Resolution of 2,2-Disubstituted Epoxides via Biocatalytic Azidolysis

Molinaro, Carmela; Guilbault, Audrey-Anne; Kosjek, Birgit

doi:10.1021/ol101406k

Cited by 63 publications

(40 citation statements)

References 67 publications

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“…Azide was a frequently used nucleophile for kinetic resolution of epoxides by HHDHs Hasnaoui-Dijoux et al 2008;Molinaro et al 2010;Spelberg et al 2001). In this study, azide was used as a nucleophile and involved in the continuous dehalogenation and ring-opening processes (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, HHDHs have exhibited excellent practicability in the synthesis of optically active pharmaceutical intermediates and fine chemicals such as enantiopure halohydrins, epoxides, and β-substituted alcohols (Elenkov et al 2006aMolinaro et al 2010;Tang et al 2012). However, the reported natural HHDHs with low activity could not meet the requirements of industrial application, and most studies were still in the stage of experiment (de Vries and Janssen 2003).…”

Section: Discussionmentioning

confidence: 99%

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An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of β-substituted alcohols

Wan

Liu

Xue

et al. 2015

Appl Microbiol Biotechnol

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Halohydrin dehalogenases (HHDHs) are an important class of enzymes for preparing optically active haloalcohols, epoxides, and β-substituted alcohols. However, natural HHDHs rarely meet the requirements of industrial applications. Here, a novel high-throughput screening (HTS) methodology for directed evolution of HHDH was developed based on the colorimetric determination of azide. In this method, azide was involved in the HHDH-catalyzed ring-opening process and the decrease of azide was used to quantitatively evaluate HHDH activity. The HTS methodology was simple and sensitive (ε460 = 1.2173 × 10(4) L mol(-1) cm(-1)) and could be performed in a microplate format using whole cells. To verify the efficiency of the HTS methodology, it was adopted to engineer a HHDH (HHDH-PL) from Parvibaculum lavamentivorans DS-1, which was applied in the process for ethyl (R)-4-cyano-3-hydroxybutanoate (HN) by the conversion of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE)). A random mutant library containing 2500 colonies was screened using the HTS methodology, and three beneficial mutants F176M, A187R, and A187S were obtained. By combining the beneficial mutated residues, the variant F176M/A187R was identified with 2.8-fold higher catalytic efficiency for preparation of HN. The high-throughput colorimetric assay would be very useful for directed evolution of HHDH for preparing β-substituted alcohols.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of β-substituted alcohols

Wan

Liu

Xue

et al. 2015

Appl Microbiol Biotechnol

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“…Both 1,2-epoxydodecane and 1,2-epoxy-9-decene showed poor conversions, 7% and 16%, respectively, but with good regioselectivities (β/α) of 13 and 15, respectively, using ACN-H 2 O. Apparently, the presences of long carbon chains make the substrates less reactive [25]. The conversions did not improve using ACN-H 2 O with 10% AcOH in the aqueous phase, and the regioselectivity decreased compared to ACN-H 2 O, which is ascribed to the fact that, upon protonation of the substrates ( Table 2, entries 4 and 5), the azide ion prefers to attack the α-carbon.…”

Section: Influence Of Process Conditions In the T-buoac-h 2 Omentioning

confidence: 97%

Regioselectivity Control of the Ring Opening of Epoxides With Sodium Azide in a Microreactor

et al. 2012

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The reaction of different types of aromatic and aliphatic epoxides with sodium azide to give vicinal azido alcohols was studied in a microreactor with and without pillars in the channels. Dependent on the substrate, the regioselectivity of the ring opening is affected by the used solvent system, viz. acetonitrile-water (sometimes with 10% acetic acid to promote the reactivity of substrates) or t-butyl acetate-water containing Tween80 as a surfactant. For styrene oxide and α-methylstyrene oxide, the α/β regioselectivity changes from 4 to 10 and 1.7 to 6.2, respectively, going from acetonitrile-water to Tween80-containing t-butyl acetate-water. The addition of a surfactant (Tween80) stabilizes the interface in the biphasic t-butyl acetate-water. Pillar-containing microreactors gave better conversions than microreactors without pillars and lab scale reactions, probably due to better mixing.

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“…Halohydrin dehalogenase HheC has been studied extensively because of its high enzymatic activity and high R-enantioselectivity toward some short-chain aliphatic and aromatic substrates (14)(15)(16)(17). The enzyme has been applied in the production of optically pure epoxides and most of the R-enantiomers of ␤-substituted alcohols through kinetic resolution, with a maximum yield of 50% (18)(19)(20). Although kinetic resolution has a maximum yield of 50%, both enantiomers could be obtained by the resolution of a racemic mixture using enantiocomplementary enzymes (21,22).…”

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confidence: 99%

Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

Guo

Chen

Zheng

et al. 2015

Appl Environ Microbiol

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Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and ␤-substituted alcohols. The wild-type (WT) enzyme displays a high R-enantiopreference toward most aromatic substrates, whereas no S-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/ Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement in R-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from an E R of 65 [WT] to an E S of 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of its R-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.T he application of biocatalysts in the production of optically pure compounds has attracted much attention during the past few decades. Enantioselectivity (E) is one of the key parameters that define the usefulness of enzymes in the corresponding industrial synthesis of fine chemicals. Thus, identification of enzymes with high enantioselectivity for a desired transformation is important. In nature, biocatalysts with high enantioselectivity for specific industrial applications are not readily available. Directed evolution has become a routine approach for developing such novel biocatalysts, and a plethora of successful examples have been reported (1-6). A recent survey regarding the location of mutations that improve enzyme properties shows that to manipulate the enantioselectivity of enzymes, closer mutations are more effective than distant mutations (7). With the availability of structural information, the methodology of CASTing (combinatorial activesite saturation test) used in an iterative manner has been particularly successful in tailoring enzyme enantioselectivity (8-11).Microbial halohydrin dehalogenases attract a great deal of attention for their ability to produce optically pure epoxides and halohydrins (12, 13). The enzymes which are involved in the biodegradation pathway of halopropanols catalyze the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing epoxide and HCl. Halohydrin dehalogenase HheC has been studied extensively because of its high enzymatic act...

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Resolution of 2,2-Disubstituted Epoxides via Biocatalytic Azidolysis

Cited by 63 publications

References 67 publications

An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of β-substituted alcohols

An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of β-substituted alcohols

Regioselectivity Control of the Ring Opening of Epoxides With Sodium Azide in a Microreactor

Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

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