酵母不对称催化制备R-1,2-丙二醇

研究了采用面包酵母还原丙酮醇制备R-1,2-丙二醇的工艺。采用摇瓶对转化条件进行单因素实验,确定最优转化条件：丙酮醇浓度0.3mmol/mL,pH7.0,酵母质量浓度150g/L,乙醇浓度为0.3mmol/mL,转化时间25h。在此条件下,采用分批流加策略进行1.5L规模发酵罐转化试验,转化25h后,发酵液的R-1,2-丙二醇浓度为0.27mmol/mL。     

New evidence for the mechanism of the tin(II) chloride catalyzed reactions of vicinal diols with diazodiphenylmethane in 1,2-dimethoxyethane

A kinetic study of the tin(II) chloride catalyzed reaction of diazodiphenylmethane with ethylene glycol in dimethoxyethane is reported. The preparation and characterization of ethylene glycol monodiphenylmethyl ether, the main product from this reaction, is also reported as well as the preparation of the two diphenylmethyl monoethers of methyl 4,6-O-benzylidene-alpha-D-glucopyranoside. An unexpected relationship between the concentration of ethylene glycol and the pseudo first-order rate constant, k', was observed in these reactions. For low concentrations of ethylene glycol (below 0.06 M), k' increases with increasing concentration of the diol. This trend is reversed for high concentrations of ethylene glycol (from about 0.06 to about 0.2 M). The apparent rate constant was also inversely related to the initial concentration of diazodiphenylmethane for the concentrations investigated. These results make the previously proposed involvement of a 1,3,2-dioxastannolane intermediate very unlikely [Petursson, S.; Webber, J.M. Carbohydr. Res. 1982, 103, 41-52]. The results suggest that more likely intermediates for these reactions involve tin(II) chloride complexes in a dynamic equilibrium with the diol.     

Initial reactions in the oxidation of 1,2-dihydronaphthalene by Sphingomonas yanoikuyae strains.

The substrate oxidation profiles of Sphingomonas yanoikuyae B1 biphenyl-2,3-dioxygenase and cis-biphenyl dihydrodiol dehydrogenase activities were examined with 1,2-dihydronaphthalene and various cis-diols as substrates. m-Xylene-induced cells of strain B1 oxidized 1,2-dihydronaphthalene to (-)-(1R,2S)-cis-1,2-dihydroxy-1,2-3,4-tetrahydronaphthalene as the major product (73% relative yield). Small amounts of (+)-(R)-2-hydroxy-1,2-dihydronaphthalene (15%), naphthalene (6%), and alpha-tetralone (6%) were also formed. Strain B8/36, which lacks an active cis-biphenyl dihydrodiol dehydrogenase, formed (+)-(1R,2S)-cis-1,2-dihydroxy-1,2-dihydronaphthalene (51%), in addition to (-)-(1R,2S)-cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene (44%) and (+)-(R)-2-hydroxy-1,2-dihydronaphthalene (5%). The cis-biphenyl dihydrodiol dehydrogenase of strain B1 oxidized both enantiomers of cis-1,2-dihydroxy-1,2-dihydronaphthalene, but only the (+)-(1S,2R)-enantiomers of cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and cis-1,2-dihydroxy-3-phenylcyclohexa-3,5-diene. The results show that biphenyl dioxygenase expressed by S. yanoikuyae catalyzes dioxygenation, monooxygenation, and desaturation reactions with 1,2-dihydronaphthalene as the substrate, and cis-biphenyl dihydrodiol dehydrogenase catalyzes the enantioselective dehydrogenation of (+)-(1S,2R)-cis-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and (+)-(1S,2R)-cis-1,2-dihydroxy-3-phenylcyclohexa-3,5-diene.     

