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.
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.
Mugford
Paul F
PF
McGill University, Department of Chemistry, 801 Sherbrooke Street West, Montréal, Québec, H3A 2K6, Canada.
Lait
Susan M
SM
Keay
Brian A
BA
Kazlauskas
Romas J
RJ
D000179:Acrylates / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002087:Butyrates / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D003198:Computer Simulation; D004952:Esters / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D011487:Protein Conformation; D013237:Stereoisomerism; D002787:Sterol Esterase / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism
D000179:Acrylates; D002087:Butyrates; D004952:Esters; D006639:Histidine; D002787:Sterol Esterase; D013381:Subtilisins; C036658:acrylic acid