Production of (R )-3-pentyn-2-ol through stereospecific hydrolysis of racemic 3-pentyn-2-ol esters with microbial enzymes

Microorganisms and commercial enzymes were screened for their ability to produce (R)-3-pentyn-2-ol from racemic 3-pentyn-2-ol esters through stereospecific hydrolysis. Among the esters formed with acetic acid, propionic acid, hexanoic acid and benzoic acid, the acetate was most effectively hydrolyzed by microbial cells and commercial lipases with high stereospecificity. Rhodococcus rubropertinctus AKU NOC082 was a good catalyst for (R)-3-pentyn-2-ol production through the hydrolytic resolution of racemic 3-pentyn-2-yl acetate. With 15%, 25% and 50% (v/v) racemic 3-pentyn-2-yl acetate as the substrate, 42.6%, 40.8% and 40.0% was hydrolyzed in 5 h, 10 h and 98 h respectively, under the optimized conditions (pH 7.0, 30 °C, 7.5% wet cell concentration), the (R) enantiomer of 3-pentyn-2-ol being formed with an optical purity of 97.8%, 98.0% and 94.2% respectively. Received: 2 June 1998 / Received revision: 3 August 1998 / Accepted: 3 September 1998     

(S)-3-Pentyn-2-ol production through microbial enzyme-catalyzed, highly enantioselective hydrolysis of racemic 3-pentyn-2-ol esters

Air-dried cells of Hansenula nonfermentans AKU 4332 catalyzed the production of (S)-3-pentyn-2-ol from (RS)-3- pentyn-2-ol acetate ester at 10% (v/v). The product was formed at 96.6% e.e. with a molar yield of 45% in 24 h. © Rapid Science Ltd. 1998     

Production of (R)-3-pentyn-2-ol through stereoinversion of racemic 3-pentyn-2-ol by Nocardia fusca AKU 2123

Wet cells of Nocardia fusca AKU 2123 are good catalysts for the production of (R)-3-pentyn-2-ol (PYOH) from (RS)-PYOH through a stereoinversion reaction. Under optimal conditions (350 mM potassium phosphate buffer, pH 8.0, 30% (w/v) wet cells, 0.12% NADPH, 10% glucose, and 30 U/ml glucose dehydrogenase) (R)-PYOH of high optical purity (98.7% e.e.) was produced from 2% (v/v) (RS)-PYOH with a yield of 70.4% by 140 h incubation. Received: 22 January 1999 / Received revision: 23 April 1999 / Accepted: 1 May 1999     

NAD(+)-dependent (S)-specific secondary alcohol dehydrogenase involved in stereoinversion of 3-pentyn-2-ol catalyzed by Nocardia fusca AKU 2123

An NAD(+)-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fusca AKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The K(m) and Vmax for (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mM and 53 mumol/min/mg, and 0.33 mM and 130 mumol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The K(m) and Vmax for 2-hexanone reduction were 2.5 mM and 260 mumol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH2-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolis and phenylacetaldehyde reductase from Corynebacterium sp.     

Synthesis of (–)-Muricatacin

The synthesis of (–)-muricatacin starting from 1-bromododecane and 2-pentyn-l-ol is described. 2-Pentadecyn-1-ol (4), which was prepared from 1-bromododecane (2) and 2-pentyn-1-ol (3), was converted to epoxy alcohol 6 through a two-step reaction sequence, 6 being successively submitted to tosylation, iodination, chain extension with tert-butyl lithioacetate, and acid-catalyzed cyclization to give (–)-muricatacin (1a). Recrystallization afforded optically pure 1a.     

Epimerisation of 3β-ol to 3α-ol steroid alkaloids by Nocardia restrictus

(22S,25S)-5α-tomatanin-3β-ol, N-acetyl-(22S,25S)-5α-tomatanin-3β-ol, (22R,25R)-5α-tomatanin-3β-ol and (22R,25S)-22,26-epimino-5α-cholestane-3β,16β-diol are transformed by Nocardia restrictus into corresponding 3α-ol compounds with yields from 70 to 5%.     

