Mechanisms of killing of spores of Bacillus subtilis by dimethyldioxirane

AIMS: To determine the mechanisms of Bacillus subtilis spore resistance to and killing by a novel sporicide, dimethyldioxirane (DMDO) that was generated in situ from acetone and potassium peroxymonosulfate at neutral pH. METHODS AND RESULTS: Spores of B. subtilis were effectively killed by DMDO. Rates of killing by DMDO of spores lacking most DNA protective alpha/beta-type small, acid-soluble spore proteins (alpha- beta- spores) or the major DNA repair protein, RecA, were very similar to that of wild-type spore killing. Survivors of wild-type and alpha- beta- spores treated with DMDO also exhibited no increase in mutations. Spores lacking much coat protein due either to mutation or chemical decoating were much more sensitive to DMDO than were wild-type spores, but were more resistant than growing cells. Wild-type spores killed with this reagent retained their large pool of dipicolinic acid (DPA), and the survivors of spores treated with DMDO were sensitized to wet heat. The DMDO-killed spores germinated with nutrients, albeit more slowly than untreated spores, but germinated faster than untreated spores with dodecylamine. The killed spores were also germinated by very high pressures and by lysozyme treatment in hypertonic medium, but many of these spores lysed shortly after their germination, and none of these treatments were able to revive the DMDO-killed spores. CONCLUSIONS: DMDO is an effective reagent for killing B. subtilis spores. The spore coat is a major factor in spore resistance to DMDO, which does not kill spores by DNA damage or by inactivating some component needed for spore germination. Rather, this reagent appears to kill spores by damaging the spore's inner membrane in some fashion. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates that DMDO is an effective decontaminant for spores of Bacillus species that can work under mild conditions, and the killed spores cannot be revived. Evidence has also been obtained on the mechanisms of spore resistance to and killing by this reagent.     

Evidence for the DNA binding and adduct formation of estrone and 17β-estradiol after dimethyldioxirane activation

Estrogens, used widely from hormone replacement therapy to cancer treatment, are themselves carcinogenic, causing uterine and breast cancers. However, the mechanism of their carcinogenic action is still not known. Recently, we found that estrone (E₁) and 17β-estradiol (E₂) could be activated by the versatile epoxide-forming oxidant dimethyldioxirane (DMDO), resulting in the inhibition of rat liver nuclear and nucleolar RNA synthesis in a dose-dependent manner in vitro. Since epoxidation is often required for the activation of chemical carcinogens, we proposed that estrogen epoxidation is the underlying mechanism for the initiation of estrogen carcinogenesis (Carcinogenesis 17 (1996) 1957–1961). It is known that initiation requires the binding of a carcinogen to DNA with the formation of DNA adducts. One of the critical tests of our hypothesis is therefore to determine whether E₁ and E₂ after activation are able to bind DNA. This paper reports that after DMDO activation, [³H]E₁ and [³H]E₂ were able to bind to both A-T and G-C containing DNAs. Furthermore, the formation of E₁–DNA and E₂–DNA adducts was detected by ³²P-postlabeling analysis.     

Manganese Tetraphenylporphyrins Catalyzed Selective Oxidation of Purine Derivatives

Abstract

The oxidation of purine derivatives using porphyrins as catalysts and dimethyldioxirane (DMDO) as oxygen atom donor is reported. The regioselectivity of the oxidation was found to be dependent on the presence of a free OH moiety on the N(9)-side chain of the substrate and on the structure of the catalyst.     

Synthesis of substituted septanosyl-1,2,3-triazoles

A carbohydrate-based oxepine, derived from 2-deoxy-d-arabino-hexopyranose, was used to prepare a family of septanosyl-1,2,3-triazoles in four steps. DMDO mediated epoxidation of the oxepine followed by trapping of the intermediate 1,2-anhydroseptanose by sodium azide gave the β-substituted glycosyl azide. The septanosyl azide was then reacted with a number of alkynes under thermal Huisgen or copper(I) mediated reaction conditions. Hydrogenolysis of benzyl protecting groups gave substituted septanosyl-1,2,3-triazoles. The new septanose-based structures were then evaluated as potential glycosidase inhibitors.     

