Metallic zinc reduction of disulfide bonds between cysteine residues in peptides and proteins

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 mereapto 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 I 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).