Fermentation of 1,2-Propanediol and 1,2-Ethanediol by Some Genera of Enterobacteriaceae, Involving Coenzyme B12-Dependent Diol Dehydratase

Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 was able to grow anaerobically on 1,2-propanediol and 1,2-ethanediol as carbon and energy sources. Whole cells of the bacterium grown anaerobically on 1,2-propanediol or on glycerol catalyzed conversion of 1,2-diols and aldehydes to the corresponding acids and alcohols. Glucose-grown cells also converted aldehydes, but not 1,2-diols, to acids and alcohols. The presence of activities of coenzyme B(12)-dependent diol dehydratase, alcohol dehydrogenase, coenzyme-A-dependent aldehyde dehydrogenase, phosphotransacetylase, and acetate kinase was demonstrated with crude extracts of 1,2-propanediol-grown cells. The dependence of the levels of these enzymes on growth substrates, together with cofactor requirements in in vitro conversion of these substrates, indicates that 1,2-diols are fermented to the corresponding acids and alcohols via aldehydes, acyl-coenzyme A, and acyl phosphates. This metabolic pathway for 1,2-diol fermentation was also suggested in some other genera of Enterobacteriaceae which were able to grow anaerobically on 1,2-propanediol. When the bacteria were cultivated in a 1,2-propanediol medium not supplemented with cobalt ion, the coenzyme B(12)-dependent conversion of 1,2-diols to aldehydes was the rate-limiting step in this fermentation. This was because the intracellular concentration of coenzyme B(12) was very low in the cells grown in cobalt-deficient medium, since the apoprotein of diol dehydratase was markedly induced in the cells grown in the 1,2-propanediol medium. Better cell yields were obtained when the bacteria were grown anaerobically on 1,2-propanediol. Evidence is presented that aerobically grown cells have a different metabolic pathway for utilizing 1,2-propanediol.     

Cellular physiology controls photoautotrophic production of 1,2-propanediol from pools of CO2 and glycogen

Synechocystis sp. PCC 6803 PG is a cyanobacterial strain capable of synthesizing 1,2-propanediol from carbon dioxide (CO₂) via a heterologous three-step pathway and a methylglyoxal synthase (MGS) originating from Escherichia coli as an initial enzyme. The production window is restricted to the late growth and stationary phase and is apparently coupled to glycogen turnover. To understand the underlying principle of the carbon partitioning between the Calvin-Benson-Bassham (CBB) cycle and glycogen in the context of 1,2-propanediol production, experiments utilizing ¹³C labeled CO₂ have been conducted. Carbon fluxes and partitioning between biomass, storage compounds, and product have been monitored under permanent illumination as well as under dark conditions. About one-quarter of the carbon incorporated into 1,2-propanediol originated from glycogen, while the rest was derived from CO₂ fixed in the CBB cycle during product formation. Furthermore, 1,2-propanediol synthesis was depending on the availability of photosynthetic active radiation and glycogen catabolism. We postulate that the regulation of the MGS from E. coli conflicts with the heterologous reactions leading to 1,2-propanediol in Synechocystis sp. PCC 6803 PG. Additionally, homology comparison of the genomic sequence to genes encoding for the methylglyoxal bypass in E. coli suggested the existence of such a pathway also in Synechocystis sp. PCC 6803. These findings are critical for all heterologous pathways coupled to the CBB cycle intermediate dihydroxyacetone phosphate via a MGS and reveal possible engineering targets for rational strain optimization.     

Syntheses and reactions of 5-O-acetyl-1,2-anhydro-3-O-benzyl-alpha-D-ribofuranose and beta-D-lyxofuranose, 5-O-acetyl-1,2-anhydro-3,6-di-O-benzyl- and 1,2-anhydro-5,6-di-O-benzoyl-3-O-benzyl-beta-D-mannofuranose, and 6-O-acetyl-1,2-anhydro-3,4-di-O-benzyl-alpha-D-glucopyranose and -beta-D-talopyranose

