Effect of cationic and non-ionic surfactants on the hydrolysis of N-glutaryl- -phenylalanine catalysed by chymotrypsin iso-enzymes

The hydrolysis of N-glutaryl- -phenylalanine p-nitroanilide catalysed by various chymotrypsin (CT) iso-enzymes (α-CT, β-CT, δ-CT, and γ-CT) has been studied in the presence of cationic and non-ionic surfactants at concentration higher than the critical micellar concentration. The enzyme activity was tested in the presence of the following surfactants: cetyltrimethylammonium bromide (CTABr), cetyldimethylethylammonium bromide (CDMEABr), cetyltripropylammonium bromide (CTPABr), Triton X100 (TX100) and polyoxyethylene 9 lauryl ether (PO9). The activity of the iso-enzymes depends on the surfactant concentration and it varies with the surfactant head group dimensions (CTPABr>CDMEABr>CTABr). For all the iso-enzymes, superactivity has been detected only in the presence of CTPABr and CDMEABr. The extent of superactivity depends on the enzyme used (δ-CT>β-CT>γ-CT>α-CT). The observed reaction rate has been compared with the prediction of a theoretical model for enzymatic activity in the presence of surfactant aggregates in aqueous media developed in a previous paper. The results can be explained by introducing an equilibrium relation between the enzyme confined in the free bulk water and in the bound water pseudo-phase, and by allowing for different catalytic behaviours of the two forms of enzyme.The theoretical model enables the initial reaction rate to be related to the substrate concentration with an overall Michaelis–Menten equation. Good agreement has been found between experimental and model predicted values of the kinetic parameters.     

Synthesis,characterization and kinetics of formation of silver nanoparticles by reduction with adrenaline in the micellar media

The present paper describes about the easy, simple and convenient procedure for the synthesis of silver nanoparticles (Ag-NPs) in aqueous solutions by the reduction of silver nitrate with adrenaline. The surfactant molecules of cetyltrimethylammonium bromide (CTABr) and sodium dodecyl ate (SDS) behaved differently during the reduction of Ag⁺ ions by adrenaline. The obtained data suggest that the variation of [CTABr] gave a maxima-like curve for rate constant versus [CTABr], while, the values of rate constant decreased with the increase in [SDS]. The addition of surfactant molecules stabilized the Ag-NPs. The UV–Visible spectra were analyzed to deduce the particle size. The calculated sizes of the nanoparticles were further compared by the TEM images. The XRD spectrum confirmed the crystalline nature of silver nanoparticles having the face-centered cubic crystal structure. The edge length of unit cell was found 4.076 Å. The kinetics of formation of Ag-NPs was performed at different concentrations of adrenaline, AgNO₃, NaOH and [surfactant]. The values of rate constant were independent on [adrenaline] and [AgNO₃]. The increase in [NaOH] increased the rate of agglomeration of silver particles to form Ag-NPs. A linear relationship was obtained for the plot of rate constant versus [NaOH].     

The structures of six urinary oligosaccharides that are characteristic for a patient with morquio syndrome type b

Morquio syndrome type B is an inherited, lysosomal storage disease characterised by a marked deficiency in acid β-d-galactosidase, while the 2-acetamido-2-deoxy-β-d-galactose 6-sulphate sulphatase activity is normal. Urinary oligosaccharides were studied in order to evaluate the effect of the diminished β-d-galactosidase activity on the catabolism of glycoconjugates and to compare their structures with those excreted by patients with GM₁-gangliosidosis. The following oligosaccharides were isolated: β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-β-d-Manp-(1→4)- d-GlcpNAc (1), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-[α-d-Manp- (1→3)]-β-d-Manp-(1→4)-d-GlcpNAc (2a), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)- α-d-Manp-(1→3)-[α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-GlcpNAc (2b), β-d-Galp- (1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→3)-[β-d-Galp-(1→4)-β-d-GlcpNAc-(1→ 2)-α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-GlcpNAc (3), β-d-Galp-(1→4)-β-d-Glcp- NAc-(1→2)-α-d-Manp-(1→3)-{β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-[β-d-Galp- (1→4)-β-d-GlcpNAc-(1→6)]-α-d-Manp-(1→6)}-β-d-Manp-(1→4)-d-GlcpNAc (4), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→3)-[β-d-GlcpNAc-(1→4)]-[β- d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-Glcp- NAc (5). Significant differences between Morquio syndrome type B and GM₁-gangliosidosis have been observed, with regard to the excretion rate and the specific structures of urinary oligosaccharides. Compounds 2a, 2b, and 5 are novel members of the series of oligosaccharides isolated from the urine of patients with inherited, lysosomal storage diseases.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

