Fmoc-based synthesis of glycolate ester peptides for the assembly of de novo designed multimeric proteins using subtiligase

Tetrahedron Letters

Volumn 37, Issue 37, 1996, Pages 6653-6656

  Suich, Daniel J ^a   Ballinger, Marcus D ^b   Wells, James A ^b   DeGrado, William F ^a  

^a DUPONT COMPANY   (United States)

^b GENENTECH INC   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

SUBTILIGASE; SUBTILISIN; UNCLASSIFIED DRUG;

ARTICLE; ENZYME ACTIVITY; PROTEIN SYNTHESIS; TECHNIQUE;

EID: 16044366985     PISSN: 00404039     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0040-4039(96)01444-X     Document Type: Article

References (20)

1. 1. Abrahamsén, L.; Tom, J.; Burnier, J.; Butcher, K. A.; Kossiakoff, A.; Wells, J. A. Biochemistry 1991, 30, 4151-4159.
2. 2. Jackson, D. Y.; Burnier, J.; Quan, C.; Stanley, M.; Tom, J.; Wells, J. A. Science 1994, 266, 243-247.
3. 3. Jackson, D. Y.; Burnier, J. P.; Wells, J. A. J. Am. Chem. Soc. 1995, 117, 819-820.
4. 4. Chang, T. K.; Jackson, D. Y.; Burnier, J. P.; Wells, J. A. Proc. Natl. Acad. Sci. USA 1994, 91, 12544-12548.
5. 5. The benzyl 2-bromoacetate used in this study was prepared in 57% yield by treatment of bromoacetic acid with DCC (1.2 equiv.), DMAP (0.3 equiv.), and benzyl alcohol (2 equiv.) in THF for 16 hr. Benzyl 2-bromoacetate is also available commercially from Aldrich.
6. 6. All compounds exhibited satisfactory spectroscopic data.
7. 7. Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.; Masada, R. I.; Hudson, D.; Barany, G. J. Org. Chem. 1990, 55, 3730-3743.
8. 1B-G3-expected: 2772.6, found: 2772.5.
9. 9. Lovejoy, B.; Choe, S.; Cascio, D.; McRorie, D. K.; DeGrado, W. F.; Eisenberg, D. Science 1993, 259, 1288-1293.
10. 10. Betz, S.; Fairman, R.; O'Neil, K.; Lear, J.; DeGrado, W. Phil. Trans. R. Soc. Lond. B 1995, 348, 81-88.
11. 11. Ho, S. P.; DeGrado, W. F. J. Am. Chem. Soc. 1987, 109, 6751-6758.
12. 1B-G3 reactions were run at a peptide concentration of 2.0 mM and a subtiligase concentration of 5.8 μM for 3.0 hr. Reactions were terminated by the addition of neat TFA to a final concentration of 2%(v/v). In each case, all or nearly all of the unligated peptide ester had hydrolyzed after the specified reaction time. Control reactions in which subtiligase was omitted yielded only hydrolyzed and unhydrolyzed monomer.
13. 1B-G3-hydrolyzed dimer expected: 5157.2, found: 5158.3; hydrolyzed trimer expected: 7726.8, found: 7728.6; cyclic dimer expected: 5139.2, found: 5140.8.
14. 14. The tester peptides were synthesized by the same methods described for the full length peptides. Acetylation was carried out using 0.5 M acetic anhydride and 0.5 M pyridine in DMF.
15. 15. See for example: (a) Wuttke, D. S.; Gray, H.; Fisher, S. L.; Imperiali, B. J. Am. Chem. Soc. 1993, 115, 8455-8456.
16. (b) Hoogerhaut, P.; Kerling, K. E. T. J. R. Neth. Chem. Soc. 1981, 100, 215-216.
17. (c) Hoogerhaut, P.; Kerling, K. E. T. J. R. Neth. Chem. Soc. 1982, 102, 246-253.
18. (d) Vogel, K.; Chmielewski, J. J. Am. Chem. Soc. 1994, 116, 11163-11164.
19. (e) Graf, L.; Li, C. H. Proc. Natl. Acad. Sci. USA 1981, 78, 6135-6138.
20. 16. This result is obtained for ligation reactions performed at CoilSer1 concentrations of 2.5-10 mM. As the peptide concentration is lowered to 1 mM and below, yields of dimer and trimer decrease; at a peptide concentration of 10 μM, no ligated products can be detected by HPLC.