Glucopyranosiduronic Acid Research Articles

Structural basis of catalysis and substrate recognition by the NAD(H)-dependent α-d-glucuronidase from the glycoside hydrolase family 4.

Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and β-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product d-glucuronic acid, at 2.15, 1.97 and 1.85 Å resolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure-function relationships within the unique GH4 family.

Reverse-phase HPLC purification for an extremely unstable glucuronide metabolite

A pure β-​D-​Glucopyranosiduronic acid metabolite (≥98.0 % purity and a single impurity ≤0.50 %) was requested for biological studies. Due to its unusual instability, the purification of the glucuronide metabolite was extremely challenging. Initially, the crude sample (89 % HPLC area purity) was purified on a Waters SunFire C8 OBD column with 40 mM ammonium acetate buffer and acetonitrile as the mobile phase under a gradient program. The purified glucuronide metabolite solid was obtained by evaporation and lyophilization. However, this procedure yielded the target compound with 97.6 % HPLC area purity and did not meet the requirements. Through the investigation, lyophilization was identified as the key step for the purity of the metabolite, and further lyophilization resulted in an increased amount of the degraded impurities. To better understand the compound, stability studies of the purified metabolite were conducted under sample media, organic solvent, acid, base, and light exposure. The compound was observed to be extremely unstable in water, acid, base and methanol, and sensitive to light, but relatively stable in ammonium acetate buffer (pH 5.0). Taking into account compound stability and the initial purification method, the improved purification procedure was successfully developed and the purified glucuronide metabolite was obtained with 99.2 % HPLC area purity and 0.39 % of the largest single impurity.

13C-Labeled Idohexopyranosyl Rings: Effects of Methyl Glycosidation and C6 Oxidation on Ring Conformational Equilibria.

An ensemble of JHH, JCH, and JCC values was measured in aqueous solutions of methyl α- and β-d-idohexopyranosides containing selective 13C-enrichment at various carbons. By comparing these J-couplings to those reported previously in the α- and β-d-idohexopyranoses, methyl glycosidation was found to affect ring conformational equilibria, with the percentages of 4C1 forms based on 3JHH analysis as follows: α-d-idopyranose, ∼18%; methyl α-d-idopyranoside, ∼42%; methyl β-d-idopyranoside, ∼74%; β-d-idopyranose, 82%. JCH and JCC values were analyzed with assistance from theoretical values obtained from density functional theory (DFT) calculations. Linearized plots of the percentages of 4C1 against limiting JCH and JCC values in the chair forms were used to (a) determine the compatibility of the experimental JCH and JCC values with 4C1/1C4 ratios determined from JHH analysis and (b) determine the sensitivity of specific JCH and JCC values to ring conformation. Ring conformational equilibria for methyl idohexopyranosides differ significantly from those predicted from recent molecular dynamics (MD) simulations, indicating that equilibria determined by MD for ring configurations with energetically flat pseudorotational itineraries may not be quantitative. J-couplings in methyl α-l-[6-13C]idopyranosiduronic acid and methyl α-d-[6-13C]glucopyranosiduronic acid were measured as a function of solution pH. The ring conformational equilibrium is pH-dependent in the iduronic acid.

Synthesis of novel glycophanes derived from glucuronic acid by ring closing alkene and alkyne metathesis

Cyclophanes and their analogues are of interest in bioactive molecule development and in biomimetic, supramolecular and materials chemistry. Novel hybrids of sugars and cyclophanes (glycophanes) have been prepared via the coupling of alkenyl and alkynyl glucopyranosiduronic acids with phenylene-1,4-diamine and xylene-1,4-diamine and subsequent intramolecular metathesis. Structural studies showed that the geometric arrangements of the sugar groups in the macrocycles containing secondary amides differ from those in macrocycles that contain tertiary amides. This is due to the amides adopting different configurational preferences. The compounds had low solubility in water, precluding an investigation of their recognition phenomena in this medium.