Cooperative effect of the camphor imine ligand in alkynols activation by trans-[MCl2(YNC10H14O)2] (M = Pd or Pt)

The cooperative effect of the camphor imine ligand (YNC₁₀H₁₄O) in the cyclization of 5-hexyn-1-ol and 4-pentyn-1-ol promoted by complexes trans-[MCl₂(YNC₁₀H₁₄O)₂] (M = Pd, Y = NH₂, NHMe, NMe₂, OH, Ph; Pt, Y = NH₂, NHMe, NMe₂) is established from a direct relation between the constants calculated for conversion of 5-hexyn-1-ol (A) into 2-methyl-2-pent-4-ynyloxy-tetrahydropyran (B) and 4-pentyn-1-ol (C) into 2-methyl-2-pent-4-ynyloxy-tetrahydrofuran (D) and the basic character of the camphor imine substituent (Y). In the catalytic process acid-base interactions between the alkynol and the coordinated camphor imine are supported by the structural characterization of [PdCl₄][Me₂NHNC₁₀H₁₄O]₂.     

NAD+-Dependent (S)-Specific Secondary Alcohol Dehydrogenase Involved in Stereoinversion of 3-Pentyn-2-ol Catalyzed by Nocardia fusca AKU 2123

An NAD⁺-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fusca AKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The K _m and V _max for (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mM and 53 μmol/min/mg, and 0.33 mM and 130 μmol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The K _m and V _max for 2-hexanone reduction were 2.5 mM and 260 μmol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH₂-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolis and phenylacetaldehyde reductase from Corynebacterium sp.     

Genetic and Cytogenetic Analysis of the ADH Region in DROSOPHILA MELANOGASTER

Eighteen Adh-negative mutations were selected with 1-pentyn-3-ol after feeding of formaldehyde. Twelve of the 18 were shown by cytological and genetic analysis to be deletions. Cytological examination of the deletions allowed us to localize the Adh gene to a region including bands 35B3-5 on the left arm of chromosome 2. The deletions were also used to order known visible loci located near Adh.--The vital loci near Adh were also investigated. A total of 109 lethal mutations were generated with EMS and 33 of these, localized within a region defined by the overlap of two of the deletions, were found to belong to 13 complementation groups. If one includes three other loci known to belong there (el, Adh and Sco) a total of 16 complemetation groups have been identified in the region close to Adh.     

Protective group-free syntheses of (±)-frontalin, (±)-endo-brevicomin, (±)-exo-brevicomin, and (±)-3,4-dehydro-exo-brevicomin: racemic pheromones with a 6,8-dioxabicyclo[3.2.1]octane ring

Protective group-free syntheses of four racemic pheromones with a 6,8-dioxabicyclo[3.2.1]octane ring were achieved in five or six steps from commercially available (±)-3-butyn-2-ol (6) and 2-alkenyl halides or 2-alken-1-ol by employing Lewis acid-catalyzed acetalization of δ, ε-epoxy ketones as the key reaction. (±)-Frontalin (1) was prepared in a 25% overall yield in five steps from methallyl chloride (5a), (±)-endo-brevicomin (2) was prepared in a 23% overall yield in five steps from (E)-2-pentenyl bromide (5b), and (±)-exo-brevicomin (3) and (±)-3,4-dehydro-exo-brevicomin (4) were both prepared in a 4% overall yield in six steps based on (Z)-2-penten-1-ol (12).     

Involvement of the cell wall ofNocardia restricta on the bioconversions of steroid sapogenins

The bioconversion of tigogenin, (25R)-5-spirostan-3-ol byNocardia restricta to (25R)-4-spirosten-3-one and (25R)-1,4-spirostadien-3-one was strongly increased using protoplasts instead of intact cells. The same effect could be achieved by adding-cyclodextrin to the medium. Since 5-steroid sapogenins pass through the cell wall without hindrance, it can be concluded that the cell wall ofNocardia restricta is a rate-limiting factor only for 5-isomers.     

Newly discovered polyamine, 2-hydroxyspermidine, in Pseudomonas acidovorans.