Direct epoxidation of D-glucal and D-galactal derivatives with in situ generated DMDO

A multi-gram epoxidation of 3,4,6-tri-O-benzyl-D-glucal and D-galactal with dimethyldioxirane (DMDO) generated in situ from Oxone/acetone in a biphasic system (CH(2)Cl(2)-aqueous NaHCO(3)) resulted in the formation of the corresponding 1,2-anhydrosugars in a 99% yield and 100% selectivity. In a similar way, 3,4,6-tri-O-acetyl-D-glucal afforded a 7:1 mixture of the corresponding gluco and manno derivatives in an 87% overall yield.     

Synthesis, crystal structure, and reactivity of a D-xylose based oxepine

The synthesis and X-ray crystal structure of a D-xylose-based oxepine are reported. The oxepine was prepared from 2,3,4-tri-O-benzyl-D-xylose by the three-step sequence (Wittig olefination, vinyl ether formation, and ring closing metathesis) we recently reported. Epoxidation of this cyclic enol ether using dimethyldioxirane (DMDO) gave 1,2-anhydro-beta-D-idoseptanose, which was trapped by a number of nucleophiles to give alpha-idoseptanosides. The stereochemistry of epoxidation was assigned based on product analysis. Spectroscopic data of methyl 2,3,4,5-tetra-O-acetyl-alpha-D-idoseptanoside, derived from the methanolysis product 11, was compared to data of its enantiomer, the known methyl 2,3,4,5-tetra-O-acetyl-alpha-L-idoseptanoside.     

Transesterification/acetylation of long chain alcohols with alkyl acetate

Gas chromatographic characterizations of fatty alcohols are generally carried out as the free alcohols, trimethyl silyl or acetyl derivatives. In this study, transesterification/acetylation of long chain fatty alcohols is simply carried out by dissolving the alcohol in ethyl/methyl acetate and passing through a micro-column packed with solid NaOH. Reaction times are slightly different for alcohols of different chain length. Rice bran alcohols of 24–34 carbon atom are successfully acetylated. Also, castor oil methyl ester can be interesterified but with longer reaction time.     

脂肪醇制备研究进展

脂肪醇是合成表面活性剂、洗涤剂、增塑剂及其他多种精细化学用品的基础化工原料,广泛应用于纺织、日化、造纸、食品、医药、皮革等领域。本文介绍了脂肪醇的市场现状,综述了工业制备脂肪醇的传统方法,阐述了以可再生非粮生物质为原料,利用生物法制备生物基脂肪醇的方法,并对生物基脂肪醇的新合成路线的发展方向进行展望。     

Interaction of aliphatid alcohols with cytochrome P-450 from rat liver microsomes]

The interaction of alyphatic alcohols and cyclohexanol with cytochrome P-450 in microsomes has been investigated. All alchohols induced the modified 11 type spectral changes by mixing with microsomes. These changes are characterized by lambdamax = 412 and lambdamin = 380-382 nm in difference spectra. The dissociation constants of the alcohol cytochrome P-450 complexes are determined. On this dissociation constants influence pH and Triton X-100 presence. The interaction of the alcohols with cytochrome P-450 in phosphate buffer pH = 6,0 in the detergents absence is characterized by one dissociation constant for MeOH, EtOH, n-BuOH and cyclohexanol and by two dissociation constants for i-PrOH, i-BuOH and tert.-BuOH. The interaction of the alcohols with cytochrome P-450 in Tris-HCL-buffer (pH 7.5) in the Triton X-100 presence is characterized for all above alcohols by the dissociations constants, which are described by Taft equation with coefficient rho =-1.55. This fact confirms the interaction of alcohols HO-groups with heme iron of cytochrome P-450. The scheme of interaction of alcohols with cytochrome P-450 is discussed.     

