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The synthesis of novel cyclic glycosyl imidates and an investigation of their potential as donors in glycosidation reactions is described. The results show that 1,2-cis glycosides obtained from the reactions of glycosyl acetates or cyclic imidates, each derived from amides of glucuronic acid, result from the anomerisation of initially formed 1,2-trans glycosides.
Research was undertaken to effect the oxidative decarboxylation of glycuronosides. Experiments with free D-glucuronic acid and aldonic acids were also executed. Both anodic decarboxylation and variants of the Ruff degradation reaction were investigated. Anodic decarboxylation was found to be the only successful method for the decarboxylation of glucuronosides. It was, therefore, proposed that glycuronosides can only undergo a one-electron oxidation to form an acyloxy radical, which decomposes to form carbon dioxide and a C-5 radical, that is, a Hofer-Moest decarboxylation. The radical is subsequently oxidized to a cation by means of a second one-electron oxidation. The cation undergoes nucleophilic attack from the solvent (water), whose product (a hemiacetal) undergoes a spontaneous hydrolysis to yield a dialdose (xylo-pentodialdose from D-glucuronosides).
Altrenogest (17alpha-allyl-17beta-hydroxyestra-4,9,11-trien-3-one) is a steroid used for the control of estrus in horses. This drug can potentially be abused in racehorses as the occurrence of estrus can alter their performance. This work describes an analytical method based on liquid chromatography-tandem mass spectrometry for the detection of altrenogest in horse urine down to a concentration of 13 pg/mL (0.042 nM). Furthermore, the qualitative aspect of metabolism of altrenogest in the horse has been studied. The main transformations that were found for this species were conjugation with glucuronic acid and sulfate. These phase II metabolites were identified by molecular mass and by comparison of their collision-induced dissociation product ion spectra with that of the synthetic aglycone at positive and negative potential, respectively. No phase I metabolites were discovered. In order to increase the ionisation in positive electrospray, a derivatisation procedure forming a basic oxime was tested. This process significantly increased the detection sensitivity for altrenogest glucuronide in horse urine.
Ependymins, a family of extracellular glycoproteins of goldfish and mammalian brain, were shown to contain N-linked complex glycan chains. These glycoproteins reacted with a monoclonal antibody, HNK-1 which recognizes a membrane antigen on a subset of human lymphocytes, myelin-associated glycoprotein glycoprotein epitope reacting with HNK-1 antibody was previously shown to include a terminal 3-sulfoglucuronosyl residue present in certain glycolipids of the nervous tissue (Chou et al., Biochem. Biophys. Res. Commun. 1985, 128, 383-388). In this report, the presence of glucuronic acid in ependymins was demonstrated by gas-liquid chromatography and mass spectrometry. We suggest that a 3-sulfoglucuronosyl residue may be the common epitope on HNK-1-reactive glycoproteins.
The O-polysaccharide of Proteus vulgaris O44, strain PrK 67/57 was studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H, 13C HMQC, HMQC-TOCSY and HMBC experiments. The polysaccharide was found to contain an amide of D-glucuronic acid with L-alanine [D-GlcA6(L-Ala)], and the following structure of the linear pentasaccharide repeating unit was established: [structure: see text]. The structural data of the O-polysaccharide and the results of serological studies with P. vulgaris O44 O-antiserum showed that the strain studied is unique among Proteus bacteria, which is in agreement with its classification in a separate Proteus serogroup, O44.
A novel human nucleotide sugar transporter (NST) which transports both UDP-glucuronic acid (UDP-GlcA) and UDP-N-acetylgalactosamine (UDP-GalNAc) has been identified, cloned and characterized. The strategy for the identification of the novel NST involved a search of the expressed sequence tags database for genes related to the human UDP-galactose transporter-related isozyme 1, followed by heterologous expression of a candidate gene (hUGTrel7) in Saccharomyces cerevisiae and biochemical analyses. Significantly more UDP-GlcA and UDP-GalNAc were translocated from the reaction medium into the lumen of microsomes prepared from the hUGTrel7-expressing yeast cells than into the control microsomes from cells not expressing hUGTrel7. The possibility that this transporter participates in glucuronidation and/or chondroitin sulfate biosynthesis is discussed.