Glycosidation–Anomerisation Reactions of 6,1‐Anhydroglucopyranuronic Acid and Anomerisation of β‐D‐Glucopyranosiduronic Acids Promoted by SnCl4

The reaction of silylated nucleophiles with 6,1-anhydroglucopyranuronic acid (glucuronic acid 6,1-lactones) catalysed by tin(IV) chloride provides 1,2-trans or 1,2-cis (deoxy)glycosides in a manner dependent on the donor structure. The alpha-glycoside was obtained for reactions of the donor with the 2-acyl group and 2-deoxydonors, whereas the 2-deoxy-2-iodo donor gave the beta-glycoside. Experimental evidence shows that when 1,2-cis-glycoside formation occurs, the anomerisation of initially formed 1,2-trans-glycosides catalysed by SnCl(4) is possible. The anomerisation of beta-D-glucopyranosiduronic acids was found to be faster, in some cases, than anomerisation of related beta-D-glucopyranosiduronic acid esters and beta-D-glucopyranoside derivatives and the rates are dependent on the structure of the aglycon. Moreover, the rates of anomerisation of beta-D-glucopyranuronic acid derivatives can be qualitatively correlated with rates of hydrolysis of beta-D-glucopyranosiduronic acids. Mechanistic possibilities for the reactions are considered.

Convenient syntheses of metabolically important quercetin glucuronides and sulfates

Synthetic approaches to the major human plasma metabolites of quercetin, quercetin 3′-sulfate and the β- d -glucopyranosiduronic acid derivatives 3′-methylquercetin 3-glucuronide (isorhamnetin 3-glucuronide), quercetin 3-glucuronide and quercetin 3′-glucuronide are described. This is the first report of the chemical synthesis of quercetin 3′-glucuronide. All procedures start from the same precursor, 4′,7-di- O -benzylquercetin, and all are more convenient than existing methods.

Differently Glycosidated 2‐Amino‐2‐deoxy‐d‐glucopyranosiduronic Acids as Building Blocks in Peptide Synthesis

AbstractSeven differently glycosidated sugar amino acids (SSAs) derived from glucosamine have been prepared. Following standard solution‐phase peptide‐coupling procedures, the glycosidated 2‐amino‐2‐deoxy‐D‐glucopyranosiduronic acids were condensed with natural amino acids to furnish useful heterodi‐ and ‐trimeric building blocks to be used in peptide synthesis. Combinations of these building blocks yielded hetero‐oligomeric peptides with two sugar amino acid units in different distances to each other. These were prepared to evaluate the influence of glycosidic side chains on the peptide backbone. Conformations of selected examples were examined by means of ROESY spectroscopy in combination with molecular dynamics (MD) simulations and circular‐dichroism (CD) studies.

Synthesis of two hyaluronan trisaccharides

[formula: see text] The synthesis of two hyaluronan trisaccharides, methyl O-(beta-D-glucopyranosyluronic acid)-(1,3)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1,4)-O-beta-D- glucopyranosiduronic acid and methyl O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1,4)-O-beta- D-glycopyranosyluronic acid)-(1,3)-O-(2-acetamido-2-deoxy-beta-D-glucopyranoside, are described. Construction of the target molecules was achieved though a combination of the phenyl sulfoxide and trichloroacetimidate glycosylation methodologies. This is the first report on the synthesis of the beta-methyl derivatives, which represent the smallest fragments that incorporate all the structural features of polymeric hyaluronan.

Medicinal foodstuffs. VII. On the saponin constituents with glucose and alcohol absorption-inhibitory activity from a food garnish "Tonburi", the fruit of Japanese Kochia scoparia (L.) Schrad.: structures of scoparianosides A, B, and C.

The methanolic extract of a food garnish "Tonburi", the fruit of Japanese kochia scoparia (L.) Schrad. (Chenopodiaceae), was found to inhibit the increase in serum glucose-loaded rats. Through bioassay-guided separation, momordin Ic and its 2'-O-beta-D-glucopyranoside were isolated as the active principles from this medicinal foodstuff together with three new saponins named scoparianosides A, B, and C. The structures of scoparianosides A, B, and C were elucidated on the basis of chemical and physicochemical evidence as 3 beta, 22 alpha-dihydroxyolean-12en-28-oic acid (22 alpha-hydroxyoleanolic acid), 3-O-beta-D-xylopyranosyl (1 --> 3)-beta-D-glucopyranosiduronic acid, 3 beta-hydroxyolean -18-en-28-oic acid (morolic acid), 3-O-beta-D-xylopyranosyl(1 --> 3)-beta-D -glucopyranosiduronic acid, and 3 beta-hydroxyolean-13(18)-en-28-oic acid, 3-O-beta-D-xylopyranoxyl(1 --> 3) -beta-D-glucopyranosiduronic acid. Momordin Ic and its 2'-O-beta-D-glucopyranoside, both of which are the principal saponin constituents of this medicinal foodstuffs, were found to potently inhibit glucose and ethanol absorption in rats.

