Alicyclic Alcohols Research Articles

Purification and Characterization of NAD-Dependent Morphine 6-Dehydrogenase from Hamster Liver Cytosol, a New Member of the Aldo–Keto Reductase Superfamily

Morphine 6-dehydrogenase, which catalyzes the dehydrogenation of morphine to morphinone, was purified 815-fold to a homogeneous protein from the soluble fraction of hamster liver with a yield of 15%. The enzyme was a monomeric protein with a molecular weight of 38 kDa and an isoelectric point of 5.6. Although both NAD and NADP served as cofactors, the enzyme activity with NADP was less than 5% that found with NAD at pH 7.4. With NAD, the enzyme gave the maximal activity at pH 9.3, and the Km and Vmax values toward morphine were 1.0 mM and 0.43 unit/mg protein, respectively. Among morphine congeners, normorphine exhibited higher activity than morphine, but codeine and ethylmorphine were poor substrates, and dihydromorphine and dihydrocodeine showed no detectable activity. The enzyme also exhibited significant activity for a variety of cyclic and alicyclic alcohols. In addition to xenobiotics, the enzyme catalyzed the dehydrogenation of 17β-hydroxysteroids with much higher affinities than morphine. In the reverse reaction, the enzyme exhibited high activity for o-quinones, but morphinone, naloxone, and aromatic aldehydes and ketones were reduced at slow rates. Sulfhydryl reagents and ketamine strongly inhibited the enzyme, whereas pyrazole, barbital, and indomethacin had little effect on enzyme activity. 17β-Hydroxysteroids inhibited the enzyme in a competitive manner against morphine. A total of 302 amino acid residues, which comprised approximately 94% of whole protein, were identified by sequencing of the peptides obtained by proteolytic digestion. This amino acid sequence of the enzyme showed significant homology to members of the aldo–keto reductase (AKR) superfamily and shared 63–64% identity with members of the AKR1C subfamily. These findings indicate that the enzyme is a new member of the AKR superfamily that is involved in steroid metabolism as 17β-hydroxysteroid dehydrogenase as well as xenobiotic metabolism.

Quinolinium Fluorochromate (QFC) Supported on Alumina as a Selective Oxidant for Alcohols

Quinolinium fluorochromate(QFC) supported on alumina (neutral) prepared by a new method oxidises selectively aliphatic primary and secondary alcohols, substituted benzylic alcohols and alicyclic alcohols to their corresponding carbonyl compounds in good yield.

Purification and characterization of rat liver naloxone reductase that is identical to 3alpha-hydroxysteroid dehydrogenase

1. Rat liver cytosol produced exclusively 6β-naloxol from naloxone in the presence of either NADPH or NADH at pH 7.4. The amount of 6β-naloxol formed with NADPH was about four times that with NADH. The enzyme responsible for this reaction, termed naloxone reductase, was purified to a homogeneous protein by various chromatographic techniques. 2. The purified enzyme is a monomeric protein with a molecular weight of 34000 and an isoelectric point of 5.9, and it has a dual co-factor specificity for NADPH and NADH. The enzyme catalysed the reduction of various carbonyl compounds as well as naloxone analogues, and the dehydrogenation of 3α-hydroxysteroids and alicyclic alcohols. Indomethacin, quercetin and sulphhydryl reagents potently inhibited the enzyme, but pyrazole and barbital had no effect on the enzyme activity. 3. Identity of naloxone reductase and 3alpha-hydroxysteroid dehydrogenase in rat liver was demonstrated by comparing the elution profiles of the two enzyme activities during purification, the ratios of the two enzyme activities at each purification steps, and thermal stability and susceptibility to inhibitors for the two enzyme activities. 4. Amino acid sequences of five peptides obtained by proteolytic digestion of the purified enzyme were completely identical to the corresponding regions of previously reported 3alpha-hydroxysteroid dehydrogenase.

Roles of the C-terminal domains of human dihydrodiol dehydrogenase isoforms in the binding of substrates and modulators: probing with chimaeric enzymes.

