Regiospecific hydroxylation of 3-(methylaminomethyl) pyridine to 5-(methylaminomethyl) -2 (1H) -pyridinone byArthrobacter ureafaciens

Microbial production of a 6-hydroxy-3-pyridylmethyl compound from 3-pyridylmethyl compound was investigated. The hydroxylation of 3-(methylaminomethyl)pyridine to 5-(methylaminomethyl)-2(1H)-pyridinone, tautomer of 2-hydroxy-5(methylaminomethyl)pyridine, by resting cells ofArthrobacter ureafaciens JCM3873 was found to proceed regio- and chemo-selectively with an almost quantitative yield. The addition of molybdate ion and nicotine as an inducer to the culture medium was required for the preparation of cells containing high hydroxylation activity. The optimal temperature and pH for the hydroxylation by using resting cells were 35°C and around 7, respectively. This hydroxylation enzyme does undergo inhibition by the substrate. The inhibitory effect could be eliminated by stepwise feeding of the substrate. Under adequate conditions, 23 mg/ml of 5-(methylaminomethyl)-2(1H)-pyridinone was produced with a molar yield of nearly 100% from 3-(methylaminomethyl)pyridine.     

Industrial production of (R)-1,3-butanediol by new biocatalysts

We have developed the economical and convenient biocatalytic process for the preparation of (R)-1,3-butanediol (BDO) by stereo-specific microbial oxido-reduction on an industrial scale. (R)-1,3-BDO is an important chiral synthon for the synthesis of various optically active compounds such as azetidinone derivatives lead to penem and carbapenem antibiotics.

We studied on two approaches to obtain (R)-1,3-BDO. The first approach was based on enzyme-catalyzed asymmetric reduction of 4-hydroxy-2-butanone; the second approach was based on enantio-selective oxidation of the undesired (S)-1,3-BDO in the racemate. As a result of screening for yeasts, fungi and bacteria, the enzymatic resolution of racemic 1,3-BDO by the Candida parapsilosis IFO 1396, which showed differential rates of oxidation for two enantiomers, was found to be the most practical process to produce (R)-1,3-BDO with high enantiomeric excess and yield.

We characterized the (S)-1,3-BDO dehydrogenase purified from a cell-free extract of C. parapsilosis. This enzyme was found to be a novel secondary alcohol dehydrogenase (CpSADH). We have attempted to clone and characterize the gene encoding CpSADH and express it in Escherichia coli. The CpSADH activity of a recombinant E. coli strain was more than two times higher than that of C. parapsilosis. The production yield of (R)-1,3-BDO from the racemate increased by using the recombinant E. coli strain. Interestingly, we found that the recombinant E. coli strain catalyzed the reduction of ethyl 4-chloro-3-oxo-butanoate to ethyl (R)-4-chloro-3-hyroxy-butanoate with high enantiomeric excess.     

Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O⁶-(2-hydroxyethyl) 2′-deoxyguanosine (O⁶-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O⁶-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5 mM HENU resulted in mutation frequencies of 7.2±2.2×10⁻⁵, 45.2±2.9×10⁻⁵ and 120.3±24.4×10⁻⁵, respectively. Comparison of the mutation frequencies demonstrates that 1 and 5 mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C→A:T transitions (52%) and a significant number of A:T→T:A transversions (16%). We propose that the observed G:C→A:T transitions are produced by the DNA alkylation product O⁶-HOEtdG. These results suggest that the formation of O⁶-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.     

Synthesis of (E)-9-(1-pyrenyl)-4-hydroxynon-2-enal, a fluorescent probe of the (E)-4-hydroxynon-2-enal with retained biological properties

(E)-9-(1-pyrenyl)-4-hydroxynon-2-enal (FHNE), a fluorescent probe of (E)-4-hydroxynon-2-enal (HNE) is synthesised in seven steps and in 35% overall yield, starting from commercially available 1-pyrencarboxyaldehyde. When incubated with cultured HeLa cells this fluorescent probe penetrates cells and particularly concentrates in the region surrounding the nucleus. As the parent compound, HNE it is able to induce the activation of heat shock factor (HSF) and it is able to induce the binding of HSF to heat shock element (HSE).     

Hydroxylated jasmonic acid and related compounds from Botryodiplodia theobromae

From the culture filtrate of the fungus Botryodiplodia theobromae five hydroxylated cyclopentane fatty acids of the jasmonic acid type were isolated and identified as (11 S -(-)-hydroxyjasmonic acid; (11R)-(-)-hydroxyjasmonic acid; (-)-12-hydroxyjasmonic acid; (-)-8ξ-hydroxyjasmonic acid; (-)-3-oxo-2-(1ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid; (-)-3-oxo-2(4ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid. In addition, the corresponding hydroxylated iso-jasmonic acid analogues were found as minor constituents. During silica gel chromatography 11,12-didehydrojasmonic acid, 11ξ-acetoxyjasmonic acid, 3-oxo-2-(4ξ-acetoxy-2Z-pentenyl)cyclopent-1-yl-butyric acid 3-oxo-2-(2Z,4-pentadienyl)cyclopent-1-yl-butyric acid were formed as artefacts.     

