Stereoselective biocatalytic hydride transfer to substituted acetophenones by the yeast Metschnikowia koreensis

Freely suspended and variously entrapped viable cells of the yeast Metschnikowia koreensis were examined for the asymmetric reduction of prochiral acetophenone. A ketone substrate at 25 mM can be converted (92%) to the corresponding alcohol within 3 h using freely suspended cells [46 mg/mL dry cell weight (DCW)] at pH 9 (Tris buffer, 50 mM), 25 °C, in an agitated reactor (200 rpm). The reaction displayed an excellent stereoselectivity of >99%. Supplementation of the reaction mixture with glucose (20 g/L) greatly enhanced the rate of the bioreduction reaction likely because of improved cofactor recycling in the cells. The cells could successfully reduce various acetophenones substituted with electron withdrawing groups on the phenyl ring, particularly at the para-position compared to ortho- or meta-substituted acetophenones. The ketone reductase of M. koreensis showed Prelog-selectivity as the reaction exclusively yielded (S)-alcohols. The thermostability and the substrate tolerance of the yeast were improved by immobilization in calcium alginate beads. Immobilization reduced the effectiveness factor only slightly.     

(R)-1-Arylethanols from aryl iodides through a two-step one-pot enantioselective chemoenzymatic process

(R)-1-Arylethanols have been prepared in high to excellent overall yields through a two-step one-pot process that involves the palladium-catalyzed conversion of aryl iodides into the corresponding acetophenones, in the presence of acetic anhydride, EtN(i-Pr)₂, LiCl, and Pd₂(dba)₃ followed by an enantioselective reduction step catalyzed by the alcohol dehydrogenase enzyme from Lactobacillus brevis.     

Bioactive acetophenones from Plectranthus venteri

In a project to investigate the chemistry of South African Plectranthus species, from the dichloromethane extract of the aerial parts of Plectranthus venteri, we isolated two known natural product acetophenones, namely 2-hydroxy-3,4,5,6-tetramethoxy-acetophenone (1) and 2-hydroxy-4,5,6-trimethoxy-acetophenone (2). Structures were assigned using NMR spectroscopy and HRTOFESIMS. Compound 1 was previously synthesised as a precursor to the polymethoxylated flavone nobiletin. The acetophenones exhibited remarkable inhibitory activities against the transfer of the IncW plasmid R7K in a bacterial plasmid transfer inhibition assay.     

Prenylated acetophenones from Melicope obscura and Melicope obtusifolia ssp. obtusifolia var. arborea and their distribution in Rutaceae

Four prenylated acetophenones 2,6-dihydroxy-4-geranyloxyacetophenone (1), 4-geranyloxy-2,6,β-trihydroxyacetophenone (2), 2,6-dihydroxy-4-geranyloxy-3-prenylacetophenone (3), and 4-geranyloxy-3-prenyl-2,6,β-trihydroxyacetophenone (4) have for the first time been isolated from Melicope obscura (1 and 2) and Melicope obtusifolia ssp. obtusifolia var. arborea (3 and 4). The distribution of prenylated acetophenones in Rutaceae is reviewed and the results, including the new records, indicate that prenylated acetophenones are valuable as chemotaxonomic markers for the subfamily Rutoideae, tribe Xanthoxyleae sensu Engler.     

Synthesis and antioxidant properties of substituted 2-phenyl-1H-indoles

In this study, we report the design, synthesis and antioxidant activity of a series of substituted 2-(4-aminophenyl)-1H-indoles and 2-(methoxyphenyl)-1H-indoles. The new compounds are structurally related to the known indole-based antioxidant lead compound melatonin (MLT), and the antitumour 2-(4-aminophenyl)benzothiazole and 2-(3,4-dimethoxyphenyl)benzothiazole series. Efficient access to the target 2-phenylindoles was achieved via Fischer indole synthesis between substituted phenylhydrazines and acetophenones. 2-(4-Aminophenyl)indoles (such as the 6-fluoro analogue 3b) in particular showed potent antioxidant activity in the DPPH and superoxide radical scavenging assays (80% and 81% inhibition at 1 mM concentration of 3b, respectively), at a level comparable with the reference standard MLT (98% and 75% at 1 mM).     

