Glycosyl Conjugates Research Articles

Current Advances and Applications in Animals, Plants and Biological Sciences

Plants' reliance on molecular epigenetic control reflects their developmental, lifestyles, and the evolutionary histories. Plants grows by continually developing new parts from the self-sufficing stem cells population, termed as meristem, as opposed to mammals, whose tissues and organs formation is primarily determined during the embryo development. The interest in different medically active products has been developed to improve their pharmacokinetic and biological properties for utilizing glycosyl-conjugation. The conjugation of various drugs with various mono, di, or polysaccharides has boosted the therapeutic potentials of these drugs that is also manifested by a significant number of research papers. The current review article encapsulates extremely important and the up-to-date example of this conjugation, specially associated to enhancing antitumor activities of original glycoconjugates. The given examples along projected mechanisms of activities enhancement may guide to design, synthesize and evaluate new glycosyl conjugates for improved therapeutics.

Transcriptional and biochemical analyses of gibberellin expression and content in germinated barley grain.

Mobilization of reserves in germinated cereal grains is critical for early seedling vigour, global crop productivity, and hence food security. Gibberellins (GAs) are central to this process. We have developed a spatio-temporal model that describes the multifaceted mechanisms of GA regulation in germinated barley grain. The model was generated using RNA sequencing transcript data from tissues dissected from intact, germinated grain, which closely match measurements of GA hormones and their metabolites in those tissues. The data show that successful grain germination is underpinned by high concentrations of GA precursors in ungerminated grain, the use of independent metabolic pathways for the synthesis of several bioactive GAs during germination, and a capacity to abort bioactive GA biosynthesis. The most abundant bioactive form is GA1, which is synthesized in the scutellum as a glycosyl conjugate that diffuses to the aleurone, where it stimulates de novo synthesis of a GA3 conjugate and GA4. Synthesis of bioactive GAs in the aleurone provides a mechanism that ensures the hormonal signal is relayed from the scutellum to the distal tip of the grain. The transcript data set of 33 421 genes used to define GA metabolism is available as a resource to analyse other physiological processes in germinated grain.

Profiling and identification of chlorogenic acid metabolites in rats by ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer

1. Chlorogenic acids (CGAs), one kind of major bioactive constituents isolated from Flos Lonicera Japonica, possess many biological activities, such as antibacterial, antioxidant and antiviral activities. In this study, we established an efficient strategy using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) to profile the in vivo metabolic fate of CGAs in rat urine and plasma.2. The extract from Flos Lonicera Japonica was orally administrated to Sprague-Dawley (SD) rats at a dose of 1000 mg/kg body weight. Then, a combination of various post-acquisition data mining methods, including high-resolution extracted ion chromatogram (HREIC) and multiple mass defect filters (MMDFs) and diagnostic product ions (DPIs), were adopted to characterize the known and unknown CGA metabolites in SD rats.3. As a result, a total of 68 CGA metabolites were unambiguously or tentatively screened and characterized. These metabolites, including 18 prototype compounds and 50 metabolites, were deduced to be yielded via methylation, hydrogenation, demethylation, dehydration, sulfate conjugation, glucuronide conjugation, glycosylation conjugation and their composite reactions, which mainly occurred to caffeoylquinic acids, dicaffeoylquinic acids, p-coumaroylquinic acids and feruloylquinic acids.4. In conclusion, this study profiled CGA metabolites, which are useful in understanding the in vivo metabolic fate, effective forms, and pharmacological and toxic actions of CGAs.

β-Glucosidase involvement in the bioactivation of glycosyl conjugates in plants: synthesis and metabolism of four glycosidic bond conjugates in vitro and in vivo.

Mobile glucose-pesticide conjugates in the phloem are often restricted by decreases in biological activity. However, plants can bioactivate endogenous glucosides, which are assumed as able to bioactivate exogenous conjugates. In this study, four glycosidic bonds (O-, S-, N-, and C-glycosidic bonds) of glucose-pesticide conjugates were designed and synthesized, and then metabolism assays were carried out in vitro and in vivo. Results showed that β-glucosidases played a role in the hydrolysis of O-glycosidic bond conjugates in Ricinus communis L. The liberated aglycons possessed insecticidal activities against Plutella xylostella L. and Spodoptera litura F. These results could help establish methods of circumventing the mutual exclusivity of phloem mobility and biological activity by hydrolyzing endogenous β-glucosidases.