Bioreduction of 2-azido-1-arylethanones mediated by Geotrichum candidum and Rhodotorula glutinis

Enantioselective reductions of p-X-C₆H₄C(O)CH₂N₃ (X = H, Cl, Br, CH₃, OCH₃) mediated by Rhodotorula glutinis and Geotrichum candidum afforded the corresponding alcohols with complementary R and S configurations, respectively, in excellent yield and enantiomeric excesses. The obtained (R)-azidoalcohols are important starting materials for preparation of natural products and valuable pharmaceutical compounds such as (R)-Tembamide and (R)-Aegeline.     

Mutations induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lacZ and cII genes of Muta™ Mouse

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) found in chewing tobacco, snuff, cigarettes, and cigars is a tobacco-specific nitrosamine and classified as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). NNK given intraperitoneally was seen to induce lung and liver adenomas.To evaluate the genotoxicity of NNK in vivo, NNK was intraperitoneally administered to Muta™ Mouse at two concentrations (125 and 250 mg/kg, once a week for 4 weeks) followed by the measurement of mutant frequencies in the lacZ and cII genes from lung and liver in the same mice. Characterization of the types of the mutation was determined by sequencing the cII genes from mutant plaques. The mutant frequencies in both target genes from both organs dose-dependently increased up to 10 times compared to those of the control group. For the types of mutations, the ratio of the G:C to A:T mutation in the total number of mutants was less than the ratio of A:T to T:A and A:T to C:G transversion, contrary to a previous report [Cancer Res, 49 (1989) 5305]. The A:T to T:A transversion was the most highly induced mutation both in the lung and liver cII genes. The increasing rate of mutant frequencies in lung and liver over the vehicle control was 55 and 56 times, respectively, while the increasing rate of G:C to A:T transition was only 1.9 and 2.8 times, respectively.These observations show that NNK predominantly induces DNA adducts leading to A:T to T:A and/or A:T to C:G mutations in the transgene.     

Product profiles in enzymic and non-enzymic oxidations of the lignin model compound erythro-1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol

The erythro form of the lignin model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol (1) was oxidized with laccase/ABTS, lead(IV) tetraacetate (LTA), lignin peroxidase/H₂O₂, cerium(IV) ammonium nitrate (CAN) and Fenton's reagent. The product profiles obtained with the different oxidants were compared after separation, identification and quantification of the products using HPLC, UV-diode array detector and electrospray ionization mass spectrometry in positive ionization mode. The oxidants generated different product profiles that reflected their different properties. Oxidation with laccase/ABTS resulted almost exclusively in formation of 1-(3,4-dimethoxyphenyl)-3-hydroxy-2-(2-methoxyphenoxy)-1-propanone (2). Oxidation with LTA resulted in more 3,4-dimethoxybenzaldehyde (3) than ketone 2. Lignin peroxidase and CAN gave similar product profiles and aldehyde 3 was the predominant product (only small amounts of ketone 2 were formed). Oxidation with Fenton's reagent resulted in the formation of more aldehyde 3 than ketone 2 but the yields were very low. CAN served as an excellent model for the lignin peroxidase-catalyzed oxidation, while the laccase-mediator system, LTA and Fenton's reagent provided distinctly different product profiles. Erythro-1-(3,4-dimethoxyphenyl)-1,2,3-propanetriol was present among the products obtained on oxidation with LTA, lignin peroxidase, CAN and Fenton's reagent. The differences in redox potential between the oxidants afford an explanation of the diverse product patterns but other factors may also be of importance. The reactions leading to cleavage of the β-ether bond with formation of 1-(3,4-dimethoxyphenyl)-1,2,3-propanetriol (veratrylglycerol) were found to proceed without affecting the configuration at the β-carbon atom.     

Analysis of a novel antiinflammatory agent, 1-(7-tert.-butyl-2,3-dihydro-3,3-dimethylbenzo[b]furan-5-yl)-4-cyclopropylbutan-1-one (PGV-20229), in plasma matrices by stable-isotope-dilution gas chromatography-mass spectrometry

A sensitive and selective GC-MS method was developed for the determination of low levels of a novel antiinflammatory agent, 1-(7-tert.-butyl-2,3-dihydro-3,3-dimethylbenzo[b]furan-5-yl)-4-cyclopropylbutan-1-one (I), in small volumes of animal plasma. The method involved the addition of ¹³C₆-labeled-I to plasma samples, followed by a simple liquid-liquid extraction with hexane to isolate the analytes from matrix components. The levels of I in the sample extracts were determined by isotope-dilution GC-MS analysis using selected-ion monitoring. The method was linear over three orders of magnitude, with a limit of quantitation of 1.8 ng/ml I, using plasma sample volumes of 0.1 ml. The method was utilized to determine the pharmacokinetic parameters of I in rats and dogs, following intravenous administration.     