Human liver dihydrodiol dehydrogenase (DD; EC 1.3.1.20) exists in isoforms (DD1, DD2 and DD4) composed of 323 amino acids. DD1 and DD2 share 98% amino acid sequence identity, but show lower identities (approx. 83%) with DD4, in which a marked difference is seen in the C-terminal ten amino acids. DD4 exhibits unique catalytic properties, such as the ability to oxidize both (R)- and (S)-alicyclic alcohols equally, high dehydrogenase activity for bile acids, potent inhibition by steroidal anti-inflammatory drugs and activation by sulphobromophthalein and clofibric acid derivatives. In this study, we have prepared chimaeric enzymes, in which we exchanged the C-terminal 39 residues between the two enzymes. Compared with DD1, CDD1-4 (DD1 with the C-terminal sequence of DD4) had increased kcat/Km values for 3alpha-hydroxy-5beta-androstanes and bile acids of 3-9-fold and decreased values for the other substrates by 5-100-fold. It also became highly sensitive to DD4 inhibitors such as phenolphthalein and hexoestrol. Another chimaeric enzyme, CDD4-1 (DD4 with the C-terminal sequence of DD1), showed the same (S)-stereospecificity for the alicyclic alcohols as DD1, had decreased kcat/Km values for bile acids with 7beta- or 12alpha-hydroxy groups by more than 120-fold and was resistant to inhibition by betamethasone. In addition, the activation effects of sulphobromophthalein and bezafibrate decreased or disappeared for CDD4-1. The recombinant DD4 with the His314-->Pro (the corresponding residue of DD1) mutation showed intermediate changes in the properties between those of wild-type DD4 and CDD4-1. The results indicate that the binding of substrates, inhibitors and activators to the enzymes is controlled by residues in their C-terminal domains; multiple residues co-ordinately act as determinants for substrate specificity and inhibitor sensitivity.

Identification of a principal mRNA species for human 3alpha-hydroxysteroid dehydrogenase isoform (AKR1C3) that exhibits high prostaglandin D2 11-ketoreductase activity.

Human 3alpha-hydroxysteroid dehydrogenase exists in four isoforms, which belong to the aldo-keto reductase (AKR) superfamily and are named AKR1C1-AKR1C4. The properties of AKR1C3 have not been fully characterized compared to the other isoforms. In addition, a cDNA that shows more than 99% homology with AKR1C3 cDNA has been cloned from human myeloblasts. We have here expressed and purified a recombinant enzyme (designated as DBDH) from this cDNA. DBDH oxidized xenobiotic alicyclic alcohols and 3alpha- or 17beta-hydroxy-5beta-androstanes, and catalyzed the reversible conversion between prostaglandin D2 and 9alpha,11beta-prostaglandin F2 more efficiently than that of 3alpha- or 17beta-hydroxysteroids:the respective Km values were 0.6 and 6.8 microM, and kcat/Km values were about 1,000 min-1.mM-1. Anti-inflammatory drugs highly inhibited the enzyme. The recombinant AKR1C3 prepared by site-directed mutagenesis of DBDH also showed the same properties as the wild-type DBDH. Analyses of expression of mRNAs for DBDH and AKR1C3 by reverse transcription-PCR indicated that only one mRNA species for DBDH is expressed in 33 human specimens of liver, kidney, lung, brain, heart, spleen, adrenal gland, small intestine, placenta, prostate, and testis. These results suggest that AKR1C3 acts as prostaglandin D2 11-ketoreductase, and that its principal gene in the human has a coding region represented by DBDH cDNA.

Radical addition reaction of alcohols to 1-(4-nitrophenyl)-5H-pyrrolin-2-one

Quantum chemical calculations of the parameters of the 1-(4-nitrophenyl)-5H-pyrrolin-2-one molecule have been carried out by the MNDO method. The radical addition reaction of aliphatic, alicyclic, and aromatic alcohols to 1-(4-nitrophenyl)-5H-pyrrolin-2-one with the formation of 3- and 4-substituted pyrrolidones has been investigated.