A previously unknown hydroxylated polyamine has been recovered from Pseudomonas acidovorans 29. It has been identified as 2-hydroxyspermidine, N4-(3-aminopropyl)-1,4-diaminobutane-2-ol, by its chromatographic behavior, electrophoretic mobility, and reaction with metaperiodate. It can be synthesized enzymatically from 2-hydroxyputrescine by cell-free preparations from Escherichia coli or P. acidovorans 29 which contain propylamine transferase. It is interesting to note that the naturally occurring compound is the 2-hydroxyspermidine and not the 3-hydroxyspermidine, N1-(3-aminopropyl)-1,4-diaminobutane-2-ol, indicating that the propylamine transferase reacts preferentially with the amine distal to the hydroxyl group. A mixture of 2- and 3-hydroxyspermidines and hydroxyspermine was synthesized by reacting acrylonitrile with 2-hydroxyspermidine and catalytic reduction of the products with hydrogen. N-(gamma-aminopropyl)-beta-alanine, used to help identify the hydroxyspermidines, was synthesized from N-(3-aminopropyl)-3-aminopropanenitrile by hydrolysis with 10% NaOH.     

The specificities and configurations of ternary complexes of yeast and liver alcohol dehydrogenases

1. Some aspects of the substrate specificities of liver and yeast alcohol dehydrogenases have been investigated with pentan-3-ol, heptan-4-ol, (+)-butan-2-ol, (+/-)-butan-2-ol, (+/-)-hexan-3-ol and (+/-)-octan-2-ol as potential substrates. The liver enzyme is active with all substrates tested, including both isomers of each optically active alcohol. In contrast, the yeast enzyme is completely inactive towards those secondary alcohols where both alkyl groups are larger than methyl and active with only the (+)-isomers of butan-2-ol and octan-2-ol. 2. The absence of stereospecificity of liver alcohol dehydrogenase towards optically active secondary alcohols and its broad specificity towards secondary alcohols in general are explained in terms of an alkyl-binding site that will react with a variety of alkyl groups and the ability of the enzyme to accommodate a fairly large unbound alkyl group in an active enzyme-NAD(+)-secondary alcohol ternary complex. The absolute optical specificity of the yeast enzyme towards n-alkylmethyl carbinols and its unreactivity towards pentan-3-ol, hexan-3-ol and heptan-4-ol are explained by its inability to accept alkyl groups larger than methyl in the unbound position in a viable ternary complex. 3. Comparison of the known configurations of the n-alkylmethyl carbinols and [1-(2)H]ethanol and [1-(3)H]geraniol, which have been used in stereospecificity studies with these enzymes by other workers, provides strong evidence for which alkyl group of the substrate is bound to the enzyme in the oxidation of n-alkylmethyl carbinols. The conclusions reached are, for butan-2-ol oxidation with the liver enzyme, confirmed by deductions from kinetic data obtained with (+)-butan-2-ol and a sample of butan-2-ol containing 66% of (-)-butan-2-ol. 4. Initial-rate parameters for the oxidations of (+)-butan-2-ol, 66% (-)-butan-2-ol and pentan-3-ol by NAD with liver alcohol dehydrogenase are presented. The data are completely consistent with a general mechanism of catalysis previously proposed for this enzyme.     

Synthesis and in vitro evaluation of antiviral and cytostatic properties of novel 8-triazolyl acyclovir derivatives

Abstract

As a part of the research aimed on identification of new nucleobase derivatives with improved biological properties, a series of novel 8-substituted acyclovir derivatives were synthesized. The 8-azidoguanosine 4 and novel 8-azidoacyclovir 9 were synthesized from commercially available guanosine 1 and acyclovir 6 which were transformed into 8-bromopurine derivatives 2 and 7 and hydrazine derivatives 3 and 8, respectively. 8-Triazolylguanosine 5 and 8-triazolylacyclovir analogs 10–12 were successfully synthesized via the Cu(I) catalyzed 1,3-dipolar cycloaddition reaction of azides 4 and 9 with propargyl alcohol, 4-pentyn-1-ol and 5-hexyn-1-ol. The novel 1,4-disubstituted 1,2,3-triazolyl compounds 5, 10–12 were evaluated for antiviral activity against selected DNA and RNA viruses and cytostatic activity against normal Madine Darby canine kidney (MDCK I) cells, and seven tumor cell lines (HeLa, CaCo-2, NCI-H358, Jurkat, K562, Raji and HuT78). While tested compounds exerted no antiviral activity at nontoxic concentrations, the 8-triazolyl acyclovir derivative 10, with the shortest alkyl substituent at the C-4 of triazole ring, was found to be the most active against the CaCo-2 cell line.     

