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A highly efficient oxidation of cyclohexane to cyclohexanol and cyclohexanone is accomplished over calcined vanadium phosphorus oxide (VPO) catalysts in a relatively mild condition using hydrogen peroxide under a nitrogen atmosphere.
A highly efficient oxidation of cyclohexane to cyclohexanol and cyclohexanone is accomplished over calcined Au/ZSM-5 molecular sieve catalyst with oxygen as oxidant.
CAL-B catalysed the resolution of several five and six-membered cyclic beta-hydroxy esters efficiently with the exception of the cis-cyclohexanol (+/-)-4. When employing molecular modelling techniques the conformation turned out to be the most important determinant for their reactivity towards O-acetylation. In all cases, the R enantiomers reacted faster than the S enantiomers since the reactive intermediates of the former can adopt more favourable ring conformations and thus experience less steric hindrance in the active site. Furthermore, the minimised structure for the main conformer of R-4 showed that the axial hydrogens in the 3 and 5-positions with respect to the hydroxyl group prevent the enzymatic reaction.
Electron impact (70 eV) mass spectra of a series of C1-C6 alcohols encased in large superfluid liquid helium nanodroplets (approximately 60,000 helium atoms) have been recorded. The presence of helium alters the fragmentation patterns when compared with the gas phase, with some ion product channels being more strongly affected than others, most notably cleavage of the C(alpha)-H bond in the parent ion to form the corresponding oxonium ion. Parent ion intensities are also enhanced by the helium, but only for the two cyclic alcohols studied, cyclopentanol and cyclohexanol, is this effect large enough to transform the parent ion from a minor product (in the gas phase) into the most abundant ion in the helium droplet experiments. To demonstrate that these findings are not unique to alcohols, we have also investigated several ethers. The results obtained for both alcohols and ethers are difficult to explain solely by rapid cooling of the excited parent ions by the surrounding superfluid helium, although this undoubtedly takes place. A second factor also seems to be involved, a cage effect which favors hydrogen atom loss over other fragmentation channels. The set of molecules explored in this work suggest that electron impact ionization of doped helium nanodroplets does not provide a sufficiently large softening effect to be useful in analytical mass spectrometry.
A series of "organic chemical hydrides" such as cyclohexane, methylcyclohexane, cyclohexene, 2-propanol, and cyclohexanol were applied to the direct PEM fuel cell. High performances of the PEM fuel cell were achieved by using cyclohexane (OCV = 920 mV, PD(max) = 15 mW cm(-2)) and 2-propanol (OCV = 790 mV, PD(max) = 78 mW cm(-2)) as fuels without CO(2) emissions. The rates of fuel crossover for cyclohexane, 2-propanol, and methanol were estimated, and the rates of fuel permeation of cyclohexane and 2-propanol were lower than that of methanol. Water electrolysis and electro-reductive hydrogenation of acetone mediated by PEM were carried out and formation of 2-propanol in cathode side was observed. This system is the first example of a "rechargeable" direct fuel cell.
Because opioid and cannabinoid systems have been reported to interact in the modulation of addictive behaviour, this study was aimed at investigating the ability of cannabinoid agents to reinstate or prevent heroin-seeking behaviour after a prolonged period of extinction. In rats previously trained to self-administer heroin intravenously, non-contingent non-reinforced priming administrations of heroin and cannabinoids were presented after long-term extinction, and lever pressing following injections was observed. Results showed that: (i) intravenous priming infusions of heroin (0.1 and 0.2 mg/kg) lead to reinstatement of drug-seeking behaviour; (ii) intraperitoneal priming injections of the central cannabinoid receptor agonists R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazinyl) (1-naphthalenyl)methanonemesylate (WIN 55,212-2, 0.15 and 0.3 mg/kg) and (-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP 55,940, 0.05 and 0.1 mg/kg), but not delta9-tetrahydrocannabinol (delta9-THC, 0.1-1.0 mg/kg), effectively restored heroin-seeking behaviour; (iii) intraperitoneal priming injection of the central cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)4-methyl-1H-pyrazole-3-carboxamide (SR 141716A, 0.3 mg/kg) did not reinstate responding, but (iv) completely prevented heroin-induced reinstatement of drug-seeking behaviour. Moreover, heroin-seeking behaviour was still present for a few days following cannabinoid primings, indicating a long-lasting effect of cannabinoids on responding for heroin. These findings indicate that relapse to heroin after an extended drug-free period is triggered by cannabinoid agonists and that SR 141716A prevents drug-seeking behaviour, suggesting that the use of the cannabinoid antagonist could have some therapeutic benefits in heroin-induced relapse.
