Substrate Specificity and Enantioselectivity of 4-Hydroxyacetophenone Monooxygenase

ABSTRACT The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.

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Chemoenzymatic Synthesis of Apremilast: A Study Using Ketoreductases and Lipases

Journal of the Brazilian Chemical Society ◽

10.21577/0103-5053.20210012 ◽

2021 ◽

Author(s):

Kimberly Vega ◽

Daniel Cruz ◽

Artur Oliveira ◽

Marcos da Silva ◽

Telma de Lemos ◽

...

Keyword(s):

Aspergillus Niger ◽

Ethyl Acetate ◽

Kinetic Resolution ◽

Enantiomeric Excess ◽

The Other ◽

Chemoenzymatic Synthesis ◽

Hydrolysis Reaction ◽

Chiral Alcohol ◽

Enantiomerically Pure ◽

Enantiomeric Ratio

The key step in the chemoenzymatic synthesis of apremilast was to produce the chiral alcohol (R)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanol, (R)-3. Two enzymatic approaches were evaluated to obtain (R)-3, one using ketoreductases and the other lipases. Bioreduction of 1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanone (2), using ketoreductase KRED‑P2-D12, led to (R)-3 with 48% conversion and 93% enantiomeric excess (ee). Kinetic resolution of rac-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethyl acetate (rac-4), via hydrolysis reaction, with 20% of n-butanol, catalyzed by lipase from Aspergillus niger yielded (R)-3 with > 99% ee, 50% conversion and E-value (enantiomeric ratio) > 200. The reaction between enantiomerically pure (R)-3 and 4-acetylamino-isoindol-1,3-dione (8) afforded apremilast in 65% yield and 67% ee.

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Isolation of an esterase-producing Trichosporon brassicae and its catalytic performance in kinetic resolution of ketoprofen

Canadian Journal of Microbiology ◽

10.1139/w01-121 ◽

2001 ◽

Vol 47 (12) ◽

pp. 1101-1106 ◽

Cited By ~ 11

Author(s):

Duan Shen ◽

Jian-He Xu ◽

Peng-Fei Gong ◽

Hui-Yuan Wu ◽

You-Yan Liu

Keyword(s):

Ethyl Ester ◽

Sole Carbon Source ◽

Kinetic Resolution ◽

Enantiomeric Excess ◽

Catalytic Performance ◽

Soil Samples ◽

Enantioselective Hydrolysis ◽

Resting Cells ◽

Enantiomeric Ratio ◽

Hydrolysis Of

A yeast strain CGMCC 0574, identified as Trichosporon brassicae, was selected from 92 strains for its high (S) selectivity in the hydrolysis of ketoprofen ethyl ester. The effective strains of the microorganisms were isolated from soil samples with the ester as the sole carbon source. The ethyl ester proved to be the best substrate for resolution of ketoprofen among several ketoprofen esters examined. The resting cells of CGMCC 0574 could catalyze the hydrolysis of ketoprofen ethyl ester with an enantiomeric ratio of 44.9, giving (S)-ketoprofen an enantiomeric excess of 91.5% at 42% conversion.Key words: ketoprofen, biocatalytic resolution, enantioselective hydrolysis, microbial esterase, Trichosporon brassicae.

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Effect of Reaction Parameters on the Lipase-Catalyzed Kinetic Resolution of (RS )-Metoprolol

ASEAN Journal of Chemical Engineering ◽

10.22146/ajche.51857 ◽

2020 ◽

Vol 20 (1) ◽

pp. 20

Author(s):

Mariani Rajin ◽

Asiah Binti Zulkifli ◽

Sariah Abang ◽

S.M Anissuzzaman ◽

Azlina Harun Kamaruddin

Keyword(s):

Adrenergic Receptor ◽

Kinetic Resolution ◽

Beta Blockers ◽

Enantiomeric Excess ◽

Acyl Donor ◽

Market Demand ◽

Disease Treatment ◽

Enantiomeric Ratio ◽

Candida Antarctica B ◽

Candida Antarctica B Lipase

Racemic metoprolol is a selective ß1-blocker, which is used in cardiovascular disease treatment. It has been found that (S)-metoprolol has a higher affinity to bind the ß-adrenergic receptor compared to (R)-metoprolol. Moreover, the regulatory authorities’ high market demand and guidelines have increased the preference for single enantiomer drugs. In this work, the lipase-catalyzed kinetic resolution of racemic metoprolol was performed to obtain the desired enantiomer. The type of lipase, acyl donor, and solvent were screened out. This was achieved by Candida antarctica B lipase-catalyzed transesterification of racemic metoprolol in hexane and vinyl acetate as the solvent and an acyl donor, which gave maximum conversion of (S)-metoprolol (XS) of 52%, enantiomeric excess of substrate, (ees) of 92% and product (eeP) of 90% with enantiomeric ratio (E) of 62. This method can be considered as green chemistry, which can be applied to produce other enantiopure beta-blockers.

