New Synthetic Strategies for Protein Engineering

蛋白质工程的新合成策略

• 批准号: 6854801
• 负责人: MATTHEW B FRANCIS
• 金额: $ 28.54万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6854801
• 关键词: SDS polyacrylamide gel electrophoresis; alkenes; alkynes; carbene; chemical addition; chemical conjugate; chemical group; chemical reaction; disulfide bond; gene expression; ligands; mass spectrometry; method development; peptide chemical synthesis; protein binding; protein engineering; protein purification; protein structure function; quinones; synthetic protein; tyrosine

DESCRIPTION (provided by applicant): The central focus of the research herein is the development of new, highly site-selective reactions that can expand and complement the current state of the art in protein modification. A multidisciplinary approach has been developed that combines protein expression, purification, and characterization with the physical organic chemistry and organic synthesis techniques required for new reaction development. In particular, the reactions under study are designed to be orthogonal to existing lysine- and cysteine-based strategies, and thus can be used in concert with these tried-and-true methods. By targeting underutilized functional groups and by enhancing reaction selectivity between similar functionality, it is also anticipated that many entirely new avenues for protein labeling, protein immobilization, and de novo protein synthesis will emerge from these studies. Specifically, two powerful new strategies will be developed for the facile modification of tyrosine residues on protein surfaces. The first uses diazonium-coupling reactions to activate tyrosine residues, followed by a hetero-Diels-Alder reaction for further conjugation. Start to finish, this procedure takes as little as three hours to carry out, and can be used to couple alkenes and alkynes to tyrosines with absolute selectivity. The second strategy is a new three-component Mannich-based coupling reaction that can modify tyrosine residues with exquisite selectivity and simultaneously install two new functional groups. This reaction will be explored for as a new strategy for protein ligation. Transition-metal catalyzed reactions will also be explored, as they offer many opportunities for highly selective reactions and the activation of currently unmodifiable functional groups. In particular, the development of a metal carbene-based strategy for disulfide bonds will be explored as a means to modify antibody fragments and N-terminal methionine residues.

描述（由申请人提供）：本文研究的主要重点是开发新的，高度的现场选择性反应，可以扩展和补充蛋白质修饰中最新的最新状态。已经开发了一种多学科方法，该方法将蛋白质表达，纯化和表征与新反应发展所需的物理有机化学和有机合成技术结合在一起。特别是，所研究的反应被设计为与现有的赖氨酸和半胱氨酸策略是正交的，因此可以与这些尝试的方法一起使用。通过靶向不足的功能组并增强相似功能之间的反应选择性，还可以预见，许多全新的蛋白质标记，蛋白质固定化和从头蛋白质合成的全新途径将从这些研究中出现。 具体而言，将制定两种强大的新策略，以便于蛋白质表面上的酪氨酸残基轻松修饰。第一个使用重氮耦合反应来激活酪氨酸残基，然后是杂二烷 - alder反应，以进一步结合。开始到完成，此过程只需要三个小时才能进行，并且可以绝对选择性将烯烃和炔烃与酪氨酸搭配到酪氨酸。第二种策略是一种新的三成分基于MANNICH的耦合反应，可以以精致的选择性修饰酪氨酸残基，并同时安装两个新的功能组。该反应将作为蛋白质连接的新策略进行探讨。 还将探索过渡金属催化的反应，因为它们为高度选择性反应和目前无法解缩的官能团的激活提供了许多机会。特别是，将探讨基于金属碳纤维的二硫键键的策略作为修改抗体片段和N末端蛋氨酸残基的一种手段。