Evolution of p450's Machinery for C-H Amination

描述（由申请人提供）：β-氨基酸是许多小分子和大β-肽药物中发现的重要基序。我们致力于开发合成这些核心的通用方法。该提案的重点是开发一种使用金属酶催化 C-H 胺化合成 β-氨基酸的新方法。目前自然界中尚未发现已知的C-H胺化催化剂。我们建议改进现有的 p450 CYP102A1 (p450BM3) 机制（一种有效的 C-H 羟基化催化剂），以在羧基的 ¿ 位进行 C-H 胺化。先前的研究表明，这种酶可以将氧气引入多种底物中，以 O2 作为化学计量氧化剂。我们建议使用叠氮化物作为氮基氧化剂进行胺化。我们的具体目标是： (1) 进化现有的 p450BM3，使其胺化具有长烷氧基酸键的酯，以模拟 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)使用定向演化与氢键添加剂一起胺化烷基酸。这些目标将利用阿诺德实验室现有的大量 p450BM3 突变体来启动，然后进行进一步的进化。我们将使用荧光炔的点击反应来 确定叠氮化物的消耗程度以加速筛选过程。所有起始材料都可以快速制备或从商业供应商处购买。

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.