The baker's yeast-mediated reduction of conjugated methylene groups in an organic solvent

The baker's yeast-mediated reduction of a series of conjugated methylene compounds was conducted in a petroleum ether reaction system. The methylene ketone, 3-phenyl-3-buten-2-one (1), was stereoselectively reduced to (R)-3-phenyl-2-butanone; no reduction of the ketone carbonyl was observed. Reduction of 2-phenyl-2-propenenitrile (7) also occurred stereoselectively to give (R)-2-phenylpropanenitrile (8) in good yield. The yeast mediated reduction of the methylene aldehyde, 2-phenyl-2-propenal (3), gave a mixture of products arising from reduction of both the methylene and carbonyl groups; stereoselective reduction of the methylene group gives (R)-2-phenylpropanal (6) which is rapidly reduced to (R)-2-phenyl-1-propanol (5), whereas reduction of the carbonyl gives 2-phenyl-2-propen-1-ol (4) which is slowly reduced to racemic (5).     

Novel reactions of 5-bromouracil derivatives with active methylene compounds

5-Bromouracil derivatives (1) reacted with carbanions at room temperature to give 6-substituted uracils (6), 2,4-diazabicyclo[4,1,0] heptanes (8) and 2,4-diazabicyclo[4,3,0]nonane derivative (9), which depend largely upon the structure of the active methylene compounds employed for the generation of carbanions.     

Purification and characterization of the methylene tetrahydromethanopterin dehydrogenase MtdB and the methylene tetrahydrofolate dehydrogenase FolD from Hyphomicrobium zavarzinii ZV580

Recently, it has been shown that heterotrophic methylotrophic Proteobacteria contain tetrahydrofolate (H(4)F)- and tetrahydromethanopterin (H(4)MPT)-dependent enzymes. Here we report on the purification of two methylene tetrahydropterin dehydrogenases from the methylotroph Hyphomicrobium zavarzinii ZV580. Both dehydrogenases are composed of one type of subunit of 31 kDa. One of the dehydrogenases is NAD(P)-dependent and specific for methylene H(4)MPT (specific activity: 680 U/mg). Its N-terminal amino acid sequence showed sequence identity to NAD(P)-dependent methylene H(4)MPT dehydrogenase MtdB from Methylobacterium extorquens AM1. The second dehydrogenase is specific for NADP and methylene H(4)F (specific activity: 180 U/mg) and also exhibits methenyl H(4)F cyclohydrolase activity. Via N-terminal amino acid sequencing this dehydrogenase was identified as belonging to the classical bifunctional methylene H(4)F dehydrogenases/cyclohydrolases (FolD) found in many bacteria and eukarya. Apparently, the occurrence of methylene tetrahydrofolate and methylene tetrahydromethanopterin dehydrogenases is not uniform among different methylotrophic alpha-Proteobacteria. For example, FolD was not found in M. extorquens AM1, and the NADP-dependent methylene H(4)MPT dehydrogenase MtdA was present in the bacterium that also shows H(4)F activity.     

Structure-activity relationships of the peptide deformylase inhibitor BB-3497: modification of the methylene spacer and the P1' side chain

Structural modifications to the peptide deformylase inhibitor BB-3497 are described. In this paper, we describe the initial SAR around this lead for modifications to the methylene spacer and the P1' side chain. Enzyme inhibition and antibacterial activity data revealed that the optimum distance between the N-formyl hydroxylamine metal binding group and the P1' side chain is one unsubstituted methylene unit. Additionally, lipophilic P1' side chains that closely mimic the methionine residue in the substrate provided compounds with the best microbiological profile.     

Photooxidation of amino acids in the presence of methylene blue

A systematic study was made of the photochemical action of methylene blue on amino acids.Tyrosine, tryptophan, histidine, methionine, and cystine were highly reactive during the photoöxidation; the rest of the amino acids acted sluggishly or not at all.In tyrosine, tryptophan, and histidine, the entire oxygen uptake and CO₂ evolution were due to the cyclic nucleus, involving rupture of the rings.During the photochemical action of methylene blue on tyrosine, tryptophan, and methionine, intermediary oxidizing agents were formed; in methionine this was shown to be H₂O₂.The photoöxidation of methionine resulted in the formation of methionine sulfoxide as an end product.Iodometric titration and measurement of ultraviolet absorption during irradiation of methionine indicate the formation of an intermediary dehydrogenation product which appears to differ from Lavine's dehydromethionine.Cystine was photoöxidized, probably beyond the cysteic acid stage.Peptide bonds did not participate in the photochemical action of methylene blue.Methylation of the α-amino group of lysine to the corresponding secondary and tertiary compounds produced increased reactivity in the photoöxidation.     