In order to enhance the detection sensitivity of various glucuronic acid conjugates (phenol, menthol borneol, estrone and testosterone) in high-performance liquid chromatography (HPLC), the compounds were esterified with 4-bromomethyl-7-methoxycoumarin in the presence of potassium carbonate and 18-crown-6 in acetone. The resulting esters were chromatographed on either a normal-phase (NP) column (LiChrospher DIOL) with hexane-ethanol mixtures as eluents or a reversed-phase column (LiChrospher CH-18) with methanol-water mixtures. They were detected by UV spectrophotometry at 328 nm. The structure of the derivatives was confirmed by mass spectrometry by direct introduction and chemical ionization. Prior to this step, their isolation on a semi-preparative scale was performed by NP-HPLC. The extraction of the studied glucuronides from microsomal solutions was tested by ion-suppression and ion-pair liquid-liquid partition and liquid-solid chromatography (on octadecylsilica cartridges). Extraction and chromatographic data are discussed with regard to the determination of glucuronyltransferase activity towards the aglycones cited above.
Measurement of hepatic glucose production (HGP) by standard isotope dilution reveals only the net release of glucose from the liver, not the flux across glucose-6-phosphatase ([G6Pase] or total hepatic glucose output), hepatic glucose cycling (HGC), irreversible glucose disposal into glycogen in the liver (hepatic Rd), or net hepatic glucose balance. We describe two independent isotopic techniques for measuring these parameters in vivo, both of which use secreted glucuronate (GlcUA). HGC can be quantified by measuring a correction factor for glucose label retained in hepatic glucose-6-phosphate (G6P), sampled as GlcUA. A complementary technique for measuring total hepatic glucose output is also described (reverse dilution), requiring administration of no labeled glucose but instead a labeled gluconeogenic precursor and unlabeled glucose. Hepatic Rd is calculated by multiplying the rate of appearance (Ra) of hepatic UDP-glucose ([UDP-glc] based on dilution of labeled galactose in GlcUA) times the direct entry of glucose into hepatic UDP-glc and the fraction of labeled UDP-glc retained in the liver. The sum of hepatic Rd plus HGC represents the total hepatic glucose phosphorylation rate. Rats received intravenous (i.v.) glucose infusions at a rate of 15 to 30 mg/kg/min after a 24-hour fast. Despite a suppression of net HGP more than 50%, total hepatic glucose output was not significantly decreased, because of increased HGC. Total hepatic glucose output calculated by reverse dilution yielded similar results during i.v. glucose infusions at 15 mg/kg/min, although values were higher than obtained by the correction-factor method at 30 mg/kg/min. The fraction of labeled UDP-glc released into blood glucose, representing a hepatic glycogen cycle, decreased from 35% (fasted) to nearly 0% (i.v. glucose 30 mg/kg/min). Hepatic Rd was 1.4, 4.6, and 7.5 mg/kg/min (fasted and i.v. glucose 15 and 30 mg/kg/min, respectively); total hepatic glucose phosphorylation increased substantially (from 4.2 to 8.5 to 12.7 mg/kg/min) and net hepatic glucose balance changed from negative to positive during i.v. glucose. In conclusion, hepatic G6Pase flux, glucose phosphorylation, HGC, disposal of glucose into glycogen, and net glucose balance can be measured noninvasively in vivo under various metabolic conditions by techniques involving the GlcUA probe.