Production of (R)‐1‐phenylethanols through bioreduction of acetophenones by a new fungus isolate Trichothecium roseum

A total of 120 fungal strains were isolated from soil samples and evaluated in the bioreduction of substituted acetophenones to the corresponding (R)‐alcohols. Among these strains, isolate Trichothecium roseum EBK‐18 was highly effective in the production of (R)‐alcohols with excellent enantioselectivity (ee > 99%). Gram scale preparation of (R)‐1‐phenylethanol is reported. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Synthesis and pharmacological studies of new pyrazole analogues of podophyllotoxin

The pyrazole analogues of podophyllotoxin were synthesized by the chalcone route. This route attracts the attention because of its simple operating conditions and easy availability of the chemicals. Initially, benzylideneacetophenones (chalcones) were prepared in high yields by Claisen-Schmidt reaction of acetophenones with 4-(methylthio)benzaldehyde. The cyclopropyl ketones were prepared in good yields by the reaction of chalcones with trimethylsulfoxonium iodide. Tetralones were prepared in good yields by the Friedel-Craft’s intramolecular cyclization reaction of cyclopropyle ketones in the presence of anhyd. stannic chloride and acetic anhydride. The tetralones on formylation to give substituted hydroxylmethylene tetralones. Condensation of substituted hydroxylmethylene tetralones with hydrazine hydrate afforded target compounds. The structures of the synthesized compounds were confirmed by IR, ¹H NMR and Mass spectral technique. The title compounds were screened for their antimitotic and antimicrobial activities. Among the synthesized compounds cyclopropyl ketones and pyrazole analogues of podophyllotoxin, compound 7-(methylthio)-5-(4-(methylthio)phenyl)-4,5-dihydro-² H-benzo[g]indazole is more active than 5-(4-(methylthio)phenyl)-4,5-dihydro-² H-benzo[g]indazole, 7-methyl-5-(4-(methylthio)phenyl)-4,5-dihydro-² H-benzo[g]indazole, 7-methoxy-5-(4-(methylthio)phenyl)-4,5-dihydro-² H-benzo[g]indazole and the key intermediate tetralones in 100, 200 and 400 ppm at 12, 18 and 24 h and also showed very good activity against screened bacteria and fungi compared to their standard.     

Isolation and characterization of a novel Rhodococcus strain with switchable carbonyl reductase and para-acetylphenol hydroxylase activities

In the search for an effective biocatalyst for the reduction of acetophenones with unprotected hydroxy group on the benzene ring, a microorganism, which reduced para-acetylphenol to S-(?)-1-(para-hydroxyphenyl)ethanol under anaerobic conditions, was isolated from soil samples and the 16S rDNA study showed that it was phylogenetically affiliated with species of the genus Rhodococcus and was most similar to Rhodococcus pyridinivorans. Unexpectedly, this strain also hydroxylated para-acetylphenol to give 4-acetylcatechol in presence of oxygen, possessing para-acetylphenol hydroxylase activity. While the reduction of para-acetylphenol had an optimal reaction pH at 7 and a broad optimal temperature range (35–45 °C), the hydroxylation reached the maximum conversion at the pH range of 7–8 and 35 °C. This study identified for the first time a Rhodococcus strain with para-acetylphenol hydroxylase activity, which also contains highly enantioselective carbonyl reductase activity with potential applications for the asymmetric reduction of these less-explored but important ketones such as α-aminoacetophenone, 3′-hydroxyacetophenone and 4′-hydroxyacetophenone. The para-acetylphenol hydroxylase and carbonyl reductase activity are switchable by the reaction conditions.     

Highly Enantioselective Production of Chiral Secondary Alcohols with Candida zeylanoides as a New Whole Cell Biocatalyst

The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of biocatalysts in reaction, economic, and ecological issues. Herein, fifteen yeast strains belonging to three food originated yeast species Candida zeylanoides, Pichia fermentans, and Saccharomyces uvarum were tested for their capability for asymmetric reduction of acetophenone to 1‐phenylethanol as biocatalysts. Of these strains, C. zeylanoides P1 showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high yields. The preparative scale asymmetric bioreduction of 4‐nitroacetophenone ( 1m ) by C. zeylanoides P1 gave (S)‐1‐(4‐nitrophenyl)ethanol ( 2m ) with 89% yield and > 99% enantiomeric excess. Compound 2m has been obtained in an enantiomerically pure and inexpensive form. Additionally, these results indicate that C. zeylanoides P1 is a promising biocatalyst for the synthesis of chiral alcohols in industry.     