Design and synthesis of novel substituted quinazoline derivatives as antileishmanial agents

4-(Substituted-benzylidine)-2-substituted-5,6-dihydrobenzo[h]quinazoline (5a–p) and 4-(substituted-benzylidine)-2-substituted-3, 4, 5, 6-tetrahydrobenzo[h]quinazoline (6a–p) have been synthesized from 2-(substituted-benzylidine)tetralone-1(3a–d) and several substituted guanidine sulfates(4a–d).These compounds were tested for their in vitro antileishmanial activity. The compounds 6i, 6f, 6g show promising antileishmanial activity against Leishmania donovani.     

N-Acetylhexosamine triad in one molecule: Chemoenzymatic introduction of 2-acetamido-2-deoxy-β-d-galactopyranosyluronic acid residue into a complex oligosaccharide

A complex trisaccharide β-d-GalpNAcA-(1 → 4)-β-d-GlcpNAc-(1 → 4)-d-ManpNAc (3) was prepared in a good yield (35%) in a transglycosylation reaction catalyzed by β-N-acetylhexosaminidase from Talaromyces flavus using p-nitrophenyl 2-acetamido-2-deoxy-β-d-galacto-hexodialdo-1,5-pyranoside (1) as a donor followed by the in situ oxidation of the aldehyde functionality by NaClO₂. The disaccharide β-d-GlcpNAc-(1 → 4)-d-ManpNAc (2) was used as galactosyl acceptor. A disaccharide β-d-GalpNAcA-(1 → 4)-d-GlcpNAc (4; 39%) originated as a by-product in the reaction. Oligosaccharides comprising a carboxy moiety at C-6 are shown to be very efficient ligands to natural killer cell activation receptors, particularly to human receptor CD69. Thus, oxidized trisaccharide 3 is the best-known oligosaccharidic ligand to this receptor, with IC₅₀ = 2.5 × 10⁻⁹ M. The presented method of introducing a β-d-GalpNAcA moiety into carbohydrate structures is versatile and can be applied in the synthesis of other complex oligosaccharides.     

Syntheses and structures of vinyl and aryl derivatives of carbonyl halide iron complexes

cis,trans-Fe(CO)₂(PMe₃)₂(p-Y-C₆H₄)X [X=Br, Y=H (4a), MeO (4b), Cl (4c), F (4d), Me (4e); X=I, Y=H (5); X=Cl, Y=H (6)] and cis,trans-Fe(CO)₂(PMe₃)₂(σ-CHCH₂)X [X=Br (7); X=I (8); X=Cl (9)] are prepared by reacting dihalide complexes cis,trans,cis- Fe(CO)₂(PMe₃)₂X₂ [X=Br (1), X=I (2), X=Cl (3)] with Grignard reagents p-Y-C₆H₄-MgBr (Y=H, OMe, Cl, F, Me) or CH₂CH-MgBr and with lithium reagents PhLi, CH₂CH-Li. With both reagents, the reaction proceeds following two parallel pathways: one is the metallation reaction which yields alkyl derivatives, the other affords 17 electron complexes [Fe(CO)₂(PMe₃)₂X] via monoelectron reductive elimination. The influence of the halides and organometallic reagents on the yield of the metallation reaction is discussed. The solution structure of the complexes is assigned on the basis of IR and ¹H, ¹³C, ¹⁹F, ³¹P NMR spectra. The solid state structure of complexes 4a, 5 and 6 is determined by single crystal X-ray diffractometric methods.     

Effect of DHLA on response of isolated rat urinary bladder to repetitive field stimulation

Lipoic acid is an essential coenzyme in the oxidation of pyruvate and -ketoglutarate. It is easily converted to its reduced form, dihydrolipoic acid (DHLA), in vivo thereby forming a redox pair. DHLA is important in the maintenance and integrity of specific neuronal and subcellular membranes. In the present study we investigated the effect of DHLA on the response of isolated rat bladder strips to repetitive field stimulation (FS), a method used to exhaust synaptic stores of acetylcholine resulting in nerve and synaptic damage.Isolated strips of rat urinary bladders were separated into 4 groups. Group 1 strips were incubated with choline + acetyl-CoA; Group 2 strips with choline, acetyl-CoA + DHLA; and Group 3 with DHLA. Group 4 strips were controls. All strips in Groups 1–3 were subjected to 2 h of repetitive FS followed by 2 h of recovery.DHLA had no effect on the progressive decrease in contractile response observed during repetitive stimulation. However, strips incubated in the presence of DHLA showed a significantly greater degree of recovery than strips incubated in the absence of DHLA. We believe that the protection of the contractile response is related to DHLA's ability to protect nerve and/or muscle membranes from oxidative damage.     