The title compounds 5-O-acetyl-1,2-anhydro-3-O-benzyl-alpha-D-ribofuranose and 5-O-acetyl-1,2-anhydro-3-O-benzyl-beta-D-lyxofuranose, and 6-O-acetyl-1,2-anhydro-3,4-di-O-benzyl-alpha-D-glucopyranose and 6-O-acetyl-1,2-anhydro-3,4-di-O-benzyl-beta-D-talopyranose, and 5-O-acetyl-1,2-anhydro-3,6-di-O-benzyl-beta-D-mannofuranose and 1,2-anhydro-5,6-di-O-benzoyl-3-O-benzyl-beta-D-mannofuranose have each been synthesized from the corresponding 2-O-tosylate and 1-free hydroxyl intermediates by base-initiated intramolecular S(N)2 ring closure in almost quantitative yields. Acetyl and benzoyl groups were not affected in the ring closure reactions. Condensation of 6-O-acetyl-1,2-anhydro-3,4-di-O-benzyl-alpha-D-glucopyranose and 5-O-acetyl-1,2-anhydro-3,6-di-O-benzyl-beta-D-mannofuranose with 1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose in the presence of ZnCl2 as the catalyst afforded the 1,2-trans-linked 6-O-acetyl-3,4-di-O-benzyl-beta-D-glucopyranosyl-(1-->6)-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose and 5-O-acetyl-3,6-di-O-benzyl-alpha-D-mannofuranosyl-(1-->6)-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose as the sole products in satisfactory yields, while condensation of 5-O-acetyl-1,2-anhydro-3-O-benzyl-beta-D-lyxofuranose with 3-O-benzyl-1,2-O-isopropylidene-alpha-D-xylofuranose yielded the 1,2-trans-linked 5-O-acetyl-3-O-benzyl-alpha-D-lyxofuranosyl-(1-->5)-3-O-benzyl-1,2-O-isopropylidene-alpha-D-xylofuranose as the sole product in a good yield. The 6-O-acetyl group in the glycosyl donor, 6-O-acetyl-1,2-anhydro-3,4-di-O-benzyl-alpha-D-glucopyranose, did not influence the stereoselectivity of the ring-opening-coupling reaction.     

Accumulation of 1,2-propanediol and enhancement of aerobic stability in whole crop maize silage inoculated with Lactobacillus buchneri

Aims: To assess the effects of inoculation of Lactobacillus buchneri on the ensiling properties and aerobic stability of maize silage. Methods and Results: Chopped whole crop maize was ensiled in 0.5 litre airtight polyethylene bottles (0.4 kg per bottle) and in double-layered, thin polyethylene bags (15 kg per bag), with or without inoculation of Lact. buchneri. The silos were stored for two to four months and the chemical composition, microbial numbers and aerobic stability were determined. Inoculation lowered lactic acid and yeasts, and increased acetic acid and pH value, resulting in improved aerobic stability of the silages. Inoculated silages produced 1,2-propanediol, the content of which increased as ensiling was prolonged, and nearly 50 g kg-1 dry matter had accumulated after four months of storage. The effects of inoculation, however, were much less pronounced in silages prepared in bags. Mannitol was found in all silages; the production was lowered by Lact. buchneri treatment and appeared to be unrelated to the accumulation of 1,2-propanediol. Conclusions: Inoculation of Lact. buchneri occasionally causes accumulation of 1,2-propanediol in silages without further degradation into propionic acid and 1-propanol. Significance and Impact of the Study: Substantial amounts of 1,2-propanediol could be consumed by ruminants when fed on silages inoculated with Lact. buchneri. In addition to increasing acetic acid, attention needs to be paid to 1,2-propanediol because the two fermentation products might affect the intake and utilization of silage-based diets.     

