In vitro and in silico inhibition of angiotensin-converting enzyme by carbohydrates and cyclitols

Fifteen carbohydrates (d-mannose, d-glucose, d-galactose, methyl-α-d-glucose, l-rhamnose, d-xylose, d-fructose, d-arabinose, dulcitol, mannitol, β-maltose, α-lactose, melibiose, sucrose, and raffinose) and four cyclitols [l-(+)-bornesitol, myo-inositol, per-O-acetyl-1-l-(+)-bornesitol, and quinic acid] were assayed for in vitro ACE inhibition. Of these molecules, per-O-Acetyl-1-l-(+)-bornesitol, quinic acid, methyl-α-d-glucose, d-rhamnose, raffinose, and the disaccharides were determined to be either inactive or weak ACE inhibitors, whereas l-(+)-bornesitol, d-galactose, d-glucose, and myo-inositol exhibited significant ACE inhibition. Molecular docking studies were performed to investigate interactions between active compounds and human ACE (Protein Data Bank, PDB 1O83). The results of various calculations showed that all active sugars bind to the same enzyme region, which is a tunnel directed towards the active site. With the exception of myo-inositol (K _i = 13.95 μM, IC₅₀ = 449.2 μM), the active compounds presented similar K _i and IC₅₀ values. d-Galactose (K _i = 19.6 μM, IC₅₀ = 35.7 μM) and l-(+)-bornesitol (K _i = 25.3 μM, IC₅₀ = 41.4 μM) were the most active compounds, followed by d-glucose (K _i = 32.9 μM, IC₅₀ = 85.7 μM). Our docking calculations are in agreement with the experimental data and show a new binding region for sugar-like molecules, which may be explored for the development of new ACE inhibitors.     

Synthesis of N-glycyl-β-glycopyranosyl amines,derivatives of natural oligosaccharides — glucose analogs of Lex antigen

Treatment of the natural tri-, tetra-, and pentasaccharides, β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, α-l-Fucp-(1→2)-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, and α-l-Fucp-(1→2)-[α-d-GalNAcp-(1→3)]-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, which are glucose analogs of Le^x, with ammonium carbamate in aqueous methanol gave the corresponding β-glycopyranosyl amines. After their N-acylation with N-Z-glycine N-hydroxysuccinimidyl ester (Z is benzyloxycarbonyl) with subsequent hydrogenolytic removal of Z-group, corresponding N-glycyl-β-glycopyranosyl amines were obtained in yields up to 70%.     

The yjjN of E. coli codes for an l-galactonate dehydrogenase and can be used for quantification of l-galactonate and l-gulonate

Escherichia coli is able to utilize l-galactonate as a sole carbon source. A metabolic pathway for l-galactonate catabolism is described in E. coli, and it is known to be interconnected with d-galacturonate metabolism. The corresponding gene encoding the first enzyme in the l-galactonate pathway, l-galactonate-5-dehydrogenase, was suggested to be yjjN. However, l-galactonate dehydrogenase activity was never demonstrated with the yjjN gene product. Here, we show that YjjN is indeed an l-galactonate dehydrogenase having activity also for l-gulonate. The K _m and k _cat for l-galactonate were 19.5?±?0.6 mM and 0.51?±?0.03 s^?1, respectively. In addition, YjjN was applied for a quantitative detection of the both of these substances in a coupled assay. The detection limits for l-galactonate and l-gulonate were 1.65 and 10 μM, respectively.     

