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

DOI: 10.1007/s10989-006-9059-7

Cite this article as:
Schnölzer, M., Alewood, P., Jones, A. et al. Int J Pept Res Ther (2007) 13: 31. doi:10.1007/s10989-006-9059-7

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

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

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