Glucuronic acid conjugates of clofibrate: Four isomeric structures

After ingestion of the hypolipidemic drug, clofibrate, four glucuronic acid conjugates of 2-(4′-chlorophenoxy)-2-methylpropanoic acid have been found in the urine of man. The four conjugates differed structurally at the glucuronic acid moiety which was found to be present as the α- and β-anomers of both its pyranose and furanose forms. The structural assignments were based on gas chromatographic and mass spectral data. This is the first documentation of different anomeric forms of glucuronic acid conjugates as metabolites.     

A novel, highly modified, bacteriophage DNA in which thymine is partly replaced by a phosphoglucuronate moiety covalently bound to 5-(4',5'-dihydroxypentyl)uracil

Bacteriophage SP-15, which infects Bacillus subtilis, contains a highly modified DNA in which 62% of its thymine residues are replaced by 5-(4',5'-dihydroxypentyl)uracil to which is attached a phosphoglucuronate via a phosphodiester linkage to one of the hydroxyl groups of the pentyl side chain. Glucose is also bound to this residue probably by glycosidic linkage to the other hydroxyl group of the pentyl side chain. In 0.3 M KOH at 37 degrees C, glucuronic acid 1-phosphate is slowly released from this DNA. After enzymatic or acid-induced dephosphorylation, this sugar was identified by chromatography in two thin layer chromatography systems, conversion to glucuronolactone under conditions known to lactonize glucuronic acid, and reaction in four colorimetric assays for hexuronic acids. Phage SP-15 DNA is the first DNA found to have a uronic acid moiety or a phosphate which is not part of the phosphodiester backbone. The glucuronic acid phosphate might be derived from uridine pyrophosphoglucuronic acid, whose glucuronic acid moiety is normally destined for synthesis of teichuronic acid in the host cell wall.     

Synthesis of oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3

Di-, tri- and tetrasaccharide fragments of the linear chain of the capsular polysaccharide of Streptococcus pneumoniae type 3 consisting of glucose and glucuronic acid residues connected with beta 1----3- and beta 1----4-glycosidic linkage have been synthesised. A new method for selective deprotection of C3-hydroxyl group in the glucopyranuronic acid moiety is proposed.     

The copolymeric structure of pig skin dermatan sulphate. Characterization of d-glucuronic acid-containing oligosaccharides isolated after controlled degradation of oxydermatan sulphate

Selective periodate oxidation of unsubstituted l-iduronic acid residues in copolymeric dermatan sulphate chains was followed by reduction-hydrolysis or alkaline elimination. By this procedure the glucuronic acid-containing periods were isolated in oligosaccharide form; general formula: [Formula: see text] Further degradation of these oligosaccharides with chondroitinase-AC yielded three types of products: (a) sulphated trisaccharide containing an unsaturated uronosyl moiety in the non-reducing terminal and a C(4) fragment in the reducing terminal, DeltaUA-GalNAc-(-SO(4))-R; (b) monosulphated, unsaturated disaccharide, DeltaUA-GalNAc-SO(4) when n is greater than or equal to 2; and (c) N-acetylgalactosamine with or without sulphate. Oligosaccharides containing a single glucuronic acid residue (n=1) comprised more than half of the glucuronic acid-containing oligosaccharides. The terminal N-acetylgalactosamine moiety of the shortest oligosaccharide was largely 4-sulphated, whereas higher oligosaccharides primarily contained 6-sulphated or unsulphated hexosamine moieties in the same position. Moreover, IdUA-SO(4)-containing oligosaccharides were encountered. These oligosaccharides were resistant to the action of chondroitinase-ABC.     

The isolation and characterization of bilirubin diglucuronide, the major bilirubin conjugate in dog and human bile.

