Liver-microsome-mediated formation of alkylating agents from vinyl bromide and vinyl chloride.

When a mixture of vinyl chloride/oxygen or vinyl bromide/air was passed through a mouse-liver microsomal system, volatile alkylating metabolites were trapped by reaction with excess 4-(4-nitrobenzyl)pyridine. The absorption spectra of the adducts, either from vinyl bromide or vinyl chloride, were identical with that obtained by reaction of chloroethylene oxide with 4-(4-nitrobenzyl) pyridine. Chloroethylene oxide decomposes in aqueous solution with a half-life of 1.6 minutes. After reaction of chloroethylene oxide and 2-chloroacetaldehyde with adenosine and Sephadex chromatography the binding products were compared with those formed in the presence of vinyl chloride, mouse-liver microsomes and adenosine. A common product of these reactions was tentatively characterized as 3-β-ribofuranosyl-imidazo-[2,1-i]purine.     

Regioselective acylation of several polyhydroxylated natural compounds by Candida antarctica lipase B

Regioselective acylation of four polyhydroxylated natural compounds, deacetyl asperulosidic acid (1), asperulosidic acid (2), puerarin (3) and resveratrol (4) by Candida antarctica Lipase B in the presence of various acyl donors (vinyl acetate, vinyl decanoate or vinyl cinnamoate) was studied. Compounds 1, 2 and 4 were regioselectively acetylated with vinyl acetate to afford products, 3'-O-acetyl-10-O-deacetylasperulosidic acid (1a), 3',6'-O-diacetyl-10-O-deacetylasperulosidic acid (1b), 3'-O-acetylasperulosidic acid (2a), 3',6'-O-diacetylasperulosidic acid (2b), 4'-O-acetylresveratrol (4a), respectively, with yields of 22 to 50%, while reactions with vinyl decanoate and vinyl cinnamoate were slow with lower yields. Compound 3 was readily acylated with all three acyl donors and quantitatively converted to products 6'-O-acetylpuerarin (3a), 6'-O-decanoylpuerarin (3b), 6'-O-cinnamoylpuerarin (3c), respectively. The structures of these acylated products were determined by spectroscopic methods (MS and NMR).     

Synthesis and evaluation of 4-(substituted styryl/alkenyl)-3,5-bis(4-hydroxyphenyl)-isoxazoles as ligands for the estrogen receptor

A series of 3,5-bis (4-hydroxyphenyl) isoxazoles bearing a styryl/alkyl vinyl group at the 4-position were prepared and evaluated as ligands for the estrogen receptor-alpha (ERα). The target compounds were prepared using the Suzuki reaction to couple an iodo-isoxazole intermediate with a series of styryl/alkenyl boronic acids, followed by O-demethylation. The products were evaluated for their estrogen receptor-α ligand binding domain (ERα-LBD) binding affinity using a competitive binding assay. The 4-(4-hydroxystyryl) derivative 4h displays binding properties similar to those of the previously described pyrazole class of ER ligands, indicating that the ERα-LBD tolerates the presence of the added vinyl group at the 4-position of the isoxazole ring.     

Synthesis of a new fluorine-18-labeled bexarotene analogue for PET imaging of retinoid X receptor

The reference standard 2-fluoro-4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)vinyl)benzoic acid was synthesized from 2,5-dimethyl-2,5-hexanediol and 2-fluoro-4-methylbenzoic acid in 10 steps with 3% overall chemical yield. The precursor 2-nitro-4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)vinyl)benzoic acid was synthesized from 2,5-dimethyl-2,5-hexanediol and dimethyl-2-nitroterephthalate in seven steps with 2% overall chemical yield. The target tracer 2-[¹⁸F]fluoro-4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)vinyl)benzoic acid was synthesized from its nitro-precursor by the nucleophilic substitution with K[¹⁸F]F/Kryptofix 2.2.2 and isolated by HPLC combined with solid-phase extraction (SPE) purification in 20–30% radiochemical yield with 37–370 GBq/μmol specific activity at end of bombardment (EOB).     