Synthesis and application of clickable and biocompatible fluorescent glycosyl labels

Two novel, biocompatible and highly stereoselective fluorescent glycosyl labels featuring azide groups have been successfully synthesized. The functional groups provide convenient routes for fluorescent glycosyl conjugates via click chemistry. Three fluorescent glycose-conjugates were synthesized. One fluorescent conjugate was used to trace the translocation in castor seedlings. The developed fluorescent labels for biomolecules are likely to provide an important method for investigating various biological events. We believe that the probes obtained are useful for studies on biological processes.

Synthesis of N‐Glycosyl Conjugates through a Multicomponent Domino Process

AbstractThe development of efficient methods for the combinatorial synthesis of N‐glycosyl conjugates is vital for many fields of modern synthetic organic chemistry. Herein, we report a multicomponent domino process for the regioselective synthesis of a large array N‐glycosyl‐Asp‐urea conjugates, which could be further functionalized in a chemoselective way, starting from easily accessible reactants such as N‐glycosylamines, fumaric acid monoesters, azides, and isocyanates. The process occurs under very mild conditions, does not require the use of strong bases/acids or high temperature, and is highly versatile, working efficiently with a range of protecting groups and substituents.

Flowers from Kalanchoe pinnata are a Rich Source of T Cell-Suppressive Flavonoids

The chemical composition and immunosuppressive potential of the flowers from Kalanchoe pinnata (Crassulaceae) were investigated. We found that the aqueous flower extract was more active than the leaf extract in inhibiting murine T cell mitogenesis in vitro. Flavonoids isolated from the flower extract were identified and quantitated based on NMR and HPLC-DAD-MS analysis, respectively. Along with quercetin, four quercetin glycosyl conjugates were obtained, including quercetin 3-O-beta-D-glucuronopyranoside and quercetin 3-O-beta-D-glucopyranoside, which are described for the first time in K. pinnata. All flavonoids inhibited murine T cell mitogenesis and IL-2 and IL-4 production without cell toxicity. This is the first report on the pharmacological activity of flowers of a Kalanchoe species, which are not used for curative purposes. Our findings show that K. pinnata flowers are a rich source of T-suppressive flavonoids that may be therapeutically useful against inflammatory diseases.

Peracetylated N-Acetylmannosamine, a Synthetic Sugar Molecule, Efficiently Rescues Muscle Phenotype and Biochemical Defects in Mouse Model of Sialic Acid-deficient Myopathy

Distal myopathy with rimmed vacuoles/hereditary inclusion body myopathy (DMRV/hIBM), characterized by progressive muscle atrophy, weakness, and degeneration, is due to mutations in GNE, a gene encoding a bifunctional enzyme critical in sialic acid biosynthesis. In the DMRV/hIBM mouse model, which exhibits hyposialylation in various tissues in addition to muscle atrophy, weakness, and degeneration, we recently have demonstrated that the myopathic phenotype was prevented by oral administration of N-acetylneuraminic acid, N-acetylmannosamine, and sialyllactose, underscoring the crucial role of hyposialylation in the disease pathomechanism. The choice for the preferred molecule, however, was limited probably by the complex pharmacokinetics of sialic acids and the lack of biomarkers that could clearly show dose response. To address these issues, we screened several synthetic sugar compounds that could increase sialylation more remarkably and allow demonstration of measurable effects in the DMRV/hIBM mice. In this study, we found that tetra-O-acetylated N-acetylmannosamine increased cell sialylation most efficiently, and in vivo evaluation in DMRV/hIBM mice revealed a more dramatic, measurable effect and improvement in muscle phenotype, enabling us to establish analysis of protein biomarkers that can be used for assessing response to treatment. Our results provide a proof of concept in sialic acid-related molecular therapy with synthetic monosaccharides.

Glycosyl conjugates of biotinylated diaminopyridine applied for study of carbohydrate-to-carbohydrate interaction