The chemical structure of the major conjugate of bilirubin was unequivocally elucidated by structural analysis. The conjugated bilirubins were first separated from the lipid components of human duodenal aspirates or dog gall-bladder bile, and then resolved by t.l.c. into a series of tetrapyrroles. The major tetrapyrrole was then converted into its more stable dipyrrolic azo derivative for further analysis. The conjugated moiety of the azopigment was characterized after methanolysis with sodium methoxide. This reaction yields two types of product, those soluble in water and those soluble in organic solvents. The organic-soluble fraction was shown by t.l.c. and mass spectrometry to contain the methyl esters of the dipyrrolic azo derivatives of bilirubin. The water-soluble materials were analysed by enzymic procedures, t.l.c., n.m.r. spectrometry and combined g.l.c. and mass spectrometry. This analysis showed that the only water-soluble product resulting from the methanolysis was glucuronic acid. The structure was identical with that of pure standards, on both mass spectrometry and n.m.r. spectroscopy. No contaminating moieties were found. Quantitative measurement indicated that the glucuronic acid had been released in a 1:1 molar ratio with the resulting methyl esters of the dipyrrolic azo derivatives of bilirubin. This unequivocally establishes bilirubin diglucuronide as the major pigment present in bile. Past problems with identification of bilirubin diglucuronide were shown to originate from procedures which resulted in incomplete separation and isolation of the azopigments of the conjugated bilirubins, owing to contamination by biliary lipids.     

Functional specialization of UDP‐glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin,glycyrrhizin

Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side‐effects. Here, we report that UDP‐glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP‐glucuronic acid to glycyrrhetinic acid 3‐O‐monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin‐deficient (83‐555) strain of G. uralensis. The natural variant showed loss of specificity for UDP‐glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83‐555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP‐glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP‐glucuronic acid. The functional arginine residue and resultant specificity for UDP‐glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP‐sugar selectivity for the rational engineering of sweet triterpenoid saponins.     

Characterization of anti-complementary acidic heteroglycans from the seed of Coix lacryma-jobi var. ma-yuen

The anti-complementary polysaccharides, CA-1 and CA-2, were purified from the seed of Coix lacryma-jobi L. var. ma-yuen. CA-1 consists of rhamnose, arabinose, xylose, galactose, galacturonic acid and glucuronic acid in molar ratios of 1.8:43.8:10.8:33.2:3.2:7.2, and CA-2 consists of rhamnose, arabinose, xylose, mannose, galactose, glucose, galacturonic acid and glucuronic acid in molar ratios of 2.4:37.0:11.8:1.7:35.6:2.9:2.6:6.0. CA-1 and CA-2 contained 8 ∼ 11% protein. Their M_rs were estimated to be 160 000 in CA-1 and 70 000 in CA-2 by gel filtration. CA-2 showed more potent anti-complementary activity than CA-1 in low dose.Methylation analysis of CA-1, its carboxyl-reduced products (reduced CA-1a and CA-1b) and CA-2 were carried out by the use of GC/MS and the results suggested that CA-1 has a very complicated and highly branched structure, and CA-2 is also composed of the same glycosidic linkages as CA-1 in different molar proportions. The results of exo α-L-arabinofuranosidase treatment and partial acid hydrolysis suggested that CA-1 and CA-2 contained arabino 3,6-galactan moiety and most of the arabinose was present as an α-L-furanosyl residues in the non-reducing terminals and (1 → 5)-linked side chains which mostly attached to the O-3 of (1 → 6)-linked galactopyranosyl residues. The results also suggested that CA-1 and CA-2 contained rhamnogalacturonan moiety which has a main chain consisting of (1 → 4)-linked galacturonic acid and (1 → 2)-linked rhamnose, and arabino 3,6- and 4-galactan might be attached to the rhamnosyl residue at the O-4. All glucuronic acid residues were present at the non-reducing terminals.     

Characterization of sulfated glucuronic acid containing glycolipids reacting with IgM M-proteins in patients with neuropathy

In some patients with neuropathy and plasma cell dyscrasia, the serum IgM M-proteins are known to bind to the myelin associated glycoprotein and to peripheral nerve glycolipids. We have isolated two acidic glycolipids which bind to the M-protein from human cauda equina by DEAE-Sephadex, Iatrobeads, and high performance liquid column chromatographies. The major acidic glycolipid migrated between GM1 and GD1a and the minor acidic glycolipid migrated between GD1a and GD1b. Their structures were elucidated by sugar analysis, enzymatic digestion, mild acid hydrolysis, permethylation, fast atom bombardment mass spectrometry, and NMR studies. Their core structure was confirmed to be paragloboside by high performance thin-layer chromatography-immunostaining using anti-paragloboside monoclonal antibody. Both acidic glycolipids lacked sialic acid but contained sulfated glucuronic acid as their acidic moiety. The sulfate group in the glucuronic acid was established by periodate oxidation and permethylation studies to be attached to the 3 position. The structures of the two acidic glycolipids are therefore consistent with the following: IV3GlcUA(3-sulfate)nLcOse4Cer and VI3GlcUA(3-sulfate)nLcOse6Cer. Additionally, the free carboxyl group on the glucuronic acid residue was shown to be necessary to bind the IgM M-proteins from neuropathy patients.     