Reductive dechlorination of high concentrations of tetrachloroethene to ethene by an anaerobic enrichment culture in the absence of methanogenesis.

Tetrachloroethene, also known as perchloroethylene (PCE), is a common groundwater contaminant throughout the United States. The incomplete reductive dechlorination of PCE--resulting in accumulations of trichloroethene, dichloroethene isomers, and/or vinyl chloride--has been observed by many investigators in a wide variety of methanogenic environments. Previous mixed-culture studies have demonstrated that complete dechlorination to ethene is possible, although the final dechlorination step from vinyl chloride to ethene is rate limiting, with significant levels of vinyl chloride typically persisting. In this study, anaerobic methanol-PCE enrichment cultures which proved capable of dechlorinating high concentrations PCE to ethene were developed. Added concentrations of PCE as high as 550 microM (91-mg/liter nominal concentration; approximately 55-mg/liter actual aqueous concentration) were routinely dechlorinated to 80% ethene and 20% vinyl chloride within 2 days at 35 degrees C. The methanol level used was approximately twice that needed for complete dechlorination of PCE to ethene. The observed transformations occurred in the absence of methanogenesis, which was apparently inhibited by the high concentrations of PCE. When incubation was allowed to proceed for as long as 4 days, virtually complete conversion of PCE to ethene resulted, with less than 1% persisting as vinyl chloride. An electron balance demonstrated that methanol consumption was completely accounted for by dechlorination (31%) and acetate production (69%). The high volumetric rates of PCE dechlorination (up to 275 mumol/liter/day) and the relatively large fraction (ca. one-third) of the supplied electron donor used for dechlorination suggest that reductive dechlorination could be exploited for bioremediation of PCE-contaminated sites.     

Rapidly in situ-forming degradable hydrogels from dextran thiols through Michael addition

Thiol-functionalized dextrans (dex-SH) (M(n,dextran) = 14K or 31K) with degrees of substitution (DS) ranging from 12 to 25 were synthesized and investigated for in situ hydrogel formation via Michael type addition using poly(ethylene glycol) tetra-acrylate (PEG-4-Acr) or a dextran vinyl sulfone conjugate with DS 10 (dex-VS DS 10). Dex-SH was prepared by activation of the hydroxyl groups of dextran with 4-nitrophenyl chloroformate and subsequent reaction with cysteamine. Hydrogels were rapidly formed in situ under physiological conditions upon mixing aqueous solutions of dex-SH and either PEG-4-Acr or dex-VS DS 10 at polymer concentrations of 10 to 20 w/v%. Rheological studies showed that these hydrogels are highly elastic. By varying the DS, concentration, dextran molecular weight, and type of cross-linker, hydrogels with a broad range of storage moduli of 9 to 100 kPa could be obtained. Varying the ratio of thiol to vinyl sulfone groups from 0.9 to 1.1 did not alter the storage modulus of the hydrogels, whereas larger deviations from equimolarity (thiol to vinyl sulfone ratios of 0.75 and 1.5) considerably decreased the storage modulus. The plateau value of hydrogel storage modulus was reached much faster at pH 7.4 compared to pH 7, due to a higher concentration of the thiolate anion at higher pH. These hydrogels were degradable under physiological conditions. Degradation times were 3 to 7 weeks for dex-SH/dex-VS DS 10 hydrogels and 7 to over 21 weeks for dex-SH/PEG-4-Acr hydrogels, depending on the DS, concentration, and dextran molecular weight.     

