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，高度位点选择性的反应，可以扩展和补充当前蛋白质修饰的艺术状态。一种多学科的方法已经发展起来，它将蛋白质的表达、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相结合。特别是，研究中的反应被设计成与现有的赖氨酸和半胱氨酸为基础的策略正交，因此可以与这些经过验证的方法一起使用。通过靶向未充分利用的官能团，并通过增强类似功能之间的反应选择性，也可以预期，许多全新的蛋白质标记、蛋白质固定和从头蛋白质合成途径将从这些研究中出现。

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.