Production of Enantiopure Chiral Epoxides with E. coli Expressing Styrene Monooxygenase

Styrene monooxygenases are a group of highly selective enzymes able to catalyse the epoxidation of alkenes to corresponding chiral epoxides in excellent enantiopurity. Chiral compounds containing oxirane ring or products of their hydrolysis represent key building blocks and precursors in organic synthesis in the pharmaceutical industry, and many of them are produced on an industrial scale. Two-component recombinant styrene monooxygenase (SMO) from Marinobacterium litorale was expressed as a fused protein (StyAL2StyB) in Escherichia coli BL21(DE3). By high cell density fermentation, 35 gDCW/L of biomass with overexpressed SMO was produced. SMO exhibited excellent stability, broad substrate specificity, and enantioselectivity, as it remained active for months and converted a group of alkenes to corresponding chiral epoxides in high enantiomeric excess (˃95–99% ee). Optically pure (S)-4-chlorostyrene oxide, (S)-allylbenzene oxide, (2R,5R)-1,2:5,6-diepoxyhexane, 2-(3-bromopropyl)oxirane, and (S)-4-(oxiran-2-yl)butan-1-ol were prepared by whole-cell SMO.

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Production of Optically Pure Aryl Epoxides by Recombinant E. coli Carrying Styrene Monooxygenase

Recombinant Protein Production with Prokaryotic and Eukaryotic Cells. A Comparative View on Host Physiology ◽

10.1007/978-94-015-9749-4_11 ◽

2001 ◽

pp. 141-149

Author(s):

A. Colmegna ◽

E. Galli ◽

G. Sello ◽

G. Bestetti

Keyword(s):

E Coli ◽

Styrene Monooxygenase ◽

Optically Pure

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Room Temperature Flow Electrochemistry as a Means of Retaining the “Memory of Chirality” via Hofer Moest Type Reaction

10.26434/chemrxiv.11516967.v2 ◽

2020 ◽

Author(s):

Tomas Hardwick ◽

Rossana Cicala ◽

Nisar Ahmed

Keyword(s):

Continuous Flow ◽

Room Temperature ◽

Biological Activities ◽

Supporting Electrolyte ◽

Enantiomeric Excess ◽

Flow Chemistry ◽

Batch Processes ◽

Enabling Technology ◽

Chiral Compounds ◽

Memory Of Chirality

<p>Many chiral compounds have become of great interest to the pharmaceutical industry as they possess various biological activities. Concurrently, the concept of “memory of chirality” has been proven as a powerful tool in asymmetric synthesis, while flow chemistry has begun its rise as a new enabling technology to add to the ever increasing arsenal of techniques available to the modern day chemist. Here, we have employed a new simple electrochemical microreactor design to oxidise an L-proline derivative at room temperature in continuous flow. Flow performed in microreactors offers up a number of benefits allowing reactions to be performed in a more convenient and safer manner, and even allow electrochemical reactions to take place without a supporting electrolyte due to a very short interelectrode distance. By the comparison of electrochemical oxidations in batch and flow we have found that continuous flow is able to outperform its batch counterpart, producing a good yield (71%) and a better enantiomeric excess (64%) than batch with a 98% conversion. We have, therefore, provided evidence that continuous flow chemistry has the potential to act as a new enabling technology to replace some aspects of conventional batch processes. </p>

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Altering the Stereoselectivity of Whole-Cell Biotransformations via the Physicochemical Parameters Impacting the Processes

Catalysts ◽

10.3390/catal11070781 ◽

2021 ◽

Vol 11 (7) ◽

pp. 781

Author(s):

Agnieszka Raczyńska ◽

Joanna Jadczyk ◽

Małgorzata Brzezińska-Rodak

Keyword(s):

