Engineered myoglobin catalysts for selective carbene transfer reactions

Expanding the reaction scope of biological catalysts beyond the realm of enzymatic transformations occurring in nature can create new opportunities for the exploitation of biocatalysis for organic synthesis. In this lecture, we will present recent progress made by our group toward the design, investigation, and application of engineered myoglobins for catalyzing abiological carbene transfer reactions. These efforts have recently led to the development of efficient and stereoselective biocatalysts for the asymmetric construction of carbon-carbon and carbon-heteroatom bonds via carbene insertion into olefins, heteroatom-hydrogen bonds, C—H bonds, and carbonyls. These myoglobin-based catalysts could be successfully applied for the stereoselective synthesis of chiral building blocks and drug molecules at the multigram scale. Presentation of these results will be complemented with a discussion of our current understanding of the mechanism of these reactions and of the structural determinants of reactivity and stereoselectivity in this new class of ‘carbene transferases’

Chemo-enzymatic fluorination of unactivated organic compounds

Fluorination has gained an increasingly important role in drug discovery and development. Here we describe a versatile strategy that combines cytochrome P450–catalyzed oxygenation with deoxofluorination to achieve mono- and polyfluorination of nonreactive sites in a variety of organic scaffolds. This procedure was applied for the rapid identification of fluorinated drug derivatives with enhanced membrane permeability

A Continuing Career in Biocatalysis: Frances H. Arnold

On the occasion of Professor Frances H. Arnold’s recent acceptance of the 2018 Nobel Prize in Chemistry, we honor her numerous contributions to the fields of directed evolution and biocatalysis. Arnold pioneered the development of directed evolution methods for engineering enzymes as biocatalysts. Her highly interdisciplinary research has provided grounds not only for understanding the mechanisms of enzyme evolution but also for developing commercially viable enzyme biocatalysts and biocatalytic processes. In this Account, we highlight some of her notable contributions in the past three decades in the development of foundational directed evolution methods and their applications in the design and engineering of enzymes with desired functions for biocatalysis. Her work has created a paradigm shift in the broad catalysis field

Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions

The surge in reports of heme-dependent proteins as catalysts for abiotic, synthetically valuable carbene and nitrene transfer reactions dramatically illustrates the evolvability of the protein world and our nascent ability to exploit that for new enzyme chemistry. We highlight the latest additions to the hemoprotein-catalyzed reaction repertoire (including carbene Si–H and C–H insertions, Doyle–Kirmse reactions, aldehyde olefinations, azide-to-aldehyde conversions, and intermolecular nitrene C–H insertion) and show how different hemoprotein scaffolds offer varied reactivity and selectivity. Preparative-scale syntheses of pharmaceutically relevant compounds accomplished with these new catalysts are beginning to demonstrate their biotechnological relevance. Insights into the determinants of enzyme lifetime and product yield are providing generalizable cues for engineering heme-dependent proteins to further broaden the scope and utility of these non-natural activities

Evolutionary History of a Specialized P450 Propane Monooxygenase

The evolutionary pressures that shaped the specificity and catalytic efficiency of enzymes can only be speculated. While directed evolution experiments show that new functions can be acquired under positive selection with few mutations, the role of negative selection in eliminating undesired activities and achieving high specificity remains unclear. Here we examine intermediates along the ‘lineage’ from a naturally occurring C12–C20 fatty acid hydroxylase (P450BM3) to a laboratory-evolved P450 propane monooxygenase (P450PMO) having 20 heme domain substitutions compared to P450BM3. Biochemical, crystallographic, and computational analyses show that a minimal perturbation of the P450BM3 fold and substrate-binding pocket accompanies a significant broadening of enzyme substrate range and the emergence of propane activity. In contrast, refinement of the enzyme catalytic efficiency for propane oxidation (not, vert, similar 9000-fold increase in kcat/Km) involves profound reshaping and partitioning of the substrate access pathway. Remodeling of the substrate-recognition mechanisms ultimately results in remarkable narrowing of the substrate profile around propane and enables the acquisition of a basal iodomethane dehalogenase activity as yet unknown in natural alkane monooxygenases. A highly destabilizing L188P substitution in a region of the enzyme that undergoes a large conformational change during catalysis plays an important role in adaptation to the gaseous alkane. This work demonstrates that positive selection alone is sufficient to completely respecialize the cytochrome P450 for function on a nonnative substrate