The N-glycan acceptor specificity of a glucuronyltransferase, GlcAT-P, associated with biosynthesis of the HNK-1 epitope

The acceptor specificity of a rat brain glucuronyltransferase, GlcAT-P, associated with biosynthesis of the HNK-1 epitope on glycoproteins, was investigated using asialoorosomucoid as a model acceptor substrate. Structural analysis of N-linked oligosaccharides, to which glucuronic acid was transferred by GlcAT-P, by means of two-dimensional mapping of pyridylamino-oligosaccharides and MS spectrometry, demonstrated that the enzyme transferred glucuronic acid to bi-, tri-, and tetra-antennary complex type sugar chains, with almost equal efficiency, indicating that the enzyme has no preference as to the number of acceptor sugar branches. Next, we studied the branch specificity of this enzyme by means of the selective branch scission method involving two step exoglycosidase digestion using authentic pyridylamino-oligosaccharides. The GlcAT-P is highly specific for the terminal N-acetyllactosamine structure and no glucuronic acid was incorporated into a Gal1-3GlcNAc moiety. The GlcAT-P transferred glucuronic acid to the galactose residues in the N-acetyllactosamine branches of bi-, tri-, and tetra-antennary oligosaccharide chains, with different efficiencies and most preferentially to those in the Gal1-4GlcNAc1-4Man1-3 branch.     

Studies on the biosynthesis of striatal-type diterpenoids and the biological activity of herical

Feeding experiments with specifically 13C-labeled glucose disclosed that the diterpenoid part of the striatals/striatins is formed via the mevalonate pathway, whereas the pentose moiety originates either via glucuronic acid (70%) or the pentose phosphate cycle (30%). Application of radioactively labeled herical to cultures of Hericium ramosum demonstrates the pivotal role of this cyathane-xyloside in striatal biosynthesis. Herical inhibits a large spectrum of fungi and bacteria and shows cytotoxic and hemolytic properties.     

Functional characterization of GumK, a membrane-associated beta-glucuronosyltransferase from Xanthomonas campestris required for xanthan polysaccharide synthesis

Xanthomonas campestris is a Gram-negative bacterium that produces an exopolysaccharide known as xanthan gum. Xanthan is involved in a variety of biological functions, including pathogenesis, and is widely used in the industry as thickener and viscosifier. Although the genetics and biosynthetic process of xanthan are well documented, the enzymatic components have not been examined and no data on glycosyltransferases have been reported. We describe the functional characterization of the gumK gene product, an essential protein for xanthan synthesis. Immunoblots and complementation studies showed that GumK is a 44-kDa protein associated to the membrane fraction. This value corresponds to the expected molecular mass for GumK encoded by an extended open reading frame than proposed from previous genetic data and in X. campestris published complete genome. The protein was expressed in Escherichia coli cells. The purified protein catalyzed the transfer of a glucuronic acid residue from UDP-glucuronic acid to mannose-alpha-1,3-glucose-beta-1,4-glucose-P-P-polyisoprenyl with formation of a glucuronic acid-beta-mannose linkage. We examined the acceptor substrate specificity. GumK was unable to use the trisaccharide acceptor freed from the pyrophosphate lipid moiety. Replacement of the natural lipid moiety by phytanyl showed that the catalytic function could proceed with glucuronic acid transfer. These results suggest the enzyme does not show specificity for the lipidic portion of the acceptor. GumK showed diminished activity when tested with 6-O-acetyl-mannose-alpha-1,3-glucose-beta-1,4-glucose-P-P-polyisoprenyl, a putative intermediate in the synthesis of xanthan. This could indicate that acetylation of the internal mannose takes place after the formation of the GumK product.     

