Design and synthesis of silicon-containing tubulin polymerization inhibitors: Replacement of the ethylene moiety of combretastatin A-4 with a silicon linker

Silicon-containing combretastatin analogs were designed, synthesized and evaluated for stability and biological activities. Among them, compound 31 exhibited strong tubulin polymerization-inhibitory activity and very potent tumor cell growth-inhibitory activity (IC₅₀ = 0.007 μM) in MCF-7 cell proliferation assay. This compound also potently inhibited [³H]colchicine binding (90.7% inhibition at 3 μM). These activities were comparable to those of combretastatin A-4 (CA-4) (1). In addition, compound 31 was physico-chemically more stable than 1. These results suggest that a silicon linker can act as a bioisoster of a cis carbon–carbon double bond.     

Effect of acyl donor chain length on isoquercitrin acylation and biological activities of corresponding esters

The enzymatic acylation of isoquercitrin with fatty acid esters of various carbon chain lengths was carried out in 2-methyl-2-butanol using Novozym 435^®. The conversion yield and the initial rate decreased from 66% to 38% and from 17.7 to 10.1 μmol/h respectively, as the carbon chain of the acyl donor increased from C4 to C18. Isoquercitrin acylated derivatives showed higher xanthine oxidase inhibition activities than isoquercitrin. This property increased with the lipophilicity of the derivative esters. The scavenging activity of isoquercitrin esters against ABTS and DPPH radicals decreased with the acyl chain length. Conversely, for esters from C6 to C18, a linear growing relationship can be established between the chain length and the superoxide radical scavenging activity. Furthermore, an improved antiproliferative effect on Caco2 cancer cells was induced by addition of isoquercitrin esters comparing with isoquercitrin.     

Ability of Vasconcellea × heilbornii lipase to catalyse the synthesis of alkyl esters from vegetable oils

Lipase-catalysed synthesis of alkyl esters is regarded as a potential alternative to chemical catalysis. Owing to its availability as a waste material from the babaco fruit production, its strong lipolytic activity and its natural immobilization, the dried latex of Vasconcellea × heilbornii appears as a good candidate to produce alkyl esters. The ability and performance of this lipase to catalyse the alcoholysis of sunflower oil with various primary alcohols was evaluated in a solvent-free system. A linear correlation between the final reaction rate and the alcohol polarity was established. For methanolysis, the influence of substrates ratio on final conversion rate was studied at different temperatures. At 30 °C, the lipase was inactivated by shaking in a mixture containing more than 0.5 molar equivalents of methanol; the minimum methanol concentration for enzyme deactivation increased with temperature. Moreover, for a 0.5:1 methanol/TAG molar ratio, conversion rates of 73, 66 and 55% were obtained at 30, 40 and 55 °C respectively, showing that the increase of temperature diminished the final methanolysis conversion rate. These facts were associated to the miscibility of methanol in oil and to the thermodynamic state of the medium. To overcome the inactivation of the lipase by methanol, alcoholysis was carried out by fractionated addition of methanol. In those conditions, Vasconcellea × heilbornii latex could catalyse the conversion of 70% of sunflower TAGs in methyl esters at 30 °C.     

Structural and Mechanistic Analysis of the Choline Sulfatase from Sinorhizobium melliloti: A Class I Sulfatase Specific for an Alkyl Sulfate Ester

