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.     

Carbohydrate recognition of symmetrical tripodal receptor type tris(2-aminoethyl)amine derivatives

Carbohydrate recognition of some bioactive symmetrical tripodal receptor type tris(2-aminoethyl)amine (TAEA) derivatives was investigated. In calorimetric experiments, the highest binding constant (Ka) of compound C (C₃₅H₄₉N₅O₄S) with methyl α-d-mannopyranoside was Ka = 858 M^?1 with 1:1 stoichiometry. Formation of hydrogen bonds in binding between symmetrical tripodal receptor type compound C and sugars was suggested by the large negative values of ?H° (=?34 to ?511 kJ mol^?1). In a comparison of each set of α- and β-anomers of some monosaccharides (methyl α/β-d-galactopyranoside, methyl α/β-d-glucopyranoside, and methyl α/β-l-fucopyranoside), compound C showed that the binding constant of β-anomer was larger than that of the corresponding α-anomer, indicating higher β-anomer selectivity. The calculated energy-minimized structure of the complex of compound C with guest methyl α-d-mannopyranoside is also presented. The experimental results obtained from this work indicated that symmetrical tripodal receptor type TAEA derivative C has a lectin-like carbohydrate recognition property.     

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.

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.     

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%.     

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.     

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).     

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.

     

Interactions of some protonated α-amino acid methyl esters with hexaethyl p-tert-butylcalix[6]arene hexaacetate in nitrobenzene saturated with water

The exchange extraction constants corresponding to the general equilibrium C⁺(aq) + 1·Cs⁺(nb) ? 1·C⁺ (nb) + Cs⁺(aq) occurring in the two-phase water–nitrobenzene system (C⁺ = protonated α-amino acid methyl ester, 1 = hexaethyl p-tert-butylcalix[6]arene hexaacetate; aq = aqueous phase, nb = nitrobenzene phase) were evaluated on the basis of extraction experiments and γ-activity measurements. Further, the stability constants of the 1·C⁺ cationic complex species in nitrobenzene saturated with water were calculated; they were found to increase in the following cation order: protonated l-tryptophan methyl ester < protonated l-phenylalanine methyl ester < protonated l-leucine methyl ester < protonated l-methionine methyl ester < protonated l-valine methyl ester.     

Untersuchungen über die Biosynthese der Cyclite, 25. Mitt.: Vorkommen und Bildung von Condurit inMarsdenia-Arten

A systematic study of tropical Asclepiadaceae shows thatMarsdenia abyssinica, M. zambesica, M. erecta, M. angolensis, andDragea Faulknerae contain conduritol as their main cyclitol component. Taking earlier reports into consideration, these findings mean that conduritol can be found only in the subfamily Cynanchoideae of the Asclepiadaceae. This very restricted distribution is in contrast to that of its isomer,l-leucanthemitol, which—at least in traces—can be detected in almost all green plants. Infusion experiments with¹⁴C-labelled possible precursors of conduritol inMarsdenia abyssinica showed incorporation ofd-glucose andd-galactose into the cyclitol. The hexose molecules seem to be built in into conduritol as such, i.e. without being fragmented previously.l-Leucanthemitol is incorporated into conduritol with a much better radiochemical yield than the two hexoses. This result suggests that the last step in the biosynthesis of conduritol is an epimerization ofl-leucanthemitol to conduritol.     

The crystal structures of chiral (R/S) (C8H11N)2·Cu(OAc)2

The two title chiral complexes (R/S) (C₈H₁₁N)₂·Cu(OAc)₂ were synthesized by the reaction of chiral d(+)/l(?)-??-ethylphenyl amine with copper(II) acetate monohydrate in dry ethanol. The crystal structures of 1a and 1b were obtained and determined by X-ray crystallography.     

Reagentless d-sorbitol biosensor based on d-sorbitol dehydrogenase immobilized in a sol–gel carbon nanotubes–poly(methylene green) composite

A reagentless d-sorbitol biosensor based on NAD-dependent d-sorbitol dehydrogenase (DSDH) immobilized in a sol–gel carbon nanotubes–poly(methylene green) composite has been developed. It was prepared by durably immobilizing the NAD⁺ cofactor with DSDH in a sol–gel thin film on the surface of carbon nanotubes functionalized with poly(methylene green). This device enables selective determination of d-sorbitol at 0.2 V with a sensitivity of 8.7?μA?mmol^?1?L?cm^?2 and a detection limit of 0.11 mmol?L^?1. Moreover, this biosensor has excellent operational stability upon continuous use in hydrodynamic conditions.

d- and l-amino acids in Antarctic lakes: assessment of a very sensitive HPLC-MS method