A cold-active esterase with a substrate preference for vinyl esters from a psychrotroph, Acinetobacter sp. strain no. 6: gene cloning, purification, and characterization

It has recently been shown that fatty acid vinyl esters serve as effective acylating agents for the synthesis of esters by enzymatic transesterification in high yields. To enhance the usefulness of this system at low temperatures, we have searched for the gene coding for a cold-active lipolytic enzyme with a substrate preference for fatty acid vinyl esters and obtained it from the genomic library of Acinetobacter sp. strain no. 6, a psychrotroph isolated from Siberian soil. The gene (termed aelh, 777 bp) encoded a protein of 258 amino acids, and sequence analysis revealed that the enzyme shows a high sequence similarity to β-ketoadipate enol-lactone hydrolase involved in the β-ketoadipate pathway for the bacterial catabolism of benzoic acid. The aelh gene was expressed in the E. coli C600 cells under the control of lac promoter and the expression product was purified to homogeneity and characterized. It was a monomeric esterase preferentially catalyzing the hydrolysis of enol esters, such as fatty acid vinyl esters with a short-chain acyl group. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, a specific inhibitor for serine hydrolases. The enzyme could also catalyze transesterification, for example, between vinyl propionate and propanol yielding propyl propionate at 4 °C. These results indicate the usefulness of an esterase (termed AELH) for the enzymatic synthesis of esters by transesterification using vinyl esters as an acyl donor.     

Reductive dechlorination of high concentrations of tetrachloroethene to ethene by an anaerobic enrichment culture in the absence of methanogenesis.

Tetrachloroethene, also known as perchloroethylene (PCE), is a common groundwater contaminant throughout the United States. The incomplete reductive dechlorination of PCE--resulting in accumulations of trichloroethene, dichloroethene isomers, and/or vinyl chloride--has been observed by many investigators in a wide variety of methanogenic environments. Previous mixed-culture studies have demonstrated that complete dechlorination to ethene is possible, although the final dechlorination step from vinyl chloride to ethene is rate limiting, with significant levels of vinyl chloride typically persisting. In this study, anaerobic methanol-PCE enrichment cultures which proved capable of dechlorinating high concentrations PCE to ethene were developed. Added concentrations of PCE as high as 550 microM (91-mg/liter nominal concentration; approximately 55-mg/liter actual aqueous concentration) were routinely dechlorinated to 80% ethene and 20% vinyl chloride within 2 days at 35 degrees C. The methanol level used was approximately twice that needed for complete dechlorination of PCE to ethene. The observed transformations occurred in the absence of methanogenesis, which was apparently inhibited by the high concentrations of PCE. When incubation was allowed to proceed for as long as 4 days, virtually complete conversion of PCE to ethene resulted, with less than 1% persisting as vinyl chloride. An electron balance demonstrated that methanol consumption was completely accounted for by dechlorination (31%) and acetate production (69%). The high volumetric rates of PCE dechlorination (up to 275 mumol/liter/day) and the relatively large fraction (ca. one-third) of the supplied electron donor used for dechlorination suggest that reductive dechlorination could be exploited for bioremediation of PCE-contaminated sites.     

Coordination of a disubstituted alkene in a chelated d-metal-alkyl-alkene complex. Evidence for equilibrium between complexes with coordinated and free alkenes

Addition of (Cp*₂YH)₂ (4) to 2-methyl-1,4-pentadiene produced the yttrium-alkyl-alkene chelate complex Cp*₂YCH₂CH₂CH₂C(CH₃)=CH₂ (2) in which a disubstituted alkene is complexed to the metal center. Evidence for coordination of the alkene unit of 2 comes from the ¹H and ¹³C NMR chemical shifts of the vinyl units and from observation of nOe effects between Cp* protons and vinyl hydrogens. The disubstituted alkene ligand of 2 is weakly bound, and evidence for an equilibrium with substantial amounts of complex 3 with a free alkene was obtained from variable temperature ¹H NMR spectroscopy.     