Microbial formation, biotechnological production and applications of 1,2-propanediol

This short review covers metabolic pathways, genetics and metabolic engineering of 1,2-propanediol formation in microbes. 1,2-Propanediol production by bacteria and yeasts has been known for many years and two general pathways are recognized. One involves the metabolism of deoxyhexoses, where lactaldehyde is formed during the glycolytic reactions and is then reduced to 1,2-propanediol. The second pathway derives from the formation of methylglyoxal from dihydroxyacetonephosphate and its subsequent reduction to 1,2-propanediol. The enzymes involved in the reduction of methylglyoxal can generate isomers of lactaldehyde or acetol, which can be further reduced by specific reductases, giving chiral 1,2-propanediol as the product. The stereospecificity of the enzymes catalyzing the two reduction steps is important in deriving a complete pathway. Through genetic engineering, appropriate combinations of enzymes have been brought together in Escherichia coli and yeast to generate 1,2-propanediol from glucose. The optimization of these strains may yield microbial processes for the production of this widely used chemical. Received: 25 May 2000 / Received revision: 24 July 2000 / Accepted: 25 July 2000     

Regioselective O-glucosylation by sucrose phosphorylase: a promising route for functional diversification of a range of 1,2-propanediols

1,2-Propanediol and 3-aryloxy/alkyloxy derivatives thereof are bulk commodities produced directly from glycerol. Glycosylation is a promising route for their functional diversification into useful fine chemicals. Regioselective glucosylation of the secondary hydroxyl in different 1,2-propanediols was achieved by a sucrose phosphorylase-catalyzed transfer reaction where sucrose is the substrate and 2-O-α-d-glucopyranosyl products are exclusively obtained. Systematic investigation for optimization of the biocatalytic synthesis included prevention of sucrose hydrolysis, which occurs in the process as a side reaction of the phosphorylase. In addition to ‘nonproductive’ depletion of donor substrate, the hydrolysis also resulted in formation of maltose and kojibiose (up to 45%) due to secondary enzymatic glucosylation of the glucose thus produced. Using 3-ethoxy-1,2-propanediol as the acceptor substrate (1.0 M), the desired transfer product was obtained in about 65% yield when employing a moderate (1.5-fold) excess of sucrose donor. Loss of the glucosyl substrate to ‘glucobiose’ by-products was minimal (<7.5%) under these conditions. The reactivity of other acceptors decreased in the order, 3-methoxy-1,2-propanediol > 1,2-propanediol > 3-allyloxy-1,2-propanediol > 3-(o-methoxyphenoxy)-1,2-propanediol > 3-tert-butoxy-1,2-propanediol. Glucosylated 1,2-propanediols were not detectably hydrolyzed by sucrose phosphorylase so that their synthesis by transglucosylation occurred simply under quasi-equilibrium reaction conditions.     

The metabolism of 1,2-propanediol by the propylene oxide utilizing bacterium Nocardia A60

A bacterium designated Nocardia A60 was isolated for its capacity to utilize propylene oxide (1,2-epoxypropame) aerobically as a carbon and energy source for growth. Extracts of cells grown on the epoxide catalyzed the conversion of propylene oxide to 1,2-propanediol This epoxidase activity was absent in cells grown on 1,2-propanediol or succinate. During growth of the organism on propylene oxide and 1,2-propanediol it contained high levels of diol dehydratase (EC 4.2.1.28). Enhanced levels of propionyl-CoA carboxylase during growth on propylene oxide and 1,2-propanediol suggest that these compounds are metabolized via propionate and succinate.     

Isolation of a bacterium assimilating (R)-3-chloro-1,2-propanediol and production of (S)-3-chloro-1,2-propanediol using microbial resolution

A bacterium capable of assimilating 3-chloro-1,2-propanediol was isolated from soil by enrichment culture. The strain was identified as Alcaligenes sp. by taxonomic studies. The crude extracts of the cells had dehalogenating activities and converted various halohydrins to the corresponding epoxides. 3-Chloro-1,2-propanediol was degraded stereospecifically by the strain, liberating chloride ion. The residual isomer was found to be the (S)-form (99.4% enantiomeric excess). (S)-3-Chloro-1,2-propanediol was obtained from the racemate by use of this strain in 38% yield, and (S)-glycidol (99.4% enantiomeric excess) was subsequently synthesized from the obtained (S)-3-chloro-1,2-propanediol by alkaline treatment.     

