, K.; Kihara, K.; Nonaka, Y. J. Chem. Soc., Perkin Trans. II 1981, 356. (v) Oka, T.; Morihara. K. J. Biochem. 1978, 84, 1277. (w) Morihara, K.; Oka, T. Biochern. J. 1977, 163, 531.CrossRefGoogle Scholar
10. Organic solvent systems are required to prevent enzymatic hydrolysis of the starting peptide fragments and also to promote peptide bond formation.5 Google Scholar
11. Hill, T. G.; Wang, P. W.; Oehler, L.M.; Huston, M. E.; Warrchow, C. A.; Smith, M. B.; Bednarski, M. D.; Cailstrom, M. R. previous paper in this issue.Google Scholar
12. The 1-CPC(ENZYME) materials were purified by gel filtration chromatography using 0.05 M sodium borate solution at pH 8 on Sephacryl HR-200 at a flow rare of 1.5 mL/min. Alternatively, isolation by dialysis of the reaction solution using Spectra Por CE 100K MWCO membrane against 2 × 250 mL of 0.05 M sodium borate at pH 8 for approximately 12 h gave approximately 40% yields for α-chymotrypsin (E.C. 3.4.21.1, Sigma) and thermolysin (Type X, Sigma) conjugates and apprioximately 10% yields (80% recovered activity) for subtilisin BPN' (Type XXVII, Sigma) conjugates. The yields were determined by measurement of their relative activity with N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide as the substrate in 0.05 M sodium borate at pH 8 containing 10% methanol.Google Scholar
13. Previous investigations using α-chymotrypsin as a catalyst have focused on the use of water immiscible organic solvents such as toluene or methylene chloride, or water miscible systems such as mixtures of dimethyl formamide in water. The problem with the former system is that most large peptide fragments are not soluble in water immiscible solvent systems while the problem with the latte approach is that occurs if the reaction is allowed to proceed to completion. The use of high concentrations of organic solvents in which peptide fragments art soluble would eiminate these problems, but most proteases are not highly catalytically actve-in organic media.Google Scholar
14. Barany, G.; Merrifield, R. B. In The Peprider, Gross, E., Meienhofer, J., Eds.; Academic Press: New York, 1980; Vol.2 p 1.Google Scholar
15. All compounds were fully characterized by 1H and 13C NMR and high resolution mass spectroscopy. N-Ac-L-Phe-L-Ala-NH2: mp 234–235 °C (lit. 9m 232–234 °C); [α]25D +3.4° (c 1.62, MeOH) (lit.9m [α]25D +5.4° (c 0.33, MeOH)); 1H NMR (500 MHz, D2O) δ 1.13 (d. 3 H), 1.77 (s. 3 H), 2.88 (m, 2 H), 4.06 (q, 1 H), 4.37 (t, 1 H), 7.15 (m. 5 H); 13C NMR (125 MHz, CD3OD) δ 16.61, 20.83, 37.20, 54.76. 126.33, 127.98. 128.79. 136.89, 171.89: HRMS FAB m/z for C14H2ON3O3, calcd 278.1504 [(M+H)+], found 278.1515. N-Cbz-L-Leu-L-Leu-L-Phe-L-Leu-O'Bu: mp 185–187 °C, [α]25D-59.17° (c 0.60, MeOH). 1H NMR (500 MHz, CDCl3): δ 0.84–0.96 (m, 18 H), 1.44 (s. 9 H). 1.47–1.56 (m. 9 H), 2.72 (dd, 1 H), 3.04 (m. 1 H), 3.65 (m. 1 H), 4.13 (m 1 H), 5.10 (dd. 2 H), 6.53 (m. 1 H), 6.81 (d, 1 H), 7.66 (d, 1 H), 7.18–7.36 (m, 10 H); 13C NMR (125 MHz. CD3OD) δ 20.44, 21.81. 21.86. 21.94, 24.26, 24.32, 26.72, 29.14, 40.21, 40.32, 51.43, 53.61, 53.88, 66.22, 81.17, 126.19, 127.28, 127.50, 127.89, 127.96, 128.84; HRMS FAB m/z for C39H59N4 O7, calcd 695.4383 [(M+H)+], found 695.4364. N-Cbz-L-Val-L-Leu-L-Phe-L-Leu-O'Bu: mp 184–186 °C, [α]25D +3.4° (c 1.62, MeOH). 