Self-sufficient P450-reductase chimeras for biocatalysis.

In recent years, cytochromes P450 have emerged as powerful, versatile biocatalysts for the site-selective functionalization of small molecules. Catalyzing an impressive range of chemical transformations, these enzymes have been widely used to effect C-H oxidation, biaryl coupling, and carbon-heteroatom bond formation, among many other reactions. However, the majority of P450s are multi-protein systems that employ secondary redox partners in key steps of the catalytic cycle, which limits their broader applicability. In response, the discovery of self-sufficient P450s, such as P450BM3 and P450RhF, has provided a template for the construction of artificial, self-sufficient P450-reductase fusions. In this chapter, we describe a procedure for the design, assembly, and application of two engineered, self-sufficient P450s of Streptomyces origin via fusion with an exogenous reductase domain. In particular, we generated artificial chimeras of P450s PtmO5 and TleB by linking them covalently with the reductase domain of P450RhF. Upon verification of their activities, both enzymes were employed in preparative-scale biocatalytic reactions. This approach can feasibly be applied to any P450 of interest, thereby laying the groundwork for the production of self-sufficient P450s for diverse chemical applications.

Charting the Evolution of Chemoenzymatic Strategies in the Syntheses of Complex Natural Products.

Bolstered by recent advances in bioinformatics, genetics, and enzyme engineering, the field of chemoenzymatic synthesis has enjoyed a rapid increase in popularity and utility. This Perspective explores the integration of enzymes into multistep chemical syntheses, highlighting the unique potential of biocatalytic transformations to streamline the synthesis of complex natural products. In particular, we identify four primary conceptual approaches to chemoenzymatic synthesis and illustrate each with a number of landmark case studies. Future opportunities and challenges are also discussed.

Reinvigorating the Chiral Pool: Chemoenzymatic Approaches to Complex Peptides and Terpenoids.

Biocatalytic transformations that leverage the selectivity and efficiency of enzymes represent powerful tools for the construction of complex natural products. Enabled by innovations in genome mining, bioinformatics, and enzyme engineering, synthetic chemists are now more than ever able to develop and employ enzymes to solve outstanding chemical problems, one of which is the reliable and facile generation of stereochemistry within natural product scaffolds. In recognition of this unmet need, our group has sought to advance novel chemoenzymatic strategies to both expand and reinvigorate the chiral pool. Broadly defined, the chiral pool comprises cheap, enantiopure feedstock chemicals that serve as popular foundations for asymmetric total synthesis. Among these building blocks, amino acids and enantiopure terpenes, whose core structures can be mapped onto several classes of structurally and pharmaceutically intriguing natural products, are of particular interest to the synthetic community.In this Account, we summarize recent efforts from our group in leveraging biocatalytic transformations to expand the chiral pool, as well as efforts toward the efficient application of these transformations in natural products total synthesis, the ultimate testing ground for any novel methodology. First, we describe several examples of enzymatic generation of noncanonical amino acids as means to simplify the synthesis of peptide natural products. By extracting amino acid hydroxylases from native biosynthetic pathways, we obtain efficient access to hydroxylated variants of proline, lysine, arginine, and their derivatives. The newly installed hydroxyl moiety then becomes a chemical handle that can facilitate additional complexity generation, thereby expanding the pool of amino acid-derived building blocks available for peptide synthesis. Next, we present our efforts in enzymatic C-H oxidations of diverse terpene scaffolds, in which traditional chemistry can be combined with strategic applications of biocatalysis to selectively and efficiently derivatize several commercial terpenoid skeletons. The synergistic logic of this approach enables a small handful of synthetic intermediates to provide access to a plethora of terpenoid natural product families. Taken together, these findings demonstrate the advantages of applying enzymes in total synthesis in conjunction with established methodologies, as well as toward the expansion of the chiral pool to enable facile incorporation of stereochemistry during synthetic campaigns.