Enzymatic Oxidation and Separation of Various Saccharides with Immobilized Glucose Oxidase

Glucose oxidase from Aspergillus niger, the specific enzyme for β-d-glucose oxidation, can also oxidize other related saccharides at very slow or negligible rates. The present study aimed to compare the kinetics of d-glucose oxidation using immobilized glucose oxidase on bead cellulose for the oxidation of related saccharides using the same biocatalyst. The significant differences were observed between the reaction rates for d-glucose and other saccharides examined. As a result, k _cat/K _M ratio for d-glucose was determined to be 42 times higher than d-mannose, 61.6 times higher than d-galactose, 279 times higher than d-xylose, and 254 times higher than for d-fructose and d-cellobiose. On the basis of these differences, the ability of immobilized glucose oxidase to remove d-glucose from d-cellobiose, d-glucose from d-xylose, and d-xylose from d-lyxose was examined. Immobilized catalase on Eupergit and mixed with immobilized glucose oxidase on bead cellulose or co-immobilized with glucose oxidase on bead cellulose was used for elimination of hydrogen peroxide from the reaction mixture. The accelerated elimination of d-glucose and d-xylose in the presence of co-immobilized catalase was observed. The co-immobilized glucose oxidase and catalase were able to decrease d-glucose or d-xylose content to 0–0.005% of their initial concentrations, while a minimum decrease of low oxidized saccharides d-xylose, d-cellobiose, and d-lyxose, respectively, was observed.     

Quantitative Analysis of Caffeoylquinic Acids and Styrylpyrones in Sweetia panamensis Bark by UPLC

Six secondary metabolites from the methanolic extract of Sweetia panamensis (Fabaceae) bark were isolated and characterised. Along with the pyrones desmethylangonine β-d-O-glucopyranoside and desmethylangonine β-d-O-glucopyranosyl-(1→6)-O-β-d-glucopyranoside, already reported in this species, 5-O-caffeoylquinic acid (chlorogenic acid), 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid and the isoflavonoid 5-O-methylgenistein 7-O-β-d-glucopyranoside were isolated for the first time from S. panamensis. Additionally, an LC-ESI-MS qualitative analysis was performed and an ultra performance liquid chromatography (UPLC) method was developed and validated for the determination of these compounds. The UPLC method was applied to the quantitative analysis of plant samples. Pyrones and caffeoylquinic acids resulted to be the main compounds in the extract; in particular desmethylangonine β-d-O-glucopyranosyl-(1→6)-O-β-d-glucopyranoside was the most abundant compound.     

Characterization of a d-Stereoselective Aminopeptidase (DamA) Exhibiting Aminolytic Activity and Halophilicity from Aspergillus oryzae

β-Aminopeptidases exhibit both hydrolytic and aminolytic (peptide bond formation) activities and have only been reported in bacteria. We identified a gene encoding the β-aminopeptidase homolog from a genome database of the filamentous fungus Aspergillus oryzae. The gene was overexpressed in A. oryzae, and the resulting recombinant enzyme was purified. Apart from bacterial homologs [β-Ala-para-nitroanilide (pNA)], the enzyme preferred d-Leu-pNA and d-Phe-pNA as substrates. Therefore, we designated this gene as d-stereoselective aminopeptidase A (damA). The purified recombinant DamA was estimated to be a hexamer and was composed of two subunits with molecular masses of 29.5 and 11.5 kDa, respectively. Optimal hydrolytic activity of DamA toward d-Leu-pNA was observed at 50 °C and pH 8.0. The enzyme was stable up to 60 °C and from pH 4.0–11.0. DamA also exhibited aminolytic activity, producing d-Leu-d-Leu-NH₂ from d-Leu-NH₂ as a substrate. In the presence of 3.0 M NaCl, the amount of pNA liberated from d-Leu-pNA by DamA was 3.1-fold higher than that in the absence of NaCl. Thus, DamA is a halophilic enzyme. The enzyme was utilized to synthesize several hetero-dipeptides containing a d-amino acid at the N-terminus as well as physiologically active peptides.     

