Metallic Zinc Reduction of Disulfide Bonds between Cysteine Residues in Peptides and Proteins

Article

DOI: 10.1007/s10989-005-8113-1

Cite this article as:
Erlandsson, M. & Hällbrink, M. Int J Pept Res Ther (2005) 11: 261. doi:10.1007/s10989-005-8113-1

Abstract

The use of powdered metallic zinc in acidic solution for the reduction of disulfide bonds in peptides and proteins has been investigated. The method has several advantages over the traditional mercapto based reducing methods currently used; the reducing agent is readily available and inexpensive; reduction can be performed in weakly acidic solutions of water and/or acetonitrile; work up simply consists of a centrifugation step followed by pipeting the supernatant from the metal pellet, thereby greatly diminishing the risk of reoxidation as a more elaborate work up procedure could result in. As no mercapto compounds are added, there is no risk that the reducing agent will interfere in subsequent modification of the thiol functionality. Disulfides in a model peptide are reduced within 5 min in any mixture of water/acetonitrile containing 1% TFA, all disulfides in insulin is reduced within 1 h in any mixture of water/acetonitrile containing 5% acetic acid. To stress the convenience of the metallic zinc reduction method, the resulting thiol compound was subjected to two commonly employed reactions in peptide chemistry: Cys(Npys) directed disulfide formation (70% yield) and native chemical ligation between the reduced model peptide and Boc-Ala-p-metylthiobenzyl ester (65% yield of the ligation product plus disulfide formation between Cys and p-thiocresol).

Keywords

Disulfides reduction metallic zinc native chemical ligation directed disulfide formation 

Abbreviations

AcN

acetonitrile

HPLC

high pressure liquid chromatography

TFA

trifluoroacetic acid

Boc

tert-butyloxycarbonyl

NPys

3–nitro-2-pyridinesulfenyl

DIPEA

N,N-diisopropylethylamine

DCM

dichloromethane

DCC

N,N-dicyclohexylcarbodiimide

TBTU

2-(1H-benzotriazole-1-yl)1,1,3,3-tetramethyluronium tetrafluoroborate

HOBt

N-hydroxybenzotriazole

HF

hydrofluoric acid

MS

mass spectrometry

NCL

native chemical ligation

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Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  1. 1.Department of Neurochemistry and NeurotoxicologyStockholm UniversityStockholmSweden

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