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In Situ Neutralization in Boc-chemistry Solid Phase Peptide Synthesis

Rapid, High Yield Assembly of Difficult Sequences

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International Journal of Peptide Research and Therapeutics Aims and scope Submit manuscript

A Bruce Merrifield Commemorative Issue to this article was published on 05 December 2006

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).

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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

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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.

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Authors and Affiliations

  1. The Scripps Research Institute, La Jolla, CA, 92037, USA

    Martina Schnölzer, Alun Jones & Stephen B. H. Kent

  2. Drug Design and Development Center, University of Queensland, Brisbane, Queensland, Australia

    Paul Alewood & Dianne Alewood

  3. RoomW204, GordonCIS, 929East 57th Street, Chicago, IL, USA

    Stephen B. H. Kent

Authors
  1. Martina Schnölzer
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  2. Paul Alewood
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  3. Alun Jones
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  4. Dianne Alewood
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  5. Stephen B. H. Kent
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Correspondence to Stephen B. H. Kent.

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Dedicated to Professor Bruce Merrifield on the occasion of his 70th birthday.

Republished with the permission of Blackwell Publishing from International Journal of Peptide Protein Research, volume 40, pp. 180–193, 1992.

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Schnölzer, M., Alewood, P., Jones, A. et al. In Situ Neutralization in Boc-chemistry Solid Phase Peptide Synthesis. Int J Pept Res Ther 13, 31–44 (2007). https://doi.org/10.1007/s10989-006-9059-7

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  • DOI: https://doi.org/10.1007/s10989-006-9059-7

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