Vector-borne diseases pose a rising global health challenge, necessitating the development of safe and effective pest protection agents. Here, we report a highly selective biocatalytic direct Prins cyclohydration for the synthesis of (1R)-cis-p-menthane-3,8-diol (PMD), a natural insect repellent with high efficacy. By strategically engineering squalene-hopene cyclases (SHCs), we achieved >96% diastereomeric excess, surpassing previous synthetic methods. Structural and mechanistic analyses suggest direct Prins cyclohydration and a precisely positioned water molecule within the enzyme's active pocket adjacent to the final carbocation that drives hydration and catalytic efficiency. Fine-tuning the biocatalytic setup enabled preparative scale production, without losing much product selectivity. Moreover, we demonstrate access to the other naturally occurring PMD isomers from (R)- and (S)-citronellal, as well as a one-pot cascade starting from E/Z-citral. This study paves the way for highly selective access to stereodefined terpene-derived repellents and establishes engineered squalene-hopene cyclases as a tool for direct asymmetric Prins cyclohydration.

The development of synthetic routes to produce enantiopure (R)-citronellal as a key intermediate for the synthesis of (–)-menthol and other valuable terpenoids is highly relevant in the pharmaceutical, flavor, and fragrance industries. Herein, we showcase a cascade with two consecutive biocatalytic steps performed separately using the inherent selectivity of a short-chain alcohol dehydrogenase (SDR) and an ene reductase (ERED) from the Old Yellow Enzyme (OYE) family. The first reaction involves the AaSDR1-catalyzed oxidation of relatively inexpensive geraniol in a biphasic system, providing geranial as an intermediate. The organic phase containing geranial is then extracted and transferred to the second step, where the ERED variant OYE2_Y83V catalyzes the asymmetric reduction of geranial to produce (R)-citronellal, achieving >90% conversion and >99% enantiomeric excess. The use of n-heptane in a two-liquid phase system not only facilitates substrate and product solubilization but also minimizes geranial isomerization. This biocatalytic cascade therefore enables the synthesis of enantiopure (R)-citronellal.

Biocatalytic asymmetric reduction of alkenes in organic solvent is attractive for enantiopurity and product isolation, yet remains under developed. Herein we demonstrate the robustness of an ene reductase immobilised on Celite for the reduction of activated alkenes in micro-aqueous organic solvent. Full conversion was obtained in methyl t-butyl ether, avoiding hydrolysis and racemisation of products. The immobilised ene reductase showed reusability and a scale-up demonstrated its applicability.