Glucosyl Conjugates Research Articles

Triclosan uptake and transformation by the green algae Euglena gracilis strain Z

Triclosan is an antimicrobial chemical present in consumer products that is frequently detected in aquatic environments. In this research, we investigated the role of a common freshwater microalgae species, Euglena gracilis for triclosan uptake and transformation in open–water treatment wetlands. Lab–scale wetland bioreactors were created under various conditions of light (i.e., continuous (white) light, red light, and in the dark), media (i.e., wetland, autoclaved wetland, Milli–Q, and growth media water), and presence or absence of algae. Triclosan and its potential transformation products were identified in the water and algae phases. Triclosan transformation occurred most rapidly with reactors that received continuous (white) light, with pseudo first–order rate constants, k, ranging from 0.035 to 0.292 day−1. This indicates that phototransformation played a major role in triclosan transformation during the day, despite light screening by algae. Algae contributed to the uptake and transformation of triclosan in all reactors, and algae and bacteria both contributed to triclosan biotransformation under dark conditions, representative of nighttime conditions. Some transformation products were formed and further transformed, e.g., triclosan–O–sulfate, methoxy and diglucosyl conjugate of hydroxylated triclosan, and dimethoxy and glucosyl conjugate of 2,4–dichlorophenol, suggesting their minimal accumulation over the 25 days of the experiments. This study shows that the combined action of light, microbes, and algae allows the safe transfer and transformation of triclosan in open–water treatment wetlands.

Salicylic acid involved in chilling-induced accumulation of calycosin-7-O-β-d-glucoside in Astragalus membranaceus adventitious roots

Calycosin and calycosin-7-O-β-d-glucoside (CG) are major isoflavonoids in Astragalus membranaceus and have multiple beneficial activities. Adventitious roots (ARs) are becoming attractive resources to obtain biologically active compounds. Salicylic acid (SA) is an important endogenous phytohormone, which is involved in the regulation of biotic and abiotic stresses. However, little is known about the potential role of SA on isoflavonoid accumulations under chilling stress. In the present study, calycosin was found to accumulate mostly in its glucosyl conjugate (CG) form in A. membranaceus ARs (AMARs). Compared to control conditions (25 °C), chilling (5 °C) induced the accumulation of CG, which was confirmed by increased expression levels of gene-encoding enzymes in the CG biosynthetic pathway. Furthermore, chilling triggered the accumulation of SA prior to CG accumulation. In addition, the inhibition of SA biosynthesis with paclobutrazol (PAC) in chilling-exposed AMARs suppressed the accumulation of CG and gene expressions, while exogenous addition of SA to PAC-treated AMARs restored CG content and gene expressions. These results indicated that in AMARs, SA involved in chilling-induced CG accumulation by regulating the expression levels of genes in the CG biosynthetic pathway.

Synthesis of saccharin-glycoconjugates targeting carbonic anhydrase using a one-pot cyclization/deprotection strategy

Carbonic anhydrase IX (CA IX) has been identified as a biomarker and drug target for several malignant tumors due to its role in cancer cell growth and proliferation. Simple cyclic sulfonamides, like saccharin (SAC), have shown up to a 60-fold selectivity towards CA IX over other ubiquitous CA isoforms, with greater selectivity obtained applying the “tail-approach” to derivatize SAC with a methylene triazole linker that connected to a “tail” beta glucoside. These modifications of SAC led to an increased selectivity of more than 1000-fold towards CA IX, whereas clinically available CA inhibitors show little to no isoform selectivity. As part of our interest in the development of new CA inhibitors, we found the existing synthetic protocol, which relies on a N-tert-butyl saccharin intermediate, to be problematic in the final deprotection steps. We therefore describe an alternative approach to the synthesis of these compounds featuring a gentle “one pot” deprotection/cyclization as the final synthetic step, and report new galactosyl and glucosyl conjugates with low to mid nM inhibition of CA IX.

Role of Aromatic Moiety in the Probe Property toward Picric Acid: Synthesis, Crystal Structure, Spectroscopy, Microscopy, and Computational Modeling of a Knoevenagel Condensation Product of d-Glucose.

