文献：Novel mono-lipidated dimeric glucagon-like peptide-1 receptor agonist with improved longacting and hypoglycemic activity

文献链接：https://pubmed.ncbi.nlm.nih.gov/35520704/

作者：Lidan Sun, Jing Han, Xinyu Chen,Yue Han,Lingling Wua,Xia Ea

相关产品：Bis-maleimide amine 双马来酰亚胺-胺基

原文摘要：Dimerization is a useful tool to boost ligand–receptor interaction. Both lipidation and dimerization effectively prolong the short half-life (t1/2) of peptides by facilitating binding with serum albumin and increasing hydrodynamic size. Here, we described two novel GLP-1 conjugates with high glucagon-like peptide-1 (GLP-1) receptor activation potencies, dimerized GLP-1 (Di-GLP-1) and lipidated Di-GLP-1 (Lip-Di-GLP-1). Di-GLP-1 and Lip-Di-GLP-1 were prepared through cysteine–maleimide specific coupling reactions using Gly8-Cys31-GLP-1, bis-maleimide amine, and activated palmitic acid. The receptor activation potencies of Di-GLP-1 and Lip-Di-GLP-1 were 13.6-fold and 9.5-fold higher than GLP-1, respectively. The in vivo hypoglycemic and insulinotropic activities of Di-GLP-1 and Lip-Di-GLP-1 were also better than GLP-1 in db/db mice. Furthermore, Lip-Di-GLP-1 was found to have greater circulating t1/2 than synthesized liraglutide by 1.8-fold. Accordingly, the improved pharmacokinetic profiles of Lip-Di-GLP-1 resulted in protracted antidiabetic effects as confirmed by hypoglycemic duration test. Moreover, Lip-Di-GLP-1 administered in mice potently inhibits gastric emptying and reduce food intake. Chronic Lip-Di-GLP-1 treatment in db/db mice resulted in significant improvements in food intake, body weight, pancreatic function and corrected hyperglycemia, which was more effective than synthesized liraglutide. Our research indicated that combined dimerization and lipidation were effectively applied to GLP-1, and the preclinical results suggested the potential usage of Lip-Di-GLP-1 as a long-acting antidiabetic agent.

双马来酰亚胺（BMI）是一种含有两个马来酰亚胺官能团的化合物。马来酰亚胺基团具有高反应活性，其结构中的碳 - 碳双键能够参与多种加成反应。这种双官能团的结构使得双马来酰亚胺可以与含有活泼氢的化合物（如胺、醇、硫醇等）发生反应，形成交联网络结构。

通常为白色至浅黄色结晶性粉末或固体。具有较高的熔点，这与其分子间较强的作用力有关，一般熔点范围在 150 - 200℃左右。在有机溶剂中有一定的溶解性，如可溶于二甲基甲酰胺（DMF）、二甲基乙酰胺（DMAc）、N - 甲基吡咯烷酮（NMP）等极性有机溶剂，而在水和一些非极性溶剂中的溶解性较差。Di - GLP - 1 多肽是 GLP - 1 脂化多肽的二聚体或者经过特定修饰的双分子结构。它继承了 GLP - 1 的一些基本化学结构特征，比如含有肽键连接的氨基酸序列，并且这种二聚体结构可能会改变其生物活性和药代动力学特性。脂化多肽Lip - Di - GLP - 1 是脂质体包裹的 Di - GLP - 1。脂质体是一种人工合成的囊泡结构，主要由磷脂双分子层组成，内部为水相。Di - GLP - 1 被包裹在脂质体内部的水相中或者吸附在脂质体表面。双马来酰亚胺有着多重应用，例如在Di-GLP-1和Lip-Di-GLP-1的制备中。

图为：Di-GLP-1和Lip-Di-GLP-1的结构和生物活性试验

双马来酰亚胺在Di-GLP-1和Lip-Di-GLP-1制备中的应用：

利用双马来酰亚胺和半胱氨酸残基，在硫醇和马来酰亚胺基团之间进行化学选择性迈克尔加成，合成了Di-GLP-1。简而言之，采用Fmoc合成策略，采用固相合成法合成了Gly8-Cys31-GLP-1。对于Di-GLP-1，Gly8-Cys31-GLP-1与双马来酰亚胺胺反应。在含有DIPEA的甲醇中。对于Lip-Di-GLP-1，NHS活化的棕榈酸与双马来酰亚胺││首先反应，然后是Gly8-Cys31-GLP-1。被添加并使用相同的合成程序。将反应溶液直接注入制备的反相HPLC中得到纯化产物，用ESI电离法Bruker MicroTOF││2法对产物进行表征。产生较高的GLP-1R激活能力，长效GLP-1类似物，即Di-GLP-1（二聚GLP- 1）和Lip-Di-GLP-1（脂化Di-GLP-1）。首先，将GLP-1中的Ala8替换为Gly，以避免DPP-IV降解，并在GLP-1的端引入一个Cys，最后构建Gly8-Cys31-GLP-1。然后利用Gly8-Cys31-GLP-1、双马来酰亚胺胺和活化棕榈酸的半胱氨酸-马来酰亚胺特谱偶联反应成功获得Di-GLP-1和Lip-Di-GLP-1。

图为：Di-GLP-1 (A)和Lip-Di-GLP-1 (B)的体外有害性作用

结论：通过在Di-GLP-1和Lip-Di-GLP-1的制备过程中，双马来酰亚胺由于其特殊的构成——两个马来酰亚胺基团连接而成的双官能团化合物，具有羰基和含氮环氧树脂的结构特点。还具有良好的稳定性和生物活性，可以作为交联剂，与含有氢键或氨基的化合物发生反应，形成稳定的交联结构。最终得到的Di-GLP-1和Lip-Di-GLP-1相比Lip-Di-GLP-1具有更好的药代动力学特性。