Novel alkyl alkanethiolsulfonate sulfhydryl reagents. Modification of derivatives ofl-cysteine

A simple, general scheme for the synthesis of sulfhydryl-specific alkyl alkanethiolsulfonate (RSSO₂R′) reagents where R′ is methyl, has been developed. Two new reagents, methyl aminoethanethiolsulfonate (2) and methyl benzylthiolsulfonate (3) were synthesized. These were used to modify stoichiometrically and selectively under mild conditions the sulfhydryl groups ofN-acetyl-l-cysteine ethyl ester (4),N-acetyl-l-cysteinep-nitroanilide (7), glutathione, and the A chain of bovine insulin. The corresponding β-S-(β-aminoethanethiol) and β-S-(benzylthiol) derivatives ofl-cysteine and of the peptides were afforded. The characteristics and significance of these reactions and products are discussed.     

Imitator strategies

An imitator strategy in an evolutionary game is a pair (u, p) consisting of a conventional and generally-known strategy u (the personal strategy) and a probability p of using the opponent's personal strategy rather than one's own. The question of whether imitator strategies (p > 0) can be used to invade populations using conventional ESSs is explored, and is found to depend in part on the variability of strategy present in the initial population. Interior values of p (0 < p < 1) are found never to be optimal. Examples are used repeatedly to illustrate the theory. The imitator strategy is also compared with the successful Tit-for-Tat strategy considered in the Axelrod-Hamilton study of the Prisoner's dilemma.     

Biotransformation of p-, m-, and o-hydroxybenzoic acids by Panax ginseng hairy root cultures

The regioselective glycosylation of three isomers of hydroxybenzoic acids was observed in Panax ginseng hairy root cultures. p-Hydroxybenzoic acid (1) and m-hydroxybenzoic acid (2) were converted into their corresponding glycosides (1a and 2a) and glycosyl esters (1b and 2b) while no metabolite of o-hydroxybenzoic acid (3) was detected. A new compound, m-hydroxybenzoic acid β-d-xylopyranosyl (1 → 6)-β-d-glycopyranosyl ester (2c) was identified as a biotransformation product of 2. Further time-course studies of the biotransformation reactions showed that the glycosides were major products in the latter stage. The addition of carbohydrates or antioxidants increased glycosyl esters formation.     

A new route for the synthesis of Streptococcus pneumoniae 19F and 19A capsular polysaccharide fragments avoiding the β-mannosamine glycosylation step

The recently described [Attolino, E.; Bonaccorsi, F.; Catelani, G.; D’Andrea, F. Carbohydr. Res. 2008, 343, 2545–2556.] β-d-MaNAcp-(1→4)-β-d-Glcp thiophenyl glycosyl donor 3 was used in α-glycosylation reactions of OH-2 and OH-3 of the suitably protected p-MeO-benzyl α-l-rhamnopyranoside acceptors 7 and 8. Glycosylation of the axial OH-2 of 7 took place in high yield (76%) and with acceptable stereoselectivity (α/β = 3.4) leading to the protected trisaccharide α-11, corresponding to the repeating unit of Streptococcus pneumoniae 19F. The same reaction on equatorial OH-3 of acceptor 8 gave the trisaccharide α-15, a constituent of the repeating unit of S. pneumoniae 19A, but in lower yield (41%) and without stereoselection (α/β = 1:1.3). Utilizing the introduced orthogonal protection of OH-1 and OH-4″, the trisaccharide α-11 was transformed into a trisaccharide building block suitable for the synthesis of its phosphorylated oligomers.     

Synthesis of pentopyranosyl-containing thiodisaccharides. Inhibitory activity against β-glycosidases