Effect of competitive terminal electron acceptor processes on dechlorination of cis-1,2-dichloroethene and 1,2-dichloroethane in constructed wetland soils

Thermodynamic calculations were coupled with time-series measurements of chemical species (parent and daughter chlorinated solvents, H(2), sulfite, sulfate and methane) to predict the anaerobic transformation of cis-1,2-dichloroethene (cis-1,2-DCE) and 1,2-dichloroethane (1,2-DCA) in constructed wetland soil microcosms inoculated with a dehalorespiring culture. For cis-1,2-DCE, dechlorination occurred simultaneously with sulfite and sulfate reduction but competitive exclusion of methanogenesis was observed due to the rapid H(2) drawdown by the dehalorespiring bacteria. Rates of cis-1,2-DCE dechlorination decreased proportionally to the free energy yield of the competing electron acceptor and proportionally to the rate of H(2) drawdown, suggesting that H(2) competition between dehalorespirers and other populations was occurring, affecting the dechlorination rate. For 1,2-DCA, dechlorination occurred simultaneously with methanogenesis and sulfate reduction but occurred only after sulfite was completely depleted. Rates of 1,2-DCA dechlorination were unaffected by the presence of competing electron-accepting processes. The absence of a low H(2) threshold suggests that 1,2-DCA dechlorination is a cometabolic transformation, occurring at a higher H(2) threshold, despite the high free energy yields available for dehalorespiration of 1,2-DCA. We demonstrate the utility of kinetic and thermodynamic calculations to understand the complex, H(2)-utilizing reactions occurring in the wetland bed and their effect on rates of dechlorination of priority pollutants.     

Identification of the 1,2-propanediol-1-yl radical as an intermediate in adenosylcobalamin-dependent diol dehydratase reaction

The reaction catalyzed by adenosylcobalamin-dependent diol dehydratase proceeds by a radical mechanism. A radical pair consisting of the Co(II) of cob(II)alamin and an organic radical intermediate formed during catalysis gives EPR spectra. The high-field doublet and the low-field broad signals arise from the weak interaction of an organic radical with the low-spin Co(II) of cob(II)alamin. To characterize the organic radical intermediate in the diol dehydratase reaction, several deuterated and (13)C-labeled 1,2-propanediols were synthesized, and the EPR spectra observed in the catalysis were measured using them as substrate. The EPR spectra with the substrates deuterated on C1 showed significant line width narrowing of the doublet signal. A distinct change in the hyperfine coupling was seen with [1-(13)C]-1,2-propanediol, but not with the [2-(13)C]-counterpart. Thus, the organic radical intermediate observed by EPR spectroscopy was identified as the 1,2-propanediol-1-yl radical, a C1-centered substrate-derived radical.     

C(2)-Symmetric dialkoxyphosphoramide and dialkoxythiophosphoramide derivatives of (1R, 2R)-1,2-diaminocyclohexane as chiral ligands for the titanium(IV) alkoxide-promoted asymmetric addition reactions of diethylzinc to arylaldehydes.

C(2)-Symmetric chiral diethoxyphosphoramide 4, diethoxythiophosphoramide 5, and diisopropoxyphosphoramide 6 of (1R, 2R)-1,2-diaminocyclohexane were prepared by the reactions of diethoxyphosphinic chloride, diethoxythiophosphinic chloride, and diisopropoxyphosphinic chloride with (1R, 2R)-1,2-diaminocyclohexane, respectively. They were used as catalytic chiral ligands in the asymmetric addition reactions of diethylzinc to aldehydes in the presence of titanium(IV) isopropoxide to give the corresponding sec-alcohols with 43-70% ee. Chiral ligands 4 and 5 gave the sec-alcohols with opposite absolute configuration.     

