The squalene hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC) is a highly efficient enzyme catalyst for stereoselective Brønsted acid catalysis. We engineered AacSHC to catalyze the selective Prins cyclization of citronellal. Four active site variants were identified for the diastereoselective cyclization of (S)‐citronellal to stereoisomers (−)‐iso‐isopulegol, (+)‐isopulegol and (−)‐neo‐isopulegol, respectively. The replacement of active site residues resulted in two triple variants that catalyzed the transformation of (R)‐citronellal to give the isomers (+)‐neo‐isopulegol and (−)‐isopulegol with up to >99 % de, respectively. The newly designed library of functionally diverse active site geometries exhibits high selective control during citronellal cyclization, leading exclusively to a single diastereomer of the desired isopulegol. Whereas the cyclization of citronellal with chemical catalysts was observed to produce the isopulegol isomer with the lowest energy, the reaction with AacSHC variants proceeded with higher product selectivity. The results of this study show that variants of AacSHC are excellent catalysts for the highly selective formation of isopulegol stereoisomers.

Squalene-hopene cyclases (SHCs) are the biocatalytic pendant to asymmetric Brønsted-acid catalysis and thus comprise enormous synthetic potential. Nevertheless, their substrate scope is currently limited to terpenes. Herein, we present how to tailor the SHC's cation cage for an enantioselective semipinacol rearrangement of bicyclic allylic alcohols to produce valuable oxa-carbon spirocyclic compounds. Exploiting the subtle divergence of SHC active sites combined with structure-guided semirational engineering, we designed a biocatalyst with a high catalytic performance of ∼4500 TTN and excellent enantioselectivity of 99.5% enantiomeric excess (ee). In silico studies suggest that a broadened active site is pivotal for catalysis. This intricate cationic rearrangement is easily scalable, employing lyophilized cell powder in water. Furthermore, our substrate scope studies demonstrate the acceptance of diverse ring-sized substrates but also reveal that the naturally confined active site limits the function as a general "semipinacolase." This study showcases the ability to harness the SHC's cation cage to tap into the broader field of asymmetric Brønsted-acid catalysis.

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