基于多肽巯基脯氨酸硫酯的多肽连接方法学研究

Chemical syntheses and modifications of large biologics, such as polypeptides and proteins, has become a hot area in the discipline of chemical biology. While obtaining biomolecules in a homogeneous form can usually be a formidable task using biochemical methods, utilizing chemical synthesis provides a highly selective and controllable approach. In this proposal, we will explore new ligation methods towards the preparation of peptides and proteins. By introducing a mercapto group at the 4-position of proline-thioester, it is hoped that the peptides containing such moiety could undergo ligation reactions under standard native chemical ligation conditions. Such transformation, in conjunction with metal-free desulfurization, would solve the problem in peptide ligation involving low reactivity of peptidyl prolyl thioester, thus providing a solution to prepare peptides and proteins at feasible proline sites. We will further utilize the developed methodology in the synthesis of a representative proline-rich peptide. We believe that the success of this research will extend the reach of ligation methods, and expand the tools for synthesizing large biological molecules via chemical approaches. It will also provide requisite materials for the related biological studies in the allied field such as biology, and make irreplaceable contribution to building the foundation for the area of chemical biology.

以多肽及蛋白质等生物大分子为对象的化学合成和修饰改造，是当今化学生物学研究的热点领域。应用化学方法，可以制备生物方法无法得到的均一结构的目标化合物，并且在进行修饰时具有可控性、选择性好等特点。本项目中我们将针对多肽及蛋白化学合成的连接方法进行探索，通过引入脯氨酸4位巯基，活化在常规自然化学连接条件下难以反应的多肽脯氨酸硫酯，从而实现温和条件下在脯氨酸位点的多肽连接。运用这一策略，结合无金属脱硫的方法，一系列含脯氨酸的多肽将能够得到高效的合成。在此基础上，我们将能够对一些运用其他方法难以合成的具有挑战性的富脯氨酸多肽进行制备。该项目的研究，将扩展已有的多肽连接方法，实现在脯氨酸位点进行多肽连接的突破；通过该策略进行生物大分子的合成，将为人们进一步研究相关分子的生物活性和作用，产生积极的促进作用，也将会对化学生物学这一新兴学科以及生命科学相关领域的发展产生意义深远的积极影响。

本项目在《资助项目计划书》基础上开展工作，在执行期内已全面完成预期目标，三年内针对多肽片段在大位阻困难位点的连接难题，开展了以下几方面工作：1）提出多肽硫酯“内活化”策略，通过多肽4-巯基脯氨酸硫酯实现了温和条件下多肽片段在大位阻脯氨酸位点的高效连接；2）进一步发展“内活化”策略，设计并应用多肽青霉胺衍生物，显著提高多肽片段在大位阻缬氨酸位点的连接效率；3）运用“内活化”缬氨酸位点连接的方法对药用多肽——甲状旁腺激素实现精确化学改造。这些工作为多肽片段在困难氨基酸位点的连接提供了高效可行的方案，也为多肽类药物的精准改造与优化提供了重要手段。项目执行期内，研究团队在J. Am. Chem. Soc.、Chem. Sci.等期刊上发表SCI论文3篇；项目负责人获得国家自然科学基金面上、优秀青年基金等项目资助。