To clone novel type 1 Baeyer-Villiger monooxygenase (BVMO) genes, we isolated or collected 25 bacterial strains able to grow on alicyclic compounds. Twelve of the bacterial strains yielded polymerase chain reaction (PCR) fragments with highly degenerate primers based on the sequences of known and putative BVMOs. All these fragments were found to encode peptides homologous to published BVMO sequences. The complete BVMO genes and flanking DNA were cloned from a Comamonas, a Xanthobacter and a Rhodococcus strain using the PCR fragments as probes. BVMO genes cloned from the first two strains could be expressed to high levels in Escherichia coli using standard expression vectors, and the recombinants converted cyclopentanone and cyclohexanone to the corresponding lactones. The Rhodococcus BVMO, a putative steroid monooxygenase, could be expressed after modification of the N-terminal sequence. However, recombinants expressing this protein did not show activity towards progesterone. An esterase homologue located directly upstream of the Xanthobacter BVMO gene and a dehydrogenase homologue encoded directly downstream of the Comamonas sp. NCIMB 9872 BVMO gene were also expressed in E. coli and shown to specify lactone hydrolase and cyclohexanol dehydrogenase activity respectively.
Odorant receptors (ORs) constitute the molecular basis for the detection of volatile odorous molecules and the perception of smell. Our understanding of chemical senses has been greatly expanded by the discovery of the OR gene families in vertebrates and in the nematode Caenorhabditis elegans. Recently, candidate Drosophila OR genes have been identified. The putative ORs do not possess any primary sequence identity with known vertebrate or C. elegans receptors, but belong to the family of G protein-coupled receptors according to their predicted seven transmembrane topology. To prove olfactory function of these proteins, we expressed a member of the putative Drosophila OR gene family, Or43a, in Xenopus laevis oocytes. Using two-electrode voltage-clamp recording we identified four odors (cyclohexanone, cyclohexanol, benzaldehyde, and benzyl alcohol) that activated the receptor at low micromolar concentration and structurally related substances that did not. This report shows the function and specificity of a member of the recently identified family of Drosophila ORs expressed in a heterologous system.
Experiments were performed to investigate the effects on the spontaneous, nonquantal release of acetylcholine (AcCho) from motor nerve terminals of substances known to inhibit the AcCho transport system present in cholinergic synaptic vesicles. In mouse diaphragms, the hyperpolarization normally produced by d-tubocurarine in the endplate area of muscle fibers that had been treated by an anticholinesterase was partly or completely blocked by 2-(4-phenylpiperidino)cyclohexanol (AH5183, 0.1-1 microM), quinacrine (0.1 microM), and tetraphenylborate (1 microM). Since the sensitivity of the subsynaptic area to AcCho was not changed, the block of the hyperpolarizing action of d-tubocurarine indicated in inhibition of the spontaneous, nonquantal release of AcCho. This was confirmed in experiments on rat diaphragm using direct radioenzymatic measurement of the AcCho released into the incubation medium. The release of AcCho from the innervated diaphragm was decreased by about 50% in the presence of AH5183 (0.01-1 microM) and by 42% in the presence of quinacrine (0.1-1 microM). The AcCho released was presumably neural, since the release of AcCho from 4-day denervated diaphragms was not diminished by either AH5183 or quinacrine. The results indicate that the spontaneous release of AcCho from the motor nerve terminals is highly sensitive to low concentrations of specific inhibitors and is probably mediated by a carrier. It is proposed that spontaneous release is due to the incorporation into the membrane of the nerve terminal during exocytosis of the vesicular transport system responsible for moving AcCho into the vesicle.
