Synthesis of Six Natural Products Enabled by Selective Hydroxylation of Orcinaldehyde-Like Substrates With an Enzyme Library.

Achieving selective oxygenation chemistry offers a powerful strategy for streamlining routes to complex target molecules, yet the development of site-selective C-H functionalization reactions is challenging. Several enzyme families are capable of catalyzing C-H hydroxylation reactions and, in principle, provide an attractive solution for achieving selective oxidative transformations. In practice, however, the difficulty of identifying a biocatalyst suited to a specific synthetic target often renders chemoenzymatic strategies impractical. For example, biosynthetic enzymes such as CitB and ClaD can mediate site-selective benzylic hydroxylation, enabling access to select natural products that share structural features with their native substrates. Nonetheless, such approaches typically lack generality and do not readily extend to broader collections of natural products. Here, we explore the previously unstudied natural sequence space surrounding biosynthetic hydroxylases CitB and ClaD to generate a bespoke panel of uncharacterized enzymes that collectively expand the substrate scope of biocatalytic benzylic hydroxylation reactions. Development of this curated biocatalyst panel enabled the hydroxylation of a set of orcinaldehyde-like substrates, which were further elaborated to six natural products.