Determination of Tryptophan in Lysozyme and Lysozyme Hydrolysate by Chiral LC Using d-Tryptophan as Internal Standard

In the present study, a new LC method is described for the quantitation of tryptophan (Trp) in lysozyme and enzymatic lysozyme hydrolysate. To compensate for partial breakdown of Trp during hydrolysis with 4 M methanesulfonic acid, an enantiomer dilution method was developed. The method makes use of free d-Trp or a d-Trp-containing dipeptide as internal standard for the quantitation of l-tryptophan in these matrices. After acid hydrolysis in 4 M methanesulfonic acid, LC analysis is performed on a Crownpak CR chiral column in combination with fluorescence detection. Optimum time and temperature for the acid hydrolysis were investigated in order to obtain complete hydrolysis of the source materials. A comparison of the l-Trp recoveries was made for d-Trp and Gly-d-Trp as internal standards. By choosing a hydrolysis time of 150 min at 150 °C, 93% recovery of l-Trp from lysozyme was achieved. Under these conditions, no racemization occurred. When choosing d-Trp as internal standard, a direct LC method for l-Trp in lysozyme and enzymatic lysozyme hydrolysate was established without the need for pre-column derivatization and without the need to use Trp protecting agents during acid hydrolysis.     

Enzymatic synthesis of tryptamine and its halogen derivatives selectively labeled with hydrogen isotopes

Nine isotopomers of tryptamine and its halogen derivatives, labeled with deuterium, tritium in side chain, i.e., [(1R)-²H]-, [(1R)-³H]-, 5-F-[(1R)-²H]-, 5-F-[(1R)-³H]-, 5-Br-[(1R)-²H]-, double labeled [(1R)-²H/³H]-, 5-F-[(1R)-²H/³H]-, and ring labeled [4-²H]-, and [5-²H]-tryptamine, were obtained by enzymatic decarboxylation of l-Trp and its appropriate derivatives in deuteriated or tritiated media, respectively. Intermediates: [5′-²H]-l-Trp used for further decarboxylation was synthesized by enzymatic coupling of [5-²H]-indole with S-methyl-l-cysteine, and [4′-²H]-l-Trp was obtained by isotope exchange ¹H/²H of the authentic l-Trp dissolved in heavy water induced by UV-irradiation. Doubly labeled [(1R)-²H/³H]- and 5-F-[(1R)-²H/³H]-tryptamine were obtain by decarboxylation of l-Trp or [5′-F]-l-Trp carried out in ²H³HO incubation medium.     

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.     

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.     

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%.     

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.

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.     

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.

     

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.     

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).     

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.     

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.     

Enantioselective extraction of fenvaleric acid enantiomers by two-phase (W/O) recognition chiral extraction

To establish an extraction method for fenvaleric acid (FA) enantiomers using l-iso-butyl-l-tartaric esters and hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector, the distribution of FA enantiomers was examined in methanol aqueous solution containing HP-β-CD and 1,2-dichloroethane organic solution containing l-iso-butyl-l-tartaric esters. The influences of the concentration of l-iso-butyl-l-tartaric esters and HP-β-CD, organic diluent, pH, extraction temperature and the concentration of methanol aqueous solution on the partition coefficient (k) and separation factor (α) of FA were investigated. The experiment results showed that the complex formed by l-iso-butyl-l-tartaric esters with S-enantiomer is stabler than that with R-enantiomer. With the increase of the concentration of l-iso-butyl-l-tartaric ester, k and α increased; With the increase of the concentration of HP-β-CD, k increased firstly, and then decreased, but α increased all the while, k was the highest when the concentration of HP-β-CD was 4 mmol L^?1. 1,2-dichloroethane organic diluent was better than the others. With the increase of pH, k and α decreased; with further increasing concentration of methanol aqueous solution, k and α decreased, k and α were the highest when the concentration of methanol aqueous solution was 10%. The extraction temperature had a great influence on k and α, too.     