The glucuronic acid conjugate of N-hydroxy-4-aminobiphenyl believed to be the carrier form responsible for transporting the active N-hydroxy compound from its site of formation in the liver to its site of carcinogenic action in the bladder has been prepared synthetically. The synthetic conjugate is identical by infrared and chromatographic analyses with the conjugate isolated from urine, thus unequivocally establishing its structure as an N--C conjugate, sodium (N,4-biphenyl-N-hydroxy-D-glucuronosylamine).
Chemical modification of rat hepatic microsomes with N-ethylmaleimide (NEM) resulted in inactivation of UDP-N-acetylglucosamine (UDP-GlcNAc)-dependent stimulation of glucuronidation of p-nitrophenol. Inactivation kinetics and pH dependence were in agreement with the modification of a single sulfhydryl group. NEM also inactivated the uptake of UDP-glucuronic acid (UDP-GlcUA) but not UDP-glucose. With various sulfhydryl-modifying reagents, the inactivation of UDP-GlcUA uptake was linked to that of glucuronidation. UDP-GlcUA protected against NEM-sensitive inactivation of both UDP-GlcNAc-dependent stimulation of glucuronidation and UDP-GlcUA uptake, suggesting that the sulfhydryl group is located within or near the UDP-GlcUA binding site of the microsomal protein involved in the stimulation. Using microsomes labeled with biotin-conjugated maleimide and immunopurification with anti-peptide antibody against UDP-glucuronosyltransferase family 1 (UGT1) isozymes, immunopurified UGT1s were found to be labeled with the maleimide and UDP-GlcUA protected against the labeling as it did with the NEM-sensitive inactivation. These data suggest the involvement of a sulfhydryl residue of microsomal protein in the UDP-GlcNAc-dependent stimulation mechanism via the stimulation of UDP-GlcUA uptake into microsomal vesicles.
The pH-rate profiles for the hydrolysis of uridine 5'-diphospho-alpha-D-glucose (UDPG) and uridine 5'-diphospho-alpha-D-glucuronic acid (UDPGA) in aqueous solution have been measured. The results obtained and a comparison with other data suggests that the mechanism of hydrolysis of each activated glycosyl-donor at pH 1-4 probably involves the slow ionisation, via an S(N)1 process, of the neutral molecule to a glycosyl ion and UDP. From these data, the catalytic power (k(cat)/k(uncat)) of the glycosyltransferases has been estimated for the first time to be in the order of 10(11-13).
It has been suggested that the FGF-2 binding site on heparan sulfate chains is a trisulfated pentasaccharide containing three hexuronic acid units. The configuration at C-5 of two of them being undetermined, we have synthesized the four possible pentasaccharides, and have evaluated their FGF-2 binding affinity through in vitro biological assays. The pentasaccharide containing L-iduronic acid as the sole hexuronic acid showed higher affinity for FGF-2 than the other pentasaccharides, where one hexuronic acid unit at least is D-glucuronic acid.
Previous studies have documented the presence of protein-mediated transport of UDP-glucuronic acid (UDP-GlcUA) in rat liver endoplasmic reticulum (ER). To determine the crucial amino acids of the membrane transporter and evaluate their function in regulating the glucuronidation reaction, we examined the effect of histidyl-specific irreversible inhibitors on the uptake of radiolabeled UDP-GlcUA in rat liver ER. Inactivation of uptake (initial rate) was more pronounced with hydrophobic reagents [diethyl pyrocarbonate (DEPC), p-bromophenacyl bromide] as compared to the more hydrophilic reagent (p-nitrobenzenesulfonic acid methyl ester). DEPC was used to further characterize the inhibition because of its greater specificity for protein histidyl residues. While initial [14C]UDP-GlcUA uptake rates were diminished by DEPC treatment of intact microsomes, the accumulation of isotope at equilibrium was not significantly affected, indicating no loss of vesicle integrity. A pKa of approximately 7 for the modified residue(s) of the transporter supported the alkylation of imidazole moieties. Protection against inactivation was observed with UDP-GlcUA as well as other nucleotide-sugars known for their interaction with this transporter. Uptake activity of the transporter (Vmax) but not UDP-GlcUA binding (Km) was affected by a limited inactivation. Furthermore, a partial inactivation of the transporter impaired the binding of the photoaffinity label [beta-32P]5-azido-UDP-GlcUA to UDP-glucuronosyltransferases (UGTs) in intact, but not in detergent-disrupted, ER vesicles. These results demonstrate the involvement of histidyl residue(s) in the UDP-GlcUA uptake process in rat liver ER, provide additional evidence for the lumenal orientation of the UGT active site, and support the view that translocation of the UGT cosubstrate is a rate-limiting step of the glucuronidation reaction.
