Evolution of p450's Machinery for C-H Amination

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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.