Simultaneous Determination of Safingol and d-erythro-Sphinganine in Human Plasma by LC with Fluorescence Detection

l-threo-Sphinganine (safingol) is a putative synthetic sphingosine kinase inhibitor currently being tested in clinical trials as an anticancer agent. To enable defining the pharmacokinetic properties of safingol in humans, we developed a sensitive analytical method to simultaneously quantitate safingol and its naturally-occurring diastereomer, d-erythro-sphinganine in human plasma. Of the two different fluorogenic derivatization agents (NDA and OPA) and several pH conditions compared for the derivatization, we found that NDA derivatization achieved more than 20 times greater sensitivity compared with OPA derivatization, and pH 9.0 showed the highest sensitivity for both compounds. An analytical method for liquid chromatography (LC) with a fluorescence detector (FLD) was developed and validated with calibration curve ranges of 20–1,000 ng mL^?1 for safingol and d-erythro-sphinganine. Our LC-FLD method using NDA-derivatization enabled simultaneous quantification of safingol and its naturally-occurring diastereomer, d-erythro-sphinganine with satisfactory sensitivity in human plasma.     

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

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

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.     

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%.     

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.     

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.     

Asteropsiside A and other asterosaponins from the starfish Asteropsis carinifera

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

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.     

Preparative Isolation and Purification of Flavonoids and Protocatechuic Acid from Sea Buckthorn Juice Concentrate (Hippophaë rhamnoides L. ssp. rhamnoides) by High-Speed Counter-Current Chromatography

High-speed counter-current chromatography (HSCCC)—a support free all liquid–liquid chromatography technique—has been successfully used for the preparative isolation of isorhamnetin 3-O-β-d-glucoside, isorhamnetin 3-O-β-rutinoside, quercetin 3-O-β-d-glucoside, syringetin 3-O-β-d-glucoside and protocatechuic acid from sea buckthorn juice concentrate (Hippophaë rhamnoides L. ssp. rhamnoides, Elaeagnaceae). The preparative HSCCC instrument was a multilayer coil planet centrifuge equipped with three preparative coils. Separation was performed with a two phase solvent system (n-hexane–n-butanol–water, 1:1:2 v/v/v) in ‘head-to-tail’ mode. Each injection of 4.1 g crude ethyl acetate extract yielded isorhamnetin 3-O-β-d-glucoside (95 mg), isorhamnetin 3-O-β-rutinoside (10 mg), quercetin 3-O-β-d-glucoside (5 mg), and protocatechuic acid (34 mg) with purities >98%. The flavonoid syringetin 3-O-β-d-glucoside (2 mg) was a novel compound for H. rhamnoides. Chemical structures of all compounds were determined by HPLC–ESI–MS–MS, 1D-NMR (¹H, ¹³C, DEPT 135) spectroscopy and for elucidation of glycosidic linkages 2D-NMR (HMBC) spectroscopy was used.     

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

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

β-d-Xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-Directed Mutagenesis, Alternate Substrates, and Active-Site Inhibitor

β-d-Xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-β-d-xylooligosaccharides to d-xylose. Catalysis and inhibitor binding by the GH43 β-xylosidase are governed by the protonation states of catalytic base (D14, pK _a 5.0) and catalytic acid (E186, pK _a 7.2). Biphasic inhibition by triethanolamine of E186A preparations reveals minor contamination by wild-type-like enzyme, the contaminant likely originating from translational misreading. Titration of E186A preparations with triethanolamine allows resolution of binding and kinetic parameters of the E186A mutant from those of the contaminant. The E186A mutation abolishes the pK _a assigned to E186; mutant enzyme binds only the neutral aminoalcohol $ \left( {{\text{pH}} - {\text{independent}}\;K_{\text{i}}^{\text{triethanolamine}} = 19\,{\text{mM}}} \right) $ , whereas wild-type enzyme binds only the cationic aminoalcohol $ \left( {{\text{pH}} - {\text{independent}}\;K_{\text{i}}^{\text{triethanolamine}} = 0.065\,{\text{mM}}} \right) $ . At pH 7.0 and 25°C, relative kinetic parameter, $ k_{\text{cat}}^{\text{4NPX}}/k_{\text{cat}}^{\text{4NPA}} $ , for substrates 4-nitrophenyl-β-d-xylopyranoside (4NPX) and 4-nitrophenyl-α-l-arabinofuranoside (4NPA) of E186A is 100-fold that of wild-type enzyme, consistent with the view that, on the enzyme, protonation is of greater importance to the transition state of 4NPA whereas ring deformation dominates the transition state of 4NPX.     

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.     

Thermal analysis of new glycopolymers derived from monosaccharides

A novel monomer carrying carbohydrate moiety was prepared by simple reaction of methacrylic acid with 3-O-(2′,3′-epoxy-propyl)-1,2:5,6-di-O-isopropylidene-α-d-glucofuranose. Another d-glucose oligomer was synthesized by the polycondensation of a dicarboxylic acid including the carbohydrate residue into the main polymeric chain, 3-O-benzyl-5,6-(bis-O-(acryloyloxy))-1,2-di-O-isopropylidene-α-d-glucofuranose, with propane-1,3-diol using p-toluenesulfonic acid as catalyst. These products were copolymerized with styrene and 2-hydroxypropyl methacrylate, respectively, at different mass ratios, using benzoyl peroxide as initiator. Differential scanning calorimetry was performed in order to study the copolymerization process of the monomer and oligomer into the chosen co-monomers, respectively, and the activation energy of this process was evaluated using Kissinger–Akahira–Sunose (KAS) method. The storage and loss modulus of the obtained glycopolymers were evaluated using dynamic mechanical analysis. The thermal stability of the obtained products was studied via thermogravimetry.     