Purification, Characterization and Partial Primary Structure of Morphine 6-Dehydrogenase from Rabbit Liver Cytosol

Morphinone, a toxic metabolite, was formed from morphine by NAD(P)-dependent morphine 6-dehydrogenase(s) in both the cytosol and microsomal fractions of the rabbit liver at pH 7.4. The enzyme activity in the cytosol fraction was about twice that in the microsomal fraction and NAD served as the preferred cofactor in both fractions. The enzyme in the cytosol fraction was purified to a homogeneous protein by the use of various chromatographic techniques. The enzyme is a monomeric protein with a molecular weight of 36,000 and an isoelectric point of 6.4. The enzyme had a dual cofactor specificity but NAD was more efficiently utilized than NADP. With NAD, the enzyme showed an optimal pH of 9.4, and theKmandVmaxvalues toward morphine were 0.72 mmand 0.59 unit/mg protein, respectively. The enzyme also exhibited a significant activity for morphine analogs having an unsaturated bond at C-7,8 (codeine, ethylmorphine, and normorphine), alicyclic alcohols (3-hydroxyhexobarbital, 1-indanol, and cyclohexene-2-ol) and benzenedihydrodiol. In the reverse reaction, the enzyme exhibited highly restricted specificity foro-quinones. Sulfhydryl reagents and quercetin inhibited the enzyme but pyrazole, barbital, and indomethacin had little effect on the enzyme activity. Androstanes, lithocholic acid, and estradiol potently inhibited the enzyme in a competitive manner toward morphine binding. The partial amino acid sequence of the random peptides obtained by the proteolytic digestion of the enzyme, which comprised about 40% of the whole protein, revealed a significant homology to the corresponding regions in the members of the aldo-keto reductase family. These results therefore indicate that the present enzyme is a new and unique member of the aldo-keto reductase family.

Excess volumes, speeds of sound and transport properties of mixtures of 2-methoxyethanol with branched and alicyclic alcohols at 303.15 K

Excess volumes, speeds of sound and viscosities at 303.15 K and atmospheric pressure are reported for six binary liquid mixtures containing 2-methoxyethanol as common component and propan-2-ol, 2-methylpropan-1-ol, 3-methylbutan-1-ol, butan-2-ol, 2-methylpropan-2-ol and cyclohexanol as non-common components. The sign and magnitude of these quantities have been used to discuss the type and nature of binary interactions. Furthermore, the computed results are fitted to the Redlich-Kister polynomial relation. The results are interpreted on the basis of intermolecular interactions between unlike molecules.

Synthesis of ultra‐high molecular weight polyacrylonitrile with highly isotactic content (mm > 0.60) using dialkylmagnesium/polyhydric alcohol system as catalyst

AbstractAn attempt was made to synthesize a catalyst having high activity for synthesis of highly isotactic polyacrylonitrile (PAN) with high molecular weight by anionic polymerization. The reaction products of di‐n‐hexylmagnesium [(n‐C6H13)2Mg] with alicyclic alcohols such as 1,4‐cyclohexanediol or 1,5‐decalindiol were found to be effective as polymerization catalysts for preparation of PAN samples whose triad isotacticity [the content of mm (m, meso configuration)] is about 0.64 and viscosity‐average molecular weight (Mv) exceeds 1 × 105. The triad tacticities [mm, mr, and rr contents (r, racemo configuration)] of the polymers obtained were analysed by 13C NMR and their Mvs were determined by viscometry in dimethylsulphoxide at 25°C using Kamide et al.'s Mark–Houwink–Sakurada equation.Mv of PAN increased significantly by after‐treatment of the catalysts, prepared from (n‐C6H13)2Mg and alicyclic alcohols, with triethylalyminium (Et3Al). Ultra‐high molecular weight PAN samples ranging in Mv from 2 × 106 to 6 × 106 [(mm) = 0.57–0.66] were obtained in anisole at 135°C by using the above catalyst system. A main role of Et3Al in these catalyst systems is considered to reduce, in advance, OH groups on the catalyst, resulting in a significant suppression of termination reactions of propagating PAN chains during the polymerization.

