By leveraging enzyme evolution technologies, the enantioselectivity of a KetoREDuctase (KRED) towards the nearly spatially symmetrical ketone tetrahydrothiophene-3-one was increased from 63% ee to 99.3% ee. The biocatalytic process gives (R)-tetrahydrothiophene-3-ol in one step from a commodity chemical and supplants the original multistep hazardous processes starting from the chiral pool. The biocatalytic process has been successfully scaled to 100 kg.

(R)-2-Methylpentanol is an important chiral intermediate for the synthesis of certain medicinally important compounds, natural products, and liquid crystals. Here we describe the development of a practical kinetic resolution utilizing an enantiospecific biocatalytic reduction of racemic 2-methylvaleraldehyde. The process utilizes an evolved ketoreductase enzyme to selectively reduce the (R)-enantiomer of racemic 2-methylvaleraldehyde to the desired product with high volumetric productivity. A scaleable method for separating the desired product from the off-enantiomer of the starting material is also described. The process is cost-effective, green, and amenable to manufacturing scale.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.