Mutant d-amino acid oxidase with higher catalytic efficiency toward d-amino acids with bulky side chains

d-Amino acid oxidase from the yeast Trigonopsis variabilis (TvDAAO) is widely used in fine organic synthesis, including the preparation of unnatural l-amino acids and α-keto acids. The analysis of the three-dimensional structure of TvDAAO was carried out with the aim of producing the enzyme specific to d-amino acids with bulky side chains. The analysis revealed the residue Phe54 at the entrance to the active site, which controls the substrate access to this site. The residue Phe54 was replaced by residues Ala, Ser, and Tyr. The cultivation of recombinant E. coli strains expressing TvDAAO mutants showed that the mutein with the Phe54Ala substitution had very low stability. Thus, the inactivation of the enzyme occured within 10 min after the cell disruption. The Phe54Ser TvDAAO and Phe54Tyr TvDAAO mutants were obtained as homogeneous preparations, and their thermal stability and catalytic properties were investigated. The introduction of Phe54Ser and Phe54Tyr substitutions resulted in additional stabilization of the protein macromolecule compared to the wild-type TvDAAO. Thus, the half-inactivation time for the mutant enzymes at 54 °C increased by a factor of 1.5 and 2, respectively. As in the case of wild-type TvDAAO, the thermal inactivation of the muteins proceeds via a two-step dissociative mechanism. The introduction of mutations led to a strong change in the substrate specificity profile. The mutants have no activity toward a series of d-amino acids (Phe54Ser TvDAAO toward d-Ala, d-Ser, d-Val, and d-Thr; Phe54Tyr TvDAAO toward d-Ser, d-Tyr, d-Thr, and d-Lys). The catalytic efficiency (the k _cat/K _M ratio) of the Phe54Ser TvDAAO mutant toward d-amino acids with bulky side chains (d-Lys, d-Asn, d-Phe, d-Tyr, d-Trp, and d-Leu) increased from 2.4 to 7.3 times.     

Cis-Cycloprolylprolin-Anion-ein konfigurationsstabiles Carbanion

The racemisation ofcyclo-(l-Pro?l-Pro) (2) with metal amides in liq. ammonia was examined. The K-kation causes more extensive racemisation than Na-kation, which in turn is more effective than Li⁺. This, the racemisation of2 int-butyl alcohol with K⁺C₆H₅O^? and the data gained from corresponding deuterated medium show that the racemisation of2 proceeds in two steps: in the first, the less stabletrans-cyclo-(l-Pro?d-Pro) (3) is formed, followed by the rapid conversion of3 to a mixture ofcyclo-(l-Pro?l-Pro) andcyclo-(d-Pro?d-Pro) in the second step.     

A method for the determination of d-kynurenine in biological tissues

d-Kynurenine (d-KYN), a metabolite of d-tryptophan, can serve as the bioprecursor of kynurenic acid (KYNA) and 3-hydroxykynurenine, two neuroactive compounds that are believed to play a role in the pathophysiology of several neurological and psychiatric diseases. In order to investigate the possible presence of d-KYN in biological tissues, we developed a novel assay based on the conversion of d-KYN to KYNA by purified d-amino acid oxidase (d-AAO). Samples were incubated with d-AAO under optimal conditions for measuring d-AAO activity (100 mM borate buffer, pH 9.0), and newly produced KYNA was detected by high-performance liquid chromatography (HPLC) with fluorimetric detection. The detection limit for d-KYN was 300 fmol, and linearity of the assay was ascertained up to 300 pmol. No assay interference was noted when other d-amino acids, including d-serine and d-aspartate, were present in the incubation mixture at 50-fold higher concentrations than d-KYN. Using this new method, d-KYN was readily detected in the brain, liver, and plasma of mice treated systemically with d-KYN (300 mg/kg). In these experiments, enantioselectivity was confirmed by determining total kynurenine levels in the same samples using a conventional HPLC assay. Availability of a sensitive, specific, and simple method for d-KYN measurement will be instrumental for evaluating whether d-KYN should be considered for a role in physiology and pathology.     