Amino acids represent a fraction of organic matter in marine and freshwater ecosystems, and a source of carbon, nitrogen and energy. l-Amino acids are the most common enantiomers in nature because these chiral forms are used during the biosynthesis of proteins and peptide. To the contrary, the occurrence of d-amino acids is usually linked to the presence of bacteria. We investigated the distribution of l- and d-amino acids in the lacustrine environment of Terra Nova Bay, Antarctica, in order to define their natural composition in this area and to individuate a possible relationship with primary production. A simultaneous chromatographic separation of 40 l- and d-amino acids was performed using a chiral stationary phase based on teicoplainin aglycone (chirobiotic tag). The chromatographic separation was coupled to two different mass spectrometers—an LTQ-Orbitrap XL (Thermo Fisher Scientific) and an API 4000 (ABSciex)—in order to investigate their quantitative performance. High-performance liquid chromatography coupled with mass spectrometry methods were evaluated through the estimation of their linear ranges, repeatability, accuracy and detection and quantification limits. The high-resolution mass spectrometer LTQ-Orbitrap XL presented detection limits between 0.4 and 7 μg?l ^?1, while the triple quadrupole mass spectrometer API 4000 achieved the best detection limits reported in the literature for the quantification of amino acids (between 4 and 200 ng?l ^?1). The most sensitive method, HPLC-API 4000, was applied to lake water samples. Figure

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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.     

Enantioselective Separation and Determination of Carnosine in Rat Plasma by Fluorescence LC for Stereoselective Pharmacokinetic Studies

A sensitive fluorescence liquid chromatographic analytical method was developed for the simultaneous determination of carnosine enantiomers in rat plasma. The method was applied to pharmacokinetic studies. Chiral separation of carnosine enantiomers was achieved by pre-column derivatization with o-phthaldialdehyde and the thiol N-acety-l-cysteine as derivating reagents. They were separated on an ODS column and detected by fluorescence detection (λ_ex = 350 nm, λ_em = 450 nm). γ-Aminobutyric acid was used as internal standard. The method was linear up to 6,000 ng mL^?1 for l-carnosine, 4,000 ng mL^?1 for d-carnosine. Low limit of quantitation (LLOQ) was 40 ng mL^?1 for each isomer. The relative standard deviations obtained for intra- and inter-day precision were lower than 12% and the recoveries were higher than 75% for both enantiomers. The method was applied to a stereoselective study on the pharmacokinetics of carnosine after oral administration with a single dose (carnosine, 75 mg kg^?1 for each isomer) to a rat. The initial data indicated that l-carnosine had a larger value of the highest plasma concentration than d-carnosine (C _max 5,344 vs. 1,914 ng mL^?1), and that of l-carnosine had a lower value of AUC_(0?∞) and t _1/2(h) (AUC_(0?∞) 5,306 vs. 6,321 ng h mL^?1, t _1/2 1.43 vs. 3.37 h). Our results indicated that the pharmacokinetic of l-carnosine and d-carnosine revealed enantioselective properties significantly.     

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.     

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.     

Numerical modelling of inclusion-type complexes formed by chiral 18-crown-6 ethers bearing sugar moieties with enantiomers of phenylalanine methyl ester cations

The crystal structure of α-d-mannosido-benzo-18-crown-6·KSCN (1) was solved by X-ray single crystal diffractometry. C₂₈H₃₆O₁₀·KSCN is orthorhombic, space groupP2₁2₁2₁ withZ=4,a=8.035(4),b=9.960(2),c=38.83(2) Å,M _r =629.8,V=3103.6 ų,D _x =1.347 g cm^?3, μ(CuKα)=2.53 mm^?1, λ=1.54178 Å,F(000)=1324. FinalR=0.043 for 1139 unique observed reflections measured at room temperature. The potassium ion is surrounded by a nearly planar hexagon of oxygen atoms of the macrocyclic ring and lies on the plane formed by those atoms. Hexagonal pyramidal coordination is completed by the nitrogen atom of the thiocyanate anion. The SCN ion was found on the face of the macrocyclic ring opposite that for the chiral mannopyranoside moiety. The molecular structure of α-d-mannosido-18-crown-6 (2) and the structure of molecular complexes of2 and α-d-glucosido-benzo-18-crown-6 (3) were studied by molecular mechanics methods. The results suggest enthalpy driven selectivity of complexation of the phenylalanine methyl ester (4) by2 and both enthalpy and entropy effects in selective complexation of4 by3.     

Synthesis ofS-(Carboxymethyl)-d-cysteine by 3-chloro-d-alanine chloride-lyase ofPseudomonas putida CR 1-1

S-(Carboxymethyl)-d-cysteine, which is an important component of semisynthetic cephalosporin, MT-141, was enzymatically synthesized.S-(Ethoxy-carbonyl-methyl)-d-cystein was synthesized from 3-chloro-d-alanine and ethyl thioglycolate by the β-replacement reaction of 3-chloro-d-alanine chloride-lyase fromPseudomonas putida CR 1-1 and subsequently hydrolyzed by alkali. The synthesizedS-(carboxymethyl)-d-cysteine was isolated from a large scale reaction mixture and identified physicochemically. The reaction conditions for the synthesis ofS-(ethoxycarbonylmethyl)-d-cysteine were optimized using resting cells ofP. putida CR 1-1.