Synthesis of Short-Chain Diols and Unsaturated Alcohols from Secondary Alcohol Substrates by the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

The Rieske nonheme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading bacterial strain Aquincola tertiaricarbonis L108 has been described to attack short-chain tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, whereas an mdpJ knockout mutant did not show such activity. In addition, wild-type cells converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-propanol resulted in an enantiomeric excess of about 70% for the (R)-enantiomer, indicating that this reaction was favored. Likewise, desaturation of (R)-2-butanol to 3-buten-2-ol was about 2.3-fold faster than conversion of the (S)-enantiomer. The biotechnological potential of MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block chemicals is discussed.     

A Model to Evaluate the Cytotoxicity of the Fungal Volatile Organic Compound 1-octen-3-ol in Human Embryonic Stem Cells

Microbial growth in damp indoor environments has been correlated with risks to human health. This study was aimed to determine the cytotoxicity of 1-octen-3-ol (“mushroom alcohol”), a major fungal volatile organic compound (VOC) associated with mushroom and mold odors. Using an airborne exposure technique, human embryonic stem cells were exposed for 1 h to different concentrations (0–1,000 ppm) of racemic 1-octen-3-ol and its enantiomers, (R)-(−)-1-octen-3-ol and (S)-(+)-1-octen-3-ol. Cytotoxicity was assayed using both the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and the fluorescently tagged Calcein AM-mediated “live and dead” assay. Racemic 1-octen-3-ol and (S)-(+)-1-octen-3-ol exhibited greater cytotoxicity to the undifferentiated human cell line H1 than did (R)-(−)-1-octen-3-ol. The inhibition concentration 50 (IC₅₀) values assessed by the MTS assay for racemic 1-octen-3-ol, (S)-(+)-1-octen-3-ol and (R)-(−)-1-octen-3-ol were, respectively, 109, 98, and 258 ppm. These IC₅₀ values were 40–80-fold lower than that of vapor phase toluene, an industrial chemical used as a positive control in this study. Our report pioneers the modeling of human embryonic stem cells as an in vitro approach to screen the potential toxicity of fungal VOCs. Human embryonic stem cells exposed to 1-octen-3-ol, and its enantiomers in the vapor phase showed more cytotoxicity than those exposed to toluene.     

Lipase-catalyzed asymmetric synthesis of naphtho[2,3-c]furan-1(3H)-one derivatives by a one-pot dynamic kinetic resolution/intramolecular Diels–Alder reaction: Total synthesis of (−)-himbacine

One-pot sequential reactions using the acyl moieties installed by enzymatic dynamic kinetic resolution of alcohols have been little investigated. In this work, the acryloyl moiety installed via the lipase/oxovanadium combo-catalyzed dynamic kinetic resolution of a racemic dienol [4-(cyclohex-1-en-1-yl)but-3-en-2-ol or 1-(cyclohex-1-en-1-yl)but-2-en-1-ol] with a (Z)-3-(phenylsulfonyl)acrylate underwent an intramolecular Diels–Alder reaction in a one-pot procedure to produce an optically active naphtho[2,3-c]furan-1(3H)-one derivative (98% ee). This method was successfully applied to the asymmetric total synthesis of (?)-himbacine.     

Synthesis and fungicidal activity of enantiomerically pure (R)- and (S)-silicon-containing azole fungicides

Enantiomerically pure (R)- and (S)-1-(1H-1,2,4-triazol-1-yl)-2-(4-fluorophenyl)-3-trimethylsilylpropan-2-ol 1 were prepared via an enantioselective Grignard reaction. The absolute stereochemistry of 1 was determined by X-ray analysis. In a comparison of in vitro antifungal activities of the enantiomers, the (-)-enantiomer with the R-absolute configuration was far more potent than the (+)-enantiomer.     