Synthesis of β-d-glucopyranosyl- and β-d-galactopyranosylamines from 4-bromo-3-methylaniline and 2-amino-5-bromopyridine

The possibility of coupling of d-glucose and d-galactose with 4-bromo-3-methylaniline, 2,4,6-tribromoaniline, and 2-amino-5-bromopyridine was studied. The substituent in the aromatic ring was found to influence the conditions and possibility of the reaction. The yields of β-d-glucopyranosyl- and β-d-galactopyranosylamines from 4-bromo-3-methylaniline and 2-amino-5-bromopyridine were 50–65%; 2,4,6-tribromoaniline did not react at all.     

Molecular dynamics simulation study of xyloglucan adsorption on cellulose surfaces: effects of surface hydrophobicity and side-chain variation

The effect of surface hydrophobicity and side-chain variation on xyloglucan adsorption onto cellulose microfibrils (CMF) is investigated via molecular dynamics simulations. A molecular model of CMF with (100), (010), (1–10), (110) and (200) crystal faces was built. We considered xylogluco-oligosaccharides (XGO) with three repeating units, namely (XXXG)_3, (XXLG)₃, and (XXFG)₃ (where each (1,4)-β-d-glucosyl residue in the backbone is given a one-letter code according to its substituents: G = β-d-Glc; X = α-d-Xyl-(1,6)-β-d-Glc; L = β-d-Gal-(1,2)-α-d-Xyl-(1,6)-β-d-Glc; F = α-l-Fuc-(1,2)-β-d-Gal-(1,2)-α-d-Xyl-(1,6)-β-d-Glc). Our work shows that (XXXG)₃ binds more favorably to the CMF (100) and (200) hydrophobic surfaces than to the (110), (010) and (1–10) hydrophilic surfaces. The origin of this behavior is attributed to the topography of hydrophobic CMF surface, which stabilizes (XXXG)₃ in flat conformation. In contrast, on the rough hydrophilic CMF surface (XXXG)₃ adopts a less favorable random-coil conformation to facilitate more hydrogen bonds with the surface. Extending the xyloglucan side chains from (XXXG)₃ to (XXLG)₃ hinders their stacking on the CMF hydrophobic surface. For (XXFG)₃, the interaction with the hydrophobic surface is as strong as (XXXG)₃. All three XGOs have similar binding to the hydrophilic surface. Steered molecular dynamics simulation was performed on an adhesive model where (XXXG)₃ was sandwiched between two CMF hydrophobic surfaces. Our analysis suggests that this sandwich structure might help provide mechanical strength for plant cell walls. Our study relates to a recently revised model of primary cell walls in which extensibility is largely determined by xyloglucan located in limited regions of tight contact between CMFs.     

Novel aromatic polymers for immobilizing Β-D-glucosidase and their possible application to cellulolysis

We have synthesized, by enzymic and chemical means, a variety of novel polyaromatic-enzyme complexes that are extremely stable and show promise in the conversion of cellulose to glucose. Thus we have prepared a number of homo- and heteropolymeric supports (involvingl-tyrosine, pyrogallol, resorcinol, phloroglucinol, orcinol, catechol, protocatechuic acid, and various hydroxybenzoic acids) and discovered that, for example, a resorcinol-Β-d-glucosidase copolymer has high stability combined with lowK _m (10.5 mM vs commercial soluble (3-d-glucosidase 9.3 mM) and high V_max values (104 Μmol ρNP mg^-1H^-1 vs 85 Μmol ρNP mg^-1H^-1). These properties are enhanced when the copolymer is complexed with bentonite clay. The kinetic constants of the resorcinol-Β-d-glucosidase copolymer-bentonite complex wereK _m = 9.6 mM andV _max = 73.5 Μmol ρNP mg^-1H^-1. Stability has been assessed against proteolysis, organic solvents, elevated temperatures, storage, and incorporation into fresh soil. A cellulase preparation fromTrichoderma viride has also been copolymerized with a variety of phenolic macromolecules and displays varying degrees of stability and activity against carboxymethyl cellulose. The resorcinol Β-d-glucosidase-copolymer was immobilized on a PM10 ultrafiltration membrane (K _m = 16.8 mM; V_max = 42.4 (Μmol ρNP mg^-1H^-1) and showed enhanced thermostability, a broader pH range for maximal activity, and could be reused without loss of activity. An ultrafiltration cell, containing the membrane-immobilized resorcinol-Β-d-glucosida se copolymer, can be operated as a continuous reactor with substrate flow rates from 0.1 to 0.7 mL min^-1 without decrease in product formation.     

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.     

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.     

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.     

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.