Duocarmycin-based prodrugs for cancer prodrug monotherapy

The synthesis and biological evaluation of novel prodrugs based on the cytotoxic antibiotic duocarmycin SA (1) for a selective treatment of cancer using a prodrug monotherapy (PMT) are described. Transformation of the phenol 8 with the glucuronic acid benzyl ester trichloroacetimidate 9b followed by reaction with DMAI x HCl (10) gives the glucuronide 11b, which is deprotected to afford the desired prodrug 4a containing a glucuronic acid moiety. In addition, the prodrug 4b with a glucuronic methyl ester unit is prepared. The cytotoxicity of the glucuronides is determined using a HTCFA-assay with IC(50) values of 610 nM for 4a and 3300 nM for 4b. In the presence of beta-glucuronidase, 4a expresses an IC(50) value of 0.9 nM and 4b of 2.1 nM resulting in QIC(50) values of about 700 for 4a and 1600 for 4b.     

Sargassan: A sulphated heteropolysaccharide from Sargassum linifolium

A new sulphated heteropolysaccharide containing glucuronic acid, mannose, galactose, xylose, fucose and a protein moiety has been extracted from Sargassum linifolium. The polysaccharide extracted with HCl was richer in its carbohydrate and protein contents and contained lower amounts of ash than that extracted with oxalic acid.     

Structural interactions in chondroitin 4-sulfate mediated adherence of Plasmodium falciparum infected erythrocytes in human placenta during pregnancy-associated malaria

Infection with Plasmodium falciparum during pregnancy results in the adherence of infected red blood cells (IRBCs) in placenta, causing pregnancy-associated malaria with severe health complications in mothers and fetuses. The chondroitin 4-sulfate (C4S) chains of very low sulfated chondroitin sulfate proteoglycans (CSPGs) in placenta mediate the IRBC adherence. While it is known that partially sulfated but not fully sulfated C4S effectively binds IRBCs, structural interactions involved remain unclear and are incompletely understood. In this study, structurally defined C4S oligosaccharides of varying sulfate contents and sizes were evaluated for their ability to inhibit the binding of IRBCs from different P. falciparum strains to CSPG purified from placenta. The results clearly show that, with all parasite strains studied, dodecasaccharide is the minimal chain length required for the efficient adherence of IRBCs to CSPG and two 4-sulfated disaccharides within this minimal structural motif are sufficient for maximal binding. Together, these data demonstrate for the first time that the C4S structural requirement for IRBC adherence is parasite strain-independent. We also show that the carboxyl group on nonreducing end glucuronic acid in dodecasaccharide motif is important for IRBC binding. Thus, in oligosaccharides containing terminal 4,5-unsaturated glucuronic acid, the nonreducing end disaccharide moiety does not interact with IRBCs due to the altered spatial orientation of carboxyl group. In such C4S oligosaccharides, 14-mer but not 12-mer constitutes the minimal motif for inhibition of IRBC binding to placental CSPG. These data have important implications for the development and evaluation of therapeutics and vaccine for placental malaria.     

Human low-molecular-mass kininogen. Amino-acid sequence of the light chain; homology with other protein sequences

The chemical structure of the polysaccharide moiety of the lipopolysaccharide Rhodopseudomonas sphaeroides ATCC 17023 was established. Mild acetic acid hydrolysis of isolated lipopolysaccharide, followed by preparative high-voltage paper electrophoresis afforded three oligosaccharides. They were characterized by chemical and physicochemical studies to be: GlcA(alpha 1----4)dOclA8P, Thr(6') GlcA(alpha 1----4)GlcA and GlcA(alpha 1----4)dOclA, where GlcA is D-glucuronic acid and dOc1A is 3-deoxy-D-manno-octulosonic acid. Carboxyl-reduction of the lipopolysaccharide followed by acid hydrolysis gave a trisaccharide: GlcA(alpha 1----4)Glc(alpha 1----4)Glc, showing the presence of three residues of glucuronic acids in the O-specific chain and indicating that only two of them are reducible by NaBH4. The linkage between the polysaccharide and lipid A was shown to be through a single 1,4-linked residue of dOc1A attached by a 2,6'-linkage to the lipid A moiety.     