Hydrolysis of organic sulfate esters proceeds by two distinct mechanisms, water attacking at either sulfur (S–O bond cleavage) or carbon (C–O bond cleavage). In primary and secondary alkyl sulfates, attack at carbon is favored, whereas in aromatic sulfates and sulfated sugars, attack at sulfur is preferred. This mechanistic distinction is mirrored in the classification of enzymes that catalyze sulfate ester hydrolysis: arylsulfatases (ASs) catalyze S–O cleavage in sulfate sugars and arylsulfates, and alkyl sulfatases break the C–O bond of alkyl sulfates. Sinorhizobium meliloti choline sulfatase (SmCS) efficiently catalyzes the hydrolysis of alkyl sulfate choline-O-sulfate (k_cat/K_M = 4.8 × 10³ s^? 1 M^? 1) as well as arylsulfate 4-nitrophenyl sulfate (k_cat/K_M = 12 s^? 1 M^? 1). Its 2.8-Å resolution X-ray structure shows a buried, largely hydrophobic active site in which a conserved glutamate (Glu386) plays a role in recognition of the quaternary ammonium group of the choline substrate. SmCS structurally resembles members of the alkaline phosphatase superfamily, being most closely related to dimeric ASs and tetrameric phosphonate monoester hydrolases. Although > 70% of the amino acids between protomers align structurally (RMSDs 1.79–1.99 Å), the oligomeric structures show distinctly different packing and protomer–protomer interfaces. The latter also play an important role in active site formation. Mutagenesis of the conserved active site residues typical for ASs, H₂¹⁸O-labeling studies and the observation of catalytically promiscuous behavior toward phosphoesters confirm the close relation to alkaline phosphatase superfamily members and suggest that SmCS is an AS that catalyzes S–O cleavage in alkyl sulfate esters with extreme catalytic proficiency.     

Synthesis of short chain alkyl esters using cutinase from Burkholderia cepacia NRRL B2320

Short chain alkyl esters are well appreciated for fruity flavors they provide. These are mainly applied to the fruit-flavored products like jam, jelly, beverages, wine and dairy. Cutinase from Burkholderia cepacia NRRL B 2320 was found to be active in catalyzing the synthesis of alkyl esters in organic solvent. The optimal temperature range for the enzyme catalyzed synthesis was found to be from 35 °C to 40 °C. The maximum conversion (%) during synthesis of ester was obtained for butyric acid (C4) and valeric acid (C5) with butanol reflecting the specificity of the enzyme for short-chain length fatty acids. In case of alcohol specificity, butanol was found to be most preferred substrate by the enzyme and conversion (%) decreased with increasing carbon chain length of alcohol used in the esterification reaction. The kinetic analysis for the synthesis of butyl butyrate by varying concentration of one substrate at a time (butanol or butyric acid), showed that Ping–Pong Bi Bi model with acid inhibition and influence of initial water is most suitable model for the prediction of the reaction kinetics.     

Application of carbon fiber composite minielectrodes for measurement of kinetic constants of nitric oxide decay in solution

Carbon fiber microelectrodes and carbon fiber composite minielectrodes (CFM/CFCM) have been generally used for measurements of nitric oxide (NO) concentration in chemical and biological systems. The response time of a CFM/CFCM is usually from milliseconds to seconds depending on the electrode size, the thickness of coating layers on the electrode, and NO diffusion coefficients of the coating layers. As a result, the time course of recoded current changes (I–t curves) by the CFM/CFCM may be different from the actual time course of NO concentration changes (c–t curves) if the half-life of NO decay is close to or shorter than the response time of the electrode used. This adds complexity to the process for determining rate constants of NO decay kinetics from the recorded current curves (I–t curves). By computer simulations based on a mathematical model, an approximation method was developed for determining rate constants of NO decay from the recorded current curves. This method was first tested and valuated using a commercial CFCM in several simple reaction systems with known rate constants. The response time of the CFCM was measured as 4.7 ± 0.7 s (n = 5). The determined rate constants of NO volatilization and NO autoxidation in our measurement system at 37 °C are (1.9 ± 0.1) × 10^?3 s^?1 (n = 4) and (2.0 ± 0.3) × 10³ M^?1 s^?1 (n = 7), which are close to the reported rate constants. The method was then applied to determine the rate of NO decay in blood samples from control and smoking exposed mice. It was observed that the NO decay rate in the smoking group is >20% higher than that in control group, and the increased NO decay rate in the smoking group was reversed by 10 μM diphenyleneiodonium chloride (DPI), an inhibitor of flavin enzymes such as leukocyte NADPH oxidase.     

