New Synthetic Strategies for Protein Engineering

蛋白质工程的新合成策略

基本信息

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
项目类别：
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

项目摘要

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）：本文研究的中心焦点是开发新的、高度位点选择性的反应，该反应可以扩展和补充蛋白质修饰领域的现有技术。已经开发出一种多学科方法，将蛋白质表达、纯化和表征与新反应开发所需的物理有机化学和有机合成技术相结合。特别是，正在研究的反应被设计为与现有的基于赖氨酸和半胱氨酸的策略正交，因此可以与这些经过验证的方法配合使用。通过针对未充分利用的官能团并增强相似功能之间的反应选择性，预计这些研究将出现许多全新的蛋白质标记、蛋白质固定和蛋白质从头合成途径。具体来说，将开发两种强大的新策略来轻松修饰蛋白质表面的酪氨酸残基。第一种方法使用重氮偶联反应来激活酪氨酸残基，然后通过杂狄尔斯-阿尔德反应进行进一步缀合。从开始到结束，该过程仅需三个小时即可完成，并且可用于以绝对选择性将烯烃和炔烃与酪氨酸偶联。第二种策略是一种新的三组分曼尼希偶联反应，可以以精细的选择性修饰酪氨酸残基，并同时安装两个新的官能团。该反应将作为蛋白质连接的新策略进行探索。过渡金属催化反应也将得到探索，因为它们为高选择性反应和当前不可修饰的官能团的激活提供了许多机会。特别是，将探索开发基于金属卡宾的二硫键策略，作为修饰抗体片段和 N 末端甲硫氨酸残基的手段。