Studies on the continuous production of (R)-(−)-phenylacetylcarbinol in an enzyme-membrane reactor

The optimization of a continuous enzymatic reaction yielding (R)-(−)-phenylacetylcarbinol ((R)-PAC), a key intermediate of the (1R,2S)-(−)-ephedrine synthesis, is presented. We compare the suitability of different mutants of the pyruvate decarboxylase (PDC) from Zymomonas mobilis with respect to their application in biotransformation using pyruvate or acetaldehyde and benzaldehyde as substrates, respectively. Starting from 90 mM pyruvate and 30 mM benzaldehyde, (R)-PAC was obtained with a space time yield of 27.4 g/(L·day) using purified PDCW392I in an enzyme-membrane reactor. Due to the high stability of the mutant enzymes PDCW392I and PDCW392M towards acetaldehyde, a continuous procedure using acetaldehyde instead of pyruvate was developed. The kinetic results of the enzymatic synthesis starting from acetaldehyde and benzaldehyde demonstrate that the carboligation to (R)-PAC is most efficiently performed using a continuous reaction system and feeding both aldehydes in equimolar concentration. Starting from an inlet concentration of 50 mM of both aldehydes, (R)-PAC was obtained with a space-time yield of 81 g/(L·day) using the mutant enzyme PDCW392M. The new reaction strategy allows the enzymatic synthesis of (R)-PAC from cheap substrates free of unwanted by-products with potent mutants of PDC from Z. mobilis in an aqueous reaction system.     

Enantiomeric oxidation of organic sulfides by the filamentous fungi Botrytis cinerea, Eutypa lata and Trichoderma viride

The biotransformations of a series of substituted sulfides were carried out with the filamentous fungi Botrytis cinerea, Eutypa lata and Trichoderma viride. Several products underwent microbial oxidation of sulfide to sulfoxide with medium to high enantiomeric purity. With regard to sulfoxide enantioselectivity, the (R)-enantiomer was favoured in biotransformations by T. viride and E. lata while the (S)-enantiomer was favoured in those by B. cinerea. A minor amount of sulfone product was also obtained.     

3-(3,4-Dichlorophenyl)-1, 1-dimethylurea-insensitive oxygen production in a cell-free preparation from Phormidium luridum that shows redox potential dependent coupling to one-electron oxidants

A cell-free preparation has been isolated from Phormidium luridum that evolves oxygen when coupled to one-electron oxidants, that is insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and that yields oxygen at a rate dependent on redox potential. In this preparation the Hill oxidant couples closer to the oxygen-producing apparatus than in any other cell-free system. Light saturation curve data for the cell-free preparation shows a stabilization, by the Hill oxidant, of intermediates in oxygen synthesis. In whole cells coupled to CO₂ or to K₃ Fe(CN)₆ no such stabilization occurs and a 2nd order light intensity dependence of the oxygen-production rate is observed.     

(3S,4S,5R)-3,4,5-trihydroxy-1-cyclohexene-carboxylic acid from Sequoiadendron giganteum

From the leafy lateral branchlets of Sequoiadendron giganteum, (3S,4S,5R)-3,4,5-trihydroxy-1-cyclohexenecarboxylic acid has been isolated. Its structure was proved spectroscopically.     

pH control of the chlorophyll a fluorescence in algae

The pH of the suspension medium was found to have a remarkable influence on the “slow” (min) time course of Chlorophyll a fluorescence yield in the green alga Chlorella pyrenoidosa and in the blue-green alga Anacystis nidulans. In Chlorella, the decay of fluorescence yield, in the 1- to 5-min region, is strongly retarded at alkaline pH; this decay rate shows an optimum at pH 6–7. In Anacystis, the rise of fluorescence yield, in the same time range, is decreased optimally at pH 6–7; poisoning with 3(3,4-dichlorophenyl)-1,1-dimethylurea reverses the direction of this pH effect. These observations suggest a correlation of the H⁺ status (or the processes associated with it such as photophosphorylation and resulting conformational changes) of the chloroplast to the yield of chlorophyll a fluorescence in vivo.     

