Syntheses of biotinylated and dethiobiotinylated insulins

The 600-MHz proton spectrum of dethiobiotin (prepared from d-biotin with Raney nickel) was measured in order to gain information pertaining to its stereochemical homogeneity. The spectrum demonstrated clearly that the material is a 6:1 mixture of two stereoisomers. The cis compound, corresponding to the stereochemistry of d-biotin, is the major isomer. Two biotinyl- and two dethiobiotinylinsulins were prepared in which the distance between the biotins and insulin was varied by interposition of spacer arms. The synthesis of these compounds involved repeated N-hydroxysuccinimido ester condensations. Biotin N-hydroxysuccinimido ester, dethiobiotin N-hydroxysuccinimido ester, 6-aminohexanoic acid, and N-[3-[(3-aminopropyl)carboxyamino]-propyl]succinamic acid N-tert-butyl ester served as the building blocks for the spacers. The latter compound was prepared from N-[3-[(3-aminopropyl)amino]propyl]succinamic acid sulfate by the use of a selective amino-protecting method based on the differential stability toward acid of citraconyl and tert-butoxycarbonyl amino-protecting groups. The structure of N-[3-[(3-aminopropyl)amino]propyl]succinamic acid sulfate was established unequivocally by X-ray diffraction. The attachment of the biotinylated spacers to the insulin was exclusively at the N alpha, B1 position. Homogeneity of the final products as well as of the intermediates used in their synthesis was established by thin-layer chromatography, by high-pressure liquid chromatography, and in most instances by elemental analysis. The ratio of 6-aminohexanoic acid to lysine in hydrolysates of the insulin derivatives was in agreement with theory. The insulin derivatives were required for a study on the effect of avidin on their ability to interact with insulin receptors on rat epididymal adipocytes, which is described in the accompanying paper.