Advertisement

In Situ Neutralization in Boc-chemistry Solid Phase Peptide Synthesis

Rapid, High Yield Assembly of Difficult Sequences
  • Martina Schnölzer
  • Paul Alewood
  • Alun Jones
  • Dianne Alewood
  • Stephen B. H. Kent
Article

Simple, effective protocols have been developed for manual and machine-assisted Boc-chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (>0.2 m) of Boc-amino acid-OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30 s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10 min coupling times were used throughout. Overall cycle times were 15 min for manual and 19 min for machine-assisted synthesis (75 residues per day). No racemization was detected in the .base-catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA-DMF; chain-termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc-amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for “difficult” sequences arising from sequence-dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc-chemistry SPPS protocols or in Fmoc-chemistry SPPS. Reported syntheses include HIV-1 protease(1–50,Cys.amide), HIV-1 protease(53–99), and the full length HIV-l protease(1–99).

Keywords

difficult sequences in situ neutralization ion-spray mass spectrometry solid phase peptide synthesis (SPPS) 

Abbreviations

ACP

acyl carrier protein

API

atmospheric pressure ionization

BOP

benzotriazolyloxy tris-(dimethylamino)phosphonium hexafluorophosphate

Boc

tert.-butyloxycarbonyl

DCC

dicyclohexylcarbodiimide

DCM

dichloromethane

DIC

diisopropylcarbodiimide

DIEA

diisopropylethylamine

DMF

N,N-di-methylformamide

Fmoc

fluorenylmethyloxycarbonyl

HBTU

2-(1-H-benzotriazol-l-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate

HIV-1 PR

human immunodeficiency virus type 1 protease

HOBt

1-hydroxybenzotriazole

HPLC

high performance liquid chromatography

SPPS

solid phase peptide synthesis

TFA

trifluoroacetic acid

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Notes

Acknowledgments

We thank G. Lu for excellent technical assistance in the machine-assisted syntheses, H.-R. Rackwitz for the syntheses of [l-Cys66]ACP(65–74) and [d-Cys66]ACP(65–74), S. Clark for the design of the 20 mL reaction vessel, and B. Garnham, M. Baca, S.C.F. Milton and R.C. deL. Milton for their helpful advice and discussions. Support of this work by the Markey Foundation is gratefully acknowledged. M. Schnölzer is supported by an AIDS Scholarship from the Bundesministerium fur Forschung und Technologie, Germany.

