Optimized Methods for the Production and Bioconjugation of Site-Specific, Alkyne-Modified Glucagon-like Peptide-1 (GLP-1) Analogs to Azide-Modified Delivery Platforms Using Copper-Catalyzed Alkyne-Azide Cycloaddition.,Bioconjugate Chemistry

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Optimized Methods for the Production and Bioconjugation of Site-Specific, Alkyne-Modified Glucagon-like Peptide-1 (GLP-1) Analogs to Azide-Modified Delivery Platforms Using Copper-Catalyzed Alkyne-Azide Cycloaddition.
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2020-06-16 , DOI: 10.1021/acs.bioconjchem.0c00291
Seyed Ebrahim Alavi ₁ , Peter John Cabot ₁ , Gee Yi Yap ₁ , Peter Michael Moyle ₁

Affiliation

This study aimed to develop and optimize chemistries to produce alkyne-modified glucagon-like peptide-1(7–36)-amide (GLP-1(7–36)-NH₂) libraries, which could be rapidly and efficiently conjugated to other components and screened to identify compounds with the best drug delivery properties, as potential treatments for type 2 diabetes or obesity. For this purpose, the Lys26 (K26) side-chain, and the amino (N)- and carboxy (C)-termini of a dipeptidyl peptidase 4 (DPPIV)-resistant GLP-1 sequence (GLP-1(7–36;A8G)-NH₂), were modified with an alkyne (4-pentynoic acid or propiolic acid). These analogs were characterized with respect to human GLP-1 receptor (hGLP-1R) agonist activity, effects on cell viability and human serum stability, revealing that these modifications maintained low (N-terminal; EC₅₀ 1.5 × 10^–9 M) to subnanomolar (C-terminal and K26, ∼4 × 10^–10 M) agonist activity toward hGLP-1, had no effect on cell viability, and for the N-terminal and K26 modifications, increased human serum proteolytic stability (t_1/2 > 24 h). Copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction conditions were investigated using the C-terminal modified GLP-1 analog and an azide-modified model lipid peptide, with respect to the effects of altering the azide/alkyne ratio, cosolvents, temperature, reducing agents, Cu(I)-stabilizing ligand, copper source, and the concentrations of reagents/reactants, in order to identify general conditions that provide fast reactions and high yields. A 1:2 azide–alkyne (lipid:GLP-1 peptide) and 4:1 sodium ascorbate/copper sulfate molar ratio in 65% v/v DMSO–water at room temperature, in the absence of Cu(I)-stabilizing ligands (THPTA or l-histidine) and buffers (phosphate, pH 7), provided the best yields. This work reports a library of characterized GLP-1 analogs and chemistries for their attachment to other species, providing useful tools to improve GLP-1 delivery and pharmacology (e.g., through conjugation to other species that lower blood glucose, increase the duration of action, or enable delivery via a nonparenteral route).

中文翻译：

使用铜催化的炔烃-叠氮化物环加成法生产特定位置的炔烃修饰的胰高血糖素样肽1（GLP-1）类似物的最佳方法，类似于叠氮化物修饰的递送平台。

这项研究旨在开发和优化化学方法，以生产炔烃修饰的胰高血糖素样肽-1（7–36）-酰胺（GLP-1（7–36）-NH ₂）库，该库可以快速有效地与其他化合物偶联成分进行筛选，以鉴定具有最佳药物递送特性的化合物，作为2型糖尿病或肥胖症的潜在治疗方法。为此，耐二肽基肽酶4（DPPIV）的GLP-1序列的Lys26（K26）侧链和氨基（N）-和羧基（C）-末端（GLP-1（7–36; A8G）-NH ₂），用炔烃（4-戊酸或丙酸）修饰。这些类似物针对人类GLP-1受体（hGLP-1R）激动剂的活性，对细胞活力和人类血清稳定性的影响进行了表征，表明这些修饰保持了低水平（N末端； EC ₅₀ 1.5×10 ^–9 M）。亚纳摩尔（C末端和K26，〜4×10 ^–10 M）对hGLP-1的激动剂活性，对细胞活力没有影响，并且对于N末端和K26修饰，增加了人类血清蛋白水解稳定性（t _1/2> 24小时）。使用C端修饰的GLP-1类似物和叠氮化物修饰的模型脂质肽研究了铜催化的叠氮化物-炔烃环加成（CuAAC）反应条件，研究了改变叠氮化物/炔烃比，助溶剂，温度，还原剂，稳定Cu（I）的配体，铜源以及试剂/反应物的浓度，以便确定提供快速反应和高收率的一般条件。在室温下，在没有Cu（I）稳定配体的情况下，在65％v / v DMSO-水中，存在1：2的叠氮化物-炔烃（脂质：GLP-1肽）和4：1的抗坏血酸钠/硫酸铜摩尔比（THPTA或l-组氨酸）和缓冲液（磷酸盐，pH 7），收率最高。这项工作报告了一个具有特征性的GLP-1类似物和化学物质与其他物种的依附关系的库，为改善GLP-1的递送和药理作用提供了有用的工具（例如，通过与降低血糖，增加作用持续时间的其他物种结合，或启用通过非胃肠外途径递送）。

更新日期：2020-07-15

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