31 P-MRS of the healthy human brain at 7 T detects multiple hexose derivatives of uridine diphosphate glucose.

Nucleotide sugars are required for the synthesis of glycoproteins and glycolipids, which play crucial roles in many cellular functions such as cell communication and immune responses. Uridine diphosphate-glucose (UDP-Glc) was previously believed to be the only nucleotide sugar detectable in brain by 31 P-MRS. Using spectra of high SNR and high resolution acquired at 7 T, we showed that multiple nucleotide sugars are coexistent in brain and can be measured simultaneously. In addition to UDP-Glc, these also include UDP-galactose (UDP-Gal), -N-acetyl-glucosamine (UDP-GlcNAc) and -N-acetyl-galactosamine (UDP-GalNAc), collectively denoted as UDP(G). Coexistence of these UDP(G) species is evident from a quartet-like multiplet at -9.8 ppm (M-9.8 ), which is a common feature seen across a wide age range (24-64 years). Lineshape fitting of M-9.8 allows an evaluation of all four UDP(G) components, which further aids in analysis of a mixed signal at -8.2 ppm (M-8.2 ) for deconvolution of NAD+ and NADH. For a group of seven young healthy volunteers, the concentrations of UDP(G) species were 0.04 ± 0.01 mM for UDP-Gal, 0.07 ± 0.03 mM for UDP-Glc, 0.06 ± 0.02 mM for UDP-GalNAc and 0.08 ± 0.03 mM for UDP-GlcNA, in reference to ATP (2.8 mM). The combined concentration of all UDP(G) species (average 0.26 ± 0.06 mM) was similar to the pooled concentration of NAD+ and NADH (average 0.27 ± 0.06 mM, with a NAD+ /NADH ratio of 6.7 ± 2.1), but slightly lower than previously found in an older cohort (0.31 mM). The in vivo NMR analysis of UDP-sugar composition is consistent with those from tissue extracts by other modalities in the literature. Given that glycosylation is dependent on the availability of nucleotide sugars, assaying multiple nucleotide sugars may provide valuable insights into potential aberrant glycosylation, which has been implicated in certain diseases such as cancer and Alzheimer's disease.

Novel derivatives of UDP-glucose: concise synthesis and fluorescent properties.

A series of novel 5-substituted UDP-glucose derivatives with interesting fluorescent properties and potential applications as sensors for carbohydrate-active enzymes is reported. An efficient synthesis of the target molecules was developed, centred around the Suzuki-Miyaura reaction of (hetero)arylboronic acids with 5-iodo UDP-glucose. Interestingly, the optimised cross-coupling conditions could also be applied successfully to 5-bromo UMP, but not to 5-bromo UDP-glucose.

Synthesis of UDP-glucose derivatives modified at the 3-OH as potential chain terminators of beta-glucan biosynthesis.

A series of UDP-D-glucose derivatives and precursors that have been modified at C-3 were synthesised from D-glucose as potential chain terminators of beta-glucan biosynthesis. None of the UDP-derivatives or the precursors tested displayed significant anti-fungal activity in a series of germination assays on the dermatophyte Trichophyton rubrum.

Structure-activity relationship of uridine 5'-diphosphoglucose analogues as agonists of the human P2Y14 receptor.

UDP-glucose (UDPG) and derivatives are naturally occurring agonists of the Gi protein-coupled P2Y14 receptor, which occurs in the immune system. We synthesized and characterized pharmacologically novel analogues of UDPG modified on the nucleobase, ribose, and glucose moieties, as the basis for designing novel ligands in conjunction with modeling. The recombinant human P2Y14 receptor expressed in COS-7 cells was coupled to phospholipase C through an engineered Galpha-q/i protein. Most modifications of the uracil or ribose moieties abolished activity; this is among the least permissive P2Y receptors. However, a 2-thiouracil modification in 15 (EC50 49 +/- 2 nM) enhanced the potency of UDPG (but not UDP-glucuronic acid) by 7-fold. 4-Thio analogue 13 was equipotent to UDPG, but S-alkylation was detrimental. Compound 15 was docked in a rhodposin-based receptor homology model, which correctly predicted potent agonism of UDP-fructose, UDP-mannose, and UDP-inositol. The hexose moiety of UDPG interacts with multiple H-bonding and charged residues and provides a fertile region for agonist modification.

A convenient gram-scale synthesis of uridine diphospho(13C6)glucose.

A simple gram-scale synthesis of uridine diphospho(13C6)glucose is presented from D-(13C6)glucose. The critical step uses a 1H-tetrazole-catalyzed coupling of 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl-1-phosphate and UMP-morpholidate. The uridine diphospho(13C6)glucose was used in the structural identification of (1-->3)-beta-D-glucan from Lolium multiflorum.

Cyclic uridine diphosphate glucose: a new pyrimidine analog of cyclic ADP ribose.

Novel compound 1, as the first example of cyclic ADP-ribose analogs containing a pyrimidine residue, was synthesized by a chemical strategy employing a Mitsunobu reaction for the condensation of the glucosyl moiety on protected uridine, and a Matsuda procedure for the cyclization step.

Fluorescent analogs of UDP-glucose and their use in characterizing substrate binding by toxin A from Clostridium difficile.

