Hofmann K, Zhang W J, Romovacek H, Finn F M, Bothner-By A A, Mishra P K
Biochemistry. 1984 Jun 5;23(12):2547-53. doi: 10.1021/bi00307a002.
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.
为了获取有关去硫生物素立体化学纯度的信息，对（由d - 生物素与雷尼镍制备的）去硫生物素的600兆赫质子谱进行了测量。该谱清晰地表明该物质是两种立体异构体的6:1混合物。对应于d - 生物素立体化学的顺式化合物是主要异构体。制备了两种生物素基和两种去硫生物素基胰岛素，其中生物素与胰岛素之间的距离通过间隔臂的插入而改变。这些化合物的合成涉及重复的N - 羟基琥珀酰亚胺酯缩合反应。生物素N - 羟基琥珀酰亚胺酯、去硫生物素N - 羟基琥珀酰亚胺酯、6 - 氨基己酸和N - [3 - [(3 - 氨基丙基)羧基氨基] - 丙基]琥珀酰胺酸N - 叔丁酯用作间隔臂的构建单元。后一种化合物是由N - [3 - [(3 - 氨基丙基)氨基]丙基]琥珀酰胺酸硫酸盐通过基于柠康酰基和叔丁氧羰基氨基保护基团对酸的稳定性差异的选择性氨基保护方法制备的。N - [3 - [(3 - 氨基丙基)氨基]丙基]琥珀酰胺酸硫酸盐的结构通过X射线衍射明确确定。生物素化间隔臂与胰岛素的连接仅在Nα，B1位置。通过薄层色谱法、高压液相色谱法以及在大多数情况下通过元素分析确定了最终产物以及用于其合成的中间体的纯度。胰岛素衍生物水解产物中6 - 氨基己酸与赖氨酸的比例与理论相符。这些胰岛素衍生物用于一项关于抗生物素蛋白对其与大鼠附睾脂肪细胞上胰岛素受体相互作用能力影响的研究，该研究在随附的论文中有所描述。
