Evolution of p450's Machinery for C-H Amination

描述（由申请人提供）：氨基酸是许多小分子和大分子肽药物中发现的重要基序。我们努力开发一种合成这些核的通用方法。本提案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。我们建议改进现有的p450 CYP102A1 （p450BM3）机制，以在羧基的¿-位置进行C-H胺化，p450 CYP102A1 （p450BM3）是一种有效的C-H羟基化催化剂。以前的研究表明，这种酶可以将氧引入到各种底物中，O2作为化学计量氧化剂。我们建议使用叠氮化物作为氨化的氮基氧化剂。我们的具体目标是：(1)将现有的p450BM3进化为具有长烷氧酸键的胺化酯，以模拟p450BM3的天然底物；然后用定向进化法将该方法扩展到简单酯类；(2)利用定向进化与氢键添加剂协同作用使氨基化烷基酸。这些目标将在Arnold实验室中使用大量现有的p450BM3突变体，然后进行进一步的进化。我们将使用一个点击反应与荧光炔

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.