RP-LC Resolution of (R,S)-Atenolol via Diastereomerization with Marfey’s Reagent and Its Structural Variants Under Conventional and Microwave Heating

(R,S)-Atenolol was derivatized with Marfey’s reagent, (MR; 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide or FDNP-l-Ala-NH₂) and its four structural variants (FDNP-l-Phe-NH₂, FDNP-l-Val-NH₂, FDNP-l-Leu-NH₂ and FDNP-l-Pro-NH₂). MR reacts quantitatively with 1° and 2° amino groups and atenolol has a secondary amino group. The derivatization reactions were carried out under conventional and microwave heating and compared. The resulting diastereomers were separated on RP-TLC and on a C18 column with detection at 340 nm. (R)-Isomer eluted before (S). The conditions of derivatization and chromatographic separation were optimized. The method was validated for linearity, repeatability, limits of detection and limit of quantification.     

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.     

Analysis of multicomponent mixture and simultaneous enantioresolution of proteinogenic and non-proteinogenic amino acids by reversed-phase high-performance liquid chromatography using chiral variants of Sanger’s reagent

Four chiral derivatizing reagents (CDR 1–4), namely, FDNP-l-Ala, FDNP-l-Val, FDNP-l-Phe, and FDNP-l-Leu, were synthesized using microwave (MW) irradiation by substituting one of the fluorine atoms in difluoro dinitro benzene (DFDNB) with l-Ala, l-Val, l-Phe, and l-Leu (CDR 1–4). The other set of CDRs, namely, FDNP-l-Phe-NH₂, FDNP-l-Val-NH₂, and FDNP-l-Leu-NH₂, was also prepared. These reagents were used for synthesis of diastereomers of 18 proteinogenic and 08 non-proteinogenic amino acids, which were resolved by reversed-phase high-performance liquid chromatography using C18 column and gradient eluting mixture of aq.TFA and acetonitrile with UV detection at 340 nm. The reagents were used for resolution of a complex mixture of 18 racemic proteinogenic amino acids in a single chromatographic run of 65 min and to determine concentration of the d-amino acid in a solution of dl-amino acid. The resolution (R _S) and selectivity (α) obtained for the two sets of diastereomers were compared among themselves and among the two groups. The method was validated for accuracy, precision, limit of detection (LOD), and limit of quantification. LOD is 0.001% impurity of d-enantiomer.     

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).     

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.     

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.     

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.

     

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%.     

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.     

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.     

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.     

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.

Chiral resolution of l- and d-alanine and a racemic macrocyclic nickel(II) complex: synthesis and crystal structures

The reactions of a racemic four-coordinate Ni(II) complex [Ni(rac-L)](ClO₄)₂ with l- and d-alanine in acetonitrile/water gave two six-coordinate enantiomers formulated as [Ni(RR-L)(l-Ala)](ClO₄)·2CH₃CN (1) and [Ni(SS-L)(d-Ala)](ClO4) (2) (L = 5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclo-tetradecane, Ala^? = alanine anion), respectively. Evaporation from the remaining solutions gave two four-coordinate enantiomers characterized as [Ni(SS-L)](ClO₄)₂ (S-3) and [Ni(RR-L)](ClO₄)₂ (R-3), respectively. Single-crystal X-ray diffraction analyses of complexes 1 and 2 revealed that the Ni(II) atom has a distorted octahedral coordination geometry, being coordinated by four nitrogen atoms of L in a folded configuration, plus one carboxylate oxygen atom and one nitrogen atom of l- or d-Ala^? in mutually cis-positions. Complexes 1 and 2 are supramolecular stereoisomers, constructed via hydrogen bonding between [Ni(RR-L)(l-Ala)]⁺ or [Ni(SS-L)(d-Ala)]⁺ monomers to form 1D hydrogen-bonded zigzag chains. The homochiral natures of complexes 1 and 2 have been confirmed by CD spectroscopy.     

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.     

Comparative investigation on the enthalpy relaxation of four amorphous pentose isomers

The enthalpy relaxation of the pentose isomers (d-xylose, d-ribose, l-arabinose, and d-arabinose) was investigated in terms of the Tool–Narayanaswamy–Moynihan and Adam–Gibbs–Vogel models using differential scanning calorimetry. Single set of parameters for each model was obtained from the multicurve-fitting procedure. The differences between the glass transition and relaxation parameters were discussed in terms of isomeric effect. The cooperativity determined from curve-fitting results was further compared to those obtained via Donth’s formula.     

Stereospecific electrophoretically mediated microanalysis assay for methionine sulfoxide reductase enzymes

An electrophoretically mediated microanalysis assay (EMMA) for the determination of the stereoselective reduction of l-methionine sulfoxide diastereomers by methionine sulfoxide reductase enzymes was developed using fluorenylmethyloxycarbonyl (Fmoc)-l-methionine sulfoxide as substrate. The separation of the diastereomers of Fmoc-l-methionine sulfoxide and the product Fmoc-l-methionine was achieved in a successive multiple ionic-polymer layer-coated capillary using a 50 mM Tris buffer, pH 8.0, containing 30 mM sodium dodecyl sulfate as background electrolyte and an applied voltage of 25 kV. 4-Aminobenzoic acid was employed as internal standard. An injection sequence of incubation buffer, enzyme, substrate, enzyme, and incubation buffer was selected. The assay was optimized with regard to mixing time and mixing voltage and subsequently applied for the analysis of stereoselective reduction of Fmoc-l-methionine-(S)-sulfoxide by human methionine sulfoxide reductase A and of the Fmoc-l-methionine-(R)-sulfoxide by human methionine sulfoxide reductase B. The Michaelis–Menten constant, K _m, and the maximum velocity, v _max, were determined. Essentially identical data were determined by the electrophoretically mediated microanalysis assay and the analysis of the samples by CE upon offline incubation. Furthermore, it was shown for the first time that Fmoc-methionine-(R)-sulfoxide is a substrate of human methionine sulfoxide reductase B.

A Validated Chiral LC Method for the Enantiomeric Separation of Nateglinide and the Quantitative Determination of Its l-Enantiomer

A simple and accurate chiral liquid chromatographic method was developed for the enantiomeric purity determination of d-nateglinide and quantitative determination of l-nateglinide in bulk drug samples. Good resolution (R _s  > 6.0) between d-enantiomer and l-enantiomer of nateglinide were achieved with Chiralpak AD-H (250 × 4.6 mm, 5 μm particle size) column using hexane and ethanol (90:10 v/v) as mobile phase at 25 °C temperature. Flow rate was kept as 1.0 mL min^?1 and elution was monitored at 210 nm. The effects of the mobile phase composition, the flow rate and the temperature on the chromatographic separation were investigated. Developed method is capable to detect (LOD) and quantitate (LOQ) l-nateglinide to the levels of 0.3 and 1.0 μg mL^?1 respectively, for 10 μL injection volume. The percentage RSD of the peak area of six replicate injections of l-nateglinide at LOQ concentration was 5.2. The percentage recoveries of l-nateglinide from d-nateglinide ranged from 97.9 to 99.7. The test solution and mobile phase was found to be stable up to 24 h after preparation. The developed method was validated with respect to LOD, LOQ, precision, linearity, accuracy, robustness and ruggedness.     

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.