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Lipase-Catalyzed Kinetic Resolution of Dimethyl and Dibutyl 1-Butyryloxy-1-Carboxymethylphosphonates

Catalysts ◽

10.3390/catal11080956 ◽

2021 ◽

Vol 11 (8) ◽

pp. 956

Author(s):

Paulina Majewska

Keyword(s):

Burkholderia Cepacia ◽

Kinetic Resolution ◽

Wide Spectrum ◽

Candida Rugosa ◽

Enantiomeric Excess ◽

Optically Active ◽

Enantiomeric Ratio ◽

Catalyzed Reactions ◽

Hydrolysis Of ◽

Different Sources

The main objective of this study is the enantioselective synthesis of carboxyhydroxyphosphonates by lipase-catalyzed reactions. For this purpose, racemic dimethyl and dibutyl 1-butyryloxy-1-carboxymethylphosphonates were synthesized and hydrolyzed, using a wide spectrum of commercially available lipases from different sources (e.g., fungi and bacteria). The best hydrolysis results of dimethyl 1-butyryloxy-1-carboxymethylphosphonate were obtained with the use of lipases from Candida rugosa, Candida antarctica, and Aspergillus niger, leading to optically active dimethyl 1-carboxy-1-hydroxymethylphosphonate (58%–98% enantiomeric excess) with high enantiomeric ratio (reaching up to 126). However, in the case of hydrolysis of dibutyl 1-butyryloxy-1-carboxymethylphosphonate, the best results were obtained by lipases from Burkholderia cepacia and Termomyces lanuginosus, leading to optically active dibutyl 1-carboxy-1-hydroxymethylphosphonate (66%–68% enantiomeric excess) with moderate enantiomeric ratio (reaching up to 8.6). The absolute configuration of the products after biotransformation was also determined. In most cases, lipases hydrolyzed (R) enantiomers of both compounds.

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A FACILE SYNTHESIS AND REACTIONS OF AMINO SELENOLO[2,3-b]PYRIDINE CARBOXYLATE

JOURNAL OF ADVANCES IN CHEMISTRY ◽

10.24297/jac.v12i1.845 ◽

2015 ◽

Vol 12 (1) ◽

pp. 3910-3918 ◽

Cited By ~ 1

Author(s):

Dr Remon M Zaki ◽

Prof Adel M. Kamal El-Dean ◽

Dr Nermin A Marzouk ◽

Prof Jehan A Micky ◽

Mrs Rasha H Ahmed

Keyword(s):

Biological Activity ◽

Carbonyl Compounds ◽

Mass Spectra ◽

The Other ◽

Pyridine Derivatives ◽

Facile Synthesis ◽

High Biological Activity ◽

Basic Hydrolysis ◽

Pyridine Carboxylate ◽

Hydrolysis Of

Incorporating selenium metal bonded to the pyridine nucleus was achieved by the reaction of selenium metal with 2-chloropyridine carbonitrile 1 in the presence of sodium borohydride as reducing agent. The resulting non isolated selanyl sodium salt was subjected to react with various α-halogenated carbonyl compounds to afford the selenyl pyridine derivatives 3a-f which compounds 3a-d underwent Thorpe-Ziegler cyclization to give 1-amino-2-substitutedselenolo[2,3-b]pyridine compounds 4a-d, while the other compounds 3e,f failed to be cyclized. Basic hydrolysis of amino selenolo[2,3-b]pyridine carboxylate 4a followed by decarboxylation furnished the corresponding amino selenolopyridine compound 6 which was used as a versatile precursor for synthesis of other heterocyclic compound 7-16. All the newly synthesized compounds were established by elemental and spectral analysis (IR, 1H NMR) in addition to mass spectra for some of them hoping these compounds afforded high biological activity.

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Lipase Catalyzed Synthesis of Enantiopure Precursors and Derivatives for β-Blockers Practolol, Pindolol and Carteolol

Catalysts ◽

10.3390/catal11040503 ◽

2021 ◽

Vol 11 (4) ◽

pp. 503

Author(s):

Morten Gundersen ◽

Guro Austli ◽

Sigrid Løvland ◽

Mari Hansen ◽

Mari Rødseth ◽

...

Keyword(s):