Hyperconjugation and charge distribution in alicyclic alcohols and exo- and endo-norbornol

Ab initio and MM2 calculations are used to investigate charge distributions in selected alcohols (methanol, ethanol, 2-propanol and t-butanol and exo- and endo-norbornol). The calculated net atomic charges obtained using full optimized geometries with a range of basis sets (STO-3G, 4-31G, 6-31G ∗∗) indicates that selected carbon and hydrogen atoms in the proximity of a nonbonding oxygen lone pair are more negatively charged and the carbon-hydrogen bonds are longer than usual. Analysis of the fully optimized geometries, HOMO population and net atomic charges suggests that the differences in charge distribution on the carbon and hydrogen atoms and their bond lengths can be explained in terms of hyperconjugation.

The Room Temperature Dielectric Spectrum of 2,2-Dimethyl-1 -butanol

Abstract The dielectric spectrum of the title substance ('neohexanol') in its pure liquid state is reported for 293 K up to 71 GHz and, for a restricted frequency range, also for lower temperatures (down to 253 K). The room temperature spectrum resembles that of alicyclic alcohols, in particular cyclopentanol, with respect to spectral shape, main relaxation time and the relation of the latter to viscosity, which similarity may be connected with the fact that these alcohols are able to form 'plastic crystals'.

Soft drugs—XVI. Design, evaluation and transdermal penetration of novel soft anticholinergics based on methatropine

Atropine has been reported to produce unwanted systemic side effects on topical administration into the eye. The same problem could arise when atropine is used topically as a suppressant of eccrine sweating. In this study, the principles of soft drug design were applied to methatropine. A hypothetical carboxylate metabolite of methatropine was reactivated by esterification with cyclic and alicyclic alcohols to yield a series of compounds ( 3a–g). In vitro evaluation by guinea pig ileum assay indicated that the compounds are potent anticholinergics and the lead carboxylate metabolite is about 60 times less potent than the most active compound of the series. The activity was found to decrease with the increasing side chain length. The n-octanol/water partition coefficients were found to be directly dependent on the chain length for the compounds made with straight chain alcohols. The transdermal permeability coefficients across the hairless mice skin were found to be directly dependent on the partition coefficients. The soft drugs are found to metabolize extensively during the penetration process compared to the unmetabolizable nature of methatropine. The soft drugs reported in this study will probably be able to elicit a local action at the site of application but will probably be metabolized rapidly in the systemic circulation, thereby avoiding the systemic side effects with a consequent increase in the therapeutic index.

Configurational effects in 17O NMR chemical shifts of hydroxyl‐substituted, bicyclo [2.2.1]heptenes and ‐heptanes, their methylated derivatives and some related compounds

AbstractWell resolved natural abundance 17O NMR spectra of 27 mono‐, di‐, tri‐ and tetrahydroxybicyclo[2.2.1]heptenes and ‐heptanes (norbornenes or norbornanes), their methylated derivatives and some related compounds were measured for 0.5 M or saturated solutions in acetonitrile at 65°C. In addition, the 17O NMR chemical shifts of 14 aliphatic and alicyclic alcohols were also determined for reference purposes. The 17O NMR chemical shifts of all compounds were assigned. Both methyl‐ and hydroxyl‐induced deshielding β‐effects were consistent with those reported in the literature. Similarly, observed shielding γ‐effects were in agreement with the earlier observations and characteristic especially for mutual γ‐gauche and ‐eclipsed orientations of substituents. In addition, clear shielding δ‐effects (up to 10 ppm) were observed in tri‐ or tetrahydroxynorbornanes. These δ‐effects can be related to the attractive van der Waals interactions observed earlier in 13C NMR chemical shifts.

Reactions of furan aldehydes 4. Oxidation-reduction reactions of furfural with monohydric alcohols

Furfural oxidizes aliphatic and alicyclic alcohols at 70–100°C under the action of sodium alcoholate to aldehydes and ketones, and then participates in a crotonate condensation. At 0–10°C the alcohol is not oxidized but the adduct with alkoxide ion is oxidized to form an alkyl furoate.