References

  1. Alewood, P., Croft, M., Schnölzer, M. and Kent, S.B.H.: 1991, in E. Giralt and D. Andreu (eds.), Peptides 1990, Proceedings of the 21st European Peptide Symposium, ESCOM, Leiden, pp. 174–175Google Scholar
  2. Bartl, R., Klöppel, K.-D. and Frank, R. (1992) in Peptides, Proceedings of the 12th American Peptide Symposium (Smith, J.A., Rivier, J.E., eds.), pp. 505–506, ESCOM, LeidenGoogle Scholar
  3. Bayer E. (1991) Angew. Chem. Int. Ed. Engl. 30:113–129CrossRefGoogle Scholar
  4. Bayer, E. and Goldammer, C.: 1992, in J.A. Smith and J.E. Rivier (eds.), Peptides, Proceedings of the 12th American Peptide Symposium. ESCOM, Leiden, pp. 589–590Google Scholar
  5. Beyermann M., Henklein P., Klose A., Sohr R., Bienert M. (1991) Int. J. Peptide Protein Res. 37:252–256CrossRefGoogle Scholar
  6. Briand, J.P., Coste, J., Van Dorsselaer, A., Raboy, B., Neimark, J., Castro, B. and Muller, S.: 1991, in E. Giralt and D. Andreu (eds.), Peptides 1990, Proceedings of the 21st European Peptide Symposium. ESCOM, Leiden, pp. 80–81Google Scholar
  7. Caporale L., Nutt R., Levy J., Smith J., Arison B., Bennett C., Albers-Schönberg G., Pitzenberger S., Rosenblatt M., Hirschmann R. (1989) J. Org. Chem. 54:343–346CrossRefGoogle Scholar
  8. Dimarchi R.D., Tam J.P., Kent S.B.H., Merrifield R.B. (1982) Int. J. Peptide Protein Res. 19:88–93CrossRefGoogle Scholar
  9. Due Larsen, B., Larsen, C. and Holm, A.: 1991, in E. Giralt and D. Andreu (eds.), Peptides 1990, Proceedings of the 21st European Peptide Symposium. ESCOM, Leiden, pp. 183–184Google Scholar
  10. Due Larsen, B., Holm, A., Christensen, D.H. and Faurskov Nielsen, O.: 1992, in R. Epton (ed.), Innovation and Perspectives in Solid Phase Synthesis 1992, Intercept, Andover, U.K., pp. 363–366Google Scholar
  11. Fields G.B., Otteson K.M., Fields C.G., Noble R.L. (1990) in Epton R. (ed.), Innovation and Perspectives in Solid Phase Synthesis SPCC Ltd, Birmingham, pp. 241–260Google Scholar
  12. Felix A.M., Wang C.-T., Heimer E.P., Fournier A. (1988) Int. J. Peptide Protein Res. 31:231–238CrossRefGoogle Scholar
  13. Gausepohl H., Kraft M., Frank R.W. (1989) Int. J. Peptide Protein Res. 34:287–294CrossRefGoogle Scholar
  14. Gausepohl H., Frank R.W., Rapp W., Bayer E. (1992) in R. Epton (ed) Innovation and Perspectives in Solid Phase Synthesis 1992, Intercept, Andover, U.K., pp. 381–385Google Scholar
  15. Gausepohl, H., Pieles, U. and Frank, R.W.: 1992, in J.A. Smith and J.E. Rivier (eds.), Peptides, Proceedings of the 12th American Peptide Symposium. ESCOM, Leiden, pp. 523–524Google Scholar
  16. Henklein, P., Beyermann, M., Costisella, B., Sohr, R. and Haußner. M.: 1992, in R. Epton (ed.), Innovation and Perspectives in Solid Phase Synthesis 1992, Intercept, Andover, U.K., pp. 401–403Google Scholar
  17. Hoeprich P., Zeiske, L., Chen, L., Salto, R., DeCamp, D.L. and Craik, C.S.: 1992, in J.A. Smith and J.E. Rivier (eds.), Peptides, Proceedings of the 12th American Peptide Symposium. ESCOM, Leiden, pp. 705–706Google Scholar
  18. Hudson D. (1988) J. Org. Chem. 53:617–624CrossRefGoogle Scholar
  19. Jezek, J. and Houghten, R.A.: 1990, in E. Giralt and D. Andreu (eds.), Peptides 1990, Proceedings of the 21st European Peptide Symposium. ESCOM, Leiden, pp. 74–75Google Scholar
  20. Kent, S.B.H., Hood, L.E., Beilan, H., Meister, S. and Geiser, T.: 1984, in U. Ragnarsson (ed.), Peptides 1984, Proceedings of the 18th European Peptide Symposium. Almquist, Wiksell International, Stockholm, pp. 185–188Google Scholar
  21. Kent S.B.H. (1988) Ann. Rev. Biochem. 57:957–989PubMedCrossRefGoogle Scholar