Tartaric acid and its O-acyl derivatives. 7. Crystal structure of O-p-anisoyl-D-tartaric acid and its dimethylammonium salt trihydrate

Monoacylated derivatives O-p-anisoyl-d-tartaric acid and its N,N-dimethylammonium salt are synthesized by the partial hydrolysis of O,O′-di-p-anisoyl-d-tartaric acid. Crystal and molecular structures of both compounds have been determined and analyzed. In both of them, some strong and moderate strength [O-H…O] hydrogen bonds exist between the carboxylic units. The intermolecular hydrogen bonds link the adjacent fragments forming infinite one-dimensional chains parallel to the X-axis.     

Two-dimensional high-performance liquid chromatographic determination of day–night variation of d-alanine in mammals and factors controlling the circadian changes

d-Alanine (d-Ala) is one of the naturally occurring d-amino acids in mammals, and its amount is known to have characteristic circadian changes. It is a candidate for a novel physiologically active substance and/or a biomarker, and the regulation mechanisms of the intrinsic amounts of d-Ala are expected to be clarified. In the present study, the effects of the possible factors controlling the d-Ala amounts, e.g., diet, d-amino acid oxidase (DAO) and intestinal bacteria, on the day–night changes in the intrinsic d-Ala amounts have been investigated using a highly sensitive and selective two-dimensional high-performance liquid chromatographic system combining a reversed-phase column and an enantioselective column. The circadian rhythm was not changed under fasting conditions. In the mice lacking d-amino acid oxidase activity (ddY/DAO^- mice), clear day–night changes were still observed, suggesting that the factors controlling the d-Ala rhythm were not their food and DAO activity. On the other hand, in the germ-free mice, quite low amounts of d-Ala were detected compared with those in the control mice, indicating that the main origin of d-Ala in the mice is intestinal bacteria. Because the d-Ala amounts in the digesta containing intestinal bacteria did not show the day–night changes, the controlling factor of the circadian changes of the d-Ala amount was suggested to be the intestinal absorption.     

Asteropsiside A and other asterosaponins from the starfish Asteropsis carinifera

Three asterosaponins were isolated from the tropical starfish Asteropsis carinifera: a new one, asteropsiside A, and two known ones, regularoside A and thornasteroside A. The structure of the new compound was established using 2D NMR spectroscopy and ESI mass spectrometry as the sodium salt of 3-O-sulfonato-(20E)-6-O-{β-d-fucopyranosyl-(1→2)-β-d-galactopyranosyl-(1→4)-[β-d-quinovopyranosyl-(1→2)]-β-d-xylopyranosyl-(1→3)-β-d-quinovopyranosyl}-3β,6α-dihydroxy-5α-cholesta-9(11),20(22)-dien-23-one. Regularoside A and thornasteroside A were shown to display the ability to inhibit the growth of the T-47D and RPMI-7951 tumor cell colonies in vitro.     

Bifidobacterium longum l-Arabinose Isomerase—Overexpression in Lactococcus lactis, Purification, and Characterization

Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum l-AI was characterized using d-galactose and l-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0?C6.5. The enzyme showed the highest activity at 55?°C during a 20-min incubation at pH 6.5. The K _m value was 120?mM for l-arabinose and 590?mM for d-galactose. The V _max was 42?U mg^?1 with l-arabinose and 7.7?U mg^?1 with d-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg²⁺, Mn²⁺). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum l-AI as the catalyst at 35?°C, equilibrium yields of 36?% d-tagatose and 11?% l-ribulose with 1.67?M d-galactose and l-arabinose, respectively, as the substrates were reached.     

