Synthesis and crystal structures of the heptafluoroacetylacetonatocopper(ii) and 4-iminoheptafluoropent-2-ene-2-aminatocopper(ii) complexes with triphenylphosphine oxide and pyridine

New chelate heptafluoroacetylacetonatocopper(ii) complexes with triphenylphosphine oxide and pyridine as well as the 4-iminoheptafluoropent-2-ene-2-aminatocopper(ii) complex with triphenylphosphine oxide were synthesized. The molecular and crystal structures of the complexes were studied by X-ray diffraction analysis. The reaction of 2-amino-4-imino-heptafluoropent-2-ene with CuO affords bis(4-imino-1,1,1,3,5,5,5-heptafluoropent-2-ene-2-aminato)copper(ii) in ～90% yield.     

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.     

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.     

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.     

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.     

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.     

Exploiting thermodynamic data to optimize the enantioseparation of underivatized amino acids in ligand exchange capillary electrophoresis

Stereoselective amino acid analysis has increasingly moved into the scope of interest of the scientific community. In this work, we report a study on the chiral separation of underivatized d,l-His by ligand exchange capillary electrophoresis (LECE), utilizing accurate ex ante calculations. This has been obtained by the addition to the background electrolytes (BGE) of NaClO₄ which renders the separations “all in solution processes”, allowing to accurately calculate in advance the concentrations of the species present in solution and to optimize the system performances. To this aim, the formation of ternary complexes of Cu²⁺ ion and l-lysine (l-Lys) or l-ornithine (l-Orn) with l- and d-histidine (His), and histamine (Hm) have been studied by potentiometry and calorimetry at 25 °C and with 0.1 mol dm^?3 (KNO₃) in aqueous solution. The ternary species [Cu(L)(l-His)H]⁺ and [Cu(L)(d-His)H]⁺ (where L?=?l-Lys or l-Orn) show a slight but still detectable stereoselectivity, and the determination of ΔH° and ΔS° values allowed the understanding of the factors which determine this phenomenon. The stereoselectivity showed by the protonated ternary species has been exploited to chirally separate d,l-His in LECE, by using the binary complexes of copper(II) with l-Lys or l-Orn as background electrolytes added with the appropriate amounts of NaClO₄.

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.     

The pivotal role of copper(II) in the enantiorecognition of tryptophan and histidine by gold nanoparticles

Stereoselective amino acid analysis has increasingly moved into the scope of interest of the scientific community. In this work, we report a study on the chiral recognition of d,l-Trp and d,l-His using l-Cys-capped gold nanoparticles (AuNPs) and copper(II) ion. In the l-Cys-capped AuNPs, the thiol group of the amino acid interacts with AuNPs through the formation of Au–S bond, whereas the α-amino and α-carboxyl groups of the surface-confined cysteine can coordinate the copper(II) ion, which in turn, binds the l- or d-amino acid present in solution forming diastereoisomeric complexes. The resulting systems have been characterized by UV–Vis spectra and dynamic light scattering measurements, obtaining different results for l- and d-Trp, as well as for l- and d-His. The knowledge of the solution equilibria of the investigated systems allowed us to accurately calculate in advance the concentrations of the species present in solution and to optimize the system performances, highlighting the pivotal role of copper(II) ion in the enantiodiscrimination processes.     

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).     

Studies on enzymatic oxidation of 3′,4′-dihydroxy-l-phenylalanine to dopachrome using kinetic isotope effect methods

We report the studies on the mechanism of oxidation of 3′,4′-dihydroxy-l-phenylalanine (l-DOPA) to neurotoxic dopachrome catalyzed by enzyme horseradish peroxidase (EC 1.11.1.7) using the kinetic (KIE), and solvent (SIE), isotope effect methods. For kinetic studies two specifically deuterated isotopomers: [2′,5′,6′-²H₃]-l -DOPA was synthesized by the acid catalyzed isotopic exchange between native l-DOPA and heavy water, and [5′-²H]-l-DOPA was synthesized in two step reaction. The first step involved acid catalyzed isotopic exchange between l-tyrosine and deuterated water and resulting product [3′,5′-²H₂]-l-tyrosine was hydroxylated by enzyme tyrosinase (EC 1.14.18.1). The values of deuterium KIEs and SIE’s in the enzymatic oxidation of l-DOPA and its isotopomers are determined using non-competitive spectrophotometric method. The measured values were: KIE on V _max (1.1 and 2.2) and KIE on V _max/K _M (1.7 and 3.2) for [2′,5′,6′-²H₃]-l-DOPA and [5′-²H]-l-DOPA, respectively, while the corresponding values of SIE were: SIE on V _max (2.1, 2.4, and 2.1) and SIE on V _max/K _M (1.3. 1.6, and 1.1) for l-DOPA, [2′,5′,6′-²H₃]-l-DOPA, and [5′-²H]-l-DOPA, respectively. The size of KIE and SIE, typical for secondary isotope effects indicate that both the solvent and presence of deuterium at the 2′-, 5′, and 6′-positions of l-DOPA has the little impact on the enzymatic oxidation of this compound.     