Molecular probes for picric acid (PA) in both solution and solid states are important owing to their wide usage in industry. This paper deals with the design and development of a glucosyl conjugate of pyrene (L1) along with control molecular systems, possessing anthracenyl (L2), naphtyl (L3), and phenyl (L4) moieties, via Knoevenagel condensation of 2,4-pentanedione with d-glucose. The selectivity of L1 toward PA has been demonstrated on the basis of fluorescence and absorption spectroscopy, and the species of recognition by electrospray ionization mass spectrometry. The role of the aromatic group in the selective receptor property has been addressed among L1, L2, L3, and L4. The structural features of the {L1 + PA} complex were established by density functional theory computations. L1 was demonstrated to detect PA in solid state selectively over other nitroaromatic compounds (NACs). To study the utility of L1 in film, cellulose paper strips coated with L1 were used and demonstrated the selective detection of PA. The observed microstructural features of L1 and its complex {L1 + PA} differ distinctly in both atomic force microscopy and scanning electron microscopy, all in the support of the complex formation. Thus, L1 was demonstrated as a sensitive, selective, and inexpensive probe for PA over several NACs by visual, spectral, and microscopy methods.

Synthesis and biological evaluation of glucosyl curcuminoids.

Medicinal plants proved to be a rich source in exploring a variety of lead structures in the development of new drugs. The natural curcuminoids have gained considerable attention in recent years for their multiple beneficial pharmacological and biological activities. Clinical application of these curcuminoids is often impaired due to their poor water solubility, resulting in low in vivo bioavailability of the active compound in humans. The objective of the present study is to synthesize glucosyl conjugates of curcumin 1 and tetrahydrocurcumin 4 and to evaluate their biological activities. The study highlights the synthesis of curcumin-β-di-glucoside 3 (yield 71%) and tetrahydrocurcumin-β-di-glucoside 6 (yield 64%) in good yields in a biphasic reaction medium using a phase transfer catalyst under simple and ecofriendly conditions. Both the glucosyl curcuminoids showed enhanced antioxidant, tyrosinase enzyme inhibitory, antimicrobial and potent cytotoxic activity. The improved biological activity may be due to the increased solubility of the glucosyl conjugated compounds compared to the native curcuminoids; this was further confirmed by partition coefficient studies. Thus, the synthesized glucosyl curcumin may serve as promising future therapeutic molecule in the management of cancer, whereas glucosyl tetrahydrocurcumin can be a useful ingredient in achromatic food and in cosmetic applications.

β‐D‐Glucosyl Conjugates of Highly Potent Inhibitors of Blood Coagulation Factor Xa Bearing 2‐Chorothiophene as a P1 Motif

We synthesized a novel O-glucoside of the recently reported potent factor Xa (fXa) inhibitor 1, which bears a 5-chlorothien-2-yl moiety and 1-isopropylpiperidine as fragments that bind the S1 and S4 enzyme pockets, respectively. A β-D-glucosyl unit was conjugated through an ether-linked C3-alkyl spacer to the central phenyl ring of 1. The synthesized β-D-glucose-based compound 16 achieved picomolar inhibitory potency against human fXa (K(i)=60 pM) and high selectivity over thrombin and other serine proteases. In addition to the chlorothienyl S1 binder, a large gain in ΔG resulted from the addition of protonated 1-isopropylpiperidine (ΔΔG=29.7-30.5 kJ mol(-1)), which should bind to the aromatic S4 pocket through efficient cation-π and C-H···π interactions. Instead, the C3-alkyl-linked glucose fragment, which is likely directed toward the solvent outside the enzyme binding site, improves ΔG by an average of 2.9-3.8 kJ mol(-1) . Compound 16 showed sub-micromolar in vitro anticoagulant activity, as assessed by prothrombin time (PT) and activated thromboplastin time (aPTT) clotting assays in pooled human plasma (PT(2) and aPTT(2) equal to 0.135 and 0.389 μM, respectively). Although compound 16 was 1.4-fold less active than parent compound 1 in the ex vivo anticoagulant assay in mice, it showed a significant (1.6-fold) prolongation of PT relative to controls (P<0.05) 60 min after oral dosing (75 mg kg(-1)).

Detection, characterization and quantification of salicylic acid conjugates in plant extracts by ESI tandem mass spectrometric techniques