Differential effects of mineral and organic acids on the kinetics of arabinose degradation under lignocellulose pretreatment conditions

Sugar degradation occurs during acid-catalyzed pretreatment of lignocellulosic biomass at elevated temperatures, resulting in degradation products that inhibit microbial fermentation in the ethanol production process. Arabinose, the second most abundant pentose in grasses like corn stover and wheat straw, degrades into furfural. This paper focuses on the first-order rate constants of arabinose (5 g/L) degradation to furfural at 150 and 170 °C in the presence of sulfuric, fumaric, and maleic acid and water alone. The calculated degradation rate constants (k_d) showed a correlation with the acid dissociation constant (pK_a), meaning that the stronger the acid, the higher the arabinose degradation rate. However, de-ionized water alone showed a catalytic power exceeding that of 50 mM fumaric acid and equaling that of 50 mM maleic acid. This cannot be explained by specific acid catalysis and the shift in pK_w of water at elevated temperatures. These results suggest application of maleic and fumaric acid in the pretreatment of lignocellulosic plant biomass may be preferred over sulfuric acid. Lastly, the degradation rate constants found in this study suggest that arabinose is somewhat more stable than its stereoisomer xylose under the tested conditions.     

Four novel geminally dialkylated,non-aromatic acetophenone derivatives from Melicope coodeana

Four novel geminally dialkylated, non-aromatic acetophenone derivatives 1–4 were isolated from Melicope coodeana. The compounds, related to hop bitter acids, were named Coodeanones A–C, where Coodeanone B was found in both E and Z configuration of the 6′?–7′? double bond. Antibacterial, antifungal and antimalarial activities of the acetophenone derivaties were investigated, and Coodeanone C (4) was found to have activity toward Plasmodium falciparum (malaria) (IC₅₀ = 42.8 μM).     

Synthesis and biological evaluation of indomethacin analogs possessing a N-difluoromethyl-1,2-dihydropyrid-2-one ring system: A search for novel cyclooxygenase and lipoxygenase inhibitors

A novel class of indomethacin analogs were synthesized wherein a N-difluoromethyl-1,2-dihydropyrid-2-one moiety (5-LOX pharmacophore) was attached at its C-4 or C-5 position via either a CO (14a–b) or CH₂ (19a–b) linker to the indole N¹-position. In this regard, replacement of the 4-chlorobenzoyl group present in indomethacin by N-difluoromethyl-1,2-dihydropyrid-2-one-4-(or 5-)carbonyl and N-difluoromethyl-1,2-dihydropyrid-2-one-4-yl(or 5-yl)methylene moieties furnished compounds showing no inhibitory activities against the COX-2/5-LOX enzymes (except for the weak but selective COX-2 inhibitor 19a, COX-2 IC₅₀ = 31 μM), and moderate in vivo anti-inflammatory activities (except for the methylene compound 19a that was inactive). These structure–activity data indicate replacement of the 4-chlorobenzoyl group present in indomethacin by a N-difluoromethyl-1,2-dihydropyrid-2-one ring system connected by a CO or CH₂ linker is not a suitable approach for the design of dual COX-2/5-LOX inhibitory analogs of indomethacin.     

The cytochrome b5 reductase HPO-19 is required for biosynthesis of polyunsaturated fatty acids in Caenorhabditis elegans

Polyunsaturated fatty acids (PUFAs) are fatty acids with backbones containing more than one double bond, which are introduced by a series of desaturases that insert double bonds at specific carbon atoms in the fatty acid chain. It has been established that desaturases need flavoprotein-NADH-dependent cytochrome b5 reductase (simplified as cytochrome b5 reductase) and cytochrome b5 to pass through electrons for activation. However, it has remained unclear how this multi-enzyme system works for distinct desaturases. The model organism Caenorhabditis elegans contains seven desaturases (FAT-1, -2, -3, -4, -5, -6, -7) for the biosynthesis of PUFAS, providing an excellent model in which to characterize different desaturation reactions. Here, we show that RNAi inactivation of predicted cytochrome b5 reductases hpo-19 and T05H4.4 led to increased levels of C18:1n − 9 but decreased levels of PUFAs, small lipid droplets, decreased fat accumulation, reduced brood size and impaired development. Dietary supplementation with different fatty acids showed that HPO-19 and T05H4.4 likely affect the activity of FAT-1, FAT-2, FAT-3, and FAT-4 desaturases, suggesting that these four desaturases use the same cytochrome b5 reductase to function. Collectively, these findings indicate that cytochrome b5 reductase HPO-19/T05H4.4 is required for desaturation to biosynthesize PUFAs in C. elegans.     

