Synthesis and Biological Activity of Two Disulphide Bond Isomers of Human Insulin: [A7-A11 ,A6-B7-Cystine]- and [A6-A7,A11-B7-Cystine]insulin (Human)

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Abstract
Two unnatural disulfide bond isomers of insulin, [A7-A11,A6-B7-cystine]insulin (human) (11) and [A6-A7,A11-B7-cystine]insulin (human) (III) were synthesized utilizing a novel strategy developed recently for total synthesis of human insulin. The synthetic procedure provides an unequivocal structural assignment for II and III. The 2 isomers differ distinctly from each other as well as from human insulin in some physicochemical properties, e.g., behavior in TLC, electrophoresis and countercurrent distribution. Chemical stability at acid and neutral pH is comparable to that of insulin. In 0.1 N ammonia (pH > 10), especially in the presence of mercaptoethanol, II and III are markedly less stable than insulin. Under these unphysiological conditions, varying amounts of insulin are formed in addition to mainly polymeric products. The biological activity of the isomers is qualitatively indistinguishable from insulin in several test systems. In isolated fat cells [rat] in vitro, II and III stimulate glucose oxidation with potencies of 0.14 and 0.17, respectively (insulin = 1), and inhibit lipolysis with potencies of 0.28 and 0.26. In the rat hypoglycemia test, potencies of II and III are 0.37 and 0.21, respectively. In the mouse convulsion assay, II exhibits a potency of 0.29. Both isomers displace labeled insulin from binding to anti-insulin serum [guinea pig] in vitro with potencies of 0.20 and 0.13. The different physico-chemical behavior of the 2 isomers in comparison with insulin suggests alterations of the 3-dimensional structure of the molecule. Absence of any qualitative difference in biological activity between the isomers and insulin, together with the narrow range of potencies observed in various tests, indicates that essential regions of the tertiary structure of the isomers may be similar to corresponding regions of the insulin molecule.