New Synthetic Strategies for Protein Engineering

蛋白质工程的新合成策略

• 批准号: 7368021
• 负责人: MATTHEW B FRANCIS
• 金额: $ 26.89万
• 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/7368021
• 关键词: Address; Affinity Chromatography; Alder plant; Alkenes; Alkynes; Arts; Buffers; Catalysis; Chemicals; Complement; Condition; Coupling; Cysteine; Development; Diels Alder reaction; Disulfides; Electrons; Future; Hour; Imines; Immobilization; Immunoglobulin Fragments; Label; Ligation; Link; Lysine; Mannich Bases; Membrane Proteins; Metals; Methionine; Methods; Modification; N-terminal; Nature; Organic Chemistry; Organic Synthesis; Oxidation-Reduction; Phenazines; Post-Translational Protein Processing; Procedures; Property; Protein Biosynthesis; Protein Engineering; Proteins; Rain; Reaction; Research; Research Design; Research Personnel; Rhodium; Screening procedure; Site; Techniques; Transition Elements; Tyrosine; adduct; aqueous; base; carbene; catalyst; design; dirhodium tetraacetate; disulfide bond; fluorophore; functional group; iminoquinone; interdisciplinary approach; novel; programs; protein expression

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，这种反应可以扩展和补充蛋白质修饰的当前技术水平。发展了一种多学科的方法，将蛋白质的表达、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成与现有的基于赖氨酸和半胱氨酸的策略是正交的，因此可以与这些久经考验的方法一起使用。通过针对未被充分利用的官能团和提高相似官能团之间的反应选择性，预计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合成蛋白质的全新途径。 具体地说，将开发两种强大的新策略来方便地修饰蛋白质表面的酪氨酸残基。首先使用重氮偶联反应来激活酪氨酸残基，然后进行杂化Diels-Alder反应进行进一步的共轭反应。从开始到结束，这个过程只需要三个小时就可以完成，并且可以绝对选择性地将烯烃和炔烃偶联到酪氨酸上。第二种策略是一种新的基于Mannich的三组分偶联反应，它可以对酪氨酸残基进行高选择性的修饰，并同时安装两个新的官能团。这一反应将作为蛋白质连接的一种新策略而被探索。 还将探索过渡金属催化的反应，因为它们为高选择性反应和激活目前不可修饰的官能团提供了许多机会。特别是，将探索开发一种基于金属卡宾的二硫键策略，作为修饰抗体片段和N-末端蛋氨酸残基的手段。