Thermodynamic interpretation of effects of alcohols on membrane lipid fluidity

The effect of a series of amphiphilic compounds, the first eight n-aliphatic alcohols, on the fluidity of rat enterocyte brush border was determined by ESR using 5-doxyl stearic acid as a lipid spin probe. Packing order variations are compared to the relative hydrophobic effect of the alcohols. The concentrations, [Ci]5 of each alcohol that decrease the membrane 2T' value by 5%, vary by a factor of 1500 from methanol to octanol. From [Ci]5, the membrane concentrations Cm and the variation of free energy delta F degree due to the incorporation of the alcohols in the lipids, were calculated. These calculations were performed taking into account the respective volumes of the aqueous phase and the membrane lipids. Cm is of the order of 0.18 mol/kg for the odd chain length alcohols and of 0.27 mol/kg for the even alcohols. The value of delta F degree in cal/mol -CH2- is -687 cal on average for the eight alcohols. This work shows that for all the alcohols, the concentrations at equilibrium in the membrane and in the aqueous phase are respectively in agreement with Meyer and Overton's theory and with the gradient of free energy which constitutes the most general index of interaction of lipophilic substances with membranes.     

The kinetics and mechanism of liver alcohol dehydrogenase with primary and secondary alcohols as substrates

1. The activity of liver alcohol dehydrogenase with propan-2-ol and butan-2-ol has been confirmed. The activity with the corresponding ketones is small. Initial-rate parameters are reported for the oxidation of these secondary alcohols, and of propan-1-ol and 2-methylpropan-1-ol, and for the reduction of propionaldehyde and 2-methylpropionaldehyde. Substrate inhibition with primary alcohols is also described. 2. The requirements of the Theorell-Chance mechanism are satisfied by the data for all the primary alcohols and aldehydes, but not by the data for the secondary alcohols. A mechanism that provides for dissociation of either coenzyme or substrate from the reactive ternary complex is described, and shown to account for the initial-rate data for both primary and secondary alcohols, and for isotope-exchange results for the former. With primary alcohols, the rapid rate of reaction of the ternary complex, and its small steady-state concentration, result in conformity of initial-rate data to the requirements of the Theorell-Chance mechanisms. With secondary alcohols, the ternary complex reacts more slowly, its steady-state concentration is greater, and therefore dissociation of coenzyme from it is rate-limiting with non-saturating coenzyme concentrations. 3. Substrate inhibition with large concentrations of primary alcohols is attributed to the formation of an abortive complex of enzyme, NADH and alcohol from which NADH dissociates more slowly than from the enzyme-NADH complex. The initial-rate equation is derived for the complete mechanism, which includes a binary enzyme-alcohol complex and alternative pathways for formation of the reactive ternary complex. This mechanism would also provide, under suitable conditions, for substrate activation or substrate inhibition in a two-substrate reaction, according to the relative rates of reaction through the two pathways.     

Human alcohol dehydrogenase: dependence of secondary alcohol oxidation on the amino acids at positions 93 and 94.

The human liver alpha alpha and beta 1 beta 1 isoenzymes are straight-chain alcohol dehydrogenases with different efficiencies toward secondary alcohols. Two of the 24 amino acid substitutions in alpha alpha (A for F93 and I for T94) were made by site-directed mutagenesis of beta 1 beta 1 and the substrate specificity of beta 93A94I was examined. The Vmax/KM values of beta 93A94I for secondary alcohols (especially R enantiomers) are similar to that of alpha alpha and as much as 4000-fold greater than beta 1 beta 1, but the dependences of Vmax/KM on primary alcohol chain length are similar to beta 1 beta 1, but not alpha alpha. Thus, the substitutions of A for F93 and I for T94 in beta 1 beta 1 account for the increased efficiency towards secondary alcohols and stereoselectivity for enantiomeric alcohols, but not for the effects of chain length on the Vmax/KM for primary alcohols seen with alpha alpha.     