Inhibitory effects of 2-substituted-1-naphthol derivatives on cyclooxygenase I and II

Naphthol derivatives, 2-(3'-hydroxypropyl)-naphthalen-1-ol (2), 2-(3'-hydroxy-2'-methylpropyl)-naphthalen-1-ol (3) and 2-(3'-hydroxy-2',2'-dimethylpropyl)-naphthalen-1-ol (7) were synthesized and already reported by our group. Therefore in this paper we described further synthesis of their ether derivatives, 3-(1-methoxy-naphthalen-2-yl)-propan-1-ol (4), 3-(1-methoxy-naphthalen-2-yl)-2methyl-propan-1-ol (5), 3-(1-methoxy-naphthalen-2-yl)-2,2-dimethyl-propan-1-ol (8), 2-(3-methoxy-propyl)-naphthalen-1-ol (10) and 2-(3-methoxy-2,2-dimethyl-propyl)-naphthalen-1-ol (13). Compounds 4, 5 and 8 were prepared by methylation of compounds 2, 3 and 7, respectively while compounds 10 and 13 were prepared in good yield from naphthols 2 and 7, respectively. When tested for inhibitory activity, five compounds (2, 3, 7, 10 and 13) showed preferential inhibition of COX-2 over COX-1, while compounds 4, 5 and 8 lacked inhibitory effect on either the COX-1 or COX-2 isozyme. The structure-activity relationships of these naphthols analyzed by docking experiments, indicated that the presence of hydroxyl group at C-1 position on the naphthalene nucleus enhanced the anti-inflammatory activity towards COX-2 via hydrogen bonding to the COX-2 Val 523 side chain. When this hydroxyl group was replaced by methoxy group, there was no inhibition. C-2' Dimethyl substituents on the propyl chain also increased the inhibitory activity. All active compounds have the C-1 hydroxyl group aligned so as to form hydrogen bond with Val 523. The results provide a model for the binding of the naphthol derivatives to COX-2 and facilitate the design of more potent or selective analogs prior to synthesis.     

Synthesis and antimicrobial activity of 3-arylamino-1-chloropropan-2-ols

A series of nine 3-arylamino-1-chloropropan-2-ols 2a-2i were synthesized and their anti-fungal activity against pathogenic strains of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Candida albicans, and antibacterial activity against four pathogenic bacterial strains of Salmonella typhi, Pseudomonas aeruginosa, Streptococcus pneumonae and Staphylococcus aureus were evaluated using different assay systems. 1-Chloro-3-(4'-chlorophenylamino)-propan-2-ol was found to be the most active anti-fungal compound against three pathogenic strains under study, i.e., A. fumigatus, A. flavus and A. niger; the compound showed more than 90% inhibition of growth of A. fumigatus at a concentration of 5.85 microg/ml in disc diffusion assay. Interestingly, 1-chloro-3-(4'-chlorophenylamino)-propan-2-ol did not show any toxicity up to a concentration of 4000 microg/ml. Although 1-chloro-3-(4'-chlorophenylamino)-propan-2-ol was about 8 times less active than the standard compound amphotericin B, its toxicity was many more fold less than the toxicity of amphotericin B. Further, 1-chloro-3-(2',6'-dichlorophenylamino)-propan-2-ol and 1-chloro-3-(3',5'-dichlorophenylamino)-propan-2-ol were found to be the most active compounds against C. albicans. In the anti-microbial assay, 1-chloro-3-(2',4'-dichlorophenylamino)-propan-2-ol and 1-chloro-3-(3',5'-dichlorophenylamino)-propan-2-ol were found to be the most active compounds against Salmonella typhi and 1-chloro-3-(3',4'-dichlorophenylamino)-propan-2-ol was found to be the most active compound against P. aeruginosa. Although, the activities of 1-chloro-3-(2',4'-dichlorophenylamino)-propan-2-ol and 1-chloro-3-(3',5'-dichlorophenylamino)-propan-2-ol are about half the activity of the standard anti-bacterial compound tetracycline, these compounds also were many fold less toxic than the standard drug.