Previous studies by us and others established that cell-cell adhesion is mediated by specific carbohydrate-to-carbohydrate interaction (CCI). Those previous studies were based on various biochemical and biophysical approaches, including the use of labeled glycosyl epitopes with fluorescent tag. However, these methods ideally require that the glycosyl epitope must be fixed to a solid phase molecule, preferably with multivalency. The purpose of the present study is to establish a CCI process using specific glycosyl residues conjugated to biotinylated diaminopyridine (BAP), and to observe: (i) clear occurrence of homotypic CCI between "Os Fr.B" having 5-6 GlcNAc termini, vs. absence of such homotypic CCI between "Os Fr.1" having 2 GlcNAc termini; (ii) occurrence of heterotypic CCI between GM3 ganglioside and Os Fr.B, vs. absence of such heterotypic CCI between GM3 and Os Fr.1. Interaction between Os Fr.B-BAP conjugate and Os Fr.B-ceramide mimetic (Os Fr.B-mCer) was demonstrated based on two experiments: (i) dose-dependent binding of Os Fr.B-BAP conjugate to polystyrene plates coated with Os Fr.B-mCer was observed in the presence of bivalent cation, a prerequisite for all CCI processes, and such binding was abolished by EDTA; (ii) binding between equal nanomolar Os Fr.B-BAP and Os Fr.B-mCer was inhibited by mM concentration Os Fr.B without conjugate, in dose-dependent manner. Thus, cell adhesion processes based on homotypic CCI between N-linked glycans having multiple GlcNAc termini, and heterotypic CCI between GM3 and such glycans, were clearly observed using BAP conjugates of glycosyl epitopes.

Molecular mechanism involved in the transport of a prodrug dopamine glycosyl conjugate

We have previously demonstrated that dopamine conjugation to glucose allows it to induce therapeutic effects against Parkinson's disease after intravenous administration. In this paper we demonstrate that, unlike dopamine, the prodrug glu-dopamine is a transportable substrate of glucose transporters. Towards this, the effect of glucose-conjugation on the affinity and uptake of dopamine have been assessed in vitro, using human retinal pigment epithelium (HRPE) cells. Glucose transporter-mediated uptake was measured using [ 3H]3- O-methylglucose ([ 3H]3- O-MG) as the tracer. The uptake was found to be rapid and hyperbolically related to its concentrations ( K t = 7.8 ± 1.2 mM and V max = 54 ± 2 nmol/min mg protein). Inhibition experiments showed that dopamine was able to interact with glucose carriers only when conjugated to glucose (IC 50 = 2.6 ± 0.6 mM). HPLC analysis of HRPE cell extracts showed that both dopamine and the prodrug permeate the cell, but only the uptake of the prodrug is inhibitable by glucose. This confirms that glucose transporters mediate the transport of the prodrug glu-dopamine, but not of dopamine. HRPE cells is therefore proposed as a promising model for in vitro studies involving the glucose transporter-mediated transport of drugs and their conjugates.

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%).

Involvement of xylem sap zeatin- O-glucoside in cucumber shoot greening

A fraction of xylem sap collected from squash ( Cucurbita maxima) root and separated with gel filtration showed strong promoting activity in a greening bioassay using etiolated cucumber cotyledons. This activity decreased with waterlogging of the root. The promoting factor was purified with sequential gel-permeation, and reverse-phase and normal-phase column chromatographies. Zeatin- O-glucoside (ZOG) was identified as the promoting factor by monitoring liquid chromatography tandem mass spectrometry. Although the glycosyl conjugate of zeatin was thought to be inactive, ZOG had a promoting activity that was 100 times higher than those of zeatin and zeatin riboside between concentrations of 10 –9 to 10 –4 M in the greening bioassay. These results suggest that in some developmental stages, the conjugation of cytokinins with a sugar moiety, such as glucose, might be a key factor in the control of shoot greening by roots.

Comparative metabolism of genistin by human and rat gut microflora: detection and identification of the end-products of metabolism

1. Biotransformations by gut microflora play a pivotal role in determining the biological activity of isoflavones that occur in soya-based foods predominantly as β -glycosyl conjugates. Microflora prepared from rat caecae and human faeces were used to investigate the metabolic fate of genistein β -glycosides extracted from soya flour. The end-products of such metabolism were determined by parallel incubations of microflora with [2',3',5',6'-3H] and [4-14C]-labelled genistein. 2. Quantitative analysis by LC-MS/MS indicated very rapid and complete degradation of genistin, which was associated with a transient increase in genistein. Qualitative studies indicated that the malonyl and acetyl glycosides of genistein were also degraded by the microflora. 3. Incubation of caecal and faecal microflora with [3H] and [14C]genistein yielded similar radiolabelled metabolites, which were identified by radio-LC-MSn as the intermediates dihydrogenistein and 6'-hydroxy-O-desmethylangolensin and end-product 4-hydroxyphenyl-2-propionic acid. This profile of genistein metabolites indicated selective hydrolysis of 6'-hydroxy-O-desmethylangolensin between carbon atoms 1' and 1 to yield the end-products 4-hydroxyphenyl-2-propionic acid and 1,3,5-trihydroxybenzene. 4. The biological significance of the products of genistein metabolism warrant further investigation since they may play an important role in mediating the beneficial antioxidant health effects associated with the consumption of isoflavones in food.