An approach for the detection and characterization of SA derivatives in plant samples is presented based on liquid chromatography coupled to electrospray ionization (ESI) tandem mass spectrometric techniques. Precursor ion scan methods using an ESI triple quadrupole spectrometer for samples from plants challenged with the virulent Pseudomonas syringae pv tomato DC3000 allowed us to detect two potential SA derivatives. The criterion used to consider a potential SA derivative is based on the detection of analytes in the precursor ion scan chromatogram upon selecting m/z 137 and m/z 93 that correspond to the salicylate and its main product ion, respectively. Product ion spectra of the newly-detected analytes as well as accurate m/z determinations using an ESI Q-time-of-flight instrument were registered as means of characterization and strongly suggest that glucosylated forms of SA at the carboxylic and at the phenol functional groups are present in plant samples. The specific synthesis and subsequent chromatography of salicylic glucosyl ester (SGE) and glucosyl salicylate (SAG) standards confirmed the chemical identity of both peaks that were obtained applying different tandem mass spectrometric techniques and accurate m/z determinations. A multiple reaction monitoring method has been developed and applied to plant samples. The advantages of this LC-ESI-MS/MS methods with respect to the traditional analysis of glucosyl conjugates are also discussed. Preliminary results revealed that SA and the glucosyl conjugates are accumulated in Arabidopsis thaliana in a time dependent manner, accordingly to the up-regulation of SA-dependent defenses following P. syringae infection. This technique applied to plant hormones or fragment ions may be useful to obtain chemical family members of plant metabolites and help identify their contribution in the signaling of plant defenses.

Carbohydrate assisted fluorescence turn-on gluco–imino–anthracenyl conjugate as a Hg(ii) sensor in milk and blood serum milieu

A new anthracenyl-imino-glucosyl conjugate (L) selectively senses Hg(2+) by turn-on fluorescence with a 13 ± 1 fold enhancement by forming a 2 : 1 complex in pH 5 to 10 even in the presence of several biologically and ecologically relevant metal ions, with a 25 ± 2% fluorescence enhancement at the EPA limit of 2 ppb. L is equally sensitive towards Hg(2+) in the presence of albumin proteins and in blood serum and milk.

Carbohydrate Modified Catanionic Vesicles: Probing Multivalent Binding at the Bilayer Interface

This article reports on the synthesis, characterization, and binding studies of surface-functionalized, negatively charged catanionic vesicles. These studies demonstrate that the distribution of glycoconjugates in the membrane leaflet can be controlled by small alterations of the chemical structure of the conjugate. The ability to control the glycoconjugate concentration in the membrane provides a method to explore the relationship between ligand separation distance and multivalent lectin binding at the bilayer interface. The binding results using the O-linked glucosyl conjugate were consistent with a simple model in which binding kinetics are governed by the density of noninteracting glucose ligands, whereas the N-linked glycoconjugate exhibited binding kinetics consistent with interacting or clustering conjugates. From the noninteracting ligand model, an effective binding site separation of the sugar sites for concanavalin A of 3.6-4.3 nm was determined and a critical ligand density above which binding kinetics are zeroth order with respect to the amount of glycoconjugate present at the bilayer was observed. We also report cryo-transmission electron microscopy (cryo-TEM) images of conjugated vesicles showing morphological changes (multilayering) upon aggregation of unilamellar vesicles with concanavalin A.

Synthesis and Structural Characterization of Glucopyranosylamide Films on Gold

Self-assembled monolayers (SAMs) of glucose derivatives on gold have been prepared from alpha- and beta-glucopyranosylamide derivatives. The glucosyl conjugates were synthesized stereoselectively via the in situ generation of glucosyl isoxazolines followed by treatment with thiopyridyl esters. The resulting film structures were characterized by atomic force microscopy, reflection Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. The experimental data indicated that alpha- or beta-linked glucopyranosylamide derivatives with free hydroxyl groups attach to gold via the thiol linker. Both derivatives form monolayer films with high packing densities--comparable to those typically observed for alkanethiol monolayers on gold. Acetate analogues of these conjugates do not form SAMs on gold; they form multilayered films under identical deposition conditions.

Regulation of isoflavone production in hydroponically grown Pueraria montana (kudzu) by cork pieces, XAD-4, and methyl jasmonate

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g(-1) dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.

Isoflavone Levels in Five Soybean (Glycine max) Genotypes Are Altered by Phytochrome-Mediated Light Treatments

The objective of the present study was to determine whether concentrations of different isoflavones (puerarin, genistein, genistin, daidzein, and daidzin) in shoots and roots of five selected soybean genotypes would respond the same or differently to red (650 nm peak transmittance) and far-red (750 nm peak transmittance) light treatments given under controlled environments. Levels of isoflavones (mg g(-1) dry weight biomass) present in seeds, control roots, and shoots and 10 day light-treated seedlings (light, dark, red, and far-red wavelengths) of soybean (Glycine max) were determined by high-performance liquid chromatography analysis in comparison with known isoflavone standards. Seeds of the five soybean genotypes studied consistently stored most of their isoflavones as glucosyl conjugates (e.g., daidzin, genistin, and puerarin). For the five soybean genotypes, isoflavone levels were lower in the seeds as compared with roots plus shoots of control, time zero (first true leaf stage) seedlings. Following 10 days of the respective light treatments, we found that (i) isoflavone levels were enhanced in dark-grown plants over light-grown plants for three of the five genotypes (a new finding) and the reverse occurred for a single genotype (a typical response of legumes) and (ii) generally, far-red end of day (EOD) light treatment enhanced total isoflavone levels in roots plus shoots over red EOD light treatment. Results from the present study show that phytochrome does appear to play a role in regulating isoflavone levels in developing soybean seedlings and that this influence by red/far-red-mediated phytochrome reactions is strongly dependent on the genotypes selected for study.

