Evolution of p450's Machinery for C-H Amination

p450 C-H 胺化机械的演变

• 批准号: 8718804
• 负责人: Todd Kurt Hyster
• 金额: $ 4.4万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-19 至 2015-06-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8718804
• 关键词: Acids; Affinity; Aliphatic Acids; Alkynes; Amination; Amino Acids; Azides; Binding; Consumption; Development; Engineering; Enzymes; Esters; Evolution; Hydrogen Bonding; Hydrolysis; Hydroxylation; Iron; Lead; Methods; Mutagenesis; Nature; Nitrogen; Oxidants; Oxygen; Peptides; Pharmaceutical Preparations; Positioning Attribute; Process; Reaction; Research; Sodium Chloride; Urea; Variant; Vendor; base; carboxyl group; carboxylate; catalyst; design; directed evolution; drug candidate; metalloenzyme; mutant; public health relevance; scaffold; screening; small molecule

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.

描述（由申请人提供）：<$-氨基酸是在许多小分子和大<$-肽药物中发现的重要基序。我们奋进开发一种合成这些核的通用方法。该提案的重点是开发一种使用金属酶催化C-H胺化的新方法用于<$-氨基酸合成。目前在自然界中还没有发现已知的C-H胺化催化剂。我们建议进化p450 CYP 102 A1（p450 BM 3）的现有机制，一种有效的C-H羟基化催化剂，在羧基的<$-位置进行C-H胺化。以往的研究表明，这种酶将引入氧气到各种底物与O2作为化学计量的氧化剂。我们建议使用叠氮化物作为胺化的氮基氧化剂。我们的具体目标是：（1）使现有的p450 BM 3进化以胺化具有长烷氧基-酸键的酯以模拟p450 BM 3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键合添加剂一起胺化烷基酸。这些目标将使用阿诺德实验室中大量现有的p450 BM 3突变体启动，然后进行进一步的进化。我们将使用荧光炔的点击反应， 确定叠氮化物的消耗程度，以加速筛选过程。所有起始物料均可快速制备或从商业供应商处购买。