Alcohol Dehydrogenase Activities of Wine Yeasts in Relation to Higher Alcohol Formation

Alcohol dehydrogenase activities were examined in cell-free extracts of 10 representative wine yeast strains having various productivities of higher alcohols (fusel oil). The amount of fusel alcohols (n-propanol, isobutanol, active pentanol, and isopentanol) produced by the different yeasts and the specific alcohol dehydrogenase activities with the corresponding alcohols as substrates were found to be significantly related. No such relationship was found for ethanol. The amounts of higher alcohols formed during vinification could be predicted from the specific activities of the alcohol dehydrogenases with high accuracy. The results suggest a close relationship between the control of the activities of alcohol dehydrogenase and the formation of fusel oil alcohols. Also, new procedures for the prediction of higher alcohol formation during alcoholic beverage fermentation are suggested.     

Detection of alkanes, alcohols, and aldehydes using bioluminescence

We report a novel method for the rapid, sensitive, and quantitative detection of alkanes, alcohols, and aldehydes that relies on the reaction of bacterial luciferase with an aldehyde, resulting in the emission of light. Primary alcohols with corresponding aldehydes that are within the substrate range of the particular luciferase are detected after conversion to the aldehyde by an alcohol dehydrogenase. In addition, alkanes themselves may be detected by conversion to primary alcohols by an alkane hydroxylase, followed by conversion to the aldehyde by alcohol dehydrogenase. We developed a rapid bioluminescent method by genetically engineering the genes encoding bacterial luciferase, alcohol dehydrogenase, and alkane hydroxylase into a plasmid for simultaneous expression in an E. coli host cell line. Alkanes, alcohols, or aldehydes were detected within seconds, with sensitivity in the micromolar range, by measuring the resulting light emission with a microplate reader. We demonstrate the application of this method for the detection of alkanes, alcohols, and aldehydes and for the detection of alkane hydroxylase and alcohol dehydrogenase activity in vivo. This method is amenable to the high-throughput screening needs required for the identification of novel catalysts.     

Inhibitory effects of alcohols on the activity of human matrix metalloproteinase 7 (matrilysin)

Aliphatic alcohols inhibited the activity of human matrix metalloproteinase 7 (matrilysin) competitively with K(i) of 6.1-19.4% (v/v) or 0.66-4.80 M. From the relationship between the structures of alcohols and their K(i) values, alcohols are considered to bind the hydrophobic S1' subsite most plausibly, and the size of the pocket was estimated to be large enough to accommodate the length of 1-butanol (4-carbon chain) and the bulk of tertiary alcohols. Alcohols might be suitable probes for exploring the active-site geometry of enzymes.     

Phosphatidyl alcohols: Effect of head group size on domain forming properties and interactions with sterols

In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in membranes by the phospholipase D reaction in the presence of alcohol. The melting behavior of pure phosphatidyl alcohols and mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or cholesterol was assessed using high sensitivity differential scanning calorimetry (DSC). DPPMe had the highest melting temperature (∼ 49 °C), whereas the other phosphatidyl alcohols had similar melting temperatures as DPPC (∼ 40-41 °C). All phosphatidyl alcohols, except DPPMe, also showed good miscibility with DPPC. The effects of cholesterol on the melting behavior and membrane order in multilamellar bilayer vesicles were assessed using steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DSC. The ordering effect of cholesterol in the fluid phase was lower for all phosphatidyl alcohols as compared to DPPC and decreased with increasing head group size. The formation of ordered domains containing the phosphatidyl alcohols in complex bilayer membranes was determined using fluorescence quenching of DPH or the sterol analogue cholesta-5,7,(11)-trien-3-beta-ol (CTL). The phosphatidyl alcohols did not appear to form sterol-enriched ordered domains, whereas DPPMe, DPPEt appeared to form ordered domains in the temperature window examined (10-50 °C). The partitioning of CTL into bilayer membranes containing phosphatidyl alcohols was to a small extent increased for DPPMe and DPPEt, but in general, sterol interactions were weak or unfavorable for the phosphatidyl alcohols. Our results show that the biophysical and sterol interacting properties of phosphatidyl alcohols, having identical acyl chain structures, are markedly dependent on the size of the head group.     

