Preparation of semisynthetic insulin analogues from bis(tert-butyloxycarbonyl)-desoctapeptide-insulin phenylhydrazide: importance of the aromatic region B24-B26.

Semisynthetic analogues of insulin were prepared from derivatives of desoctapeptide-(B23-30)-insulin (DOI). A1, B1-(Boc)2-DOI (di-Boc-DOI) was converted to A1, B1-(Boc)2-DOI-B22-phenylhydrazide (di-Boc-DOI-NHNH-C6H5) by the trypsin-catalyzed addition of phenylhydrazine in aqueous organic solvents at pH 6.5 [Canova-Davis, E., & Carpenter, F. H. (1981) Biochemistry 20, 7053-7058]. Treatment of di-Boc-DOI-NHNH-C6H5 with BNPS-skatole produced the phenyldiimide. The latter was coupled with a variety of protected peptides that, after removal of protecting groups, yielded the following compounds whose biological activities were compared to that of insulin in binding, in stimulation of hexose transport (), and in the stimulation of lipogenesis [)), in terms of percent of insulin activity, all in the isolated epididymal fat cell: di-Boc-DOI 0.2, (0.1), [0.2]; di-Boc-DOI-NHNH-C6H5 0.5, (0.2), [0.5]; DOI 0.2, (0.2), [0.1]; DOI-(Gly)B23 0.2, (0.2), [0.1]; DOI-(Gly-Phe)B23-24 6.3, (6.3), [8.0]; DOI-(Gly-Phe-Phe)B23-25 17.0, (25.6), [24.7]; DOI-(Gly-Phe-Phe-Tyr)B23-26 59.0, (50.0), [69.0]. The semisynthetic derivatives represent a stepwise readdition of the aromatic residues near the C terminus of the B chain. A given analogue demonstrated comparable activity in all three biological assays. The results indicate that the stepwise addition of aromatic residues to the B-chain C terminus of DOI produces an increase in insulin-like activity. The biological activity of DOI-(Gly-Phe-Phe-Tyr)B23-26, the derivative in which the aromatic region has been completely reassembled, is the same order of magnitude as that of insulin.