Reduction of Insulin with Tributylphosphine

Abstract

Treatment of bovine insulin with 3 mole of tributylphosphine in the presence of excess ethylene imine in aqueous I-propanol at pH 8.2 leads to complete reduction and aminoethylation, affording the aminoethylated insulin A and B chains in excellent yield. Reduction with 1 mole of tributylphosphine under the same conditions is attended by disulphide exchange: The product is a mixture of partially reduced and aminoethylated species which, on further reduction and carboxymethylation, afford A-and B-ehain derivatives with varying ratios of S-aminoethyl-and S-earboxymethyl substituents.

4-pyridylmethyl, a thiol-protecting group suitable for the partial synthesis of proteins

“…Obviously, S-pyridylmethylation of insulin requires prior reduction of the three disulphide bonds. In earlier work (Ruegg & Rudinger, 1974a, 1977 we had found that tributylphosphine is an excellent reagent for the reduction of the disulphide bonds of proteins and so it was used for that purpose throughout the work reported here. Since the phosphine and its oxide do not react with pyridylmethyl chloride under the conditions used, both reagents were added together.…”

“…Most of the other chemicals and solvents were obtained from Fluka A.Gi, Buchs, Switzerland, and used without purification. Tributylphosphine was kept as a 1-5 % solution in propanol under N2; the phosphine content of the solution was determined iodometrically (Ruegg & Rudinger, 1974a, 1977 shortly before use. Propan-1-ol, referred to simply as propanol, was the standard co-solvent.…”

2-Sulphobenzyl, a new solubilizing and reversible protecting group for cysteine in proteins. Its scope and limitations

“…In the case of insulin, the disulphide bonds were first reduced with tributylphosphine as previously described (Ruegg & Rudinger, 1974a, 1977a. co-Toluenesultone was found to react specifically and in high yield with the thiol groups so generated.…”

“…As expected, S-sulphoalkylation takes place with high selectivity in aqueous, weakly alkaline solution. Since the sultone, like certain other alkylating agents (Ruegg & Rudinger, 1974a), is unaffected by tributylphosphine, reduction and alkylation were carried out in a single operation. It was found that a 10% excess of the phosphine and a 2-fold excess of the sultone were sufficient to give the S-protected insulin chains in almost quantitative yield.…”

Semi-Reversible Cross-Linking. Synthesis and Application of a Novel Heterobifunctional Reagent

“…Of particular importance could be the use of amino sulfonic acids, which, at the same time may be radioactively labelled. The sulfonic acid residue simplifies the handling of "tagged" peptides, particularly during isolation by ion exchange chromatographyl 22 !. As pointed out in the results section these aminolyses of the thiol ester are best carried out at high pH in buffer free systems which will often cause denaturation of the protein.…”