  22. Kent, S.B.H., Parker, K.F., Schiller, D.L., Woo, D.D.-L., ClarkLewis, I. and Chait, B.T.: 1988, in G.R. Marshall (ed.), Peptides: Chemistry and Biology, Proceedings of the 10th American Peptide Symposium. ESCOM, Leiden, pp. 173–178Google Scholar
  23. Kent, S.B.H., Alewood, D., Andrews, J.L., Bergman, D., Brinkworth, R. and Jones, A.: 1991, in E. Giralt and D. Andreu (eds.), Peptides 1990, Proceedings of the 21st European Peptide Symposium. ESCOM, Leiden, pp. 172–173Google Scholar
  24. Kent, S.B.H., Alewood, D., Alewood, P.F., Baca, M., Jones, A. and Schnölzer, M.: 1992, in R. Epton (ed.), Innovation and Perspectives in Solid Phase Synthesis 1992, Intercept, Andover, U.K., pp. 1–22Google Scholar
  25. Kent S.B.H., Merrifield R.B. (1983) Int. J. Peptide Protein Res. 22:57–65CrossRefGoogle Scholar
  26. Knorr R., Trzeciak A., Bannwarth W., Gillessen D. (1989) Tetrahedron Lett. 30:1927–1930CrossRefGoogle Scholar
  27. Le Nguyen, D., Seyer, R., Heitz, A. and Castro, B.: 1985, J. Chem. Soc. Perkin Trans. I, 1025–1031Google Scholar
  28. Le-Nguyen, D., Heitz, A. and Castro, B.: 1987, J. Chem. Soc. Perkin Trans. I, 1915–1919Google Scholar
  29. Mapelli, C. and Swerdloff M.D.: 1991, in E. Giralt and D. Andreu (eds.), Peptides 1990, Proceedings of the 21st European Peptide Symposium, ESCOM, Leiden, pp. 316–319Google Scholar
  30. Meister, S.M. and Kent, S.B.H.: 1984, in V.J. Hruby and D.H. Rich (eds.), Peptides: Structure and Function. Pierce Chemical Co., Rockford, IL, pp. 103–106Google Scholar
  31. Milton R.C. de L., Milton S.C.F., Adams P.A. (1990) J. Am. Chem. Soc. 112:6039–6046CrossRefGoogle Scholar
  32. Nakaie, C.R., Marchetto, R., Schreier, S. and Paiva, A.C. (1990) in J.E. Rivier and G.R. Marshall (eds.), Peptides, Proceedings of the 11th American Peptide Symposium. ESCOM, Leiden, pp. 1022–1023Google Scholar
  33. Nutt R.F., Brady S.F., Darke P.L., Ciccarone T.M., Colton C.D., Nutt E.M., Rodkey J.A., Bennett C.D., Waxman L.H., Sigal I.S., Anderson P.S., Veber D.F. (1988) Proc. Natl. Acad. Sci. USA 85:7129–7133PubMedCrossRefGoogle Scholar
  34. Reid G.E., Simpson R.J. (1992) Anal. Biochem. 200:301–309PubMedCrossRefGoogle Scholar
  35. Robey F.A., Fields R.L. (1989) Anal. Biochem. 177:373–377PubMedCrossRefGoogle Scholar
  36. Sarin V.K., Kent S.B.H., Tam J.P., Merrifield R.B. (1981) Anal. Biochem. 117:147–157PubMedCrossRefGoogle Scholar
  37. Schnolzer, M., Alewood, P., Jones, A. and Kent, S.B.H.: 1992, in J.A. Smith and J.E. Rivier (eds.), Peptides, Proceedings of the 12th American Peptide Symposium. ESCOM, Leiden, pp. 623–624Google Scholar
  38. Schnölzer, M., Jones, A., Alewood, P.F. and Kent, S.B.H.: 1992, Anal. Biochem., 204, 335–343Google Scholar
  39. Schnölzer M., Kent S.B.H. (1992) Science 256:221–225PubMedCrossRefGoogle Scholar
  40. Toth M.V., Marshall G.R. (1990) Int. J. Peptide Protein Res. 36:544–550CrossRefGoogle Scholar
  41. Van Woerkom W.J., van Nispen J.W. (1991) Int. J. Peptide Protein Res. 38:103–113Google Scholar
  42. Wlodawer A., Miller M., Jaskolski M., Sathyanarayana B.K., Baldwin E., Weber I.T., Selk L.M., Clawson L., Schneider J., Kent S.B.H. (1989) Science 245:616–621PubMedCrossRefGoogle Scholar
  43. Young J.D., Huang A.S., Ariel N., Bruins J.B., Ng D., Stevens R.L. (1990) Peptide Res. 3:194–200Google Scholar

Copyright information

© Blackwell Publishing 2007

Authors and Affiliations

  • Martina Schnölzer
    • 1
  • Paul Alewood
    • 2
  • Alun Jones
    • 1
  • Dianne Alewood
    • 2
  • Stephen B. H. Kent
    • 1
    • 3
  1. 1.The Scripps Research InstituteLa JollaUSA
  2. 2.Drug Design and Development CenterUniversity of QueenslandBrisbaneAustralia
  3. 3.ChicagoUSA

Personalised recommendations