Biocatalytic Organofluorine Synthesis Via Hemoprotein Catalyzed Carbene Transfer Reactions

"Protein engineering efforts in our laboratory led to the development of efficient hemoprotein-based biocatalysts for abiological carbene transfer reactions. These engineered ?carbene transferases' are capable of catalyzing synthetically valuable carboncarbon and carbon-nitrogen bond forming reactions with excellent catalytic efficiencies and stereoselectivities. Expanding on this area, the first goal of this dissertation research was to extend the carbene donor scope onto fluoroalkyl-substituted diazo compounds for the biocatalytic synthesis of valuable organofluorine building blocks. Toward this goal, we developed a biocatalytic protocol for the highly asymmetric and enantiodivergent synthesis of trifluoromethyl-substituted cycloclopropanes via myoglobin catalyzed cyclopropanation of aryl olefins using gaseous 2-diazo-1,1,1-trifluoroethane (DTE) as the carbene donor. The reactions were accomplished by using a two-compartment setup in which ex situ generated gaseous DTE is processed by engineered myoglobin catalysts expressed in bacterial cells. Additionally, we engineered cytochrome c from Hydrogenobacter thermophilus (Ht cyt c) into a biocatalyst capable of promoting enantioselective N-H carbene insertion reactions using acceptor-acceptor substituted alkyl 2-diazo-3,3,3-trifluoropropanoates. The dual synergistic effects of the mutations introduced to the Ht cyt c distal cavity along with the implementation of sterically demanding alkyl substituents on the diazo compound afforded high levels of enantioinduction. This novel C-N bond forming reaction, which has no precedence in chemo- or biocatalysis, affords medicinally valuable chiral a-trifluoromethyl aminoesters. The second part of this dissertation research describes the investigation of the mechanism of olefin cyclopropanation reactions catalyzed by myoglobin-based catalysts. To this extent, the basic mechanism of iron porphyrin- and hemoprotein-catalyzed cyclopropanation of styrene was studied using a combination of quantum chemical calculations and experimental mechanistic analyses. Our findings demonstrate for the first time that the synthetically useful carbon?carbon double bond functionalization reaction catalyzed by heme carbenes feature a ferrous, nonradical, concerted asynchronous mechanism with early transition state character. Furthermore, we focused on elucidating the stereochemical model and structural determinants for high stereoselectivity of the engineered myoglobin variant Mb(H64V,V68A), which catalyzes the cyclopropanation of styrene using ethyl diazoacetate with up to 10,000 TON and >99% de and ee. A combination of structural biology, computational and structure-activity relationship analyses revealed the importance of steric complementarity and weak noncovalent interactions between the first-sphere active site residues, the heme-carbene, and the olefin substrate in dictating the stereochemical outcome of the cyclopropanation reaction. These studies collectively highlight how the engineering and repurposing of metalloproteins is a powerful approach for expanding the arsenal of biocatalytic transformations useful for synthetic applications. Furthermore, these new-to-Nature biocatalytic reactions represent a new and sustainable alternative for the production of optically active building blocks of great interest in drug discovery and development"--Pages xviii-xix.