Enantiocomplementary Enzymatic Resolution of the Chiral Auxiliary: cis,cis‐6‐(2,2‐Dimethylpropanamido)spiro[4.4]nonan‐1‐ol and the Molecular Basis for the High Enantioselectivity of Subtilisin Carlsberg

Abstract

cis,cis-(+/-)-6-(2,2-Dimethylpropanamido)spiro[4.4]nonan-1-ol, 1, a chiral auxiliary for Diels-Alder additions, was resolved by enzyme-catalyzed hydrolysis of the corresponding butyrate and acrylate esters. Subtilisin Carlsberg protease and bovine cholesterol esterase both showed high enantioselectivity in this process, but favored opposite enantiomers. Subtilisin Carlsberg favored esters of (1S,5S,6S)-1, while bovine cholesterol esterase favored esters of (1R,5R,6R)-1, consistent with the approximately mirror-image arrangement of the active sites of subtilisins and lipases/esterases. A gram-scale resolution of 1-acrylate with subtilisin Carlsberg yielded (1S,5S,6S)-1 (1.1 g, 46 % yield, 99 % ee) and (1R,5R,6R)-1-acrylate (1.3 g, 44 % yield, 99 % ee) although the reaction was slow. The high enantioselectivity combined with the conformational rigidity of the substrate made this an ideal example to identify the molecular basis of the enantioselectivity of subtilisin Carlsberg toward secondary alcohols. When modeled, the favored (1S,5S,6S) enantiomer adopted a catalytically productive conformation with two longer-than-expected hydrogen bonds, consistent with the slow reaction rate. The unfavored (1R,5R,6R) enantiomer encountered severe steric interactions with catalytically essential residues in the model. It either distorted the catalytic histidine position or encountered severe steric strain with Asn155, an oxyanion-stabilizing residue.

Enantiocomplementary Enzymes: Classification, Molecular Basis for Their Enantiopreference, and Prospects for Mirror‐Image Biotransformations

“…Both active sites have limited space for one of the substituents of the secondary alcohol, but the mirror-image orientation of the catalytic centers favors opposite enantiomers. ( Figure S2 in the Supporting Information shows catalytically productive orientations for the S enantiomer of an ester of a spirocyclic secondary alcohol in the active site of subtilisin Carlsberg [15] and a (1R)-menthyl ester in the active site of Candida rugosa lipase. [16] )…”

Enantiokomplementäre Enzyme: Klassifizierung, molekulare Grundlage der Enantiopräferenz und Prognosen für spiegelbildliche Biotransformationen

“…Beide haben nur begrenzten Platz für einen der Substituenten des sekundären Alkohols, aber die spiegelbildliche Orientierung der katalytischen Zentren bevorzugt entgegengesetzte Enantiomere. (Abbildung S2 in den Hintergrundinformationen zeigt katalytisch produktive Orientierungen für das S ‐Enantiomer eines Esters eines spirocyclischen sekundären Alkohols im aktiven Zentrum von Subtilisin Carlsberg15 und einen (1 R )‐Menthylester im aktiven Zentrum der Candida‐rugosa ‐Lipase 16…”

Enantioselectivity ofd-amino acid oxidase in the presence of ionic liquids

“…[10][11][12] Enantiocomplementary enzymes favor opposite enantiomers because either the substrate or the active-site machinery is oriented differently. 16 In an oxidation example, the active site of D-amino acid oxidase (DAAO) is a mirror image of that of Lamino acid oxidase (LAAO). Both enzymes catalyze amino acid oxidation, but with opposite enantiomer preference.…”