[structures: see text] The synthesis of anilides derived from glucuronic acid is described. Secondary anilides had a Z configuration in the solid state and showed intramolecular and intermolecular hydrogen bonding. However, on the basis of NMR and IR studies, there was generally no evidence for the same hydrogen bonding in solution. Tertiary anilides showed a strong preference for the E configuration on the basis of NOE studies and molecular mechanics calculations. The alkylation of the secondary anilides induces a configurational switch that alters the orientation of the aromatic group with respect to the pyranose, which has relevance for presentation or orientation of pharmacophoric groups on carbohydrate scaffolds.
[structures: see text] Syntheses and structural analyses of bivalent carbohydrates based on anilides of glucuronic acid are described. Secondary anilides predominantly adopted the Z-anti structure; there is also evidence for population of the Z-syn isomer. Bivalent tertiary anilides displayed two signal sets in their NMR spectra, consistent with the presence of (i) a major isomer where both amides have E configurations (EE) and (ii) a minor isomer where one amide is E and the other Z (EZ). Qualitative NOE/ROE spectroscopic studies in solution support the proposal that the anti conformation is preferred for E amides. The crystal structure of one bivalent tertiary anilide showed E-anti and E-syn structural isomers; intramolecular carbohydrate-carbohydrate stacking was observed and mediated by carbonyl-pyranose, azide-azide, and pyranose-aromatic interactions. The EE to EZ isomer ratio, or the degree of folding, for tertiary amides, was greatest for a bivalent compound containing two alpha-glycosyl azide groups; this was enhanced in water, suggesting that hydrophobic interactions are partially but not wholly responsible. Computational methods predicted azide-aromatic (N...H-C interaction) and azide-azide interactions for folded isomers. The close contact of the azide and aromatic protons (N...H-C interaction) was observed upon examination of the close packing in the crystal structure of a related monomer. It is proposed that the alpha-azide group is more optimally aligned, compared to the beta-azide, to facilitate interaction and minimize the surface area of the hydrophobic groups exposed to water, and this leads to the increased folding. The alkylation of bivalent secondary anilides induces a switch from Z to E amide that alters the scaffold orientation. The synthesis of a bivalent mannoside, based on a secondary anilide scaffold, for investigation of mannose-binding receptor cross-linking and lattice formation is described.
To prepare two new glucuronide conjugates, allyl ester and allyl carbonates were used as protecting groups of the glucuronic moiety. In this way, an aniline glycosyl carbamate spacer linked to the 2'-OH of paclitaxel was obtained. By using palladium chemistry, an efficient one-step removal of all the allyl groups at the end of the synthesis afforded the desired compounds in good yields.
[reaction: see text] Participating acyl groups located at C-2 in glucosyl and related donors generally promote formation of 1,2-trans-glycosides. Reactions of some glucuronic acid donors with TMSN(3)/SnCl(4) or ROH/SnCl(4) gave only the 1,2-cis-glycoside. The stereoselectivity is consistent with participation of the C-6 group. The methodology was used for the synthesis of a Kdn2en mimetic with the alpha-configuration.
Glucuronic acid n-alkyl esters, a novel class of promising biosurfactants and their corresponding glucose esters with the same side-chain length, were synthesized by direct esterification in a non-aqueous phase (tert-butanol) using an immobilized lipase.