Synthesis and biological activity of glycosyl conjugates of N-(4-hydroxyphenyl)retinamide

Three glycosyl (glucosyl, galactosyl, mannosyl) conjugates of 4-hydroxyphenylretinamide have been synthesized and tested on a broad variety of tumor cells. All three compounds are active on promyelocytic leukemia cell lines HL60 but less than the parent compound 4-HPR. Among them, the mannosyl analog stands out by its very low toxicity.

Purification, Cloning, and Heterologous Expression of a Catalytically Efficient Flavonol 3-O-Galactosyltransferase Expressed in the Male Gametophyte of Petunia hybrida

Flavonols are plant-specific molecules that are required for pollen germination in maize and petunia. They exist in planta as both the aglycone and glycosyl conjugates. We identified a flavonol 3-O-galactosyltransferase (F3GalTase) that is expressed exclusively in the male gametophyte and controls the formation of a pollen-specific class of glycosylated flavonols. Thus an essential step to understanding flavonol-induced germination is the characterization of F3GalTase. Amino acid sequences of three peptide fragments of F3GalTase purified from petunia pollen were used to isolate a full-length cDNA clone. RNA gel blot analysis and enzyme assays confirmed that F3GalTase expression is restricted to pollen. Heterologous expression of the F3GalTase cDNA in Escherichia coli yielded active recombinant enzyme (rF3GalTase) which had the identical substrate specificity as the native enzyme. Unlike the relatively nonspecific substrate usage of flavonoid glycosyltransferases from sporophytic tissues, F3GalTase uses only UDP-galactose and flavonols to catalyze the formation of flavonol 3-O-galactosides. Kinetic analysis showed that the k(cat)/K(m) values of rF3GalTase, using kaempferol and quercetin as substrates, approaches that of a catalytically perfect enzyme. rF3GalTase catalyzes the reverse reaction, generation of flavonols from UDP and flavonol 3-O-galactosides, almost as efficiently as the forward reaction. The biochemical characteristics of F3GalTase are discussed in the context of a role in flavonol-induced pollen germination.

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The nature and magnitude of residues in apples treated with [(14)C]diphenylamine (DPA) was investigated following treatment and storage at reduced temperature. Additional apples were treated with a mixture of [(14)C]DPA and deuterium-labeled DPA (D-10-DPA) for use in metabolite identification. Stored apples were sampled at 16 different intervals throughout a 40-week period. Total radioactive residue levels remained constant over the entire testing interval. The parent chemical and several metabolites were identified on the final harvest peel and pulp samples by quantitative and qualitative analytical methods. A majority of the terminal residue, which was confined largely in the peel, consisted of unmetabolized DPA. Residues in pulp, however, were largely composed of glycosyl conjugates of several hydroxylated diphenylamine (OH-DPA) metabolites. The major polar metabolite identified in stored apples was a glucose conjugate of 4-hydroxydiphenylamine (4-OH-DPA). Additional metabolites, characterized as glycosyl conjugates of 2-OH-DPA, 3-OH-DPA, 4-OH-DPA, or dihydroxy-DPA, were also detected along with their intact (i.e., nonconjugated) forms in apple pulp.

The role of glycosylation in flavonol-induced pollen germination.

Flavonols are small (C15) plant-specific molecules that are required for petunia and maize pollen to germinate. They exist in two chemical forms: the aglycone or glycosyl conjugates. Flavonol-deficient pollen is biochemically complemented by flavonol aglycones but not by the glycosylated forms that accumulate in wild type (WT) pollen. Coincident with the biochemical induction of germination, the added flavonol aglycone is rapidly converted to a galactoside and then to a glucosyl galactoside (diglycoside) that is identical to the compound present in WT pollen. A flavonol 3-O-galactosyltransferase (F3GalTase) activity has been identified that controls the formation of glycosylated flavonols in pollen. Importantly, this enzyme also catalyzes the reverse reaction, i.e. the production of the flavonol aglycone from the galactoside and UDP (Fig. 1). F3GalTase/RevGalTase therefore has the potential to control the level of the bioactive flavonol species and as a result, pollen germination.

