Kinetic Resolution of α‐Bromoamides: Experimental and Theoretical Investigation of Highly Enantioselective Reactions Catalyzed by Haloalkane Dehalogenases

Abstract

Haloalkane dehalogenases from five sources were heterologously expressed in Escherichia coli, isolated, and tested for their ability to achieve kinetic resolution of racemic a-bromoamides, which are important intermediates used in the preparation of bioactive compounds. To explore the substrate scope, fourteen a-bromoamides, with different Caand N-substituents, were synthesized. Catalytic activity towards eight substrates was found, and for five of these compounds the conversion proceeded with a high enantioselectivity (E value > 200). In all cases, the (R)-a-bromoamide is the preferred substrate. Conversions on a preparative scale with a catalytic amount of enzyme (enzyme:substrate ratio less 1:50 w/w) were all completed within 17-46 h and optically pure a-bromoamides and a-hydroxyamides were isolated with good yields (31-50%). Substrate docking followed by molecular dynamics simulations indicated that the high enantioselectivity results from differences in the percentage of the time in which the substrate enantiomers are bound favourably for catalysis. For the preferred (R)-substrates, the angle between the attacking aspartate oxygen atom of the enzyme, the attacked carbon atom of the substrate, and the displaced halogen atom, is more often in the optimal range (> 1578) for reactivity. This can explain the observed enantioselectivity of LinB dehalogenase in a kinetic resolution experiment.

Inverting hydrolases and their use in enantioconvergent biotransformations

“…Haloalkane dehalogenases were also applied to the preparation of α-hydroxyamides via deracemization. Fourteen α-bromoamides were subjected to kinetic resolution with five heterologously expressed haloalkane dehalogenases [93], and E values of >200 were achieved for several α-haloamides. Furthermore, molecular dynamic simulations with LinB–substrate complexes showed that the preferred ( R )-enantiomer was more favorably bound in the active site.…”

Highly efficient enzymatic synthesis of tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate with a mutant alcohol dehydrogenase of Lactobacillus kefir

“…This channel appears to play an important role in positioning the substrates in such a way as to adopt a catalytic preferred conformation and therefore makes a significant contribution to the enhanced k cat . Furthermore, the k cat value is positively related to the rate of the formation of covalent intermediates (Westerbeek et al 2011) and consequently plays a major role in the increased activity (Godinho et al 2012). For the binding of the CDOH substrate to LkTADH (see Fig.…”

A Simple Enantioconvergent and Chemoenzymatic Synthesis of Optically Active α‐Substituted Amides

“…[26] Furthermore, compound 2 b, which includes a moiety derived from glycine, the most simple amino acid, was chosen to show that our methodology has potential in the preparation of peptides. Racemic compounds 2 a-c are readily accessible in more than 90 % yield in one step from simple starting materials.…”

“…[26] Upon extraction of the aqueous enzymatic reaction mixture, both products (S)-2 and (S)-3 can be obtained with high overall yields (> 80 %). [26] Upon extraction of the aqueous enzymatic reaction mixture, both products (S)-2 and (S)-3 can be obtained with high overall yields (> 80 %).…”

“…[26] To establish the best conditions for the reactions of model nucleophiles with mixtures of abromoamides (S)-2 and a-methanesulfonyloxyamides (S)-4, we performed a study in which the racemic model compound 4 a was reacted to racemic products 5 a-8 a under various conditions ( Table 1). [26] To establish the best conditions for the reactions of model nucleophiles with mixtures of abromoamides (S)-2 and a-methanesulfonyloxyamides (S)-4, we performed a study in which the racemic model compound 4 a was reacted to racemic products 5 a-8 a under various conditions ( Table 1).…”