Exclusion of alcohols from spermidine-DNA assemblies: probing the physical basis of preferential hydration

The interaction of the alcohols 2-methyl-2,4-pentanediol (MPD) and 2-propanol and of glycerol with condensed spermidine(3+)-DNA arrays are investigated with direct force measurements using osmotic stress coupled with X-ray scattering. Thermodynamic forces between DNA helices are measured from the dependence of helical interaxial spacings on the osmotic pressure applied by poly(ethylene glycol) solutions in equilibrium with the DNA phase. The sensitivity of these forces to solute concentration can be transformed into a change in the number of excess or deficit solutes or waters in the DNA phase by applying the Gibbs-Duhem equation. The alcohols examined are excluded from the condensed DNA array and strongly affect the osmotic stress force curves. DNA is preferentially hydrated. MPD is significantly more excluded than 2-propanol. The exclusion of these alcohols, however, is not due to a steric repulsion since glycerol that is intermediate in size between MPD and 2-propanol does not observably affect DNA force curves. As the distance between DNA helices varies, the change in the number of excess waters is independent of alcohol concentration for each alcohol. These solutes are acting osmotically on the condensed array. The distance dependence of exclusion indicates that repulsive water structuring forces dominate the interaction of alcohols with the DNA surface. The exclusion measured for these condensed arrays can quantitatively account for the effect of these alcohols on the precipitation of DNA from dilute solution by spermidine(3+).     

Advances in asymmetric oxidative kinetic resolution of racemic secondary alcohols catalyzed by chiral Mn(III) salen complexes

Enantiomerically pure secondary alcohols are essential compounds in organic synthesis and are used as chiral auxiliaries and synthetic intermediates in the pharmaceutical, agrochemical, and fine chemical industries. One of the attractive and practical approaches to achieving optically pure secondary alcohols is oxidative kinetic resolution of racemic secondary alcohols using chiral Mn(III) salen complexes. In the last decade, several chiral Mn(III) salen complexes have been reported with excellent enantioselectivity and activity in the homogeneous and heterogeneous catalysis of the oxidative kinetic resolution of racemic secondary alcohols. This review article is an overview of the literature on the recent development of chiral Mn(III) salen complexes for oxidative kinetic resolution of racemic secondary alcohols. The catalytic activity of monomeric, dimeric, macrocyclic, polymeric, and silica/resin supported chiral Mn(III) salen complexes is discussed in detail.     

A new approach to the synthesis of 4'-carbon-substituted nucleosides: development of a highly active anti-HIV agent 2', 3'-didehydro-3'-deoxy-4'-ethynylthymidine

Oxidation of 3'-O-TBDMS-4',5-unsaturated thymidine 3 with dimethyldioxirane (DMDO) allowed the isolation of the epoxide 4. Upon reacting with organosilicon reagents in the presence of SnCl4, 4 underwent stereoselective ring opening to give 4'-alpha-allyl (6), 4'-alpha-(2-bromoallyl) (7), 4'-alpha-(cyclopenten-3-yl) (8), and 4'-alpha-cyano (9) derivatives of thymidine. Reactions of the 3'-epimer 12 with organoaluminum reagents gave 4'-alpha-methyl (13), 4'-alpha-vinyl (14), and 4'-alpha-ethynyl (15) analogues. Compounds 13-15 were transformed into corresponding 2',3'-didehydro-3'-deoxy derivatives. Evaluation of their ability to inhibit the replication of HIV in cell culture showed that 4'-ethynyl-d4T (19) is more potent and less toxic than the parent compound d4T.