Molecular Basis for the Stereoselective Ammoniolysis of N‐Alkyl Aziridine‐2‐Carboxylates Catalyzed by Candida antarctica Lipase B

Abstract

Candida antarctica lipase B catalyzed the stereoselective ammoniolysis of N-alkyl aziridine-2-carboxylates in tBuOH saturated with ammonia and yielded the (2S)-aziridine-2-carboxamide and unreacted (2R)-aziridine-2-carboxylate. Varying the N-1 substituent on the aziridine ring changed the rate and stereoselectivity of the reaction. Substrates with a benzyl substituent or a (1'R)-1-phenylethyl substituent reacted approximately ten times faster than substrates with a (1'S)-1-phenylethyl substituent. Substrates with a benzyl substituent showed little stereoselectivity (E=5-7) while substrates with either a (1'R)- or (1'S)-1-phenylethyl substituent showed high stereoselectivity (D>50). Molecular modeling by using the current paradigm for enantioselectivity-binding of the slow enantiomer by an exchange-of-substituents orientation-could not account for the experimental results. However, modeling an umbrella-like-inversion orientation for the slow enantiomer could account for the experimental results. Steric hindrance between the methyl in the (1'S)-1-phenylethyl substituent and Thr138 and Ile189 in the acyl-binding site likely accounts for the slow reaction. Enantioselectivity likely stems from an unfavorable interaction of the methine hydrogen with Thr40 for the slow enantiomer and from subtle differences in the orientations of the other three substituents. This success in rationalizing the enantioselectivity supports the notion that an umbrella-like-inversion orientation can contribute to enantioselectivity in lipases.

Molecular Basis for the Enantio‐ and Diastereoselectivity of Burkholderia cepacia Lipase toward γ‐Butyrolactone Primary Alcohols

“…The fourth orientation is the umbrella-like inversion orientation. [1,41,42] This orientation is a mirrorimage reflection, but the mirror plane lies not between the enantiomers as is customary, but cuts through the molecule near the stereocenter perpendicular to the C À H bond. This reflection moves the hydrogen substituent to a new, previously unoccupied, region, while leaving the remaining three substituents in approximately the same location.…”

“…The primary alcohol substrates are flexible and can adjust to the binding site, but the placement of the stereocenter within a ring limits possible adjustments. Both X-ray crystal structures of enantiomers bound to enzymes [1] and molecular modeling [41,42] show that the umbrella-like orientation is common and must be considered for the slow enantiomer. Previous modeling rarely considered this possibility.…”

“…[1,39,40] Some recent modeling includes the umbrella-like orientation. [34,41,42] A third difficulty is predicting the small energy differences responsible for enantioselectivity. [34,43] Unimportant differences far from the active site change the energy of structures making it difficult to predict enantioselectivity.…”

Perspectives and Application of Halophilic Enzymes

“…Lipolytic enzymes are valuable biocatalysts due to their broad substrate specifi city and high chemo-, regio-and stereo selectivity (Park et al 2009 ). These enzymes are currently used as detergent additives, in the food and paper industries, and as enantioselective biocatalysts for the production of fi ne chemicals (Jaeger and Holliger 2010 ).…”

Identification of amino acids involved in the hydrolytic activity of lipase LipBL from Marinobacter lipolyticus

“…Microbial esterases and lipases are valuable biocatalysts due to their broad substrate specificity and high chemo-, regioand stereoselectivity (Chahinian et al, 2005;Houde et al, 2004;Jaeger & Eggert, 2002;Park et al, 2009;Rodriguez et al, 2008;Snellman & Colwell, 2004). Thus, these enzymes are currently used as detergent additives, in the food and paper industries, and as enantioselective biocatalysts for production of fine-grade chemicals (Breuer et al, 2004;Hasan et al, 2006;Jaeger & Eggert, 2002;Jaeger & Holliger, 2010;Jaeger & Reetz, 1998;Snellman et al, 2002;Schmid et al, 2001).…”