?-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.

Public Health Relevance

?-amino acids are an important motif found in numerous drug candidates. Further application of this class of molecule is dependent on a robust synthesis. The proposed research will make new variants of this scaffold accessible; which should lead to new and more effective medicinal targets.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1-F04-W (20))
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Lees, Robert G
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California Institute of Technology
Schools of Engineering
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Farwell, Christopher C; Zhang, Ruijie K; McIntosh, John A et al. (2015) Enantioselective Enzyme-Catalyzed Aziridination Enabled by Active-Site Evolution of a Cytochrome P450. ACS Cent Sci 1:89-93
Hyster, Todd K; Farwell, Christopher C; Buller, Andrew R et al. (2014) Enzyme-controlled nitrogen-atom transfer enables regiodivergent C-H amination. J Am Chem Soc 136:15505-8
Farwell, Christopher C; McIntosh, John A; Hyster, Todd K et al. (2014) Enantioselective imidation of sulfides via enzyme-catalyzed intermolecular nitrogen-atom transfer. J Am Chem Soc 136:8766-71