A Continuing Career in Biocatalysis: Frances H. Arnold

On the occasion of Professor Frances H. Arnold’s recent acceptance of the 2018 Nobel Prize in Chemistry, we honor her numerous contributions to the fields of directed evolution and biocatalysis. Arnold pioneered the development of directed evolution methods for engineering enzymes as biocatalysts. Her highly interdisciplinary research has provided grounds not only for understanding the mechanisms of enzyme evolution but also for developing commercially viable enzyme biocatalysts and biocatalytic processes. In this Account, we highlight some of her notable contributions in the past three decades in the development of foundational directed evolution methods and their applications in the design and engineering of enzymes with desired functions for biocatalysis. Her work has created a paradigm shift in the broad catalysis field

Engineered cytochromes P450 for the late-stage functionalization of natural products

Thesis (Ph. D.)--University of Rochester. Department of Chemistry, 2015.The late stage oxidation of C(sp3)-H bonds is an attractive reaction for both the late stage diversification of lead compounds as well as the synthesis of natural and synthetic organic compounds. The high bond strength of C(sp3)-H bonds, the large numbers of C(sp3)-H bonds in complex molecules, and the increased reactivity of products as compared to starting materials makes C(sp3)-H bonds challenging targets. The class of cytochromes P450 (P450s) naturally perform the selective monooxygenation of complex carbon scaffolds in the biosynthesis of natural products. The implementation of P450s in organic synthesis is hampered by the difficulty in engineering these proteins for highly regio- and stereoselective oxidations of non-native substrates. Herein we report the design and application of P450s for the late stage functionalization of natural products. First, we applied the recently developed P450 ‘fingerprinting’ method to aid in developing P450 based oxidation catalysts for the oxidation of anti-leukemic sesquiterpene lactone parthenolide. Highly regio- and stereoselective P450 catalysts were developed and applied to the chemoenzymatic synthesis of a novel class of parthenolide derivatives with increased anti-leukemic activity as compared to parthenolide. Two new ‘hotspots’ of the parthenolide scaffold were thus identified for further development, highlighting the use of P450s for the late stage development of drug like compounds. Next, we explored the use of unnatural amino acids (UAAs) to modify the activity and regioselectivity of P450 based oxidation catalysts. A set of structurally diverse UAAs were incorporated into P450s and found to yield viable oxidation catalysts with regioselectivities that differed largely from the parent enzyme. Additionally, para-amino-Phe was shown to have a general activity enhancing effect upon incorporation into P450s leading to the isolation of a variant with the highest activity toward a complex molecule reported to date. These effects could not be reproduced by incorporation of any of the 20 natural amino acids showing that UAA mutagenesis is a useful complementary method to tune the activity and regioselectivity of P450s. Recently P450s were shown to catalyze intramolecular C-H amination and, looking to expand upon the substrate scope, we chose to explore the reactivity of carbonazidates. Carbonazidates have long been a desirable substrate for C-H amination due to their high atom economy and P450s were found to be uniquely capable of activating this class of substrates. Oxazolidinones were formed via the P450 catalyzed C-H amination of benzylic and allylic C-H bonds. This reactivity was applied to the intramolecular C-H amination of two monoterpene substrates, thus showing that P450s could catalyze the C-H amination of more complex structures. Finally, the mechanism of P450 catalyzed C-H amination was explored using kinetic isotope effect experiments and radical rearrangement probe substrates showing that the C-H amination step likely proceeds through a hydrogen atom abstraction radical rebound type mechanism. A large limitation to the synthetic use of P450s for C-H amination is the low catalyst activity and large amount of reduction side product (carbamate or sulfonamide) that is formed during the reaction. The proposed mechanism for P450 catalyzed C-H amination was used to direct the engineering of P450 based catalysts. These P450 variants were found to produce less of the undesired reduction products and have greatly increased C-H amination activities, leading to a catalysts with the highest reported C-H amination activity reported to date. These studies further the applicability of P450s to the late stage amination of natural products