Although the exocytotic mechanism for quantal acetylcholine (ACh) release has been widely accepted for many years, it has repeatedly been challenged by reports that ACh released upon stimulation originates from the cytosol rather than synaptic vesicles. In this report, two independent experimental approaches were taken to establish the source of ACh released from the electromotor system of Narcine brasiliensis. Since ATP is colocalized with ACh in the cholinergic vesicle, the exocytotic theory predicts the corelease of these two components with a stoichiometry identical to that of the vesicle contents. The stimulated release of ATP from isolated synaptosomes could be accurately quantitated in the presence of the ATPase inhibitor adenosine 5'-[alpha, beta-methylene]triphosphate (500 microM), which prevented degradation of the released ATP. Various concentrations of elevated extracellular potassium (25-75 mM), veratridine (100 microM), and the calcium ionophore ionomycin (5 microM) all induced the corelease of ACh and ATP in a constant molar ratio of 5-6:1 (ACh/ATP), a stoichiometry consistent with that established for the vesicle content. In parallel to these stoichiometry studies, the compound 2-(4-phenylpiperidino)cyclohexanol (AH5183) was used to inhibit specifically the vesicular accumulation of newly synthesized (radiolabeled) ACh without affecting cytosolic levels of newly synthesized ACh in cholinergic nerve terminals. Treatment with AH5183 (10 microM) was shown to inhibit the release of newly synthesized ACh without markedly affecting total ACh release; thus, the entry of newly synthesized ACh into the synaptic vesicle is essential for its release. We conclude that ACh released upon stimulation originates exclusively from the vesicular pool and is coreleased stoichiometrically with other soluble vesicle contents.
The structure of a mammalian class IV alcohol dehydrogenase has been determined by peptide analysis of the protein isolated from rat stomach. The structure indicates that the enzyme constitutes a separate alcohol dehydrogenase class, in agreement with the distinct enzymatic properties; the class IV enzyme is somewhat closer to class I (the "classical" liver alcohol dehydrogenase; approximately 68% residue identities) than to the other classes (II, III, and V; approximately 60% residue identities), suggesting that class IV might have originated through duplication of an early vertebrate class I gene. The activity of the class IV protein toward ethanol is even higher than that of the classical liver enzyme. Both Km and kcat values are high, the latter being the highest of any class characterized so far. Structurally, these properties are correlated with replacements at the active site, affecting both substrate and coenzyme binding. In particular, Ala-294 (instead of valine) results in increased space in the middle section of the substrate cleft, Gly-47 (instead of a basic residue) results in decreased charge interactions with the coenzyme pyrophosphate, and Tyr-363 (instead of a basic residue) may also affect coenzyme binding. In combination, these exchanges are compatible with a promotion of the off dissociation and an increased turnover rate. In contrast, residues at the inner part of the substrate cleft are bulky, accounting for low activity toward secondary alcohols and cyclohexanol. Exchanges at positions 259-261 involve minor shifts in glycine residues at a reverse turn in the coenzyme-binding fold. Clearly, class IV is distinct in structure, ethanol turnover, stomach expression, and possible emergence from class I.
Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.
A novel nicotinoprotein, catalyzing the dichlorophenolindophenol-dependent oxidation of carveol to carvone, was purified to homogeneity from Rhodococcus erythropolis DCL14. The enzyme is specifically induced after growth on limonene and carveol. Dichlorophenolindophenol-dependent carveol dehydrogenase (CDH) is a homotetramer of 120 kDa with each subunit containing a tightly bound NAD(H) molecule. The enzyme is optimally active at pH 5.5 and 50 degrees C and displays a broad substrate specificity with a preference for substituted cyclohexanols. When incubated with a diastereomeric mixture of (4R)- or (4S)-carveol, CDH stereoselectively catalyzes the conversion of the (6S)-carveol stereoisomers only. Kinetic studies with pure stereoisomers showed that this is due to large differences in V(max)/K(m) values and simultaneous product inhibition by (R)- or (S)-carvone. The R. erythropolis CDH gene (limC) was identified in an operon encoding the enzymes involved in limonene degradation. The CDH nucleotide sequence revealed an open reading frame of 831 base pairs encoding a 277-amino acid protein with a deduced mass of 29,531 Da. The CDH primary structure shares 10-30% sequence identity with members of the short chain dehydrogenase/reductase superfamily. Structure homology modeling with trihydroxynaphthalene reductase from Magnaporthe grisea suggests that CDH from R. erythropolis DCL14 is an alpha/beta one-domain protein with an extra loop insertion involved in NAD binding and a flexible C-terminal part involved in monoterpene binding.
The metabolism of isopropylcyclohexane and associated renal pathology were evaluated in male Fischer 344 rats exposed by oral gavage. The rats experienced moderate proximal tubular damage similar to that produced by acyclic, branched-chain hydrocarbons. The urinary metabolites of isopropylcyclohexane included cis-4-isopropylcyclohexanol, trans-4-isopropylcyclohexanol, 2-cyclohexylpropanoic acid, 2-cyclohexyl-1,3-propanediol, 2t-hydroxy-4t-isopropylcyclohexanol, 2c-hydroxy-4c-isopropyl-cyclohexanol, and 2c-hydroxy-4t-isopropylcyclohexanol. The extent and preferred sites of oxidative metabolism of nephrotoxic hydrocarbons could potentially prove useful in elucidating the pathogenic mechanisms.