β-(1→4)-Thiodisaccharides formed by a pentopyranose unit as reducing or non reducing end have been synthesized using a sugar enone derived from a hexose or pentose as Michael acceptor of a 1-thiopentopyranose or 1-thiohexopyranose derivatives. Thus, 2-propyl per-O-acetyl-3-deoxy-4-S-(β-d-Xylp)-4-thiohexopyranosid-2-ulose (3) and benzyl per-O-acetyl-3-deoxy-4-S-(β-d-Galp)-4-thiopentopyranosid-2-ulose (11) were obtained in almost quantitative yields. The carbonyl function of these uloses was reduced with NaBH₄ or K-Selectride, and the stereochemical course of the reduction was highly dependent on the reaction temperature, reducing agent and solvent. Unexpectedly, reduction of 3 with NaBH₄–THF at 0 °C gave a 3-deoxy-4-S-(β-d-Xylp)-4-thio-α-d-ribo-hexopyranoside derivative (6) as major product (74% yield), with isomerization of the sulfur-substituted C-4 stereocenter of the pyranone. Reduction of 11 gave always as major product the benzyl 3-deoxy-4-S-(Galp)-4-thio-β-d-threo-pentopyranoside derivative 14, which was the only product isolated (80% yield) in the reduction with K-Selectride in THF at −78 °C. Deprotection of 14 and its epimer at C-2 (13) afforded, respectively the free thiodisaccharides 19 and 18. They displayed strong inhibitory activity against the β-galactosidase from Escherichia coli. Thus, compound 18 proved to be a non-competitive inhibitor of the enzyme (K_i = 0.80 mM), whereas 19 was a mixed-type inhibitor (K_i = 32 μM).     

Comparison of antioxidant effectiveness of lipoic acid and dihydrolipoic acid

The abilities of dihydrolipoic acid (DHLA) to scavenge peroxynitrite (ONOO^?), galvinoxyl radical, 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonate) cation radical (ABTS^+?), and 2,2′‐diphenyl‐1‐picrylhydrazyl radical (DPPH) were higher than those of lipoic acid (LA). The effectiveness of DHLA to protect methyl linoleate against 2,2′‐azobis(2‐amidinopropane hydrochloride) (AAPH)‐induced oxidation was about 2.2‐fold higher than that of LA, and DHLA can retard the autoxidation of linoleic acid (LH) in the β‐carotene‐bleaching test. DHLA can also trap ～0.6 radicals in AAPH‐induced oxidation of LH. Moreover, DHLA can scavenge ～2.0 radicals in AAPH‐induced oxidation of DNA and AAPH‐induced hemolysis of erythrocytes, whereas LA can scavenge ～1.5 radicals at the same experimental conditions. DHLA can protect erythrocytes against hemin‐induced hemolysis, but accelerate the degradation of DNA in the presence of Cu²⁺. Therefore, the antioxidant capacity of –SH in DHLA is higher than S‐S in LA. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 25:216–223, 2011; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20378     

Reaction of the main proteolytic fraction of Schistosoma mansoni cercarial enzymes with synthetic substrates and inhibitors of proteolytic enzymes

The purified proteolytic fraction of Schistosoma mansoni cercarial enzymes (PCF) was inhibited by Diisopropylphosphofluoridate (DFP). Its esterolytic activity was not affected by either of two specific active center reagents of proteolytic enzymes: (1) 1-chloro-3-tosylamido-7-ammo-2-heptanone (trypsin) and (2) l-1-tosylamido-2-phenylethyl chloromethyl ketone (chymotrypsin). Furthermore, PCF did not destroy the biological activity of bradykinin on the isolated guinea pig ileum, nor did it release bradykinin from purified dog plasma bradykininogen.     

Biotransformation of sesquiterpenoids having α,β-unsaturated carbonyl groups with cultured plant cells of Marchantia polymorpha

The biotransformation of sesquiterpenoids having an α,β-unsaturated carbonyl group, such as α-santonin (1), lancerodiol p-hydroxybenzoate (2), 8,9-dehydronootkatone (3), and nootkatone (4), with cultured suspension cells of Marchantia polymorpha was investigated. It was found that the CC double bond of 1 and 2 was hydrogenated to give 1,2-dihydro-α-santonin (5) and 3,4-dihydrolancerodiol p-hydroxybenzoate (6), respectively, while the allylic position of the CC double bond of 3 and 4 was hydroxylated to give 13-hydroxy-8,9-dehydronootkatone (7) and 9-hydroxynootkatone (8), respectively.     

H-nmr study on the preferred conformations of chiral pyridine-dinucleotide models

The synthesis of four chiral NAD⁺ models 1 and their 1,4-dihydro analogs 2 is described. From the temperature dependence of the ¹H-nmr spectra it is concluded that for these compounds two preferred conformations I and II, differing slightly in energy, exist. Both conformations are “folded” with the more or less parallel p-anisyl and pyridine groups mutually gauche, but in I the pyridine group is rotated by about 180° as compared with II, thus leading to a conspicuous difference in orientation of the substituent Z (NH₂CO, C₆H₅NHSO₂, (CH₂)₄NSO₂, or (C₄H₈ON)SO₂) in the pyridine ring toward the anisyl group. The most stable conformation (I) has Z closest to the center of the p-anisyl group. In 360-MHz spectra of the dihydropyridines at low temperature (−10°C), slow interconversion of I and II leads to the observation of an XY pattern for the C-4 methylene protons of the 1,4-dihydropyridine system. The anisochronity in this methylene group is caused mainly by the anisotropy of the neighboring p-anisyl group.     