Kinetic characterization of human phosphopantothenoylcysteine synthetase

Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the formation of phosphopantothenoylcysteine from (R)-phosphopantothenate and l-cysteine with the concomitant consumption of a nucleotide triphosphate. Herein, the human coaB gene encoding PPCS is cloned into pET23a and overexpressed in E. coli BL21(DE3), to yield 10 mg of purified enzyme per liter of culture. Detailed kinetic studies found that this PPCS follows a similar Bi Uni Uni Bi Ping Pong mechanism as previously described for the E. faecalis PPCS, except that the human enzyme can use both ATP and CTP with similar affinity. One significant difference for human PPCS catalysis with respect to ATP and CTP is that the enzyme shows cooperative binding of ATP, measured as a Hill constant of 1.7. PPCS catalysis under CTP conditions displayed Michaelis constants of 265 μM, 57 μM, and 16 μM for CTP, PPA, and cysteine, respectively, with a k_cat of 0.53 ± 0.01 s^? 1 for the reaction. Taking into account the cooperativity under ATP condition, PPCS exhibited Michaelis constants of 269 μM, 13 μM, and 14 μM for ATP, PPA, and cysteine, respectively, with a k_cat of 0.56 s^? 1 for the reaction. Oxygen transfer studies found that ¹⁸O from [carboxyl-¹⁸O] phosphopantothenate is incorporated into the AMP or CMP produced during PPCS catalysis, consistent with the formation of a phosphopantothenoyl cytidylate or phosphopantothenoyl adenylate intermediate, supporting similar catalytic mechanisms under both CTP and ATP conditions. Inhibition studies with GTP and UTP as well as product inhibition studies with CMP and AMP suggest that human PPCS lacks strong nucleotide selectivity.     

Purification and partial characterization of two new cold-adapted lipases from mesophilic Geotrichum sp. SYBC WU-3

Two cold-adapted lipases (Lipase-A and Lipase-B in the paper) of mesophilic Geotrichum sp. SYBC WU-3 were purified by using (NH₄)₂SO₄ fractionation, chromatography separation on a DEAE-cellulose-32 column and a Sephadex G100 column. The molecular mass of Lipase-A and Lipase-B were determined to be approximately 41.1 and 35.8 kDa, respectively by SDS-PAGE. The optimum temperature for the activity of Lipase-A was found to be 20 °C, and that of Lipase-B was 15 °C. Lipase-A and Lipase-B had good stability when temperature was below 40 °C. Both the optimum pH for the activity of the lipases was 9.5. Lipase-A retained about 80% of its activity when pH was between 3 and 6 and Lipase-B maintained over 80% activity in the pH range of 3–8. The two lipases showed hydrolysis efficiency to various p-nitrophenyl esters, but they were more active with shorter p-nitrophenyl esters (C2 and C4).     

Purification and properties of enantioselective lipase from a newly isolated Bacillus cereus C71

A lipase from Bacillus cereus C71 was purified to homogeneity by ammonium sulfate precipitation, followed by Phenyl-Sepharose chromatography, DEAE ion exchange chromatography and CIM^® QA chromatography. This purification procedure resulted in a 1092-fold purification of lipase with 18% yield. The molecular mass of the purified enzyme was determined to be approximately 42 kDa by SDS-PAGE and mass spectrometer. The lipase was stable in the pH range of 8.5–10.0, with the optimum pH 9.0. The enzyme exhibited maximum activity at 33 °C and retained 92% of original activity after incubation at 35 °C for 3 h. The protein hydrolyzed p-nitrophenyl esters with acyl chain lengths between C4 and C12. Enzyme activity was strongly inhibited in the presence of Cu²⁺ and Zn²⁺ but promoted by non-ionic surfactants. The lipase demonstrated higher enantioselectivity toward R-isomer of ethyl 2-arylpropanoate than the commercial lipases, and can be used potentially as a catalyst to prepare optically pure pharmaceuticals.     

Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production

Alkanes of defined carbon chain lengths can serve as alternatives to petroleum-based fuels. Recently, microbial pathways of alkane biosynthesis have been identified and enabled the production of alkanes in non-native producing microorganisms using metabolic engineering strategies. The chemoautotrophic bacterium Cupriavidus necator has great potential for producing chemicals from CO₂: it is known to have one of the highest growth rate among natural autotrophic bacteria and under nutrient imbalance it directs most of its carbon flux to the synthesis of the acetyl-CoA derived polymer, polyhydroxybutyrate (PHB), (up to 80% of intracellular content). Alkane synthesis pathway from Synechococcus elongatus (2 genes coding an acyl-ACP reductase and an aldehyde deformylating oxygenase) was heterologously expressed in a C. necator mutant strain deficient in the PHB synthesis pathway. Under heterotrophic condition on fructose we showed that under nitrogen limitation, in presence of an organic phase (decane), the strain produced up to 670 mg/L total hydrocarbons containing 435 mg/l of alkanes consisting of 286 mg/l of pentadecane, 131 mg/l of heptadecene, 18 mg/l of heptadecane, and 236 mg/l of hexadecanal. We report here the highest level of alka(e)nes production by an engineered C. necator to date. We also demonstrated the first reported alka(e)nes production by a non-native alkane producer from CO₂ as the sole carbon source.     

Chemical properties and antiinflammatory activity of a galactomannoglucan from Arecastrum romanzoffianum

A galactomannoglucan (GMG) with an estimated weight-average molar mass of 1.5 × 10⁵ was obtained from an aqueous extract of the mesocarp of fruits of Arecastrum romanzoffianum (Cham.) Becc. by fractionation by Sephacryl S-300 HR and Sephadex G-25. Chemical and spectroscopic studies indicated that GMG has a chain of (1 → 4)-linked β-d-mannopyranosyl residues, a chain of (1 → 3)-linked β-d-galactopyranosyl residues, a chain of (1 → 4)-linked α-d-glucopyranosyl residues, repeating units of β-d-galactopyranosyl-(1 → 4)-β-d-mannopyranosyl and β-d-mannopyranosyl-(1 → 4)-α-d-glucopyranosyl and terminal residues of d-galactopyranosyl and d-glucopyranosyl which comprised galactose, mannose and glucose in the molar ratio of 10:37:53. The polysaccharide exhibited significant antiinflammatory activity against carrageenan-induced mouse paw oedema.     

Synthesis of indole analogs as potent β-glucuronidase inhibitors

Natural products are the main source of motivation to design and synthesize new molecules for drug development. Designing new molecules against β-glucuronidase inhibitory is utmost essential. In this study indole analogs (1–35) were synthesized, characterized using various spectroscopic techniques including ¹H NMR and EI-MS and evaluated for their β-glucuronidase inhibitory activity. Most compounds were identified as potent inhibitors for the enzyme with IC₅₀ values ranging between 0.50 and 53.40 μM, with reference to standard d-saccharic acid 1,4-lactone (IC₅₀ = 48.4 ± 1.25 μM). Structure-activity relationship had been also established. The results obtained from docking studies for the most active compound 10 showed that hydrogen bond donor features as well as hydrogen bonding with (Oε1) of nucleophilic residue Glu540 is believed to be the most importance interaction in the inhibition activity. It was also observed that hydroxyl at fourth position of benzylidene ring acts as a hydrogen bond donor and interacts with hydroxyl (OH) on the side chain of catalysis residue Tyr508. The enzyme-ligand complexed were being stabilized through electrostatic π-anion interaction with acid-base catalyst Glu451 (3.96 Å) and thus preventing Glu451 from functioning as proton donor residue.     