Culture Conditions ◽

Biologically Active ◽

Enantiomerically Pure ◽

Whole Cells ◽

Chiral Compounds ◽

Physicochemical Factors ◽

Pure Compounds ◽

Genetic Level ◽

Prochiral Ketones ◽

Optically Pure

The enantioselective synthesis of organic compounds is one of the great challenges in organic synthetic chemistry due to its importance for the acquisition of biologically active derivatives, e.g., pharmaceuticals, agrochemicals, and others. This is why biological systems are increasingly applied as tools for chiral compounds synthesis or modification. The use of whole cells of “wild-type” microorganisms is one possible approach, especially as some methods allow improving the conversion degrees and controlling the stereoselectivity of the reaction without the need to introduce changes at the genetic level. Simple manipulation of the culture conditions, the form of a biocatalyst, or the appropriate composition of the biotransformation medium makes it possible to obtain optically pure products in a cheap, safe, and environmentally friendly manner. This review contains selected examples of the influence of physicochemical factors on the stereochemistry of the biocatalytic preparation of enantiomerically pure compounds, which is undertaken through kinetically controlled separation of their racemic mixtures or reduction of prochiral ketones and has an effect on the final enantiomeric purity and enantioselectivity of the reaction.

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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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High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production

Applied Microbiology and Biotechnology ◽

10.1007/s00253-012-4682-z ◽

2013 ◽

Vol 97 (9) ◽

pp. 3893-3900 ◽

Cited By ~ 26

Author(s):

Odile Francesca Restaino ◽

Ujjwal Bhaskar ◽

Priscilla Paul ◽

Lingyun Li ◽

Mario De Rosa ◽

...

Keyword(s):

Cell Density ◽

High Cell Density ◽

Coli Strain ◽

High Cell ◽

High Cell Density Cultivation ◽

E Coli ◽

Bioengineered Heparin ◽

Key Enzyme

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Synthesis of Optically Pure 2-Aziridinemethanols: Versatile Synthetic Building Blocks.

Chemical and Pharmaceutical Bulletin ◽

10.1248/cpb.42.2241 ◽

1994 ◽

Vol 42 (11) ◽

pp. 2241-2250 ◽

Cited By ~ 32

Author(s):

Nobutaka FUJII ◽

Kazuo NAKAI ◽

Hiromu HABASHITA ◽

Yuka HOTTA ◽

Hirokazu TAMAMURA ◽

...

Keyword(s):

Building Blocks ◽

Optically Pure

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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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Calcium(II)-mediated resolution of methyl o-chloromandelate with chiral O,O′-dibenzoyltartaric acid in preparative scale

Main Group Metal Chemistry ◽

10.1515/mgmc-2018-0043 ◽

2019 ◽

Vol 42 (1) ◽

pp. 19-22

Author(s):

Hong-Wu Xu ◽

Li-Huan Wu ◽

Qiang Ren ◽

Cui-Yu Liu ◽

Guan-Qing Yan

Keyword(s):

Mother Liquor ◽

Enantiomeric Excess ◽

Ethanol Solution ◽

Preparative Scale ◽

Mixed Ligands ◽

Excess Value ◽

Optically Pure

Abstract We report here the coordination-mediated resolution of methyl o-chloromandelate, which is a key intermediate for clopidogrel, in preparative scale. The reaction of CaO, optically pure (2R, 3R)-O,O′-dibenzoyltartaric acid, and methyl o-chloromandelate in ethanol solution afforded a mixed-ligands calcium(II) complex that was further purified by stirring of the crystals in hot methanol. Methyl (R)-o-chloromandelate was obtained in good enantiomeric excess value (>99.5%) and yield (71%) by treatment of the complex with acid. At the same time, (2R, 3R)-O,O′-dibenzoyltartaric acid was recovered in 72% yield. In addition, methyl (S)-o-chloromandelate was obtained in good enantiomeric excess value (>99.5%) and yield (73%) by recovery from the mother liquor and resolution with the same procedure for methyl (R)-o-chloromandelate, except that (2S, 3S)-O,O′-dibenzoyltartaric acid was used as the resolving reagent.

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