The effect of carbon dioxide on reduction of methylene blue by microorganisms

Summary and Conclusions The experiments ofHes which indicate that CO₂ is essential for the reduction of methylene blue by microorganisms presumably because it is required for the functioning of the cellular hydrogen transport mechanisms, have been repeated and results have been obtained that seemingly differ from those ofHes. We could not demonstrate any effect of CO₂ on the rate of methylene blue reduction by various bacteria in the presence of utilizable compounds such as glucose, sucrose, lactate and pyruvate. UsingE. coli and other bacteria we found, however, that the time required for the reduction of methylene blue was greatly increased by CO₂ removal when no substrates were added. This effect of CO₂ on the endogenous metabolism was observed only when the cells were depleted of endogenous reserves and of CO₂ by aeration or dilution.     

Fate and effects of methylene chloride in activated sludge.

Activated sludge obtained from a municipal wastewater treatment plant was acclimated to methylene chloride at concentrations between 1 and 100 mg/liter by continuous exposure to the compound for 9 to 11 days. Acclimated cultures were shown to mineralize methylene chloride to carbon dioxide and chloride. Rates of methylene chloride degradation were 0.14, 2.3, and 7.4 mg of CH2Cl2 consumed per h per g of mixed-liquor suspended solids for cultures incubated in the presence of 1, 10, and 100 mg/liter, respectively. Concentrations of methylene chloride between 10 and 1,000 mg/liter had no significant effect on O2 consumption or glucose metabolism by activated sludge. A hypothetical model was developed to examine the significance of volatilization and biodegradation for the removal of methylene chloride from an activated sludge reactor. Application of the model indicated that the rate of biodegradation was approximately 12 times greater than the rate of volatilization. Thus, biodegradation may be the predominant process determining the fate of methylene chloride in activated sludge systems continuously exposed to the compound.     

Synthesis and enzymatic characterization of methylene analogs of adenosine 5'-tetraphosphate (P4A)

A new methodology for synthesis of biologically important nucleoside tri- and tetraphosphates containing a bisphosphonate moiety instead of the terminal pyrophosphate bond is described. The series consists of tri- and tetraphosphate analogs of adenosine, guanosine and 7-methylguanosine (characteristic for mRNA cap). We have adopted a two-step procedure that allowed us to insert a methylene bridge into the phosphate chain. Nucleoside mono- or diphosphates were first activated (as imidazole derivatives) and then used in coupling reactions with organic salts of bisphosphonate. The resulting synthetic method enabled us to obtain the desired compounds with high yields and does not require any protective groups. This makes it very useful for the synthesis of labile compounds such as those containing the 7-methylguanosine ring. The structures of the synthesized compounds were confirmed by NMR spectroscopy. They were tested as potential substrates and inhibitors of several hydrolases.     

Structure-activity relationships of methylene or terminal side chain modified retinoids on the differentiation and cell death signaling in NB4 promyelocytic leukemia cells

New structure-activity relationships of a series of methylene or side chain modified retinoids on NB4 acute promyelocytic leukemia cells are investigated. The differentiation- and apoptosis-inducing potential of these compounds is analyzed on the basis of their selective retinoic acid receptor binding profile.     

1H N.M.R. studies of protected alpha-aminoisobutyric acid containing peptides. Chemical shift nonequivalence of benzyloxycarbonyl methylene protons

The benzylic methylene protons in a large number of benzyloxycarbonyl alpha-aminoisobutyric acid (Z-Aib) containing peptides, show chemical shift nonequivalence. The magnitude of the geminal nonequivalence is correlated with the involvement of the urethane carbonyl group, in an intramolecular hydrogen bond. Studies of the model compounds Z-Aib-Aib-Ala-NHMe, and Z-Aib-Aib-Aib-Pro-OMe clearly establish the presence of intramolecular hydrogen bonds, involving the urethane CO group. In both compounds marked anisochrony of the benzylic methylene protons is demonstrated. In Z-Aib-Aib-Pro-OMe, where a 4 leads to 1 hydrogen bonded beta-turn is not possible, the benzylic-CH2-protons appear as a singlet in CDCl3 and have a very small chemical shift difference in (CD3)2SO. The observation of such nonequivalence is of value in establishing whether the amino terminal Aib-Pro beta-turn is retained in large peptide-fragments of alamethicin.     

