Evolution of p450's Machinery for C-H Amination

描述(申请人提供)：氨基酸是许多小分子和大分子多肽药物中的一个重要基序。我们致力于开发一种合成这些核的通用方法。这项建议的重点是开发一种利用金属酶催化C-H胺化合成氨基酸的新方法。目前在自然界中还没有发现已知的C-H胺化催化剂。我们建议将现有的P450CYP102A1(P450BM3)的反应机制改进为在羧基上进行C-H胺化反应。以前的研究表明，这种酶可以将氧气引入到以O2为化学计量比氧化剂的各种底物中。我们建议使用叠氮化合物作为氨化反应的氮基氧化剂。我们的具体目标是：(1)将现有的p450BM3进化为具有长烷氧基酸键的胺化酯，以模仿p450BM3的天然底物；然后使用定向进化将该方法扩展到简单的酯；(2)使用定向进化与氢键添加剂相结合的方法来合成氨基酸酯。这些目标将利用阿诺德实验室中现有的大量p450BM3突变体来启动，随后将进行进一步的进化。我们将使用与荧光炔的点击反应来 确定叠氮化物的消耗程度，以加快筛选过程。所有起始材料都可以快速准备或从商业供应商那里购买。

DESCRIPTION (provided by applicant): ¿-amino acids are an important motif found in many small molecule and large ¿-peptide drugs. We endeavor to develop a general method for synthesizing these cores. The focus of this proposal is development of a new method for ¿-amino acid synthesis using metalloenzymes to catalyze a C-H amination. Currently there are no known C-H amination catalysts found in nature. We propose evolving the existing machinery of p450 CYP102A1 (p450BM3), an effective catalyst for C-H hydroxylation, to perform a C-H amination at the ¿-position of carboxyl groups. Previous studies have demonstrated this enzyme will introduce oxygen into a variety of substrates with O2 as the stoichiometric oxidant. We propose using azides as a nitrogen based oxidant for amination. Our specific aims are: (1) To evolve existing p450BM3 to aminate esters with long alkoxy- acid linkages to mimic the native substrate for p450BM3; then extend this method to simple esters using directed evolution; (2) Aminate alkyl acids using directed evolution in concert with a hydrogen bonding additive. These aims will be initiated using the large number of existing p450BM3 mutants in the Arnold Lab followed by further rounds of evolution. We will used a click reaction with a fluorescent alkyne to determine the degree of azide consumption in order to accelerate the screening process. All starting materials can be rapidly prepared or purchased from commercial vendors.