l-myo-Inositol-1-Phosphate Synthase Expressed in Developing Organ: Isolation and Characterisation of the Enzyme from Human Fetal Liver

l-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4) activity has been detected and partially purified for the first time from human fetal liver. Crude homogenate from the fetal liver was subjected to streptomycin sulphate precipitation and 0?C60?% ammonium sulphate fractionation followed by successive chromatography through DEAE cellulose and BioGel A 0.5-m columns. After the final chromatography, the enzyme was purified 51-fold and 3.46?% of MIPS could be recovered. The human fetal liver MIPS specifically utilised d-glucose-6-phosphte and NAD⁺ as its substrate and coenzyme, respectively. It shows pH optima between 7.0 and 7.5 while the temperature maximum was at 40?°C. The enzyme activity was remarkably stimulated by NH ₄ ⁺ , slightly stimulated by K⁺ and Ca²⁺ and highly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺. The K _m values of MIPS for d-glucose-6-phosphate and NAD⁺ were found to be as 1.15 and 0.12?mM respectively while the V _max values were 280?nM and 252?nM for d-glucose-6-phosphate and NAD⁺ correspondingly. The apparent molecular weight of the native enzyme was determined to be 170?kDa.     

Application of Hydrazino Dinitrophenyl-Amino Acids as Chiral Derivatizing Reagents for Liquid Chromatographic Enantioresolution of Carbonyl Compounds

A new series of chiral derivatizing reagents (CDRs) consisting of five hydrazino dinitrophenyl (HDNP)-amino acids (CDR 1?C5) was prepared by a two-step synthesis procedure starting from 1,5-difluoro-2,4-dinitrobenzene (DFDNB). In the first step, five fluoro-dinitrophenyl (FDNP)-reagents, namely FDNP-l-Leu, FDNP-l-Val, FDNP-l-Phe, FDNP-l-Ala and FDNP-d-Phg were synthesized by substituting one of the fluorine atoms in DFDNB moiety with amino acids l-Leu, l-Val, l-Phe, l-Ala and d-Phg, respectively. In the following step, the remaining fluorine atom of the FDNP reagents was substituted with hydrazine hydrate to obtain five HDNP reagents (i.e. CDR 1?C5; HDNP-l-Leu, HDNP-l-Val, HDNP-l-Phe, HDNP-l-Ala and HDNP-d-Phg). These five CDRs were used for synthesis of diastereomers of six racemic carbonyl compounds which were resolved by high-performance liquid chromatography using C18 column and gradient eluting mixture of acetonitrile or methanol with triethylammonium phosphate buffer with UV detection at 348 nm. Microwave irradiation was used for synthesis of both the CDRs and the diastereomers. The newly synthesized CDRs were observed to be superior in comparison to their counterparts having amino acid amides as chiral auxiliaries in terms of cost effectiveness and providing better resolution of diastereomers. The method was validated for limit of detection, linearity, accuracy and precision.     

Activity and Synergistic Antimicrobial Activity Between Diketopiperazines Against Bacteria In Vitro

The aim of the present study was to determine the synergistic effects of diketopiperazines [cyclo-(l-Pro-l-Leu) (1), cyclo-(d-Pro-l-Leu) (2), and cyclo-(d-Pro-l-Tyr) (3)] purified from a Bacillus sp. N strain associated with entomopathogenic nematode Rhabditis (Oscheius) sp. on the growth of bacteria. The minimum inhibitory concentration and minimum bactericidal concentration of the diketopiperazines was compared with that of the standard antibiotics. The synergistic antibacterial activities of the combination of diketopiperazines against pathogenic bacteria were assessed using the checkerboard assay and time?Ckill methods. The results of the present study showed that the combination effects of diketopiperazines were predominately synergistic (FIC index <0.5). Furthermore, time?Ckill study showed that the growth of the tested bacteria was completely attenuated with 4?C12?h of treatment with 50:50 ratios of diketopiperazines. These results suggest that the combination of diketopiperazines may be microbiologically beneficial. The three diketopiperazines are nontoxic to normal human cell line (L231 lung epithelial) up to 200?m???g/ml. The in vitro synergistic activity of cyclo-(l-Pro-l-Leu), cyclo-(d-Pro-l-Leu), and cyclo-(d-Pro-l-Tyr) against bacteria is reported here for the first time. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (diketopiperazines).     