Microbial metabolism of 1,2-propanediol studied by the Rumen Simulation Technique (Rusitec)

C zerkawski J.W., P iatkova M. & B reckenridge , G. 1984. Microbial metabolism of 1,2-propanediol studied by the Rumen. Simulation Technique (Rusitec). Journal of Applied Bacteriology , 56 , 81–94.
A series of experiments with the Rumen Simulation Technique (Rusitec) showed that 1,2-propanediol was metabolized efficiently by rumen micro-organisms and that the main end-products of fermentation were propionic and 2-methylbutyric acids. 'Propionaldehyde and n-propanol were also formed as intermediate compounds.' The effect of the diol on digestion of the basal diet appeared to be small with concentrate, or when the roughage was supplemented with additional nitrogen (urea). The decrease in the output of acetic and butyric acids was consistent with utilization of C₂ units for synthesis of 2-methylbutyric acid. The fermentation of 1,2-propanediol resulted in little or no increase in the output of additional microbial matter. The distribution of radioactivity from [1-¹⁴C] 1,2-propanediol confirmed that propionaldehyde and n -propanol were the primary products of metabolism of the diol and that the end-products were propionic and 2-methylbutyric acids, with very little labelling of microbial matter. Between 2% and 3% of radioactivity was found in gases and surprisingly the specific radioactivity of methane was higher than that of carbon dioxide, particularly during the initial stages of incubation. Possible pathways in the degradation of 1,2-propanediol by rumen micro-organisms are suggested and discussed in relation to similar reactions established in other systems.     

A novel enantioselective epoxide hydrolase for (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol

Bacillus sp. Z018, a novel strain producing epoxide hydrolase, was isolated from soil. The epoxide hydrolase catalyzed the stereospecific hydrolysis of (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol. Epoxide hydrolase from Bacillus sp. Z018 was inducible, and (R)-phenyl glycidyl ether was able to act as an inducer. The fermentation conditions for epoxide hydrolase were 35°C, pH 7.5 with glucose and NH₄Cl as the best carbon and nitrogen source, respectively. Under optimized conditions, the biotransformation yield of 45.8% and the enantiomeric excess of 96.3% were obtained for the product (R)-3-phenoxy-1,2-propanediol.     

Purification and characterization of an alcohol dehydrogenase from 1,2-propanediol-grownDesulfovibrio strain HDv

The sulfate-reducing bacterimDesulfovibrio strain HDv (DSM 6830) grew faster on (S)- and on (R, S)-1,2-propanediol (μ_max 0.053 h⁻) than on (R)-propanediol (0.017 h⁻¹) and ethanol (0.027 h⁻¹). From (R, S)-1,2-propanediol-grown cells, an alcohol dehydrogenase was purified. The enzyme was oxygen-labile, NAD-dependent, and decameric; the subunit mol. mass was 48 kDa. The N-terminal amino acid sequence indicated similarity to alcohol dehydrogenases belonging to family III of NAD-dependent alcohol dehydrogenases, the first 21 N-terminal amino acids being identical to those of theDesulfovibrio gigas alcohol dehydrogenase. Best substrates were ethanol and propanol (K _m of 0.48 and 0.33 mM, respectively). (R, S)-1,2-Propanediol was a relatively poor substrate for the enzyme, but activities in cell extracts were high enough to account for the growth rate. The enzyme showed a preference for (S)-1,2-propanediol over (R)-1,2-propanediol. Antibodies raised against the alcohol dehydrogenase ofD. gigas showed cross-reactivity with the alcohol dehydrogenase ofDesulfovibrio strain HDv and with cell extracts of six other ethanol-grown sulfate-reducing bacteria.     