Photooxidation of methionine with immobilized methylene blue photooxidizer

Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37°C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84% acetic acid at 4°C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94% while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21%. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.     

Borax methylene blue: a spectroscopic and staining study.

Borax methylene blue is quite stable at room temperatures of 22-25 C. At 30 C polychroming is slow; during 50 days in a water bath at this temperature the absorption peak moves from 665 to 656 nm. At 35 C, the absorption peak reaches 660 nm in 7 days, 654 nm in 14. At 60 C polychroming is rapid, the absorption peak reaching 640-620 nm in 3 days. When the pH of the borax methylene blue solutions, normally about 9.0, is adjusted to pH 6.5, the absorption peak remains at 665 nm even when incubated at 60 C for extended periods. When used as a blood stain 0.4 ml borax methylene blue (1% methylene blue in 1% borax), 4 ml acetone, 2 ml borax-acid phosphate buffer to bring the solution to pH 6.5, and distilled water to make 40 ml, with 0.2 ml 1% eosin added just before using, an excellent Nocht-Giemsa type stain is achieved after 30 minutes staining. The material plasmodia P. falciparum, P. vivax, and P. berghei stain moderate blue with dark red chromatin and green to black pigment granules. The study confirms Malachowski's 1891 results and explains Gautier's 1896-98 failure to duplicate it.     

Analysis of methylene blue in human urine by capillary electrophoresis

A capillary electrophoresis method for the determination of the dye methylene blue (tetramethylthionine, MB) in human urine depending on liquid/liquid-extraction and diode array detection has been developed, validated, and applied to samples of healthy individuals, who had been dosed with methylene blue within clinical studies. After extraction with dichloromethane and sodium hexanesulfonate, sample extracts were measured on an extended light path capillary. The dye was detected simultaneously at 292 and 592 nm using methylene violet 3 RAX as internal standard. The limit of quantification was 1.0 microg/ml. The accuracy of the method varied between -15.2 and +0.8% and the precision ranged from 2.0 to 12.0%. The method was linear at least within 1.0 and 60 microg/ml. In contrast to earlier indirect determinations no leuco methylene blue (LMB) was directly detected in urine, whereas in aqueous test solutions containing surplus amounts of ascorbic acid leuco methylene blue was well separated from MB in a single run.     

Synthesis, biological evaluation, and molecular modeling studies of methylene imidazole substituted biaryls as inhibitors of human 17alpha-hydroxylase-17,20-lyase (CYP17)--part II: Core rigidification and influence of substituents at the methylene bridge

Thirty-five novel substituted imidazolyl methylene biphenyls have been synthesized as CYP17 inhibitors for the potential treatment of prostate cancer. Their activities have been tested with recombinant human CYP17 expressed in Escherichia coli. Promising compounds were tested for selectivity against CYP11B1, CYP11B2, and hepatic CYP enzymes 3A4, 1A2, 2B6 and 2D6. The core rigidified compounds (30-35) were the most active ones, being much more potent than Ketoconazole and reaching the activity of Abiraterone. However, they were not very selective. Another rather potent and more selective inhibitor (compound 23, IC(50)=345 nM) was further examined in rats regarding plasma testosterone levels and pharmacokinetic properties. Compared to the reference Abiraterone, 23 was more active in vivo, showed a longer plasma half-life (10h) and a higher bioavailability. Using our CYP17 homology protein model, docking studies with selected compounds were performed to study possible interactions between inhibitors and amino acid residues of the active site.     

Synthesis,antielastase, antioxidant and radical scavenging activities of 4-(aza substituted) methylene substituted dihydroxy coumarines

A series of some 4-(aza substituted) methylene substituted dihydroxy coumarines were evaluated for their antioxidant and antielastase activities. Different in vitro methodologies such as total reducing power, 1,1-diphenyl-2-picryl-hydrazil (DPPH·) free radical scavenging, ABTS radical scavenging activity were used as antioxidant activity. All the tested compounds exhibited potent free radical scavenging ability and antielastase activites.     

