Lenz V, Gattner H G, Sievert D, Wollmer A, Engels M, Höcker H
Deutsches Wollforschungsinstitut, Rheinisch-Westfälischen Technischen Hochschule Aachen.
Biol Chem Hoppe Seyler. 1991 Jul;372(7):495-504. doi: 10.1515/bchm3.1991.372.2.495.
Semisynthetic des-(B27-B30)-insulins containing modified B26-tyrosine residues were prepared to refine the understanding of the importance of position B26 with regard to biological and structural properties of the hormone. The following shortened insulin analogues were synthesized by trypsin-catalysed peptide-bond formation between the C-terminal amino acid ArgB22 of des-(B23-B30)-insulin and synthetic tetrapeptides as amino components: des-(B27-B30)-insulin, des-(B27-B30)-insulin-B26-methyl ester, -B26-carboxamide with varying C-terminal hydrophobicity of the B-chain, and [Tyr(NH2)B26]-, [Tyr(NO2)B26]-, [Tyr(I2)B26]-, [D-TyrB26]des-(B27-B30)-insulin-B26-carboxamide containing non-proteinogenic amino acids in position B26. Starting from insulin and an excess of synthetic Gly-Phe-Phe-Tyr-OMe as nucleophile, des-(B27-B30)-insulin-B26-methyl ester--the formal transpeptidation product at ArgB22--was formed in one step. Biological in vitro properties (binding to cultured human IM-9 lymphocytes, relative lipogenic potency in isolated rat adipocytes) of all semisynthetic analogues are reported, ranging from slightly decreased to two-fold receptor affinity and nearly three-fold biopotency relative to insulin. If the C-terminal tetrapeptide B27-B30 is removed, full relative insulin activity is still preserved, while the shortening results in the loss of ability to associate in solution. Only after carboxamidation or methyl esterification of TyrB26 the self-association typical of native insulin can be observed, and the CD-spectral effects in the near UV spectrum related to association and hexamerization of the native hormone are qualitatively reestablished. The results of this investigation underline the importance of position B26 to the modulation of hormonal properties and solution structure of the shortened insulins.
制备了含有修饰的B26 - 酪氨酸残基的半合成去（B27 - B30）胰岛素，以加深对B26位在激素生物学和结构特性方面重要性的理解。通过胰蛋白酶催化去（B23 - B30）胰岛素的C末端氨基酸ArgB22与作为氨基组分的合成四肽之间形成肽键，合成了以下缩短的胰岛素类似物：去（B27 - B30）胰岛素、去（B27 - B30）胰岛素 - B26 - 甲酯、具有不同B链C末端疏水性的 - B26 - 羧酰胺，以及在B26位含有非蛋白氨基酸的[酪氨酸（NH2）B26] - 、[酪氨酸（NO2）B26] - 、[酪氨酸（I2）B26] - 、[D - 酪氨酸B26]去（B27 - B30）胰岛素 - B26 - 羧酰胺。以胰岛素和过量的合成甘氨酸 - 苯丙氨酸 - 苯丙氨酸 - 酪氨酸 - OMe作为亲核试剂开始，一步形成去（B27 - B30）胰岛素 - B26 - 甲酯 - ArgB22处的正式转肽产物。报道了所有半合成类似物的体外生物学特性（与培养的人IM - 9淋巴细胞结合、在分离的大鼠脂肪细胞中的相对脂肪生成能力），相对于胰岛素，受体亲和力从略有降低到两倍，生物活性接近三倍。如果去除C末端四肽B27 - B30，仍保留完全的相对胰岛素活性，而缩短导致在溶液中缔合能力丧失。只有在TyrB26进行羧酰胺化或甲酯化后，才能观察到天然胰岛素典型的自缔合现象，并定性地重建了与天然激素缔合和六聚化相关的近紫外光谱中的圆二色光谱效应。这项研究的结果强调了B26位对缩短胰岛素激素特性和溶液结构调节的重要性。