Cloning and characterization of three ketoreductases from soil metagenome for preparing optically active alcohols

Objectives

To discover novel ketoreductases (KRED) from soil metagenome preparation of chiral alcohols.

Results

Three putative KRED were cloned, heterologously expressed in Eschericha coli and characterized based on the sequence analysis of soil metagenome. All the three enzymes (KRED424, KRED432, and KRED433) had maximum activity at 55 °C and pH 7. KRED424 had a broader substrate spectrum compared with the other two. Three prochiral carbonyl compounds were used to evaluate the abilities of enantioselective reductions of the KRED. For N-Boc-3-pyrrolidone, all enzymes produced an (S)-type alcohol in enantiomeric excess (>99 % ee). For ethyl 2-oxo-4-phenylbutyrate, KRED424 showed a higher conversion (91.5 %) and enantioselectivity (S-type, >99 % ee) than KRED432 and KRED433. For ethyl 4-chloroacetoacetate (COBE), both of KRED424 and KRED433 completely converted 20 mM substrate and KRED433 could obtain an (R)-alcohol with 94 % ee.

Conclusions

The three ketoreductases have potential in the preparation of pharmaceuticals and fine chemicals.

     

Debaryomyces hansenii as a new biocatalyst in the asymmetric reduction of substituted acetophenones

Chiral secondary alcohols are convenient mediator for the synthesis of biologically active compounds and natural products. In this study fifteen yeast strains belonging to three food originated yeast species Debaryomyces hansenii, Saccharomyces cerevisiae and Hanseniaspora guilliermondii were tested for their capability for the asymmetric reduction of acetophenone to 1-phenylethanol as biocatalyst microorganisms. Of these strains, Debaryomyces hansenii P1 strain showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to the corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high conversion rates. This is the first report on the enantioselective reduction of acetophenone by D. hansenii P1 from past?rma, a fermented Turkish meat product. The preparative scale asymmetric bio reduction of 3-methoxy acetophenone 1g by D. hansenii P1 gave (R)-1-(3-methoxyphenyl) ethanol 2g 82% yield, and >99% enantiomeric excess. Compound 2g can be used for the synthesis of (+)-NPS-R-568 [3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenly) ethyl] propan-1-amine] which have a great potential for the treatment of primary and secondary hyper-parathyroidism. In addition, D. hansenii P1 successfully reduced acetophenone derivatives. This study showed that this yeast can be used industrially to produce enantiomerically pure chiral secondary alcohols, which can be easily converted to different functional groups.     

Synthesis and fungicidal activity of novel imidazole-based ketene dithioacetals

Novel imidazole-based ketene dithioacetals show impressive in planta activity against the economically important plant pathogens Alternaria solani, Botryotinia fuckeliana, Erysiphe necator and Zymoseptoria tritici. Especially derivatives of the topical antifungal lanoconazole, which bear an alkynyloxy or a heteroaryl group in the para-position of the phenyl ring, exhibit excellent control of the mentioned phytopathogens. These compounds inhibit 14α -demethylase in the sterol biosynthesis pathway of the fungi. Synthesis routes starting from either benzaldehydes or acetophenones as well as structure-activity relationships are discussed in detail.     

Didymosphaeria igniaria: a new microorganism useful for the enantioselective reduction of aryl-aliphatic ketones

Didymosphaeria igniaria is a promising biocatalyst in asymmetric reductions of prochiral aromatic-aliphatic ketones such as acetonaphthones, acetophenones, and acetylpyridines. The organism converted the substrates mainly to (S)-alcohols. Excellent results in terms of conversion and enantioselectivity (100% yield, >99% ee) were obtained with acetonaphthones. In case of acetyl pyridines, the optical purity of the product depended on the position of the carbonyl group on the pyridine ring and followed the order 2-acetyl ? 4-acetyl > 3-acetyl-pyridine. Transformation of o-methoxy-acetophenone gave optically pure (S)-(-)-1-(2-methoxyphenyl)-ethanol in 95% yield. The transformation of para-methyl ketone gave (R)-alcohol (81% ee), whereas para-bromo ketone gave (S)-alcohol (98% ee). Monitoring of the biotransformation of these substrates over time led to the conclusion that for both substrates, non-selective carbonyl group reduction occurred in the first step, followed by selective oxidation of the (R)-isomer of p-bromo-phenylethanol and selective oxidation of the (S)-isomer of p-methyl-phenylethanol. D. igniaria exhibited poor enantioselectivity in the reduction of bicyclic aryl-aliphatic ketones such as 1- and 2-tetralones. Only (S)-5-methoxy-1-tetralol was obtained in optically pure (>99% ee) form.     

