The biocatalytic transformations used by chemists are often restricted to simple functional-group interconversions. In contrast, nature has developed complexity-generating biocatalytic reactions within natural product pathways. These sophisticated catalysts are rarely employed by chemists, because the substrate scope, selectivity and robustness of these catalysts are unknown. Our strategy to bridge the gap between the biosynthesis and synthetic chemistry communities leverages the diversity of catalysts available within natural product pathways. Here we show that, starting from a suite of biosynthetic enzymes, catalysts with complementary substrate scope as well as selectivity can be identified. This strategy has been applied to the oxidative dearomatization of phenols, a chemical transformation that rapidly builds molecular complexity from simple starting materials and cannot be accomplished with high selectivity using existing catalytic methods. Using enzymes from biosynthetic pathways, we have successfully developed a method to produce ortho-quinol products with controlled site- and stereoselectivity. Furthermore, we have capitalized on the scalability and robustness of this method in gram-scale reactions as well as multi-enzyme and chemoenzymatic cascades.

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.

Generation of reactive intermediates and interception of these fleeting species under physiological conditions is a common strategy employed by Nature to build molecular complexity. However, selective formation of these species under mild conditions using classical synthetic techniques is an outstanding challenge. Here, we demonstrate the utility of biocatalysis in generating o-quinone methide intermediates with precise chemoselectivity under mild, aqueous conditions. Specifically, α-ketoglutarate-dependent non-heme iron enzymes, CitB and ClaD, are employed to selectively modify benzylic C–H bonds of o-cresol substrates. In this transformation, biocatalytic hydroxylation of a benzylic C–H bond affords a benzylic alcohol product which, under the aqueous reaction conditions, is in equilibrium with the corresponding o-quinone methide. o-Quinone methide interception by a nucleophile or a dienophile allows for one-pot conversion of benzylic C–H bonds into C–C, C–N, C–O, and C–S bonds in chemoenzymatic cascades on preparative scale. The chemoselectivity and mild nature of this platform is showcased here by the selective modification of peptides and chemoenzymatic synthesis of the chroman natural product (−)-xyloketal D.

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.

The diverse chemistry possible with flavin cofactors positions flavin-dependent enzymes as versatile synthetic tools. This focused review highlights applications of flavin-dependent enzymes in organic synthesis. Select examples of monoamine oxidases, ene-reductases, monooxygenases and halogenases in target-oriented synthesis are presented.