4-vinyl and 2,4-divinyl deuteration effects on the low frequency resonance Raman bands of myoglobin: correlation with the structure of vinyl group

Resonance Raman spectra of myoglobin (Mb) reconstituted with 4-vinyl and 2,4-divinyl deuterated protoheme IX were studied in several oxidation, spin, and ligation states (deoxyMb, MbO2, MbCO, metMbH2O, and metMbCN-) with special attention to the low frequency vibrations. Frequency shifts observed on deuteration enabled us to assign some Raman bands to vibrations specifically involving the 2- or 4-vinyl group. The most significant deuteration effect was found for a band around 410 cm-1 in the ferrous state, which loses intensity on 4-vinyl deuteration and is ascribed to a porphyrin in-plane vibration strongly coupled with the skeletal bend of the vinyl group at position 4. Such strong coupling implies that the vinyl group lies in the same plane as the pyrrole II ring, as in the crystalline state. Thus, frequency shifts on vinyl deuteration may be useful as a probe of the orientation of the vinyl group. Resonance Raman spectra of Mb coordinated with various sizes of isocyanides are interpreted in terms of vinyl orientational changes induced by ligation.     

Novel S-ribosylhomocysteine analogues as potential inhibitors of LuxS enzyme

Selective cross-coupling of the protected 6-fluoro-6-iodo-alpha-D-ribo-hex-5-enofuranose with 2 equivalents of 4-ethoxy-4-oxobutylzinc bromide in the presence of Pd[P(Ph)(3)](4) followed by deprotections gave methyl 5,6,7,8,9-pentadeoxy-6-fluoro-alpha/beta-D-ribo-dec-5(Z)-enofuranuronate; a S-ribosylhomocysteine analogue with the sulfur and carbon-5 atoms replaced by the fluoro(vinyl) unit.     

Resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase as biocatalyst

Enantioselective acylation employing vinyl alkanoates as acyl donors was exploited for the resolution of 2-(substituted phenoxy)-1-propanols carrying different substituents on the benzene ring using Achromobacter sp. lipase. These primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, based on the enantiomeric ratio E (up to 27), through acylation with vinyl butanoate in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors. Using this procedure, enantiomerically enriched (R)-2-(4-chlorophenoxy)-1-propanol was prepared in 97% e.e. and 33% yield in a gram-scale reaction.     

Haem-rotational disorder in monomeric allosteric cyano-Met insect haemoglobins monitored by resonance Raman spectroscopy

The haem-rotational disorder (insertion of haem into globin rotated about the alpha, gamma-meso axis by 180 degrees) has been investigated in the cyano-Met form of the monomeric allosteric insect haemoglobins, CTT III and CTT IV, by resonance Raman spectroscopy. The effect of haem disorder on the resonance Raman spectra has been observed in proto-IX, deutero-IX, and meso-IX CTTs. Most importantly, in the absence of overlapping vinyl vibrations, we have identified two Fe-C-N bending vibrations at 401 cm-1 and 422 cm-1 (pH 9.5) for 57Fe deutero-IX CTT IV ligated with 13C15N-, which are attributed to the two haem-rotational components. One Fe-C-N bending mode at 422 cm-1 shows a pH-induced shift to 424 cm-1 (pH 5.5) indicating the t----r conformational transition, whereas the other bending mode is pH-insensitive, representing a non-allosteric component. By replacing the unsymmetrical porphyrins with the "symmetrical" protoporphyrin-III we eliminate the haem disorder. Then, sharpening of the Fe-N epsilon(His) (at 313 cm-1) and Fe-CN (at 453 cm-1) stretching modes is observed and a single Fe-C-N bending mode (at 412 cm-1) appears. In cyano-Met proto-IX CTT III two vinyl bending vibrations at 412 cm-1 and 591 cm-1 assigned by deuteration of the vinyl groups also reflect the haem disorder. The 412 cm-1 vinyl vibration is intensity-enhanced via through-space coupling with one of the Fe-C-N bending modes (at 412 cm-1). In the cyano-Met form of proto-III CTT III this vinyl vibration is shifted to 430 cm-1 resulting in a dramatic drop in intensity. It is most likely that the specific vinyl-protein interaction at position 4 in one of the haem-rotational components is the origin of the coupling between the Fe-C-N and vinyl bending modes. The Fe-N epsilon(proximal His) and the Fe-CN stretching vibrations as well as the Fe-C-N bending vibration have been identified by 54Fe/57Fe and 13C15N/12C15N/13C14N/12C14N isotope exchange.     