Constitutive and inducible bentazon hydroxylation in shattercane ( Sorghum bicolor) and Johnsongrass ( S. halapense)

Bentazon hydroxylation was studied in excised shoots and microsomal preparations from shattercane ( Sorghum bicolor) and Johnsongrass ( S. halapense). Studies in vivo were conducted using excised shoots from untreated (constitutive) and naphthalic anhydride (NA)-treated (induced) etiolated seedlings. Both shattercane and Johnsongrass rapidly metabolized [ 14C]bentazon forming two metabolites: A minor metabolite of 6-OH-bentazon and a major metabolite, tentatively identified as the glycosyl conjugate of 6-OH-bentazon. Pretreating the seeds of both species with naphthalic anhydride increased bentazon metabolism in etiolated shoots by twofold. The cytochrome P450 inhibitor tetcyclacis (50 μ M) reduced bentazon metabolism in shoots from both NA-treated and untreated seedlings greater than 90%. Studies in vitro were conducted using microsomal preparations derived from NA-treated and untreated shattercance and Johnsongrass seedlings. Bentazon metabolism in microsomal preparations from both treated and untreated shattercane and Johnsongrass seedlings produced a single product: 6-OH-bentazon. Pretreatment of either shattercane or Johnsongrass with NA increased bentazon hydroxylation in vitro approximately twofold. Bentazon hydroxylation was nearly linear for 30 min in microsomal assays with both treated and untreated shattercane and Johnsongrass. Tetcyclacis (10 μ M) and PBO (100 μ M) inhibited bentazon hydroxylation in microsomes from treated and untreated seedlings from both species. Tridiphane (50 μ M) did not affect bentazon hydroxylation. Substrate saturation assays demonstrated that bentazon hydroxylation in vitro was saturable. Kinetic constants calculated for bentazon hydroxylation with NA-treated and untreated shattercane were K m = 234 and 317 μ M, respectively, and V max = 3.54 and 2.00 nmol · (mg protein · min) −1, respectively. Kinetic constants calculated for bentazon hydroxylation with NA-treated and untreated Johnsongrass were K m = 160 μM and 235 μ M, respectively, and V max = 1.25 and 0.68 nmol · (mg protein · min) −1, respectively. These data suggest that bentazon hydroxylation in NA-treated and untreated shattercane and Johnsongrass is mediated by constitutive and inducible cytochrome P450 monooxygenase(s).

Bile pigments in gallbladder and freshly-secreted hepatic duct bile from fed and fasted rainbow trout, Oncorhynchus mykiss

1. 1. Chromatographie analyses of bile pigments in rainbow trout reveal the presence of primarily unconjugated biliverdin (BV) and bilirubin (BR) glycosyl conjugates. Only trace amounts of unconjugated BR are present in hepatic duct (HD) bile: no β-glucuronidase activity is detectable. 2. 2. The per cent of BV and BR in HD and gallbladder biles is similar in fasted trout; however, the per cent of BV is significantly increased in HD bile from fed fish. 3. 3. Fasting decreases the rate of choleresis but does not alter the excretory rate of endogenous BV or BR. 4. 4. Erythrocyte life span is estimated to be approximately 500 days.

Effect of monooxygenase inhibitors on bentazon uptake and metabolism in maize cell suspension cultures

Maize “black Mexican sweet” (BMS) cell suspension cultures were used to study the effects of various cytochrome P450 monooxygenase inhibitors on the uptake and metabolism of the herbicide bentazon. Maize cells rapidly absorbed bentazon and metabolized it via aryl hydroxylation and glycosylation to a glycosyl conjugate of 6-hydroxybentazon. BMS cells accumulated bentazon to levels approximately 20-fold greater than those in the external medium. When BMS cells were incubated in an external medium containing 25 μ M bentazon, the formation of the glycosyl conjugate (ca. 2 nmol/min/g fresh wt) was rate-limited by aryl hydroxylation. Tetcyclacis, a plant growth retardant, phenylhydrazine, a mechanism-based cytochrome P450 inhibitor, and piperonyl butoxide, an insecticide synergist, inhibited bentazon metabolism with I 50 values of approximately 0.1, 1.0, and 1.0 μ M, respectively. Other mechanism-based cytochrome P450 inhibitors, 3(2,4-dichlorophenoxy)-1-propyne and aminobenzotriazole, also inhibited bentazon metabolism but were less effective. The results obtained with selected inhibitors are consistent with the hypothesis that aryl hydroxylation of bentazon is catalyzed by a cytochrome P450 monooxygenase.