Use of a Temporary "Solubilizing" Peptide Tag for the Fmoc Solid-Phase Synthesis of Human Insulin Glargine via Use of Regioselective Disulfide Bond Formation
Use of a Temporary "Solubilizing" Peptide Tag for the Fmoc Solid-Phase Synthesis of Human Insulin Glargine via Use of Regioselective Disulfide Bond Formation
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DOI：
10.1021/bc900181a
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发表时间：
2009-07-01
影响因子：
4.7
通讯作者：
Wade, John D.
中科院分区：
文献类型：
--
作者：
Hossain, Mohammed Akhter;Belgi, Alessia;Wade, John D.
固相肽合成已经被改进到一个阶段，其中长的和复杂的肽的有效制备现在是可实现的。然而.难溶性肽的合成后处理通常仍然是其纯化和进一步使用的显著障碍。已经开发了几种合成方案用于制备含有修饰以帮助其溶解性的此类肽。然而，这些需要使用复杂的化学或产生非天然序列。我们描述了一种简单的方法，基于使用五赖氨酸“标签”，通过一个碱基不稳定的接头连接到感兴趣的肽的C-末端。在现在自由溶解的肽的准备好的纯化之后，通过简单、短暂的碱处理除去“标签”，从而以高得多的总产率得到天然序列。通过固相合成两条链（包括众所周知的难溶性A链），然后通过区域选择性二硫键形成在溶液中结合，制备甘精胰岛素，证明了该方法的适用性。在链组合结束时，从A链中除去增溶肽标签，以接近10%的总产率提供合成人甘精胰岛素。这种方法将有助于开发新的胰岛素类似物，并广泛适用于改善纯化和收购，否则难溶性合成肽。
Solid-phase peptide synthesis has been refined to a stage where efficient preparation of long and complex peptides is now achievable. However. the postsynthesis handling of poorly soluble peptides often remains a significant hindrance to their purification and further use. Several synthetic schemes have been developed for the preparation of such peptides containing modifications to aid their solubility. However, these require the use of complex chemistry or yield non-native sequences. We describe a simple approach based on the use of penta-lysine "tags" that are linked to the C-terminus of the peptide of interest via a base-labile linker. After ready purification of the now freely solubilized peptide, the "tag" is removed by simple, brief base treatment giving the native sequence in much higher overall yield. The applicability of the method was demonstrated by the novel preparation of insulin glargine via solid-phase synthesis of each of the two chains-including the notoriously poorly soluble A-chain-followed by their combination in solution via regioselective disulfide bond formation. At the conclusion of the chain combination, the solubilizing peptide tag was removed from the A-chain to provide synthetic human glargine in nearly 10% overall yield. This approach should facilitate the development of new insulin analogues as well as be widely applicable to the improved purification and acquisition of otherwise poorly soluble synthetic peptides.