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%.     

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.     

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.     

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.     

Sensitive chemiluminescence method for the determination of glutathione, l-cysteine and 6-mercaptopurine

Glutathione (GSH), l-cysteine (l-Cys) and 6-mercaptopurine (6-MP) inhibit the CL reaction of luminol–H₂O₂ catalyzed by gold colloids. In order to explore this, GSH, l-Cys and 6-MP were injected into the chemiluminescence system of luminol and H₂O₂ catalyzed by gold colloids. The results showed that gold colloids interact with GSH, l-Cys and 6-MP and decrease the CL emission. Based on this phenomenon, a simple, sensitive and convenient flow injection CL method was developed for the determination of GSH, l-Cys and 6-MP. This method provides a novel, and effective CL assay for GSH, l-Cys and 6-MP that has been applied to the determination of GSH in human serum.     

An efficient glucose-based ligand for Heck and Suzuki coupling reactions in aqueous media

The glucose-based ligand, N-salicylidene-d-glucosamine (Sal-d-glsmN), was readily obtained by reaction of salicylaldehyde (Hsal) with the d-glucosamine hydrochloride. Ligand Sal-d-glsmN was found to be an efficient ligand in the palladium-catalyzed Suzuki and Heck C–C coupling reactions in aqueous medium under aerobic condition. It was found that the use of Sal-d-glsmN/Pd(OAc)₂ system as a catalyst, aryl halides undergo Suzuki and Heck cross-couplings, respectively, with arylboronic acids and olefins to give the desired products in moderate to excellent yields.     

A new chiral electrochemical sensor for the enantioselective recognition of penicillamine enantiomers

A new chiral electrochemical sensor has been successfully prepared through chemical linking l-methotrexate (l-Mtx) onto the gold electrode surface. Cyclic voltammetry and electrochemical impedance spectroscopy were used to investigate the enantioselective interaction between l-Mtx and Pen enantiomers. The results showed that the l-Mtx-modified gold electrode can selectively recognize penicillamine (Pen) enantiomers using Zn(II) as central ion, and larger response signal was observed from d-Pen owing to the selective formation of Zn complexes. The interaction time between the modified electrode and Pen enantiomers containing Zn(II) was considered. And the electrochemical response of the modified electrode to a series of different concentration of Pen in the presence of Zn(II) was also monitored. In addition, the enantiomeric composition of d- and l-Pen enantiomer mixtures was monitored by measuring the current responses of the sample.     

Synthesis of ruthenium(iii) and rhodium(iii) tris-acetylacetonates and palladium(ii) bis-ketoiminate using microwave heating

Preparation of ruthenium(iii) and rhodium(iii) tris-acetylacetonates and palladium(ii) bisketoiminate (Pd(i-acac)₂) under microwave irradiation using different synthetic conditions, both in the solid-phase and in solution, was studied with precise control of parameters. In the solid-phase systems, the preparation of the target product was hindered. The efficiency of the microwave heating increased when liquid phases of the reagent mixtures were used. For Pd(i-acac)₂, the highest yield was achieved under elevated temperature of the process, with the reaction time decreasing to several minutes. A laboratory procedure for the microwave synthesis of ruthenium(iii) and rhodium(iii) tris-acetylacetonates and palladium(ii) bis-ketoiminate in aqueous solutions was developed, which allowed us to obtain them in 85, 55, and 80% yields, respectively. These yields are higher than those reported in the literature, with the process becoming considerably less time consuming and laborious.     

Neue enzymatische Analysenmethoden zur Bestimmung von Maltose,Stärke und Lactat in der Lebensmittelchemie

1. Determination of Maltose. Maltose is hydrolyzed by the enzyme α-glucosidase into glucose, which is determined by the enzymes hexokinase and glucose-6-phosphate-dehydrogenase. α-Glucosidase is specific for oligosaccharides with α-1,4 and α-1,2 bonds.
2. Determination of Starch and Glycogen. Starch and glycogen are splitted to glucose by the enzyme amylo-glucosidase. Starch has to be dissolved before enzymatic cleavage. A comparison of different methods for preparing starch solutions is given.
3. Determination of d- and l-Lactate. It is possible to determine d-lactate and l-lactate with the specific enzymes d-lactate-dehydrogenase and l-lactate-dehydrogenase. By different samples it is shown that no equal quantities of d- and l-lactate were found in the analyzed foods.