Building Block ◽

Kinetic Resolution ◽

Enantiomeric Excess ◽

Catalytic Amount ◽

Building Blocks ◽

Β Blocker ◽

Β Blockers ◽

Optical Rotations ◽

Reported Data ◽

By Products

Sustainable methods for producing enantiopure drugs have been developed. Chlorohydrins as building blocks for several β-blockers have been synthesized in high enantiomeric purity by chemo-enzymatic methods. The yield of the chlorohydrins increased by the use of catalytic amount of base. The reason for this was found to be the reduced formation of the dimeric by-products compared to the use of higher concentration of the base. An overall reduction of reagents and reaction time was also obtained compared to our previously reported data of similar compounds. The enantiomers of the chlorohydrin building blocks were obtained by kinetic resolution of the racemate in transesterification reactions catalyzed by Candida antarctica Lipase B (CALB). Optical rotations confirmed the absolute configuration of the enantiopure drugs. The β-blocker (S)-practolol ((S)-N-(4-(2-hydroxy-3-(isopropylamino)propoxy)phenyl)acetamide) was synthesized with 96% enantiomeric excess (ee) from the chlorohydrin (R)-N-(4-(3-chloro-2 hydroxypropoxy)phenyl)acetamide, which was produced in 97% ee and with 27% yield. Racemic building block 1-((1H-indol-4-yl)oxy)-3-chloropropan-2-ol for the β-blocker pindolol was produced in 53% yield and (R)-1-((1H-indol-4-yl)oxy)-3-chloropropan-2-ol was produced in 92% ee. The chlorohydrin 7-(3-chloro-2-hydroxypropoxy)-3,4-dihydroquinolin-2(1H)-one, a building block for a derivative of carteolol was produced in 77% yield. (R)-7-(3-Chloro-2-hydroxypropoxy)-3,4-dihydroquinolin-2(1H)-one was obtained in 96% ee. The S-enantiomer of this carteolol derivative was produced in 97% ee in 87% yield. Racemic building block 5-(3-chloro-2-hydroxypropoxy)-3,4-dihydroquinolin-2(1H)-one, building block for the drug carteolol, was also produced in 53% yield, with 96% ee of the R-chlorohydrin (R)-5-(3-chloro-2-hydroxypropoxy)-3,4-dihydroquinolin-2(1H)-one. (S)-Carteolol was produced in 96% ee with low yield, which easily can be improved.

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Cloning, Expression, Characterization, and Biocatalytic Investigation of the 4-Hydroxyacetophenone Monooxygenase from Pseudomonas putida JD1

Applied and Environmental Microbiology ◽

10.1128/aem.02707-08 ◽

2009 ◽

Vol 75 (10) ◽

pp. 3106-3114 ◽

Cited By ~ 34

Author(s):

Jessica Rehdorf ◽

Christian L. Zimmer ◽

Uwe T. Bornscheuer

Keyword(s):

Pseudomonas Putida ◽

Open Reading Frames ◽

Gene Encoding ◽

Open Chain ◽

Aromatic Ketones ◽

Heteroaromatic Compounds ◽

Aliphatic Ketones ◽

Expression Characterization ◽

Reading Frames ◽

Villiger Oxidation

ABSTRACT While the number of available recombinant Baeyer-Villiger monooxygenases (BVMOs) has grown significantly over the last few years, there is still the demand for other BVMOs to expand the biocatalytic diversity. Most BVMOs that have been described are dedicated to convert efficiently cyclohexanone and related cyclic aliphatic ketones. To cover a broader range of substrate types and enantio- and/or regioselectivities, new BVMOs have to be discovered. The gene encoding a BVMO identified in Pseudomonas putida JD1 converting aromatic ketones (HAPMO; 4-hydroxyacetophenone monooxygenase) was amplified from genomic DNA using SiteFinding-PCR, cloned, and functionally expressed in Escherichia coli. Furthermore, four other open reading frames could be identified clustered around this HAPMO. It has been suggested that these proteins, including the HAPMO, might be involved in the degradation of 4-hydroxyacetophenone. Substrate specificity studies revealed that a large variety of other arylaliphatic ketones are also converted via Baeyer-Villiger oxidation into the corresponding esters, with preferences for para-substitutions at the aromatic ring. In addition, oxidation of aldehydes and some heteroaromatic compounds was observed. Cycloketones and open-chain ketones were not or poorly accepted, respectively. It was also found that this enzyme oxidizes aromatic ketones such as 3-phenyl-2-butanone with excellent enantioselectivity (E ≫100).

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ω-Amino Acid:Pyruvate Transaminase from Alcaligenes denitrificans Y2k-2: a New Catalyst for Kinetic Resolution of β-Amino Acids and Amines

Applied and Environmental Microbiology ◽

10.1128/aem.70.4.2529-2534.2004 ◽

2004 ◽

Vol 70 (4) ◽

pp. 2529-2534 ◽

Cited By ~ 71

Author(s):

Hyungdon Yun ◽

Seongyop Lim ◽

Byung-Kwan Cho ◽

Byung-Gee Kim

Keyword(s):

Amino Acids ◽

Kinetic Resolution ◽

Specific Activity ◽

Expression System ◽

Enantiomeric Excess ◽

Selective Enrichment ◽

Alcaligenes Denitrificans ◽

Keto Acids ◽

Optically Pure ◽

High Stereoselectivity

ABSTRACT Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed ω-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic β-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for l-β-amino-n-butyric acid (l-β-ABA). The enzyme converts various β-amino acids and amines to the corresponding β-keto acids and ketones by using pyruvate as an amine acceptor. The apparent Km and V max for l-β-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM l-β-ABA, the apparent Km and V max for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM d,l-β-ABA, producing optically pure d-β-ABA (99% enantiomeric excess) with 53% conversion.

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