Synthesis of 5,6-dideoxy-3-O-methyl-5-C-(phenylphosphinyl)-d-glucopyranose and its 1,2,4-triacetate

Methyl phenylphosphonite or dimethyl phosphite underwent acid-catalyzed addition reactions with some hexofuranos-5-ulose 5-(p-tolylsulfonylhydrazones) (7, 9, and 16), to give the corresponding adducts, 17, 18, 19, and 21. The isomer ratios of the adducts were affected by a 3-substituent in the hydrazones. Treatment of adduct 21 with sodium borohydride and sodium dihydrobis(2-methoxyethoxy)-aluminate (SDMA), followed by acid hydrolysis, gave 5,6-dideoxy-3-O-methyl-5-C-(phenylphosphinyl)-d-glucopyranose (26), which was acetylated to give the 1,2,4-tri-O-acetyl derivatives 27a and 27b. Conformational analysis of compound 27a by X-ray crystallography revealed that the compound was 1,2,4-tri-O-acetyl-5,6-dideoxy-3-O-methyl-5-C-[(S)-phenylphosphinyl]-β-d-glucopyranose in the ⁴C₁(d) form having all substituents equatorial.     

A new method of acetonation. Synthesis of 4,6-O-isopropylidene-D-glucopyranose

Ethyl isopropenyl ether reacts with D-glucose in N,N-dimethylformamide containing a trace of p-toluenesulfonic acid to give crystalline 4,6-O-isopropylidene-α,β-D-glucopyranose (2) in near-quantitative yield. The structure of 2 was established by n.m.r. spectroscopy of it and of its β-triacetate 3, and by conversion of 3 through deacetonation and subsequent acetylation into β-D-glucopyranose pentaacetate (5). The acetonation reagent operates under kinetic control, with favored attack at primary hydroxyl groups, instead of by the thermodynamic control associated with conventional acetonation methods. The reagents converts methyl α-D-glucopyranoside (7) into the 4,6-isopropylidene acetal 8, and D-mannitol (9) into a 2:1 mixture of the 1,2:5,6-di-isopropylidene acetal 10 and the 1,2:3,4:5,6-tri-isopropylidene acetal 11.     

Reactivities of the coordinated organonitriles in molybdenum(0) and tungsten(0) phosphine complexes: protonation of the nitrile carbon and cleavage of the CN triple bond

The molybdenum and tungsten dinitrogen-organonitrile complexes trans-[M(N₂)(NCR)(dppe)₂] (2, M=Mo; 4, M=W; R=Ph, C₆H₄Me-p, C₆H₄OMe-p, Me; dppe=Ph₂PCH₂CH₂PPh₂) underwent double protonation at the nitrile carbon atom with loss of N₂ and a change in oxidation state to +4 on treatment with hydrochloric acid to afford the cationic imido complexes trans-[MCl(NCH₂R)(dppe)₂]⁺. The solid-state structure of trans-[WCl(NCH₂CH₃)(dppe)₂][PF₆]·CH₂Cl₂ was determined by single-crystal X-ray analysis. Protonation of complexes 2 by fluoroboric acid or hydrobromic acid also formed the similar imido complexes trans-[MoX(NCH₂R)(dppe)₂]⁺ (X=F, Br). In contrast, the dinitrogen complex trans-[Mo(N₂)₂(dppe)₂] reacted with two equiv. of benzoylacetonitrile, a nitrile with acidic CH hydrogen atoms, to give the nitrido complex trans-[Mo(N)(NKCCHCOPh)(dppe)₂] (12), which was accompanied by evolution of dinitrogen and the formation of 1-phenyl-2-propen-1-one in high yields. For complex 12, the zwitterionic structure, where the anionic enolate ligand PhC(O⁺)=CHCN coordinates to the cationic Mo(IV) center through its nitrogen atom, was confirmed by spectroscopic measurements and single-crystal X-ray analysis. A unique intermolecular aromatic C---HO hydrogen bonding was observed in that crystal structure. Complex 12 is considered to be formed via the cleavage of the CN triple bond of benzoylacetonitrile on the metal. A reaction mechanism is proposed, which includes the double protonation of the nitrile carbon atom of the ligating benzoylacetonitrile on a low-valent molybdenum center.