Chemo- and enantioselective routes to chiral fluorinated hydroxyketones using ketoreductases

Chiral fluorinated hydroxyketones were synthesized with excellent ee (>98%) and yield by a chemo- and stereoselective reduction of prochiral methyl/trifluoromethyl diketones using commercially available ketoreductase enzymes. By using p- and m-trifluoroacetyl substituted acetophenones, we demonstrate that ketoreductases can selectively differentiate between methyl and trifluoromethyl ketones within the same molecule. As a result, useful catalysts were identified that eliminated the need for costly and time-consuming protection/deprotection of the ketone moiety, enabling a more convergent synthesis of hydroxyketones. Further, a route to chiral methyl hydroxyketones is provided where an enzyme selectively reduces the unactivated ketone.     

An alternative bioreactor concept for application of an isolated oxidoreductase for asymmetric ketone reduction

In this paper an isolated NADH dependent ketone reductase has been used to synthesise (S)-6-bromo-β-tetralol from 6-bromo-β-tetralone, together with commercially available formate dehydrogenase (FDH) as a recycle enzyme to produce preparative quantities of the product. Furthermore, initial experiments indicate potential for an alternative bioreactor concept via the use of a resin (XAD L-323) to bind the product (and residual substrate) of the conversion rather than the cofactors or enzymes, thus allowing a new method of recycle, potentially overcoming existing problems.     

Two classes of enzymes of opposite stereochemistry in an organism: one for fluorinated and another for nonfluorinated substrates

Reduction of methyl ketones by dried cells of Geotrichum candidum (APG4) afforded (S)-alcohols in excellent enantiomeric excess (ee), whereas the reduction of trifluoromethyl ketones gave the corresponding alcohols of the opposite configuration also in excellent ee. The replacement of the methyl moiety with a trifluoromethyl group alters both the bulkiness and the electronic properties, the effect of which on the stereoselectivity was examined. No inversion in stereochemistry was observed in the reduction of hindered ketones such as isopropyl ketone, while the stereoselectivity was inverted in the reduction of ketones with electron-withdrawing atoms such as chlorine. The mechanism for the inversion in stereochemistry was investigated by enzymatic studies. Several enzymes with different stereoselectivities were isolated; one of them catalyzed the reduction of methyl ketones, and another with the opposite stereoselectivity catalyzed the reduction of trifluoromethyl ketones. Furthermore, both APG4 and the isolated enzyme were applied to the reduction of fluorinated ketones on a preparative scale, which resulted in the synthesis of chiral fluorinated alcohols with excellent ee.     

Evaluation of substituent effects on activity and enantioselectivity in the enzymatic reduction of aryl ketones

The enzymatic reduction of a series of substituted aryl ketones catalyzed by 24 isolated recombinant ketoreductases was studied and the substituent effects on activity and enantioselectivity were evaluated. When comparing p- and m-substituted acetophenones, the substituent significantly affects the activity of some of the tested ketoreductases, while it has little effect on the activity of other ketoreductases. Most of the tested ketoreductases were highly enantioselective in the reduction of these aryl ketones. The electronic properties, steric factors, and the ability to form a hydrogen bond to the substituents at the ortho-position play a significant role in determining both the activity and enantioselectivity of the ketoreductase-catalyzed reductions. From an applicability point of view, both enantiomers of the product aryl alcohols could be prepared via reduction catalyzed by one or more of the ketoreductases in most cases.     

A two-step, one-pot enzymatic synthesis of 2-substituted 1,3-diols

A biocatalytic cascade reaction was designed for the stereoselective synthesis of optically pure 2-alkyl-1,3-diols employing two enzymes. The cascade process consists of two consecutive steps: a stereoselective diketone reduction and a hydroxy ketone reduction. Chiral diols were formed by the addition of ketoreductases in the same vessel, in high stereoselectivity and chemical yield, without the isolation of the intermediate β-hydroxy ketones.     