Structural and functional comparison of two human liver dihydrodiol dehydrogenases associated with 3 α-hydroxysteroid dehydrogenase activity

Two monomeric dihydrodiol dehydrogenases with pI values of 5.4 and 7.6 were co-purified with androsterone dehydrogenase activity to homogeneity from human liver. The two enzymes differed from each other on peptide mapping and in their heat-stabilities; with respect to the latter the dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase activities of the respective enzymes were similarly inactivated. The pI 5.4 enzyme was equally active towards trans- and cis-benzene dihydrodiols, and towards (S)- and (R)-forms of indan-1-ol and 1,2,3,4-tetrahydronaphth-1-ol and oxidized the 3 alpha-hydroxy group of C19-, C21- and C24-steroids, whereas the pI 7.6 enzyme showed high specificity for trans-benzene dihydrodiol, (S)-forms of the alicyclic alcohols and C19- and C21-steroids. Although the two enzymes reduced various xenobiotic carbonyl compounds and the 3-oxo group of C19- and C21-steroids, and were A-specific in the hydrogen transfer from NADPH, only the pI 5.4 enzyme showed reductase activity towards 7 alpha-hydroxy-5 beta-cholestan-3-one and dehydrolithocholic acid. The affinity of the two enzymes for the steroidal substrates was higher than that for the xenobiotic substrates. The two enzymes also showed different susceptibilities to the inhibition by anti-inflammatory drugs and bile acids. Whereas the pI-5.4 enzyme was highly sensitive to anti-inflammatory steroids, showing mixed-type inhibitions with respect to indan-1-ol and androsterone, the pI 7.6 enzyme was inhibited more potently by non-steroidal anti-inflammatory drugs and bile acids than by the steroidal drugs, and the inhibitions were all competitive. These structural and functional differences suggest that the two enzymes are 3 alpha-hydroxysteroid dehydrogenase isoenzymes.

Purification and Properties of Multiple Forms of Dihydrodiol Dehydrogenase from Human Liver1

Two acidic and three basic forms of monomeric dihydrodiol dehydrogenase with molecular weights in the range of 36,000-39,000 were purified from human liver. One acidic enzyme (pI 5.2), which was specific for NADP- and dihydrodiols of benzene and naphthalene, was immunologically identified as aldehyde reductase. The other four enzymes oxidized alicyclic alcohols as well as the dihydrodiols using both NADP+ and NAD+ as cofactors, but showed differences in specificity for hydroxysteroids and inhibitor sensitivity. Two of the basic enzymes (pI 9.7 and 9.1) exhibited a 20 alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas the third basic enzyme (pI 7.6) oxidized some 3 alpha-hydroxysteroids at low rates and was inhibited by cyclopentane-1,1-diacetic acid. Another acidic enzyme, which accounted for the largest amount of enzyme activity in the tissue and appeared in two heterogenous forms with pI values of 5.9 and 5.4, showed a high 3 alpha-hydroxysteroid dehydrogenase activity and was the most sensitive to inhibition by medroxyprogesterone acetate. The Km values of the enzymes, except the pI 5.2 enzyme, for hydroxysteroids (10(-6) to 10(-7) M) were lower than those for xenobiotic alcohols.

Dielectric Relaxation and Intermolecular Association of Alicyclic Alcohols in Liquid and Solid States

Abstract The dielectric constants and losses of cyclopentanol (CPEN), cyclohexanol (CHEX), cycloheptanol (CHEP), and cyclooctanol (COCT) have been measured at frequencies from 1.35 to 4500 MHz over a temperature range of 10–35°C. The absorption could be resolved into three Debye-like dispersion regions in liquid and solid states. The relaxation time of the first, low-frequency, dispersion region and viscosity for CHEX are slightly larger than those for CHEP. This observation can be connected with differences of the molecular geometry between a chair and a twisted chair form and an interconversion between an equatorial and an axial form. Upon solidification, the magnitude of the first dispersion region decreases from 12.67 in the liquid state to 10.25 in the solid state for CHEX and increases from 8.77 to 9.35 for COCT. A transformation from a linear chain polymer into a nonpolar cyclic polymer for CHEX and from a linear chain polymer into a chain dimer and trimer for COCT, occurs mainly upon solidification. The free energies of activation in the solid state are 26 kJ mol−1 for the first region, 23 kJ mol−1 for the second region, and 17 kJ mol−1 for the third region and are larger than those in the liquid state, respectively.