Determination of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione in plasma by direct injection into a coupled column liquid chromatographic system

The chemical substance 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) is in clinical use for the treatment of hereditary tyrosinemia type 1. In the present study, the plasma concentration of NTBC was determined by a coupled column liquid chromatographic method. A 20-μl volume of plasma was diluted with phosphate buffer, pH 2, and injected into a small precolumn (BioTrapAcid C₁₈) with a mobile phase containing sulfuric acid. The precolumn was based on the restricted access principle, i.e., retention of NTBC within the lipophilic pores, while polar and large endogenous compounds were eluted with the void volume. NTBC was transferred to the analytical column using a mobile phase with a high content of acetonitrile. The compound was monitored by UV detection at 278 nm. The standard curve was linear between 0.3 and 69 μM, and the between-day precision (RSD) was 3% (n=6 days) at 13.8 μM and 14% (n=6 days) at 0.3 μM NTBC in plasma. The quantitation limit was approximately 0.3 μM using 20 μl of plasma.     

Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid

Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups). These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid.

Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used. For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior.

More interestingly, the enantiomeric ratio (E) also depends strongly on the immobilized preparation and the conditions employed. Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 1.2 at pH 5 and 25 °C).

The other immobilized preparations, in contrast, were S selective. Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity (E=59 under optimum conditions).     

Characterization of the biological conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene in direct micellar systems

The whole cell biological conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by the E. coli JM109(pPS1778) recombinant strain carrying the naphthalene dioxygenase and regulatory genes cloned from Pseudomonas fluorescens N3 in micellar systems has been investigated using biochemical and chemico-physical techniques. Reverse and direct micellar systems have been tested. Non-ionic surfactants (Tween and Triton X series) were found not to inhibit either the growth of the bacteria and the expression of the hydroxylating dioxygenase enzyme in such systems and were utilized in order to speed up the naphthalene conversion by increasing its solubility and also its bioavailability. The phase behavior of the direct micellar system was characterized through light scattering and other chemico-physical techniques. Further addition of isopropyl-palmitate 1–2% v/v to the micellar systems resulted in an increase of the apparent substrate concentration in solution and particularly its bioavailability thus allowing faster catalytic conversions resulting in an increase in productivity for the process. Since the cis-dihydrodiols are acquiring considerable potential as chiral pool synthons in asymmetric synthesis for a variety of industrial processes, possible applications for efficient small and large-scale production of such compounds are discussed.     

Determination of 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine in plasma and whole blood by high-performance liquid chromatography

A selective and sensitive HPLC assay for the quantitative determination of a new antifilarial drug, 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine (CDR 101) is described. After extraction from plasma and blood, CDR 101 was analysed using a C₁₈ Nucleosil ODS column (250×4.6 mm, 5 μm particle size) and mobile phase of acetonitrile-0.05 M ammonium acetate adjusted to pH 3.0, with UV detection at 318 nm. The mean recoveries of CDR 101 in plasma and blood over a concentration range of 25–500 ng/ml were 95.5±2.01% and 83.3±1.87%, respectively. The within-day and day-to-day coefficient of variations for plasma were 3.23-6.21% and 2.59-9.90%, respectively, those for blood were 2.59-5.92% and 2.89-6.82%, respectively. The minimum detectable concentration for CDR 101 was 1 ng/ml in plasma and 2.5 ng/ml in whole blood. This method was found to be suitable for clinical pharmacokinetic studies.     

Influence on the plasma membrane of Candida albicans by HP (2-9)-magainin 2 (1-12) hybrid peptide

A 20-residue hybrid peptide (HP (2-9)-MA (1-12): HP-MA), incorporating 2-9 residues of Helicobacter pyroli ribosomal protein L1 (HP) and 1-12 residues of magainin 2 (MA), has more potent antibacterial activity than parent peptide HP (2-20) and magainin 2. In this study, the antifungal activity and its mechanism of HP-MA were investigated. HP-MA displayed a strong antifungal activity in an energy-dependent manner. To elucidate the antifungal mechanism(s) of HP-MA, FACScan analysis and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe of Candida albicans were examined. The results indicated that the HP-MA exerts its antifungal effect by acting on the plasma membrane. Furthermore, the peptide induced remarkable morphological change when tested for membrane disrupting activity using liposomes (PC/Cholesterol; 10:1, w/w). In C. albicans, dimorphism plays a crucial role in pathogenesis but HP-MA could disrupt the mycelial forms and exert its antifungal effect on the blastoconidia in 20% fetal bovine serum.     

Preparation of 2S-(—)-4-Amino-2-Hydroxybutanoic Acid via Yeast-Catalyzed Stereoselective Reduction

The unusual amino acid S2-(—)-4-amino-2-hydroxybutanoic acid (S-AHBA) (1), a structural component of the antibiotic Amikacin, has been prepared via yeast-catalyzed stereoselective reduction of methyl-4-benzyloxycarbonyloxyamino-2-oxobutanoate (5). The most suitable yeast was found to be Saccharomyces carlsbergensis ATCC2345 and Saccharomyces sp. Edme, which gave 40% and 54% yield, respectively, of (S)-(+)-6 (88% ee).