Synthesis and characterization of new N-phenylmaleimide thioglycosides

Thioglycosides derivatives of N-phenylmaleimide have been prepared by the reaction of derivatives of 1-thio-d-glucopyranose, d-galactopyranose, d-lactose, and d-maltose with 3,4-dichloro-N-phenylmaleimide. The reaction of 3,4-dichloro-N-phenyl maleimide with sugar thiols (protected or unprotected) took place by displacement of both chlorine atoms by sulfide nucleophile giving the corresponding bis-thioglycoside products.     

Efficient Microbial Conversion of l-Tyrosine to l-DOPA by Brevundimonas sp. SGJ

l-DOPA (3,4-dihydroxyphenyl-l-alanine), the most widely used drug for the treatment of Parkinson??s disease, was produced in buffer using biomass of Brevundimonas sp. SGJ. The effects of enhancers, such as carrageenan, diatomaceous earth, and activated charcoal, on the l-DOPA production were evaluated to obtain the maximum yield. The optimal process conditions found were pH?8, 2?g?l^?1 cell mass, 2?g?l^?1 l-tyrosine, 0.04?g?l^?1 CuSO₄, 0.02?g?l^?1 l-ascorbic acid, 0.5?g?l^?1 carrageenan, and 40?°C temperature. In addition, repeated use of cells resulted in the highest yield of 3.81?g?l^?1 (95.2%) of l-DOPA with utilization of 4?g?l^?1 l-tyrosine, and the highest tyrosinase activity (9,201?U?mg^?1) was observed at 18?h of incubation. Furthermore, the produced l-DOPA was confirmed by high-performance thin-layer chromatography, high-performance liquid chromatography, and gas chromatography?Cmass spectroscopy. Kinetic studies showed significant values of Y _p/s, Q _s, and q _s after optimization of the process. Thus, Brevundimonas sp. SGJ could be an eventual new source for large-scale production of l-DOPA.     

Stabilization ofd-amino acid oxidase from yeastTrigonopsis variabilis used for production of glutaryl-7-aminocephalosporanic acid from cephalosporin C

The studies to improve the production of glutaryl-7-ACA from cephalosporin C are described in this paper. During the conversion of cephalosporin C to keto-adipyl-7-aminocephalosporonic acid by d-amino acid oxidase (d-AAO), with the simultaneous production of equimolar amount of hydrogen peroxide, an incomplete nonenzymatic conversion of the keto form into the glutaryl form occurs, where cephalosporin C as well asd-AAO are partly destroyed in the presence of hydrogen peroxide. d-AAO was immobilized to different carriers in order to achieve better enzyme stability. The activity of immobilizedd-AAO on manganese oxide remained above 100% during the first 9 h of a semicontinuous conversion of cephalosporin C. The presence of catalase coimmobilized with D-AAO and coupled to CNBr-activated Sepharose 4B improved the operation stability ofd-AAO. An additional approach for the continuous transformation of cephalosporin C used whole cells ofTrigonopsis variabilis, containingd-AAO, immobilized to magnetic iron oxide particles.     

d-glucose Enhanced 5-Aminolevulinic Acid Production in Recombinant Escherichia coli Culture

In this study, we introduced a new strategy, feeding d-glucose, to overproduce extracellular 5-aminolevulinic acid (ALA) in the recombinant Escherichia coli. We investigated that the d-glucose concentration is dependent on extracellular ALA production. The results indicated that increasing d-glucose concentration in bacteria culture enhanced final cell density and ALA yield and simultaneously decreased the activities of ALA synthase (ALAS) and ALA dehydratase (ALAD); then, the inhibitory effect of d-glucose on ALAS activity was relieved with the metabolism of d-glucose. when 4.0 g/L d-glucose was added at late exponential phase; 1.46 g/L ALA was achieved in shaking culture, which is 47% or 109% higher than the ALA yields with 30 mM levulinic acid of ALAD inhibitor or no inhibitor. In jar fermenter, final extracellular ALA concentration reached 3.1 g/L by feeding with d-glucose.