The use of 2-deoxy-2-trichloroacetamido-?-glucopyranose derivatives in syntheses of hyaluronic acid-related tetra-, hexa-, and octa-saccharides having a methyl β-?-glucopyranosiduronic acid at the reducing end

The use of 2-deoxy-2-trichloroacetamido-?-glucopyranose derivatives in syntheses of hyaluronic acid-related tetra-, hexa-, and octa-saccharides having a methyl β-?-glucopyranosiduronic acid at the reducing end

Characterization of positional and anomeric isomers of methyl 2‐O‐ and 3‐O‐sulfo‐D‐glucopyranosiduronic acids and methyl 2‐O‐ and 3‐O‐sulfo‐D‐glucopyranosides by secondary ion mass spectrometry

AbstractSecondary ion mass spectrometry (SIMS) of anomeric pairs of methyl 2‐O‐ and 3‐O‐sulfo‐D‐glucopyranosiduronic acids and methyl 2‐O‐ and 3‐O‐sulfo‐D‐glucopyranosides show the deprotonated molecule in the negative‐ion mode and ammoniated and sodiated molecules in the positive‐ion mode. The four isomers were distinguished from each other by linked scanning at constant B/E in negative‐ and positive‐ion SIMS, which gave information characteristic of the position of the sulfate group and even the configuration of the anomeric methoxy group. The main fragmentation processes correspond to the loss of methanol and the elimination of SO3.

Enzymic synthesis of l-ascorbic acid from synthetic and biological d-glucurono-1,4-lactone conjugates

d-Glucofuranosiduronolactone conjugates, namely, semicarbazone, oxime, cyanohydrin, and thioacetal of d-glucurono-1,4-lactone, were converted to l-ascorbic acid by an enzyme system present in rat, goat, or rabbit liver microsomes. Guinea pig liver microsomes were ineffective. Neither methyl- d-glucofuranosiduronolactone nor any glucopyranosiduronic acid conjugates examined were converted into l-ascorbic acid. Methods of preparation and properties of d-glucurono-1,4-lactone semicarbazone and d-glucurono-1,4-lactone oxime have been described and a tentative mechanism of enzymic reduction of the conjugates into l-gulono-1,4-lactone has been suggested. A conjugate of d-glucurono-1,4-lactone and imidazole, which is a substrate for the microsomal enzyme leading to synthesis of l-ascorbic acid, has been identified in the urine of rat treated with chloretone, barbital, and 1,2-benzanthracene. It has been indicated that the enhanced synthesis of l-ascorbic acid after administration of various drugs and toxic chemical compounds is due to the induced formation of this endogenous substrate.

Bilirubin conjugates of human bile. The excretion of bilirubin as the acyl glycosides of aldobiouronic acid, pseudoaldobiouronic acid and hexuronosylhexuronic acid, with a branched-chain hexuronic acid as one of the components of the hexuronosylhexuronide.