Effective synthesis of (S)-3,5-bistrifluoromethylphenyl ethanol by asymmetric enzymatic reduction

The synthesis of (S)-3,5-bistrifluoromethylphenyl ethanol, a pharmaceutically important alcohol intermediate for the synthesis of NK-1 receptor antagonists, was demonstrated from a ketone via asymmetric enzymatic reduction. The isolated enzyme alcohol dehydrogenase from Rhodococcus erythropolis reduced the poorly water soluble substrate with excellent ee (>99.9%) and good conversion (>98%). The optimized process was demonstrated up to pilot scale using high substrate concentration (390 mM) using a straightforward isolation process achieving excellent isolation yields (>90%) and effective space time yield (100–110 g/L d). Process improvements, demonstrated at preparative scale, increased the substrate concentration to 580 mM achieving a space time yield of 260 g/L d.     

Practical asymmetric enzymatic reduction through discovery of a dehydrogenase-compatible biphasic reaction media

[reaction: see text] An enzyme-compatible biphasic reaction media for the asymmetric biocatalytic reduction of ketones with in situ cofactor regeneration has been developed. In this biphasic reaction media, which is advantageous for reactions at higher substrate concentrations, both enzymes (alcohol dehydrogenase and FDH from Candida boidinii) remain stable. The reductions with poorly water-soluble ketones were carried out at substrate concentrations of 10-200 mM, and the optically active (S)-alcohols were formed with moderate to good conversions and with up to >99% ee.     

Ru(II) complexes of cyclohexane diamine and monodentate phosphorus ligands for asymmetric ketone hydrogenation

The incorporation of a trans-1,2-diaminocyclohexane in place of DPEN provides improvements in enantioselectivity to asymmetric ketone hydrogenation reactions using BrXuPHOS–Ru–diamine catalysts. Substrates containing halogenated aryl rings are particularly compatible with this catalyst, however, α-chlorinated ketones remain resistant to reduction under any conditions.     

Facile Access to Chiral Alcohols with Pharmaceutical Relevance Using a Ketoreductase Newly Mined from Pichia guilliermondii

Chiral secondary alcohols with additional functional groups are frequently required as important and valuable synthons for pharmaceuticals, agricultural and other fine chemicals. With the advantages of environmentally benign reaction conditions, broad reaction scope, and high stereoselectivity, biocatalytic reduction of prochiral ketones offers significant potential in the synthesis of optically active alcohols. A CmCR homologous carbonyl reductase from Pichia guilliermondii NRRL Y‐324 was successfully overexpressed. Substrate profile characterization revealed its broad substrate specificity, covering aryl ketones, aliphatic ketones and ketoesters. Furthermore, a variety of ketone substrates were asymmetrically reduced by the purified enzyme with an additionally NADPH regeneration system. The reduction system exhibited excellent enantioselectivity (>99% ee) in the reduction of all the aromatic ketones and ketoesters, except for 2‐bromoacetophenone (93.5% ee). Semi‐preparative reduction of six ketones was achieved with high enantioselectivity (>99% ee) and isolation yields (>80%) within 12 h. This study provides a useful guidance for further application of this enzyme in the asymmetric synthesis of chiral alcohol enantiomers.     

Daucus carota and baker’s yeast mediated bio-reduction of prochiral ketones

Stereoselective reduction of prochiral ketones to the corresponding alcohols using biocatalysts has attracted much attention, from the viewpoint of green chemistry. Asymmetric reduction of indanone, tetralone and hydroxyl trimonoterpene ketones to the corresponding enantiomerically pure (S)-alcohols, using Daucus carota plant homogenate and fermented baker’s yeast cells, is described. The present study illustrates the broad substrate selectivity of the dehydrogenase enzymes present in the D. carota in the synthesis of a wide range of chiral secondary alcohols of biological importance.     

Asymmetric ketone reduction by a hyperthermophilic alcohol dehydrogenase. The substrate specificity,enantioselectivity and tolerance of organic solvents

In an effort to search for effective biocatalysts for asymmetric ketone reduction, the substrate specificity and enantioselectivity of an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus have been evaluated. This hyperthermophilic alcohol dehydrogenase catalyzes the reduction of various ketones including aryl ketones, α- and β-ketoesters. Interestingly, aryl ketones, phenyl-substituted α- and β-ketoesters were reduced to the corresponding chiral alcohols in an enantiomerically pure form, while the substrates lacking phenyl groups were reduced with a moderate enantioselectivity. It thus suggests that a phenyl group next to the carbonyl group could be very helpful for achieving an excellent enantioselectivity, and this could provide valuable guidance for the future application of this useful enzyme through rational substrate engineering. The reaction temperature increased the enzyme activity, but exerted no effect on the enantioselectivity. This alcohol dehydrogenase also showed a high tolerance of organic solvents such as dimethyl sulfoxide, iso-propanol, methyl tert-butyl ether, and hexane, a particularly important and useful feature for the reduction of ketones with a low solubility in aqueous buffers.     

