Stereodivergent, Chemoenzymatic Synthesis of Azaphilone Natural Products.

Selective access to a targeted isomer is often critical in the synthesis of biologically active molecules. Whereas small-molecule reagents and catalysts often act with anticipated site- and stereoselectivity, this predictability does not extend to enzymes. Further, the lack of access to catalysts that provide complementary selectivity creates a challenge in the application of biocatalysis in synthesis. Here, we report an approach for accessing biocatalysts with complementary selectivity that is orthogonal to protein engineering. Through the use of a sequence similarity network (SSN), a number of sequences were selected, and the corresponding biocatalysts were evaluated for reactivity and selectivity. With a number of biocatalysts identified that operate with complementary site- and stereoselectivity, these catalysts were employed in the stereodivergent, chemoenzymatic synthesis of azaphilone natural products. Specifically, the first syntheses of trichoflectin, deflectin-1a, and lunatoic acid A were achieved. In addition, chemoenzymatic syntheses of these azaphilones supplied enantioenriched material for reassignment of the absolute configuration of trichoflectin and deflectin-1a based on optical rotation, CD spectra, and X-ray crystallography.

Positioning-Group-Enabled Biocatalytic Oxidative Dearomatization.

Biocatalysts have the potential to perform reactions with exceptional selectivity and high catalytic efficiency while utilizing safe and sustainable reagents. Despite these positive attributes, the utility of a biocatalyst can be limited by the breadth of substrates that can be accommodated in the active site in a reactive pose. Proven strategies exist for optimizing the performance of a biocatalyst toward unnatural substrates, including protein engineering; however, these methods can be time intensive and require specialized equipment that renders these approaches inaccessible to synthetic chemists. Strategies accessible to chemists for the expansion of a natural enzyme's substrate scope, while maintaining high levels of site- and stereoselectivity, remain elusive. Here, we employ a computationally guided substrate engineering strategy to expand the synthetic utility of a flavin-dependent monooxygenase. Specifically, experimental observations and computational modeling led to the identification of a critical interaction between the substrate and protein which is responsible for orienting the substrate in a pose productive for catalysis. The fundamental hypothesis for this positioning group strategy is supported by binding and kinetic assays as well as computational studies with a panel of compounds. Further, incorporation of this positioning group into substrates through a cleavable ester linkage transformed compounds not oxidized by the biocatalyst SorbC into substrates efficiently oxidatively dearomatized by the wild-type enzyme with the highest levels of site- and stereoselectivity known for this transformation.

Structural basis for selectivity in flavin-dependent monooxygenase-catalyzed oxidative dearomatization.

Biocatalytic reactions embody many features of ideal chemical transformations, including the potential for impeccable selectivity, high catalytic efficiency, mild reaction conditions and the use of environmentally benign reagents. These advantages have created a demand for biocatalysts that expand the portfolio of complexity-generating reactions available to synthetic chemists. However, the tradeoff that often exists between the substrate scope of a biocatalyst and its selectivity limits the application of enzymes in synthesis. We recently demonstrated that a flavin-dependent monooxygenase, TropB, maintains high levels of site- and stereoselectivity across a range of structurally diverse substrates. Herein, we disclose the structural basis for substrate binding in TropB, which performs a synthetically challenging asymmetric oxidative dearomatization reaction with exquisite site- and stereoselectivity across a range of phenol substrates, providing a foundation for future protein engineering and reaction development efforts. Our hypothesis for substrate binding is informed by a crystal structure of TropB and molecular dynamics simulations with the corresponding computational TropB model and is supported by experimental data. In contrast to canonical class A FAD-dependent monooxygenases in which substrates bind in a protonated form, our data indicate that the phenolate form of the substrate binds in the active site. Furthermore, the substrate position is controlled through twopoint binding of the phenolate oxygen to Arg206 and Tyr239, which are shown to have distinct and essential roles in catalysis. Arg206 is involved in the reduction of the flavin cofactor, suggesting a role in flavin dynamics. Further, QM/MM simulations reveal the interactions that govern the facial selectivity that leads to a highly enantioselective transformation. Thus, the structural origins of the high levels of site-and stereoselectivity observed in reactions of TropB across a range of substrates are elucidated, providing a foundation for future protein engineering and reaction development efforts.