Biodegradation of 1,2-dichloroethane (1,2-DCA) by cometabolism in a nitrifying biofilm reactor

Biodegradation of 1,2-dichloroethane (1,2-DCA) by cometabolism was investigated in a continuous-flow nitrifying biofilm reactor over a time period of 218 days. The removal efficiency of 1,2-DCA ranged between 70 and 90%. Using the generation of chloride (Cl⁻) as an indicator of 1,2-DCA mineralization, it was shown that the cometabolic degradation of 1,2-DCA was initiated through oxidative dechlorination. However, Cl⁻ production rates were observed to be lower than the stoichiometric ones which indicated the partial mineralization of 1,2-DCA and the possibility of by-product formation due to incomplete dechlorination. At high 1,2-DCA removal rates, Cl⁻ release seemed to reach a saturation due to 1,2-DCA-dependent inactivation of NH₄–N oxidation. The cometabolic 1,2-DCA degradation capacity of nitrifiers was quite comparable to metabolic 1,2-DCA degradation capacities of pure cultures. A strong linear relationship was found between 1,2-DCA transformation yields and NH₄–N and 1,2-DCA loadings. The effect of 1,2-DCA loading on nitrifier population was monitored using molecular microbiological tools. Long-term input of 1,2-DCA to the biofilm reactor resulted in no significant changes in the quantities of Nitrosomonas, Nitrobacter and Nitrospira species and no shift in the diversities of ammonia oxidizing species. Those findings provide an insight into both the operation and the community structure in natural and managed nitrifying biofilm systems where cometabolic 1,2-DCA takes place.     

Tin and iron halogenides as additives in ruthenium-catalyzed olefin metathesis

Different metal halogenides were used as additives in metathesis of 1-octene or in several cross-metathesis reactions catalysed by first and second generation Grubbs catalyst, 1 and 2, as well as by an improved first generation-type Grubbs catalyst 3. Tin(II) chloride and bromide enhance the performance of 1 and 3. The influence on 2 is rather minor under the chosen reaction conditions. The addition of iron(II) chloride and bromide results in an improvement of 1 to a lesser extent as the tin salts. The frequently observed isomerisation of olefins in metathesis reactions catalysed by 1 is suppressed in the presence of the applied tin salts.     

Mechanistic studies of a protonolytic organomercurial cleaving enzyme: bacterial organomercurial lyase

Mechanistic studies of the protonolytic carbon-mercury bond cleavage by organomercurial lyase from Escherichia coli (R831) suggest that the reaction proceeds via an SE2 pathway. Studies with stereochemically defined substrates cis-2-butenyl-2-mercuric chloride (1) and endo-norbornyl-2-mercuric bromide (2) reveal that a high degree of configurational retention occurs during the bond cleavage, while studies with exo-3-acetoxynortricyclyl-5-mercuric bromide (3) and cis-exo-2-acetoxy-bicyclo[2.2.1]hept-5-enyl-3-mercuric bromide (4) show that the protonolysis proceeds without accompanying skeletal rearrangement. Kinetic data for the enzymatic reactions of cis-2-butenyl-2-mercuric chloride (1) and trans-1-propenyl-1-mercuric chloride (6) indicate that these substrates show enhanced reaction rates of ca. 10-200-fold over alkylvinylmercurials and unsubstituted vinylmercurials, suggesting that the olefinic methyl substituent may stabilize an intermediate bearing some positive charge. Enzymatic reaction of 2-butenyl-1-mercuric bromide (5) yields a 72/23/5 mixture of 1-butene/trans-2-butene/cis-2-butene, indicative of intervening SE2' cleavage. The observation of significant solvent deuterium isotope effects at pH 7.4 of Vmax (H2O)/Vmax(D2O) = 2.1 for cis-2-butenyl-2-mercuric chloride (1) turnover and Vmax(H2O)/Vmax(D2O) = 4.9 for ethylmercuric chloride turnover provides additional support for a kinetically important proton delivery. Finally, the stoichiometric formation of butene and Hg(II) from 1 and methane and Hg(II) from methylmercuric chloride eliminates the possibility of an SN1 solvolytic mechanism. As the first well-characterized enzymatic reaction of an organometallic substrate and the first example of an enzyme-mediated SE2 reaction the organomercurial lyase catalyzed carbon-mercury bond cleavage provides an arena for investigating novel enzyme structure-function relationships.