The vinyl ether linkages of plasmalogens are favored targets for myeloperoxidase-derived oxidants: a kinetic study

Plasmalogens, which contain a vinyl ether bond, are major phospholipids of the plasma membranes of endothelial and vascular smooth muscle cells and cardiac myocytes. These lipids, in contrast to other phospholipids, have been reported to be targets of HOCl/HOBr generated by myeloperoxidase, with elevated levels of the products of these reactions (alpha-chloro/alpha-bromo aldehydes and unsaturated lysophospholipids) having been detected in human atherosclerotic lesions. The reason(s) for the targeting of this lipid class, over other phospholipids, is poorly understood, and is examined here. It is shown that HOCl and HOBr react with a model vinyl ether (ethylene glycol vinyl ether) 200-300-fold faster ( k = 1.6 x 10 (3) and 3.5 x 10 (6) M (-1) s (-1), respectively) than with aliphatic alkenes (models of phospholipids). True plasmalogens react ca. 20-fold slower than the models. Chloramines and bromamines (from reaction of HOCl/HOBr with primary amines and alpha-amino groups) also react with vinyl ethers, unlike aliphatic alkenes, with k = 10 (-3)-10 (2) M (-1) s (-1) for chloramines (with the His side chain chloramine being the most reactive, k = 172 M (-1) s (-1)) and k = 10 (3)-10 (4) M (-1) s (-1) for bromamines. The bromamine rate constants are typically 10 (5)-10 (6) larger than those of the chloramines. Intermolecular vinyl ether oxidation by phospholipid headgroup bromamines can also occur. These kinetic data indicate that plasmalogens are significantly more susceptible to oxidation than the aliphatic alkenes of phospholipids, thereby rationalizing the detection of products from the former, but not the latter, in human atherosclerotic lesions.     

A general synthesis of 4-alkyl/aryl substituted saccharins

A new method for the synthesis of 4-alky/aryl substituted saccharins (2) has been developed. The key steps include conjugate addition/acylation of cyclohexenone (3) and reaction of the resulting β-ketoesters 4 with benzyl mercaptan to give the vinyl sulfides 5 and 6. The latter were converted to the saccharins 2 in high yield.     

Secondary alcohol dehydrogenase from a vinyl alcohol oligomer-degrading Geotrichum fermentans; stabilization with Triton X-100 and activity toward polymers with polymerization degrees less than 20

An NAD+-dependent secondary alcohol dehydrogenase has been found in the cells of Geotrichum fermentans WF9101 grown on 2,4-pentanediol (the constitutional unit of vinyl alcohol polymers). Although the enzyme was unstable and lost its activity very rapidly, we found that it could be purified after stabilization in a buffer containing 0.01% Triton X-100. The molecular weight of the purified enzyme was 88,000 with subunit size of molecular weight 42,000. The enzyme, which was specific for the (S)-(+)-enantiomers, was active on various secondary mono-ols and diols, notably, 2-hexanol. The K_m values at 30 °C, pH 8.0, were 240 m for 2-hexanol and 48.7 m for NAD⁺. This enzyme showed activity toward vinyl alcohol polymers with average degrees of polymerization of less than 20, and was assumed to contribute on the degradation of poly(vinyl alcohol) in synergism with other microorganisms.     

Sister-chromatid exchanges induced by vinyl esters and respective carboxylic acids in cultured human lymphocytes.

