Engineering Heme Proteins for Olefin and Carbon−Hydrogen Bond Functionalization Reactions

Abstract

One of the most important challenges in chemistry is the creation of new catalysts. Nature excels at this: constructed from biologically available elements, enzymes are versatile catalysts which adapt quickly to changing environments in order to sustain life. The combination of adaptable proteins with abiological reagents from synthetic chemistry affords a new direction for catalyst development. This thesis describes new enzymes, derived from a cytochrome P450 monooxygenase, which catalyze nitrogen and carbon atom transfer reactions to olefins and carbon−hydrogen bonds. Chapter 1 introduces directed evolution, a strategy for the laboratory optimization of proteins, in the context of improving metalloproteins for their native catalysis or for new reactions. Chapter 2 details the development of an enzyme-catalyzed transformation of olefins to aziridines, a valuable motif which is both present in bioactive molecules and used as a versatile building block for synthesis. This study establishes that when provided the appropriate reagents (e.g. styrenes and tosyl azide), heme proteins can adopt a nitrene transfer catalytic cycle to form aziridine products and that the turnover and selectivity of the catalyst can be optimized through mutation of its amino acid sequence. The activity of heme protein catalysts is extended to the functionalization of sp3 hybridized C−H bonds for carbon–nitrogen and carbon–carbon bond formation through nitrene and carbene insertion respectively (Chapters 3 and 4). With the exception of C−H oxygenation chemistry, iron complexes are under-utilized for sp3 C−H functionalization reactions, despite iron being readily available and non-toxic. Combining previously engineered heme proteins with suitable substrates led to initial reaction discovery. Directed evolution of these enzymes significantly improved their C−H functionalization activity (by 140-fold in Chapter 4). Characterization of evolved enzymes, including the attainment of an X-ray crystal structure (Chapter 3) and substrate scope studies (Chapters 3 and 4), were pursued. In sum, the thesis work addresses both the biological question of expanding the catalytic capabilities of existing enzymes through mutation and expands the chemistry of iron-porphyrin catalysts.