New Synthetic Strategies for Protein Engineering

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反应，其可以扩展和补充蛋白质修饰领域的当前状态。已经开发了一种多学科方法，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，研究中的反应被设计成与现有的基于赖氨酸和半胱氨酸的策略正交，因此可以与这些久经考验的方法一起使用。通过靶向未充分利用的官能团和通过增强相似官能团之间的反应选择性，还可以预期，这些研究将出现许多用于蛋白质标记、蛋白质固定化和从头蛋白质合成的全新途径。 具体而言，两个强大的新策略将开发蛋白质表面上的酪氨酸残基的简易修饰。第一种方法使用重氮偶合反应来活化酪氨酸残基，然后通过杂-Diels-桤木反应进行进一步缀合。从开始到结束，该过程只需三个小时即可完成，并且可以用于以绝对选择性将烯烃和炔偶联到酪氨酸。第二种策略是一种新的三组分的基于曼尼希的偶联反应，可以修饰酪氨酸残基与精致的选择性，同时安装两个新的功能基团。该反应可作为一种新的蛋白质连接策略。 过渡金属催化的反应也将被探索，因为它们提供了许多机会，高度选择性的反应和目前不可改性的官能团的活化。特别是，将探索基于金属卡宾的二硫键策略的开发，作为修饰抗体片段和N-末端甲硫氨酸残基的手段。

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.