Accumulation of methylene blue by erythrocytes and determination of its maximum sensitivity to cell damage

The accumulation of methylene blue in native and damaged erythrocytes treated by cetyltrimethylammonium bromide at different initial concentrations (c0) of methylene blue and different volume ratios between external solution and cells (Vs/Vc) was studied. It was shown that at low methylene blue concentrations (c0 < 200 microM), the sorption of the dye by cells made the main contribution to its accumulation. As a result, the internal concentration of methylene blue exceeded manifold its external concentration and their ratio Q was 4-6 for native and 6-9 for damaged cells. As Vs/Vc decreased and especially as c0 increased, the diffusion of methylene blue inward the cells increased concentration gradient, and Q sharply fell to 0.9-1.0. The optimal values of c0 and Vs/Vc that provide the maximum sensitivity of Q to cell damage were determined. The advantages of using Q over other parameters of methylene blue accumulation were shown.     

The purification of methylene blue and azure B by solvent extraction and crystallization.

Detailed schemes are described for the preparation of purified methylene blue and azure B from commercial samples of methylene blue. Purified methylene blue is obtained by extracting a solution of the commercial product in an aqueous buffer (pH 9.5) with carbon tetrachloride. Methylene blue remains in the aqueous layer but contaminating dyes pass into the carbon tetrachloride. Metal salt contaminants are removed when the dye is crystallized by the addition of hydrochloric acid at a final concentration of 0.25 N. Purified azure B is obtained by extracting a solution of commercial methylene blue in dilute aqueous sodium hydroxide (pH 11-11.5) with carbon tetrachloride. In this pH range, methylene blue is unstable and yields azure B. The latter passes into the carbon tetrachloride layer as it is formed. Metal salt contaminants remain in the aqueous layer. A concentrated solution oa azure B is obtained by extracting the carbon tetrachloride layer with 4.5 X 10(-4)N hydrobromic acid. The dye is then crystallized by increasing the hydrobromic acid concentration to 0.23 N. Thin-layer chromatography of the purified dyes shows that contamination with related thiazine dyes is absent or negligible. Ash analyses reveal that metal salt contamination is also negligible (sulphated ash less than 0.2%).     

Effect of subtoxic amounts of furan, acetylfuran and methylene chloride on some serum enzymes of rat

Serum transaminases (GOT and GPT) and ornithincarbamyltransferase (OCT) were determined in rats treated with subtoxic doses of furan, acetylfuran, and methylene chloride. Significant increases of all enzymes were observed in methylene chloride treated rats, while only GOT increased in rats treated with acetylfuran and with furan + methylene chloride. Calculation of the GOT/GPT ratios indicated a pattern of toxic hepatitis only for rats treated with acetylfuran and furan + methylene chloride.     

Photooxidation of methionine with immobilized methylene blue as photooxidizer.

Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37 degrees C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84% acetic acid at 4 degrees C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94%, while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21%. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.     

Spermatozoal methylene blue reduction: an indicator of mitochondrial function and its correlation with motility

Methylene blue reduction rates (MeBRR) were evaluated spectrophotometrically to study bovine spermatozoal mitochondrial function and its relation to motility. A chemical reaction (H2SO4, methylene blue solution, and zinc powder) was used to quantify methylene blue reduction. Absorbance measurements were made for 10 min at 609 nm in a narrow band spectrophotometer. In a second experiment, fresh ejaculates were assessed for concentration and motility evaluated with phase microscopy (37 degrees C). Semen was diluted to 100 million cells/mL in a sodium citrate-glucose buffer and methylene blue. Absorbance and motility were evaluated every 30 min for 2.5 h in a water-jacketed cuvette (41 degrees C). Methylene blue reduction rates and motility decreased at each subsequent period. Methylene blue reduction rates were correlated to sperm motility. Lastly, the methylene blue reduction rate was measured with a broad band spectrophotometer and compared with motility using similar conditions. Motility estimates were made on sperm from the cuvettes. Sperm motility was correlated to methylene blue reduction rates measured spectrophotometrically.