Biotransformation of biphenyl by the filamentous fungus Talaromyces helicus

The filamentous fungusTalaromyces helicus , isolated from oil-contaminated sludge, oxidizes biphenyl via 4-hydroxybiphenyl to the dihydroxylated derivatives 4,4′-dihydroxybiphenyl and 3,4-dihydroxybiphenyl, which, to a certain extent, are converted to glycosyl conjugates. The sugar moiety of the conjugate formed from 4,4′-dihydroxybiphenyl was identified as glucose. Further metabolites: 2-hydroxybiphenyl, 2,5-dihydroxylated biphenyl, and the ring cleavage product 4-phenyl-2-pyrone-6-carboxylic acid accumulated only in traces. From these results the main pathway for biotransformation of biphenyl in T. helicus could be proposed to be the excretion of dihydroxylated derivatives (>75%) and their glucosyl conjugates (<25%).

The production of L-dopa and isoflavones in seeds and seedlings of different cultivars of Vicia faba L. (fava bean)

The primary objective of this investigation is to compare the levels of L-dopa and the isoflavonoids, genistein and daidzein and their respective glucosyl conjugates, in seeds with ten-day-old seedlings of 24 different cultivars of fava bean (Vicia faba L.). Our working hypothesis is that the levels of these secondary metabolites will vary significantly in the different V. faba L. cultivars, and that seedlings will possess higher levels of both L-dopa and the isoflavonoids of interest than seeds of all of the cultivars used in this investigation. Here, we show that this hypothesis is correct. We discuss the importance of these findings for human medicine and nutrition, and their value for integrative medicine.

Synthesis of Glycosyl Derivatives as Dopamine Prodrugs: Interaction with Glucose Carrier GLUT‐1.

Glucosyl dopamine (DA) derivatives may represent a new class of DA prodrugs that would interact with glucose transporter GLUT-1, present in the blood-brain barrier, and generate DA in the brain. Therefore, compounds bearing the sugar moiety linked to either the amino group or the catechol ring of DA through amide, ester, carbamate, peptide or glycosidic bonds were synthesized. The behavior of the compounds as prodrugs was monitored in different media and the affinity of the glycoconjugates for the glucose carrier GLUT-1 using human erythrocytes was also studied. Most of the compounds were markedly stable in buffer and plasma, and several compounds released DA when incubated with brain extracts and the rate was related to the bond linking DA with glucose. The new glucosyl conjugates substituted at the C-6 position of the sugar were more potent inhibitors of glucose transport when compared to C-1 and C-3 substituted derivatives. This work provides structure-activity information about the interaction of substituted glucose with the GLUT-1 transporter.

Quantification of major isoflavonoids and l-canavanine in several organs of kudzu vine ( Pueraria montana) and in starch samples derived from kudzu roots

Isoflavonoids (daidzein, genistein, daidzin, genistin and puerarin) were extracted and analyzed quantitatively by high pressure liquid chromatography from different organs of kudzu vine ( Pueraria montana) and starch samples derived from kudzu roots. Aerial parts of kudzu plants (young shoots, leaf blades and leaf petioles) contained relatively low levels of all isoflavonoids examined, whereas seeds and seedlings were intermediate in isoflavonoid levels and roots consistently had the highest levels, particularly puerarin and the glucosyl conjugates of genistein and daidzein, namely, genistin and daidzin. Interestingly, commercially available kudzu root starch from Japan does not contain the isoflavonoids of interest, whereas homemade kudzu root starch contains all studied isoflavonoids in various amounts, and especially, high levels of puerarin. Shoots of light-grown kudzu seedlings, when compared with shoots of dark-grown seedlings had higher levels of all isoflavonoids with the exception of daidzin. In contrast, for seedling roots, such differences were not greatly different between light-grown and dark-grown plants. Light-grown intact kudzu seedlings had significantly higher levels of soluble proteins than dark-grown seedlings. Mass spectrometer analyzes of all kudzu samples for the toxic non-protein amino acid, l-canavanine, indicated it to be absent using this method of detection and level of sensitivity.