Cloning,expression and characterization of a new enantioselective esterase from a marine bacterium Pelagibacterium halotolerans B2T

An esterase, designated as PE8 (219 aa, 23.19 kDa), was cloned from a marine bacterium Pelagibacterium halotolerans B2^T and overexpressed in Escherichia coli Rosetta, resulting an active, soluble protein which constituted 23.1% of the total cell protein content. Phylogenetic analysis of the protein showed it was a new member of family VI lipolytic enzymes. Biochemical characterization analysis showed that PE8 preferred short chain p-nitrophenyl esters (C2–C6), exhibited maximum activity toward p-nitrophenyl acetate, and was not a metalloenzyme. PE8 was an alkaline esterase with an optimal pH of 9.5 and an optimal temperature of 45 °C toward p-nitrophenyl acetate. Furthermore, it was found that PE8 exhibited activity and enantioselectivity in the synthesis of methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG) from the prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG). (R)-3-MFG was obtained in 71.6% ee and 73.2% yield after 36 h reaction under optimized conditions (0.6 M phosphate buffer (pH 8.0) containing 17.5% 1,4-dioxane under 30 °C). In addition, PE8 was tolerant to extremely strong basic and high ionic strength solutions as it exhibited high activity even at pH 11.0 in 1 M phosphate buffer. Given its highly soluble expression, alkalitolerance, halotolerance and enantioselectivity, PE8 could be a promising candidate for the production of (R)-3-MFG in industry. The results also demonstrate the potential of the marine environment as a source of useful biocatalysts.     

Pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate by human serum albumin: pH-dependence of rates of individual steps

Human serum albumin (HSA) displays esterase activity reflecting multiple irreversible chemical modifications rather than turnover. Here, kinetics of the pseudo-enzymatic hydrolysis of 4-nitrophenyl acetate (NphOAc) are reported. Under conditions where [HSA] ? 5×[NphOAc] and [NphOAc] ? 5×[HSA], the HSA-catalyzed hydrolysis of NphOAc is a first-order process for more than 95% of its course. From the dependence of the apparent rate constants k_app and k_obs on [HSA] and [NphOAc], respectively, values of K_s, k₊₂, and k₊₂/K_s were determined. Values of K_s, k₊₂, and k₊₂/K_s obtained at [HSA] ? 5×[NphOAc] and [NphOAc] ? 5×[HSA] are in good agreement, the deacylation step being rate limiting in catalysis. The pH-dependence of k₊₂/K_s, k₊₂, and K_s reflects the acidic pK_a shift of the Tyr411 catalytic residue from 9.0 ± 0.1 in the substrate-free HSA to 8.1 ± 0.1 in the HSA:NphOAc complex. Accordingly, diazepam inhibits competitively the HSA-catalyzed hydrolysis of NphOAc by binding to Tyr411.     

Characterization of inducible cold-active β-glucosidases from the psychrotolerant bacterium Shewanella sp. G5 isolated from a sub-Antarctic ecosystem

The psychrotolerant bacterium Shewanella sp. G5 was used to study differential protein expression on glucose and cellobiose as carbon sources in cold-adapted conditions. This strain was able to growth at 4 °C, but reached the maximal specific growth rate at 37 °C, exhibiting similar growing rates values with glucose (μ: 0.4 h⁻¹) and cellobiose (μ: 0.48 h⁻¹). However, it grew at 15 °C approximately in 30 h, with specific growing rates of 0.25 and 0.19 h⁻¹ for cellobiose and glucose, respectively. Thus, this temperature was used to provide conditions related to the environment where the organism was originally isolated, the intestinal content of Munida subrrugosa in the Beagle Channel, Fire Land, Argentina. Cellobiose was reported as a carbon source more frequently available in marine environments close to shore, and its degradation requires the enzyme β-glucosidase. Therefore, this enzymatic activity was used as a marker of cellobiose catabolism. Zymogram analysis showed the presence of cold-adapted β-glucosidase activity bands in the cell wall as well as in the cytoplasm cell fractions. Two-dimensional gel electrophoresis of the whole protein pattern of Shewanella sp. G5 revealed 59 and 55 different spots induced by cellobiose and glucose, respectively. Identification of the quantitatively more relevant proteins suggested that different master regulation schemes are involved in response to glucose and cellobiose carbon sources. Both, physiological and proteomic analyses could show that Shewanella sp. G5 re-organizes its metabolism in response to low temperature (15 °C) with significant differences in the presence of these two carbon sources.