Identification of two dihydrodiol dehydrogenases associated with 3(17)alpha-hydroxysteroid dehydrogenase activity in mouse kidney.

Dihydrodiol dehydrogenase activity was detected in the cytosol of various mouse tissues, among which kidney exhibited high specific activity comparable to the value for liver. The enzyme activity in the kidney cytosol was resolved into one major and three minor peaks by Q-Sepharose chromatography: one minor form cross-reacted immunologically with hepatic 3 alpha-hydroxysteroid dehydrogenase and another with aldehyde reductase. The other minor form was partially purified and the major form was purified to homogeneity. These two forms, although different in their charges, were monomeric proteins with the same molecular weight of 39,000 and had similar catalytic properties. They oxidized cis-benzene dihydrodiol and alicyclic alcohols as well as trans-dihydrodiols of benzene and naphthalene in the presence of NADP+ or NAD+, and reduced several xenobiotic aldehydes and ketones with NAD(P)H as a cofactor. The enzymes also catalyzed the oxidation of 3 alpha-hydroxysteroids and epitestosterone, and the reduction of 3- and 17-ketosteroids, showing much lower Km values (10(-7)-10(-6) M) for the steroids than for the xenobiotic alcohols. The results of mixed substrate experiments, heat stability, and activity staining on polyacrylamide gel electrophoresis suggested that, in the two enzymes, both dihydrodiol dehydrogenase and 3(17)alpha-hydroxysteroid dehydrogenase activities reside on a single enzyme protein. Thus, dihydrodiol dehydrogenase existed in four forms in mouse kidney cytosol, and the two forms distinct from the hepatic enzymes may be identical to 3(17)alpha-hydroxysteroid dehydrogenases.

Monkey liver indanol dehydrogenase. Purification, properties, and kinetic mechanism.

Indanol dehydrogenase was purified to apparent homogeneity from monkey liver cytosol. The enzyme was a monomer with a molecular weight of 36,000 and pI of 8.7. The amino acid composition was determined. The enzyme oxidized alicyclic alcohols including transdihydrodiols of benzene and naphthalene in the presence of both NADP+ and NAD+, and reduced several xenobiotic carbonyl compounds in the presence of NADPH, the 4-pro-R hydrogen atom of which was transferred to the substrate. The results of fluorometric binding and kinetic studies are consistent with an ordered sequential mechanism with NADP+ binding first. The enzyme was inhibited competitively versus NADP+ and uncompetitively versus 1-indanol not only by chelating agents such as 1,10-phenanthroline and 2,2'-bipyridine but also by a nonchelating isomer, 4,4'-bipyridine, which suggests hydrophobic interaction of the aromatic compounds with the enzyme, which did not contain zinc. The enzyme was also inhibited by Cibacron blue dye, synthetic estrogens, and delta 4-3-ketosteroids. The inhibition by Cibacron blue was competitive versus NADP+ and noncompetitive versus 1-indanol, whereas those by hexestrol, medroxyprogesterone acetate, and progesterone were uncompetitive versus NADP+ and competitive versus 1-indanol, corraborating the ordered addition of the coenzyme prior to 1-indanol.

Separation and properties of multiple forms of dihydrodiol dehydrogenase from hamster liver

1. 1. Five multiple forms of dihydrodiol dehydrogenase (EC 1.3.1.20) with similar molecular weights of around 35,000 were purified from hamster liver cytosol. 2. 2. All the enzymes oxidized trons-dihydrodiols of benzene and naphthalene and reduced various carbonyl compounds, but showed clear differences in specificities for other alcohols and cofactors, and in inhibitor sensitivity. 3. 3. Two NADP +-dependent enzymes were immunologically identified with aldehyde reductase (EC 1.1.1.2) and 3α-hydroxytsteroid dehydrogenase (EC 1.1.1.50). 4. 4. The other enzymes with dual cofactor specificity oxidized xenobiotic alicyclic alcohols, and one of them was active on 3α- and 17β-hydroxysteroids with NAD + as a preferable cofactor.