Preparative access to medicinal chemistry related chiral alcohols using carbonyl reductase technology

Libraries of highly enantioenriched secondary alcohols in both enantiomeric forms were synthesised by enzymatic reduction of their parent ketones using selectAZyme? carbonyl reductase (CRED) technology. Commercially available CREDs were able to reduce a range of substrate classes efficiently and with very high enantioselectivity. Matching substrate classes to small subsets of CREDs enabled the fast development of preparative bioreductions and the rapid generation of 100–1500 mg samples of chiral alcohols in typically >95% ee and the majority in ?99.0% ee. The conditions for small scale synthesis were then scaled up to 0.5 kg to deliver one of the chiral alcohols, (S)-1-(4-bromophenyl)-2-chloroethanol, in 99.8% ee and 91% isolated yield.     

Highly Efficient Synthesis of Optically Pure (S)-1-phenyl-1,2-ethanediol by a Self-Sufficient Whole Cell Biocatalyst

Terminal vicinal diols are important chiral building blocks and intermediates in organic synthesis. Reduction of α-hydroxy ketones provides a straightforward approach to access these important compounds. In this study, it has been found that asymmetric reduction of a series of α-hydroxy aromatic ketones and 1-hydroxy-2-pentanone, catalyzed by Candida magnolia carbonyl reductase (CMCR) with glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration, afforded 1-aryl-1,2-ethanediols and pentane-1,2-diol, respectively, in up to 99 % ee. In order to evaluate the efficiency of the bioreduction, lyophilized recombinant Escherichia coli whole cells coexpressing CMCR and GDH genes were used as the biocatalyst and α-hydroxy acetophenone as the model substrate, and the reaction conditions, such as pH, cosolvent, the amount of biocatalyst and the presences of a cofactor (i.e., NADP⁺), were optimized. Under the optimized conditions (pH 6, 16 h), the bioreduction proceeded smoothly at 1.0 m substrate concentration without the external addition of cofactor, and the product (S)-1-phenyl-1,2-ethanediol was isolated with 90 % yield and 99 % ee. This offers a practical biocatalytic method for the preparation of these important vicinal diols.     

A recombinant ketoreductase tool-box. Assessing the substrate selectivity and stereoselectivity toward the reduction of β-ketoesters

The substrate selectivity and stereoselectivity of a series of ketoreductases were evaluated toward the reduction of two sets of β-ketoesters. Both the structural variety at β-position and the substituent at α-position greatly affected the activity and stereoselectivity of these ketoreductases. For the first set of β-ketoesters, at least one ketoreductase was found that catalyzed the formation of either (d) or (l) enantiomer of β-hydroxyesters from each substrate with high optical purity, with the only exception of ethyl (d)-3-hydroxy-3-phenylpropionate. For the second set of β-ketoesters with α-substituents, the situation is more complex. More commonly, a ketoreductase was found that formed one of the four diastereomers in optically pure form, with only a few cases in which enzymes could be found that formed two or more of the diastereomers in high optical purity. The continued development of new, more diverse ketoreductases will create the capability to produce a wider range of single diastereomers of 2-substituted-3-hydroxy acids and their derivatives.     

Enantioselective synthesis of ethyl (S)-2-hydroxy-4-phenylbutyrate by recombinant diketoreductase

Recombinant diketoreductase showed excellent stereoselectivity in the double reduction of β,δ-diketo esters. To investigate the substrate specificity and to broaden the applications of this new biocatalyst, a number of ketone substrates were used to evaluate the substrate spectrum and enantioselectivity of this enzyme in the present study. Among the ketone substrates tested, only this enzyme displayed high efficiency and excellent enantioselectivity in the reduction of ethyl 2-oxo-4-phenylbutyrate to ethyl (S)-2-hydroxy-4-phenylbutyrate. After optimizing the reaction conditions, the bio-reduction of ethyl 2-oxo-4-phenylbutyrate at a substrate concentration of 0.8 M (164.8 g/L) was achieved by the recombinant diketoreductase in an aqueous-toluene biphasic system coupled with formate dehydrogenase for the regeneration of cofactor, resulting in an overall hydroxyl product yield of 88.7% (99.5% ee). This new enzymatic transformation may offer a practical method for the preparation of this important chiral building block.     