Synthesis of glycosyl derivatives as dopamine prodrugs: interaction with glucose carrier GLUT-1.

Glucosyl dopamine (DA) derivatives may represent a new class of DA prodrugs that would interact with glucose transporter GLUT-1, present in the blood-brain barrier, and generate DA in the brain. Therefore, compounds bearing the sugar moiety linked to either the amino group or the catechol ring of DA through amide, ester, carbamate, peptide or glycosidic bonds were synthesized. The behavior of the compounds as prodrugs was monitored in different media and the affinity of the glycoconjugates for the glucose carrier GLUT-1 using human erythrocytes was also studied. Most of the compounds were markedly stable in buffer and plasma, and several compounds released DA when incubated with brain extracts and the rate was related to the bond linking DA with glucose. The new glucosyl conjugates substituted at the C-6 position of the sugar were more potent inhibitors of glucose transport when compared to C-1 and C-3 substituted derivatives. This work provides structure-activity information about the interaction of substituted glucose with the GLUT-1 transporter.

Constitutive elevated accumulation of phenylpropanoids in soybean roots at low temperature

Changes in phenolic acid and isoflavonoid contents in soybean ( Glycine max. (L.) Merr) roots after 24-h of low temperature cultivation were investigated. The level of total phenolic acids (represented by the sum of derivatives of benzoic and cinnamic acids) and genistein, daidzein and genistin (glucosyl conjugate of genistein) increased after 24 h of exposure to low temperature. In chill-treated roots the highest increase in genistin was observed. Application of 2-aminoindan-2-phenylphoshonic acid (AIP), a competitive inhibitor of l-phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) resulted in an increased level of endogenous free phenylalanine. An increase in the content of free phenylalanine is regarded as an indication for the inhibition of PAL in vivo. Chilling with AIP decreased valine level. AIP applied in combination with chilling decreased phenolic acids but did not effect the isoflavonoids content. These findings may indicate that the increase in the phenolics level comes from the pre-formed conjugates of phenylpropanoids .

Partial purification and characterization of a soybean β-glucosidase with high specific activity towards isoflavone conjugates

A β-glucosidase with high specific activity towards isoflavone conjugates was purified from soybean [ Glycine max] roots by high salt extraction from a low speed centrifugal pellet and subsequent anion and cation exchange chromatography. Purification required stabilization throughout fractionation in 10% glycerol. The enzyme is most likely a dimer (approximate M r 165 kDa) with potential subunits of M r 80 and/or 75 kDa. The pH and temperature optima are pH 6 and 30 °C, respectively. The enzyme was highly heat-stable. Of the various potential effectors examined, silver and mercury ions were the most inhibitory. The IC 50 of silver ions was increased from 140 μM to 14 mM in the presence of 250 μM β-mercaptoethanol. Glucono-δ-lactone was not strongly inhibitory (IC 50 24 mM). The activity was highly active against isoflavone conjugates, with a specificity constant 160–1000 fold higher for isoflavone conjugates over the generic chromogenic substrate, p-nitrophenyl β-glucoside. The enzyme was inactive against the flavonol glycosides tested. The partially purified enzyme had similar K m and k cat towards 7- O-glucosyl- and 7- O-glucosyl-6″-malonyl-isoflavones, suggesting that it may be able to cleave the esterified glucosyl conjugate. We hypothesize that the enzyme is involved in the release of daidzein and genistein, both of which play central roles in soybean defense.

Formation of glucoside conjugates during biotransformation of dibenzofuran by Penicillium canescens SBUG-M 1139.

Penicillium canescens oxidises dibenzofuran (DBF) to produce monohydroxylated derivatives and other more hydrophilic metabolites. These substances are water-soluble but unstable in organic solvents such as ethyl acetate, acetone or dichloromethane. Both extraction with ethyl acetate and enzymatic treatment of the aqueous culture filtrate with beta-glucuronidase led to decay of the hydrophilic metabolites and indicated these products to be glycoside conjugates. The glycosyl residue was identified as glucose both by liquid chromatography and by the use of glucose oxidase. The conjugate pattern formed was the same in type and amount, independent of the carbon source used for cultivation of the fungus. Clearly, DBF transformation in P canescens occurred in two phases: first the conversion to 2-, 3-, and 4-hydroxydibenzofuran (phase I), followed by the formation of the corresponding glucosyl conjugates (phase II). In contrast, 2,3-dihydroxydibenzofuran added to the cultures was transformed by ring cleavage producing a muconic acid-like dead-end product.