Engineering Hydroxylase and Ketoreductase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of Belzutifan

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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

Enzymatic C(sp³)‑H Amination: P450-Catalyzed Conversion of Carbonazidates into Oxazolidinones

Cytochrome P450 enzymes can effectively promote the activation and cyclization of carbonazidate substrates to yield oxazolidinones via an intramolecular nitrene C–H insertion reaction. Investigation of the substrate scope shows that while benzylic/allylic C–H bonds are most readily aminated by these biocatalysts, stronger, secondary C–H bonds are also accessible to functionalization. Leveraging this “non-native” reactivity and assisted by fingerprint-based predictions, improved active-site variants of the bacterial P450 CYP102A1 could be identified to mediate the aminofunctionalization of two terpene natural products with high regio- and stereoselectivity. Mechanistic studies and KIE experiments show that the C–H activation step in these reactions is rate-limiting and proceeds in a stepwise manner, namely, via hydrogen atom abstraction followed by radical recombination. This study expands the reactivity scope of P450-based catalysts in the context of nitrene transfer transformations and provides first-time insights into the mechanism of P450-catalyzed C–H amination reactions

Discovery of Potent Parthenolide-Based Antileukemic Agents Enabled by Late-Stage P450-Mediated CH Functionalization

The sesquiterpene lactone parthenolide has recently attracted considerable attention owing to its promising antitumor properties, in particular in the context of stem-cell cancers including leukemia. Yet, the lack of viable synthetic routes for re-elaborating this complex natural product has represented a fundamental obstacle toward further optimization of its pharmacological properties. Here, we demonstrate how this challenge could be addressed via selective, late-stage <i>sp</i>³ C–H bond functionalization mediated by P450 catalysts with tailored site-selectivity. Taking advantage of our recently introduced tools for high-throughput P450 fingerprinting and fingerprint-driven P450 reactivity prediction, we evolved P450 variants useful for carrying out the highly regioselective hydroxylation of two aliphatic sites (C9 and C14) in parthenolide carbocyclic backbone. By chemoenzymatic synthesis, a panel of novel C9- and C14-modified parthenolide analogs were generated in order to gain initial structure–activity insights on these previously inaccessible sites of the molecule. Notably, some of these compounds were found to possess significantly improved antileukemic potency against primary acute myeloid leukemia cells, while exhibiting low toxicity against normal mature and progenitor hematopoietic cells. By identifying two ‘hot spots’ for improving the anticancer properties of parthenolide, this study highlights the potential of P450-mediated C–H functionalization as an enabling, new strategy for the late-stage manipulation of bioactive natural product scaffolds

Evolution of a Green and Sustainable Manufacturing Process for Belzutifan: Part 1Process History and Development Strategy

An improved synthesis has been developed for belzutifan, a novel HIF-2α inhibitor for the treatment of Von Hippel–Lindau (VHL) disease-associated renal cell carcinoma (RCC). The efficiency of previous supply and commercial routes was encumbered by a lengthy 5-step sequence, needed to install a chiral benzylic alcohol by traditional methods. Identification and directed evolution of FoPip4H, an iron/α-ketoglutarate dependent hydroxylase, enabled a direct enantioselective C–H hydroxylation of a simple indanone starting material. While this enabling transformation set the stage for a greatly improved synthesis, several other key innovations were made including the development of a base-metal-catalyzed sulfonylation, a KRED-catalyzed dynamic kinetic resolution, and a facile SNAr reaction in water. Together, these improvements resulted in a significantly shorter synthesis (9 steps) versus the supply route (16 steps) and a 75% reduction in process mass intensity (PMI), while also removing the reliance on third-row transition metals and toxic solvents