Strategies for the evolution of macrocyclic peptide inhibitors of protein-protein interactions

Thesis (Ph. D.)--University of Rochester. Department of Chemistry, 2018.Macrocyclic peptides are an attractive structural class for the inhibition of protein-protein interactions, and thus appealing scaffolds for design of biologically active probes and therapeutics. Macrocyclic peptides are pre-organized, structurally diverse molecules that are capable of inhibiting difficult-to-target protein-protein interactions. With macrocyclic peptides becoming increasingly important agents in the development of new bioactive molecules and therapeutics, methods to generate, diversify and screen macrocyclic peptides for the desired activity are critical. This work introduces new strategies for the efficient generation, evolution and selection of genetically encoded macrocyclic peptides as inhibitors of protein-protein interactions. This thesis explores two main approaches for the evolution and functional selection of genetically encoded macrocyclic peptides. In both systems, macrocycles are generated as stable thioether sidechain-to-sidechain linked ring structures introduced through the spontaneous reaction of cysteine with a non-canonical amino acid, O-2-bromoethyl-tyrosine, incorporated ribosomally via amber stop codon suppression. The first strategy is a medium throughput platform, in which libraries of the macrocycles are generated in E. coli with N-terminal FLAG-tags and subject to a plate based functional assay to evaluate binding strength to a target of choice. This approach was successfully used to identify a first-in-class macrocyclic peptide inhibitor of the oncogenic Sonic Hedgehog/Patched 1 protein-protein interaction. Starting from a linear peptide known to bind Sonic Hedgehog with modest affinity, macrocyclic peptide libraries were generated in E. coli and screened in a plate-based functional assay. Over a few rounds of affinity maturation, a macrocyclic peptide was identified that is able to bind Sonic Hedgehog with high (submicromolar) affinity and reduce Hedgehog signaling in mammalian cells. In a second strategy, the production of genetically encoded macrocyclic peptides was integrated with M13 phage display to obtain a high throughput functional selection platform. This system was developed by generating large macrocyclic peptide libraries as N-terminal fusions to the viral coat protein pIII of bacteriophage M13, and subjecting these libraries to affinity selection experiments against a panel of target proteins (Streptavidin, Sonic Hedgehog, and Kelch-like ECH-associated protein 1). Through this effort, unique and potent macrocyclic peptide binders for each of the targeted proteins were obtained, thus validating the value of this platform for the rapid identification of genetically encoded macrocyclic peptide inhibitors of protein-protein interactions. Because both platforms rely on the genetic incorporation of a non-canonical amino acid to mediate peptide macrocyclization, a two-tier strategy was developed to facilitate the directed evolution of orthogonal aminoacyl tRNA synthetases (AARSs) for amber stop codon suppression. Achieving efficient amber stop codon suppression is a major bottleneck in the generation of genetically encoded macrocycles due to comparatively lower efficiency of non-canonical amino acid incorporation by engineered orthogonal aminoacyl tRNA synthetases. Work described in this thesis introduces and describes an AARS evolution platform which was successfully applied to evolve a pyrrolysyl–tRNA synthetase variant with improved efficiency toward mediating the incorporation of Nϵ‐crotonyl‐lysine and other lysine analogues via amber stop codon suppression. Mutations identified in screening were shown to be transferable and improved the respective amber stop codon suppression efficiency of other evolved AARS variants

Intramolecular C(sp3)H amination of arylsulfonyl azides with engineered and artificial myoglobin-based catalysts

Contemporary scientific perspectivism is primarily viewed as a methodological framework of how we obtain and form scientific knowledge of nature, through a broadly perspectivist process, especially, with reference to quantum mechanics. In the present study, this is implemented by representing categorically the global structure of a quantum algebra of events in terms of structured interconnected families of local Boolean probing frames, realized as suitable perspectives or contexts for measuring physical quantities. The essential philosophical meaning of the proposed approach implies that the quantum world can be consistently approached and comprehended through a multilevel structure of locally variable perspectives, which interlock, in a category-theoretical environment, to form a coherent picture of the whole in a nontrivial way