Spectrum of the Reductive Dehalogenation Activity of Desulfitobacterium frappieri PCP-1

Desulfitobacterium frappieri PCP-1 was induced for ortho- and para-dechlorinating activities by different chlorophenols. Dehalogenation rates ranging from 25 to 1,158 nmol/min/mg of cell protein were observed according to the chlorophenol tested and the position of the chlorine removed. D. frappieri shows a broad substrate specificity; in addition to tetrachloroethylene and pentachloropyridine, strain PCP-1 can dehalogenate at ortho, meta, and para positions a large variety of aromatic molecules with substituted hydroxyl or amino groups. Reactions of O demethylation and reduction of nitro to amino substituents on aromatic molecules were also observed.     

Substituent effects in the enantioselective reduction of acetophenones with nabh4 in the presence of β-cyclodextrin

The effect of substituent groups on asymmetric induction by β-cyclodextrin (β-CD) was investigated in the reduction of a series of o-, m-, and p-substituted acetophenones (X = H, Br, Cl, CH₃, NO₂, OCH₃) with aqueous NaBH₄. The inclusion of the ketones studied in β-CD led to water-insoluble compounds so that the reaction proceeded in the solid state. The substitutions resulted generally in higher enantioselectivities than that obtained for acetophenone indicating stronger host—guest interactions. Acetophenone and its m- and p-derivatives gave preponderantly the (?)-alcohol while the prevailing enantiomer was the (+)-alcohol in the case of the o-derivatives. The enantioface selectivity was found to be mainly governed by steric demands imposed by the size and the shape of the β-CD cavity in the case of the o-substituted acetophenones and by hydrophobic interactions in the case of the m-derivatives. A more complicated situation arose from the asymmetic reduction of p-derivatives where a combination of these factors with hydrogen bonding of the carbonyl group to the hydroxyls of β-CD are responsible for the enantioselectivity. © 1994 Wiley-Liss, Inc.     

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

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and β-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.     

Reductive transformation of benzoate by Nocardia asteroides and Hormoconis resinae

A variety of substituted aromatic acids were reduced to the corresponding alcohols by Nocardia asteroides JCM3016 under aerobic conditions. An isolated mold, Hormoconis resinae F328, could also reductively transform benzoate, the erythro isomer of (1R, 2S)-1-phenyl-1,2-propanediol being yielded as well as benzyl alcohol. C-1 of the diol was found to be derived from the α-carbon of benzoate by ¹³C-NMR analysis. The acyloin condensation between pyruvate and benzaldehyde formed from benzoate is assumed to participate in the diol formation. An ATP-dependent and NADPH-linked benzoate reductase (EC 1.2.1.30) and an NADPH-linked benzaldehyde reductase (EC 1.1.1.91) were demonstrated to participate in the benzoate reduction in both N. asteroides JCM 3016 and H. resinae F328.     

Comparative analysis of tertiary alcohol esterase activity in bacterial strains isolated from enrichment cultures and from screening strain libraries