Vinyl acetate--an efficient inducer of sister-chromatid exchanges (SCEs)--is known to be hydrolyzed in mammalian cells into acetic acid and acetaldehyde, the latter being the likely metabolite responsible for the SCE induction. As similar hydrolysis to acetaldehyde and to a carboxylic acid is also expected for other vinyl esters, five such compounds--vinyl formate, vinyl chloroformate, vinyl propionate, vinyl crotonate and vinyl-2-ethylhexanoate--and five carboxylic acids--formic acid, acetic acid, propionic acid, crotonic acid and 2-ethylhexanoic acid--were tested for their ability to induce SCEs in cultured (72 h) human lymphocytes with a 48-h treatment, starting at 24 h after culture initiation. Vinyl formate, vinyl propionate and vinyl crotonate induced a clear dose-dependent increase in the number of SCEs/cell at concentrations of 0.125-0.5 mM and vinyl chloroformate at 0.063-1 mM, i.e., at roughly the same concentration range as vinyl acetate and acetaldehyde. Vinyl-2-ethylhexanoate required slightly higher concentrations (0.25-4 mM) for SCE induction. All of the carboxylic acids tested also elevated SCEs, but only slightly. Formic acid and crotonic acid produced some SCE increase at a concentration of 10 mM, acetic acid at 5 and 10 mM and propionic acid at 2.5 mM. 2-Ethylhexanoic acid induced SCEs at a lower concentration range (0.63-2.5 mM) than the other acids. The positive concentrations of the first three carboxylic acids lowered the pH of the culture medium immediately after the treatment by 0.5-1.0 pH unit (lowest observed pH 6.53). The pH differences from the control cultures became smaller in measurements done 24 h and 48 h after the beginning of treatment. Propionic acid and 2-ethylhexanoic acid affected medium pH only slightly (maximum drop 0.2 pH units) at the concentrations that induced SCEs. The results lend support to the idea that the efficient SCE induction observed with the vinyl esters results from the formation of acetaldehyde, with carboxylic acids--with the possible exception of 2-ethylhexanoic acid--playing no significant role. The slight SCE induction obtained with the carboxylic acids cannot be explained by lowered pH alone.     

Nanofibrous nonmulberry silk/PVA scaffold for osteoinduction and osseointegration

Poly‐vinyl alcohol and nonmulberry tasar silk fibroin of Antheraea mylitta are blended to fabricate nanofibrous scaffolds for bone regeneration. Nanofibrous matrices are prepared by electrospinning the equal volume ratio blends of silk fibroin (2 and 4 wt%) with poly‐vinyl alcohol solution (10 wt%) and designated as 2SF/PVA and 4SF/PVA, respectively with average nanofiber diameters of 177 ± 13 nm (2SF/PVA) and 193 ± 17 nm (4SF/PVA). Fourier transform infrared spectroscopy confirms retention of the secondary structure of fibroin in blends indicating the structural stability of neo‐matrix. Both thermal stability and contact angle of the blends decrease with increasing fibroin percentage. Conversely, fibroin imparts mechanical stability to the blends; greater tensile strength is observed with increasing fibroin concentration. Blended scaffolds are biodegradable and support well the neo‐bone matrix synthesis by human osteoblast like cells. The findings indicate the potentiality of nanofibrous scaffolds of nonmulberry fibroin as bone scaffolding material. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 271–284, 2015.     

Regioselective synthesis of kojic acid esters by Bacillus subtilis protease

The lipophilicity of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one] was improved by esterifying kojic acid with either divinyl adipate, vinyl hexanoate, vinyl octanoate or vinyl decanoate using protease from Bacillus subtilis for 7 d. ¹H-NMR and ¹³C-NMR showed that the primary hydroxyl group at the C-7 position of kojic acid was regioselectively esterified to afford 7-O-vinyl adipoyl kojic acid, 7-O-hexanoyl kojic acid, 7-O-octanoyl kojic acid and 7-O-decanoyl kojic acid (13–27% yield). The kojic acid esters had radical scavenging activities, inhibited tyrosinase activity and was biodegradable.     

Preparation of optically active 4-allylazetidin-2-ones. An access to carbacephams

Transesterification of 4-allyl-1-hydroxymethylazetidin-2-ones using vinyl acetate in the presence of the lipase from Pseudomonas cepacia led to the corresponding (R)-acetates and the remaining (S)-alcohols in high yields and excellent ee's (E: 32–71). Subsequent reaction with BF₃-Et₂O can lead to the carbacepham framework.