Electrochemical reduction of α-unsaturated carbonyl compounds: Part VI. Effect of electrogenerated bases in very weakly protic solvents

Unsaturated carbonyl compounds were reduced electrochemically in carefully dried aprotic solvents. In the absence of protonating impurities, the i—V curves could be modified due to the de-activation of a part of the substrate by the action of the electrogenerated bases. In DMF solutions, the electrogenerated base which is able to deprotonate the ketone is mainly the dianion and discrepancies occur with, classical polarograms, of this class of reactants as observed at the level of the second step. For more conjugated ketones, a very cathodic step is assumed to correspond to the reduction of the conjugated anion of the ketone but the instability of this anion (inducing polymerisation reactions) prevents the acquisition of careful analytical evidence.     

Employing modular polyketide synthase ketoreductases as biocatalysts in the preparative chemoenzymatic syntheses of diketide chiral building blocks

Chiral building blocks are valuable intermediates in the syntheses of natural products and pharmaceuticals. A scalable chemoenzymatic route to chiral diketides has been developed that includes the general synthesis of α-substituted, β-ketoacyl N-acetylcysteamine thioesters followed by a biocatalytic cycle in which a glucose-fueled NADPH-regeneration system drives reductions catalyzed by isolated modular polyketide synthase (PKS) ketoreductases (KRs). To identify KRs that operate as active, stereospecific biocatalysts, 11 isolated KRs were incubated with 5 diketides and their products were analyzed by chiral chromatography. KRs that naturally reduce small polyketide intermediates were the most active and stereospecific toward the panel of diketides. Several biocatalytic reactions were scaled up to yield more than 100?mg of product. These syntheses demonstrate the ability of PKS enzymes to economically and greenly generate diverse chiral building blocks on a preparative scale.     

A novel and efficient direct aldol condensation from ketones and aromatic aldehydes catalyzed by proline-TEA through a new pathway

A novel and efficient direct aldol condensation from various ketones and a wide range of aldehydes was catalyzed by l-proline-TEA (triethylamine) in MeOH at room temperature, affording the corresponding (E)-α,β-unsaturated ketones in excellent yields. By using the method, some complicated (E)-α,β-unsaturated ketone C-glycosides were obtained from unmodified ketone C-glycosides and aldehydes. This reaction proceeds through a new pathway, in which the specific intermediate was captured and identified.     

Baeyer-Villiger单加氧酶在有机合成中的应用

综述了Baeyer-Villiger单加氧酶在有机合成中的应用.较之传统的化学反应, 氧化酶催化剂反应有较好的选择性、可控性和经济性. 环己酮加氧酶是一种还原型辅酶I (NADPH)依赖型氧化酶, 是最早被报道能够催化Baeyer-Villiger氧化的酶. 这些重要反应产生了合成化学家很感兴趣的扩环产物. 环己酮加氧酶也是有用的生物催化剂, 由于辅酶再生的问题已被工程菌克服了, 所以能像全细胞催化剂那样使用. 对酮包括杂环酮进行Baeyer-Villiger氧化和动态动力学拆分, 放大这种反应作为合成路线是很有前途的.     

Production of (R)-chiral alcohols by a hydrogen-transfer bioreduction with NADH-dependent Leifsonia alcohol dehydrogenase (LSADH)

Alcohol dehydrogenase (LSADH) isolated from Leifsonia sp. S749 was used to produce (R)-chiral alcohols. The enzyme with a broad substrate range reduced various prochiral ketones and keto esters to yield optically active secondary alcohols with a high enantiomeric excess. LSADH transferred the pro-S hydrogen of NADH to the carbonyl moiety of phenyl trifluoromethyl ketone 13 through its re face to give (S)-1-phenyl-2,2,2-trifluoroethanol 40. LSADH was able to efficiently reproduce NADH when 2-propanol was used as a hydrogen donor in the reaction mixture.