1H NMR (500 MHz, CDCl3 ): δ 0.86–0.93 (m, 18 H), 1.45 (s, 9 H), 1.47–1.56 (m, 6 H), 2.10 (m. 1 H), 3.04–3.12 (m, 3 H), 3.61 (q, 1 H), 3.99 (t, 1 H), 4.40 (m, 2 H), 5.12 (m, 1 H), 5.32 (q, 2 H), 5.64 (d. 1 H), 7.18–7.36 (m, 10 H); 13C NMR (125 MIz, CD3OD): δ 17.12. 18.31, 20.42, 21.81, 21.86, 24.24, 24.32, 26.73, 30.29, 37.14, 40.24, 40.36.51.37,51.70, 53.93, 60.80, 66.26, 81.20. 126.18. 127.32- 127.51, 127.89, 127.96, 128.82- 129.06, 136.64, 136.81: BRMS FAB m/z for C38H57N4O7, calcd 681.4226 ((M+H)+], found: 681.4204. N-Cbz-L-Phe-L-Leu-OMe: mp 101–103 °C. [α]25D-25.2° (c 1.20. MeOH); 1H NMR (400 MHz. CD3OD) δ 0.75 (dd, 6 H), 1.61 (m, 3 H), 2.83 (dd. 1 H), 3.12 (dd. I H), 3.67 (s, 3 H), 4.44 (m, 2 H), 5.00 (s, 2 H), 7.18–7.32 (m. 10 H); 13C NMR (125 MHz, CD3OD) δ 20.16, 21.64, 24.15, 37.47, 39.85,50.48, 50.52, 50.89,51.23, 51.56,55.88. 55.95, 65.69, 65.87, 66.06. 125.92. 126.24. 126.89, 127.20, 127.61. 127.95. 123.61. 128.92, 136.57, 136.85, 156.54, 172.61, 172.72, HRMS FAB m/z for C24H31N2O5, calcd 427.2232 ((M+H)+], found 427.2238. N-Cbz-L-Phe-L-Leu-O'Bu: mp 85–87 °C, [α]25D -24.7° (c 0.91, MeOH); 1H NMR (400 MRz, CD3OD) δ 0.92 (dd., 6 H), 1.45 (s, 9 H), 1.56–1.67 (m, 3 H), 2.85 (dd, 1 H), 3.17 (dd, 1 H), 4.33 (dd, 1 H), 4.44 (dd, 1 H), 5.00 (s, 2 H), 7.19–7.32 (m, 10 H); 13C NMR (125 MHz. CD3OD) δ 20.28, 20.37, 21.63, 21.70, 24.24, 24.39, 26.40, 26.54, 26.69, 26.83, 37.53, 40.08, 51.26. 51.35, 51.49, 55.88, 65.69, 65.87, 66.05, 81.07, 125.91, 126.25, 126.88, 127.20, 127.62, 127.95, 128.59, 128.91. 136.56, 136.91. 156.54, 171.59. 172.49, 172.56; HRMS FAB m/z for C27H37N2O5, calcd 469.2702 [(M+H)+], found 469.2708. N-Boc-L-Met-L-Leu-L-Phe-L-Phe-L-Leu-NH2: mp 235–237°C. [α]25D −59.17° (c 0.60. MeOH); 1H NMR (400 MHz, CD3OD) δ 0.89 (m. 12 H), 1.41 (s, 9 H), 1.55 (m. 6 H), 2.04 (s. 3 H), 2.49 (m. 2 H), 2.84–3.01 (m, 4 H), 4.13 (dd. 1 H), 4.22 (dd, 1 H), 4.30 (dd. 1 H), 4.41 (t. 1 M), 4.52 (t. 1 H), 7.02–7.38 (m. 10 H); 13C NMR (125 MHz. CD3OD) δ 13.81, 20.40, 21.68, 24.05, 27.02, 27.17, 27.31, 29.54, 30.63, 36.63, 40.18. 51.46. 52.38, 53.83, 55.01, 79.34, 127.84, 128.02, 128.65, 128.97, 136.35, 136.79, 171.69, 171.94, 173.41, 173.81; HRMS FAB m/z for C40H60N6O7S, calcd 791.4141 [(M+Na)+], found: 791.4160.Google Scholar
16. In some cases the use of a large excess of the protein catalyst can compensate for its low catalytic activity and high rate of decompositon in organic solvents. We felt that this approach is not practical for the synthesis of large peptides since the isolation of the polypeptide from the enzyme would be difficult. In addition, we observed low turnover numbers for proteases in acetonitrile and dioxane. even at high enzyme concentrations, which led to a unacceptable yields of coupled peptides (0–10%).Google Scholar
17. An analytically pure sample was obtained by HPLC using a C18 column with a 40% aqueous solution of methanol as the the mobile phase.Google Scholar
18. DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 1982, 47, 3258.Google Scholar
19. Isowa, Y.; Ohmori, M.; Ichikawa, T.; Karita, H; Sato, M.; Mori, K. Bull. Chem. Soc. Jpn. 1977, 50, 2762. Isowa, Y.; Ichikawa, T.; Ohfiori, M. Ibid. 1978, 51, 271. Isowa, Y.; Ichikawa, T. Ibid. 1979, 52, 796. Isowa, Y.; Ohmori, M.; Ichikawa, T; Mori, K.; Nonaka. Y.; Kihara. K.; Oyama. K. Satoh, H.; Nishimura, S. Teirazedron Lett. 1979, 2611.CrossRefGoogle Scholar
20. Hudson, D. J. Org. Chem. 1988, 53, 617.CrossRefGoogle Scholar