Structure elucidations have been performed on the bilirubin conjugates isolated from human hepatic bile as the phenylazo derivatives. The major bilirubin conjugates are excreted, not as was formerly thought in the form of glucuronides, but as the acyl glycosides of aldobiouronic acid, pseudoaldobiouronic acid and hexuronosylhexuronic acid. The isolated aldobiouronides are proposed to have the structures of an acyl 6-O-hexopyranosyluronic acid-hexopyranoside, an acyl 4-O-hexofuranosyluronic acid-d-glucopyranoside, and an acyl 4-O-beta-d-glucofuranosyluronic acid-d-glucopyranoside respectively, with the acyl radicals being those of the phenylazo derivative of bilirubin. The pseudoaldobiouronide is suggested to be the acyl 4-O-alpha-d-glucofuranosyl-beta-d -glucopyranosiduronic acid, with the acyl radical being that of the phenylazo derivative of vinylneoxanthobilirubinic acid. The hexuronosylhexuronide presumably is the acyl 4-O-(3-C-hydroxymethylribofuranosyluronic acid)-beta-d-glucopyranosiduronic acid, with the acyl radical being that of the phenylazo derivative of bilirubin. The 3-C-hydroxymethylriburonic acid, isolated as one of the components of the hexuronosylhexuronide, is the first natural branched-chain hexuronic acid to be detected, and the first branched-chain sugar ever detected in humans.

Steroid conjugates. V. 1 The synthesis of a suifoglucuronide derivative of estriol

The di-potassium salt of estriol-3-sulfate-16-glucuronide [17β-hydroxy-3-sulfooxyestra-l,3,5(lO)-trien-16α-yl-β-=D-glucopyrano-siduronic acid di-potassium salt] (11) has been synthesized by a pathway utilizing the direct condensation of methyl (2,3,-tri-O-acetyl-α-bromo-=D-glucopyranosid)ronate with estriol-3-benzyl ether (2). The structure of this intermediate, methyl (3-benzyloxy-17β-hydroxyestra-l,3,5(lO)-trien-16α-yl-2',3',4'-tri-O-acetyl-β-D-glucopyranosid)ronate (3) was unequivocally confirmed by its synthesis from methyl (3Ubenzyloxyestra-l,3,5(lO)-trien-17-on-16α-yl-2',3',4'-tri-O-acetyl-β-=D-glucopyranosid)ronate (5). Selective removal of the benzyl group followed by sulfation with triethylaminesulfur trioxide afforded the key compound, methyl (17β-acetoxy-3-triethylammoniosulfooxyestra-l,3,5(lO)-trien-16α-yl-2',3',4'-tri-O-acetyl-β-=D-glucopyranosid)ronate (10). Basic hydrolysis gave the sulfoglucuronide as its di-potassium salt.

The acid hydrolysis of glycosides : V. Effect of the nature of the aglycon on the hydrolysis of glycosiduronic acids (glycuronides)

Rate coefficients and kinetic parameters have been determined for the acid-catalyzed hydrolysis of fourteen β- D-glucopyranosiduronic acids containing aglycons of different electron-affinity. Energies and entropies of activation of all of these compounds were lower than those of the corresponding β- D-glucopyranosides. The glucopyranosiduronic acids were hydrolyzed at rates that were inversely proportional to the electron affinity of the aglycon, whereas the rates of hydrolysis of the glucopyranosides were independent of the polarity of the aglycon. (Carboxymethyl) β- D-glucopyranosiduronic acid was hydrolyzed at one hundredth of the rate expected. The phenomena are discussed, assuming a catalytic interaction between the carboxyl groups and the glycosidic oxygen atom. Substitution, in the aglycon, of alkyl groups for hydrogen atoms at the carbon atom adjacent to the glycosidic oxygen atom increased the rate of hydrolysis both of the glucopyranosiduronic acids and of the glucopyranosides.

Studies on the hydrolysis of glucuronosides. II. The effect of aglycon on acid hydrolysis rate of beta-D-glucopyranosiduronic acids

Effect of the electron affinity of the aglycone group in the rate of acid hydrolysis of alkyl-β-D-glucopyranosiduronic acid was examined. Electron-attracting aglycone was found to decrease the acid hydrolysis rate greatly, while the electron releasing aglycone greatly increased the rate. The Hammet-Taft rule was applied to the acid hydrolysis rate of glucopyranosiduronic acid and glucopyranoside, and the result suggested that the mechanism of hydrolysis was the same for glucopyranosiduronic acid (ρ*) and for glucopyranoside (ρ1*) having an electron releasing aglycone, but differed from that of glycopyranoside(ρ2*)having an electron-attracting aglycone. The activation energy has the greatest effect on the acid hydrolysis rate constant of glucopyranosiduronic acid and the entropy of activation has no part in it.