Evidence for an active site arginine in UDP-glucuronyltransferase

2,3-Butanedione inactivates the pure form of UDP-glucuronyltransferase used in these experiments (GT2P) (EC 2.4.1.17) purified from pig liver microsomes. The kinetics of the reaction indicates that 2,3-butanedione reacts with two amino acids that affect activity. A rapid, partial inactivation is followed by a slower rate of inactivation that leads eventually to completely inactive enzyme. UDP-glucuronic acid and glucuronic acid, as compared with UDP, are effective as protectors against the slow, secondary phase of inactivation; no ligand tested protected against the rapid phase of inactivation. The lipid environment of GT2P was a determinant of the pseudo-first order rate constant for the slow phase of inactivation, but did not affect the rate of the rapid phase of inactivation. The data suggest that GT2P contains an active site arginine that interacts with the -COO- at C-6 of the glucuronic acid moiety of UDP-glucuronic acid.     

Backbone assignment of the apo-form of the human C-terminal domain of UDP-glucuronosyltransferase 1A (UGT1A)

A major component of phase II drug metabolism is the covalent addition of glucuronic acid to metabolites and xenobiotics. This activity is carried out by UDP-glucuronosyltransferases (UGT) which bind the UDP-glucuronic acid donor and catalyze the covalent addition of glucuronic acid sugar moieties onto a wide variety of substrates. UGTs play important roles in drug detoxification and were recently shown to act in an inducible form of multi-drug resistance in cancer patients. Despite their biological importance, structural understanding of these enzymes is limited. The C-terminal domain is identical for all UGT1A family members and required for binding to UDP-glucuronic acid as well as involved in contacts with substrates. Here, we report the backbone assignments for the C-terminal domain of UGT1A. These assignments are a critical tool for the development of a deeper biochemical understanding of substrate specificity and enzymatic activity.     

Structural basis for substrate recognition by Erwinia chrysanthemi GH30 glucuronoxylanase

Xylanase A from the phytopathogenic bacterium Erwinia chrysanthemi is classified as a glycoside hydrolase family 30 enzyme (previously in family 5) and is specialized for degradation of glucuronoxylan. The recombinant enzyme was crystallized with the aldotetraouronic acid β-D-xylopyranosyl-(1→4)-[4-O-methyl-α-D-glucuronosyl-(1→2)]-β-D-xylopyranosyl-(1→4)-D-xylose as a ligand. The crystal structure of the enzyme-ligand complex was solved at 1.39 ? resolution. The ligand xylotriose moiety occupies subsites -1, -2 and -3, whereas the methyl glucuronic acid residue attached to the middle xylopyranosyl residue of xylotriose is bound to the enzyme through hydrogen bonds to five amino acids and by the ionic interaction of the methyl glucuronic acid carboxylate with the positively charged guanidinium group of Arg293. The interaction of the enzyme with the methyl glucuronic acid residue appears to be indispensable for proper distortion of the xylan chain and its effective hydrolysis. Such a distortion does not occur with linear β-1,4-xylooligosaccharides, which are hydrolyzed by the enzyme at a negligible rate. DATABASE: Structural and experimental data are available in the Protein Data Bank database under accession number 2y24 [45].     

Fragmentation pathways of acylated flavonoid diglucuronides from leaves of Medicago truncatula

Introduction – Flavonoids are important plant compounds occurring in tissues mostly in the form of glycoconjugates. Most frequently the sugar moiety is comprised of mono‐ or oligosaccharides consisting of common sugars like glucose, rhamnose or galactose. In some plant species the glycosidic moiety contains glucuronic acid and may be acylated by phenylpropenoic acids. Methodology – Flavonoid glyconjugates were extracted from leaves of Medicago truncatula ecotype R108 and submitted to analysis using high‐performance liquid chromatography combined with high‐resolution tandem (quadrupole‐time of flight, QToF) mass spectrometry. Results – The studied leaf extracts contained 26 different flavonoid glycosides among which 22 compounds were flavone (apigenin, luteolin, chrysoeriol and tricin) glucuronides and 13 were acylated with aromatic acids (p‐coumaric, ferulic or sinapic). The fragmentation pathways observed in positive and negative ion mass spectra differed substantially between each other and from these of flavonoid glycosides which did not contain acidic sugars. The application of high‐resolution MS techniques allowed unequivocal differentiation between ions with the same nominal m/z values containing different substituents (e.g. ferulic acid or glucuronic acid). Eleven of the identified flavonoids have not been reported previously in this species. Perspectives – The presented unique fragmentation pathways of flavonoid glucuronates enable detection of these compounds in tissue extracts from different plant species. Copyright © 2009 John Wiley & Sons, Ltd.