The 2004 Nobel Prize in Physiology or Medicine laureates, Richard Axel and Linda Buck, have made smell a less enigmatic sense to study. In clinical routine, olfactory function is assessed using defined concentrations of a single defined substance, a setting which is uncommon in daily life. The present study was therefore conducted to evaluate the applicability of composite odours. Air was contaminated with different quantities of cyclohexanol, cyclohexanone and cyclohexane to generate 73 gas mixtures (one component: n = 21, two components: n = 40, three components: n = 12). The intensity of perception was estimated for each mixture by an average of 60.3 healthy individuals (4,403 assessments). An artificial neural network (ANN) was trained and validated using the contaminants' concentrations with the corresponding estimated intensities. The inter-rater variability was low, as 75.7% of the assessments did not exceed a difference beyond 0.5 from the corresponding median (considered correct predictions). The ANN correctly estimated 78.1% of the gas mixtures, and in terms of the regression task the ANN demonstrated a sufficient prediction performance (Pearson's correlation coefficient r = 0.883; R(2) = 0.757) and outperformed linear regression (r = 0.770; R(2) = 0.667). Evaluating extra ANNs for gas mixtures comprising one, two or three components, the predictive power did not decrease when complexity increased. The aforementioned results reflect nonlinearity in human perception. ANN technology helps simulate human perception of composite odour intensity which may be applicable to olfactometry calibration and systems biological mathematical modelling. The use of composite odours may represent real-life problems more adequately than single substances.
An indirect high-performance liquid chromatographic method is developed for the enantioseparation of cis- and trans-2-cyanocyclopentanol and -cyclohexanol. The racemic cis-(1S,2S and 1R,2R)- or trans-(1S,2R and 1R,2S)-2-cyanocycloalkanols are converted to their diastereomers formed with (S)-(+)- or (R)-(-)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride. The diastereomers are separated on a reversed-phase column, and the conditions of derivatization and HPLC analysis are optimized.
A 22-year-old male with more than 28 needle marks on his right arm was found dead. First, he was suspected as a drug abuser. Blood, urine, spleen, and injection-site tissue was collected during autopsy. The blood and urine specimens were screened for drugs. Immunoassay studies did not show any illegal drugs. However, two unidentified peaks were isolated in both of these biological fluids by routine gas chromatography-flame-ionization detection (GC-FID) and thermionic specific detection. Additional gas chromatography-mass spectrometry analysis determined these two peaks to be tiletamine and zolazepam. These two agents are used in combination as veterinary anesthesia. The concentrations of these drugs in blood were quantitated by GC-FID and found to be 0.85 mg/L of tiletamine and 3.3 mg/L of zolazepam. In urine, tiletamine and its metabolite, 2-(ethylamino)-2-(2-thionyl) cyclohexanol, were identified to be present along with zolazepam. The concentrations of tiletamine and zolazepam in spleen were revealed to be 0.92 and 3.5 mg/kg, respectively. Injection-site tissue concentrations were determined to be 25.1 mg/kg tiletamine and 23.3 mg/kg for zolazepam. The cause of death in this case was determined to be due to the multiple drug intoxication of tiletamine and zolazepam.
A GLC assay for phencyclidine (PCP) is described, which also simultaneously measures three primary hydroxylated metabolites formed from incubating PCP in tissue homogenates. Using the FID detector, the limits of reliable detection of PCP and both monohydroxy metabolites, 4-phenyl-4-piperidino-cyclohexanol, 2, and 1-(1-phenylcyclohexyl)-4-hydroxypiperidine, 3, are 0.02 mumol per injection and 0.05 mumol for the dihydroxy metabolite, 4-(4'-hydroxypiperidino)-4-phenylcyclohexanol, 2A. Baseline separation of an compounds was achieved and coefficients of variation (between-run) was 3-6% for PCP, and both monohydroxy metabolites, and 12% for the dihydroxy metabolite. A GCMS assay is also reported herein for the analysis of PCP at low levels, and can detect 5 pmol per injection of PCP, with a linear standard curve from 50 to 2000 pmol.