Partial enzymatic elimination and quantification of sarcosine from alanine using liquid chromatography–tandem mass spectrometry

Since sarcosine and d,l-alanine co-elute on reversed-phase high-performance liquid chromatography (HPLC) columns and the tandem mass spectrometer cannot differentiate them due to equivalent parent and fragment ions, derivatization is often required for analysis of sarcosine in LC/MS systems. This study offers an alternative to derivatization by employing partial elimination of sarcosine by enzymatic oxidation. The decrease in apparent concentration from the traditionally merged sarcosine–alanine peak associated with the enzymatic elimination has been shown to be proportional to the total sarcosine present (R ²?=?0.9999), allowing for determinations of urinary sarcosine. Sarcosine oxidase was shown to eliminate only sarcosine in the presence of d,l-alanine, and was consequently used as the selective enzyme. This newly developed technique has a method detection limit of 1 μg/L (parts per billion) with a linear range of 3 ppb–1 mg/L (parts per million) in urine matrices. The method was further validated through spiked recoveries of real urine samples, as well as the analysis of 35 real urine samples. The average recoveries for low, middle, and high sarcosine concentration spikes were 111.7, 90.8, and 90.1 %, respectively. In conclusion, this simple enzymatic approach coupled with HPLC/MS/MS is able to resolve sarcosine from d,l-alanine leading to underivatized quantification of sarcosine.

Characteristics of α-l-Arabinofuranosidase from Streptomyces sp I10-1 for Production of l-Arabinose from Corn Hull Arabinoxylan

Streptomyces sp I10-1 α-l-arabinofuranosidase efficiently produced l-arabinose from high arabinose-content corn hull arabinoxylan (ratio of arabinose to xylose, 0.6). The optimum pH at 40 °C was around 6, and the enzyme was stable from pH 5 to 11. The optimum temperature was 50 °C at pH 5, and the activity was stable at 40 °C. The enzymatic activity against corn hull arabinoxylan was 2.3 times higher than towards p-nitrophenyl-α-l-arabinofuranoside. Approximately 45 % l-arabinose recovery was achieved from corn hull arabinoxylan. It was considered that l-arabinose residues not removed by the enzyme were attributable to those linked with ferulic acid. The open reading frame of the enzyme gene consisted of 1,224 bp, and the predicted peptide was 408 amino acids, which corresponded to a molecular size of 45, 248 Da. It was presumed that the smaller molecular size (31,000 Da) estimated on SDS-PAGE resulted from proteolysis by proteases. I10-1 α-l-arabinofuranosidase belongs to the Alpha-l-AF C superfamily, which is associated with glycoside hydrolase family 51, but the properties were unique.     

A new approach for quantitative analysis of l-phenylalanine using a novel semi-sandwich immunometric assay

Here, we describe a novel method for l-phenylalanine analysis using a sandwich-type immunometric assay approach for use as a new method for amino acid analysis. To overcome difficulties of the preparation of high-affinity and selectivity monoclonal antibodies against l-phenylalanine and the inability to use sandwich-type immunometric assays due to their small molecular weight, three procedures were examined. First, amino groups of l-phenylalanine were modified by “N-Fmoc-l-cysteine” (FC) residues and the derivative (FC-Phe) was used as a hapten. Immunization of mice with bovine serum albumin/FC-Phe conjugate successfully yielded specific monoclonal anti-FC-Phe antibodies. Second, a new derivatization reagent, “biotin linker conjugate of FC-Phe N-succinimidyl ester” (FC(Biotin)-NHS), was synthesized to convert l-phenylalanine to FC-(Biotin)-Phe as a hapten structure. The biotin moiety linked to the thiol group of cysteine formed a second binding site for streptavidin/horseradish peroxidase (HRP) conjugates for optical detection. Third, a new semi-sandwich-type immunometric assay was established using pre-derivatized l-phenylalanine, the monoclonal anti-FC-Phe antibody, and streptavidin/HRP conjugate (without second antibody). Using the new “semi-sandwich” immunometric assay system, a detection limit of 35 nM (60 amol per analysis) and a detection range of 0.1–20 μM were attained using a standard l-phenylalanine solution. Rat plasma samples were analyzed to test reliability. Intra-day assay precision was within 6 % of the coefficient of variation; inter-day variation was 0.1 %. The recovery rates were from 92.4 to 123.7 %. This is the first report of the quantitative determination of l-phenylalanine using a reliable semi-sandwich immunometric assay approach and will be applicable to the quantitative determination of other amino acids.     

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.     

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.     

Study of the interaction between ethanol and natural amino acids containing ionic side groups in water at T = 298.15 K

The enthalpies of solution of l-α-aspartic acid, l-α-glutamic acid, l-α-arginine, l-α-lysine, and l-α-histidine have been measured in aqueous ethanol solutions at 298.15 K. From the obtained experimental results, the standard enthalpies of solution of amino acids in water–ethanol solutions have been determined. These data were used to calculate the heterogeneous enthalpic pair interaction coefficients based on McMillan–Mayer’s formalism. These values were interpreted in the terms of the ionic or no polar effect of the side chains of l-α-amino acids on their interactions with a molecule of ethanol in water.     

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.