Preparation of partially protected des-alanineB30-insulin-B-chain-disulphide and its use in semisynthesis

The preparation of N alpha 1 N epsilon 29-bis-methylsulphonylethyloxycarbonyl-des-alanineB30-B-chain-disulphide-O gamma 13 O gamma 21-dimethyl ester is described. Starting with the di-S-sulphonate-B-chain it was prepared by i) removal of the C-terminal amino acid by digestion with carboxypeptidase A, ii) reduction and oxidation to form the cyclic disulphide, iii) esterification of the three carboxyl groups (alpha COOH = LysB29, gamma COOH - GluB13 and GluB21) followed by selective tryptic hydrolysis of the alpha-carboxyl ester, and iv) the protection of both amino groups (alpha NH2 = PheB1, epsilon NH2 = LysB29). This B-chain derivative was activated selectively at the C-terminal carboxyl group by formation of a mixed anhydride, and condensed with the dipeptide Thr-Arg. All protecting groups were removed by treatment with alkali. The human-B-chain C-terminal elongated with Arg was obtained in a yield of 30% based on the protected des-AlaB30-B-chain derivative.