The preparation of enantiopure tertiary alcohols is of great contemporary interest due to the application of these versatile building blocks in organic synthesis and as precursors towards high value pharmaceutical compounds. Herein, we describe two approaches taken towards the discovery of novel biocatalysts for the synthesis of these valuable compounds. The first approach was initiated with screening of 47 bacterial strains for hydrolytic activity towards the simple tertiary alcohol ester tert-butyl acetate. In conjunction, a second method focussed on the isolation of strains competent for growth on tert-butyl acetate as the sole source of carbon and energy. From functional screening, 10 Gram-positive Actinomycetes showed hydrolytic activity, whilst enrichment selection resulted in the identification of 14 active strains, of which five belong to the Gram-negative cell-wall type. Bacterial strains obtained from both approaches were viable for enantioselective hydrolysis of pyridine substituted tertiary alcohol esters in addition to bulky aliphatic and keto-derived substrates from the same class. Activity towards each of the test substrates was uncovered, with promising enantioselectivities of up to E = 71 in the hydrolysis of a para-substituted pyridine tertiary alcohol ester using a strain of Rhodococcus ruber. Interestingly strains of Microbacterium and Alcaligenes sp. gave opposite enantiopreference in the hydrolysis of a meta-substituted pyridine tertiary alcohol ester with E values of 17 and 54. These approaches show that via both possibilities, screening established strain collections and performing enrichment selection, it is possible to identify novel species which show activity towards sterically challenging substrates.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : IV. COMBINED ACTION OF SUBSTITUTED PHENOLS,CYANIDE, CARBON MONOXIDE,AND OTHER RESPIRATORY INHIBITORS ON RESPIRATION AND CELL DIVISION

The effects of 4,6-dinitro-o-cresol and 2,4,5-trichlorophenol on the respiration and cell division of fertilized eggs of Arbacia punctulata have been determined in the presence of each of a number of respiratory inhibitors. The experimental results obtained appear to afford some understanding of the mechanism of action of the substituted phenols on respiration and on cell division. 1. From the fact that the stimulated respiration is completely cyanide and carbon monoxide sensitive, it may be concluded that all of the extra oxygen uptake induced in Arbacia eggs by 4,6-dinitro-o-cresol passes through the metal containing oxidase system. All of the extra oxygen uptake also passes through oxidative steps which can be poisoned by non-stimulating phenols like 2,4-dinitrothymol and 4-nitrocarvacrol, by phenylurethane, by 5-isoamyl-5-ethyl barbituric acid, by malonic acid, or by iodoacetic acid. To abolish all respiratory stimulation by suboptimum concentrations of 4,6-dinitro-o-cresol, each of these inhibitors must be present in a concentration which reduces the normal respiration in the absence of substituted phenols by at least 20–40 per cent. 2. The degree of reduction of the stimulated respiration by a given concentration of carbon monoxide or potassium cyanide depends on the concentration of 4,6-dinitro-o-cresol or 2,4,5-trichlorophenol, being most marked in suboptimum concentrations and least marked in greater than optimum concentrations of the substituted phenol. In contrast to this result, the reduction of the stimulated respiration by a given concentration of 5-isoamyl-5-ethyl barbituric acid or malonic acid is least marked in suboptimum concentrations and most marked in greater than optimum concentrations of the substituted phenol. 3. The present experiments appear to indicate that the inhibition of cell division by substituted phenols is not attributable to a direct action of these agents on mitotic processes nor to an overstimulation of any respiratory process. The inhibition of cell division appears to be associated with the inhibition, by the substituted phenols, of some component of the cyanide sensitive respiratory system. This inhibition is of such a type as to allow the overall respiration to proceed at a rate in excess of the control value, even when division is completely suppressed. The dependence of the division mechanism on a respiratory step which is relatively hypersensitive to poisoning by the substituted phenols is comparable to the dependence of the Pasteur reaction in certain normal and tumor tissues on an oxidative step which is specifically poisoned by the substituted phenols (16). The substituted phenols have no inhibiting effect in vitro on the principal metal containing respiratory catalysts or the principal dehydrogenases; they also do not inhibit the fermentative reactions involved in the anaerobic glycolysis of fertilized Arbacia eggs. It is therefore suggested that the respiratory inhibiting and division inhibiting effects of the substituted phenols may be attributable to the action of these substances on one or more of the oxidation-reduction or phosphorylating steps which are involved in the transfer of hydrogen from the dehydrogenase systems to the specifically cyanide sensitive oxidase mechanism of the eggs. The identification of the respiratory step poisoned by the substituted phenol would constitute an interesting contribution to the chemistry of cell division and experiments to this end are now in progress.     

Reduction potentials of seventy-five free base porphyrin molecules: Reactivity correlations and the prediction of potentials

Phase sensitive fundamental harmonic ac voltammetric techniques have been used to measure the reduction potentials of seventy-five metal-free meso and β-pyrrole substituted porphyrins in DMF solution. Correlations are explored between the reduction potentials and porphyrin and metalloporphyrin reactivity. Such porphyrin reduction potentials can be reliably predicted using assigned substitutent partial potential values.