Uridine-5'-diphospho-1-alpha-d-glucose (UDP-Glc) is a common substrate used by glucosyltransferases, including certain bacterial toxins such as Toxins A and B from Clostridium difficile. Fluorescent analogs of UDP-Glc have been prepared for use in our studies of the clostridial toxins. These compounds are related to the methylanthraniloyl-ATP compounds commonly used to probe the chemistry of ATP-dependent enzymes. The reaction of excess methylisatoic anhydride with UDP-Glc in aqueous solution yields primarily the 2' and 3' isomers of methylanthraniloyl-UDP-Glc (MUG). As the 2' and 3' isomers readily interconvert, this isomeric mixture was copurified by HPLC away from the other isomeric products, and was characterized by a combination of NMR, fluorescence and mass spectrometric methods. TcdA binds MUG competitively with respect to UDP-Glc with an affinity of 15 +/- 2 microm in the absence of Mg2+. There is currently no evidence that the fluorescent substrate analog is turned over by the toxin in either glucosyltransferase or glucosylhydrolase reactions. Using a competition assay, the affinity of UDP-Glc was determined to be 45+/-10 microm in the absence of Mg2+. The binding of UDP-Glc and Mg2+ are highly coupled with Mg2+ affinities in the range of 90-600 microm, depending on the experimental conditions. These results imply that one of the significant roles of the metal ion might be to stabilize the enzyme-substrate complex prior to initiation of the transferase chemistry.

High yielding one-pot enzyme-catalyzed synthesis of UDP-glucose in gram scales.

Uridine diphosphoglucose is an important cofactor of glucosylating enzymes. A simple and high yielding one-pot enzymatic synthesis of UDPG on a gram scale from glucose via hexokinase, phosphoglucomutase and UDPG pyrophosphorylase (UGPase) is described. Repetitive addition of substrate was used to avoid inhibition of UGPase. The approach allows recovery of active enzymes and their re-use. The synthesis of UDP-[4-(13)C]-glucose on a 0.5 g scale resulted in a final yield of 70% and a purity of >95% after chromatographic purification.

Use of Escherichia coli polyphosphate kinase for oligosaccharide synthesis.

The Escherichia coli polyphosphate kinase (PPK) has been known to catalyze the reversible transfer of phosphate molecules between ATP and polyphosphate (poly(P)). It has also been found that the PPK catalyzes the kination of not only ADP but also other nucleoside diphosphates (NDPs) using poly(P) as a phosphate donor, yielding nucleotide triphosphates (NTPs). We used the PPK and poly(P) in place of pyruvate kinase and phosphoenol pyruvate for NTP regeneration followed by synthesis of sugar nucleotides in a cyclic synthesis system for oligosaccharides. It was confirmed that the PPK efficiently catalyzed the UTP regeneration in the cyclic system of N-acetyllactosamine synthesis. This novel activity of PPK enables us to perform the practical synthesis of oligosaccharides.

Synthesis of aryl azide derivatives of UDP-GlcNAc and UDP-GalNAc and their use for the affinity labeling of glycosyltransferases and the UDP-HexNAc pyrophosphorylase.

The chemical synthesis and utilization of two photoaffinity analogs, 125I-labeled 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc and -UDP-GalNAc, is described. Starting with either UDP-GlcNAc or UDP-GalNAc, the synthesis involved the preparation of the 5-mercuri-UDP-HexNAc and then attachment of an allylamine to the 5 position to give 5-(3-amino)allyl-UDP-HexNAc. This was followed by acylation with N-hydroxysuccinimide p-aminosalicylic acid to form the final product, i.e., 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc or UDP-GalNAc. These products could then be iodinated with chloramine T to give the 125I-derivatives. Both the UDP-GlcNAc and the UDP-GalNAc derivatives reacted in a concentration-dependent manner with a highly purified UDP-HexNAc pyrophosphorylase, and both specifically labeled the subunit(s) of this protein. The labeling of the protein by the UDP-GlcNAc derivative was inhibited in dose-dependent fashion by either unlabeled UDP-GlcNAc or unlabeled UDP-GalNAc. Likewise, labeling with the UDP-GalNAc probe was blocked by either UDP-GlcNAc or UDP-GalNAc. The UDP-GlcNAc probe also specifically labeled a partially purified preparation of GlcNAc transferase I.

The beta-glucan synthase from Lolium multiflorum. Detergent solubilization, purification using monoclonal antibodies, and photoaffinity labeling with a novel photoreactive pyrimidine analogue of uridine 5'-diphosphoglucose.

The membrane-bound beta-glucan synthase from Italian ryegrass (Lolium multiflorum L.) endosperm cells has been solubilized by both non-ionic and zwitterionic detergents. A complex relationship exists between the ratio of (1----3)-, (1----4)-, and (1----3, 1----4)-beta-glucan products of the solubilized enzyme, the cations present, and the concentration of the uridine 5'-diphosphoglucose substrate. Monoclonal antibodies directed against the beta-glucan synthase complex were generated by immunization of mice with an unfractionated microsomal reparation. Hybridoma cell lines were screened using a combination of indirect enzyme-linked immunosorbent assay followed by an enzyme-capture assay. The purified monoclonal antibodies were used with Pan-sorbin (stablized protein A-bearing staphylococcal cells) to immunoprecipitate an active beta-glucan synthase complex which had been solubilized from a microsomal preparation with 0.6% CHAPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated synthase complex revealed four major polypeptides of apparent molecular mass 30, 31, 54, and 58 kDa together with several minor components. The immunoprecipitated beta-glucan synthase complex was capable of synthesizing both (1----3)- and (1----4)-beta-glucans. A new photoreactive pyrimidine analogue of uridine 5'-diphosphoglucose, 5-[3-(p-azidosalicylamide]allyl-uridine 5'-diphosphoglucose was synthesized in a three-step reaction sequence involving mercuration of UDP-Glc, alkylation of 5-Hg-UDP-Glc, and acylation of 5-(3-amino)allyl-UDP-Glc and characterized by chemical and spectroscopic analysis. The analogue inhibits (Kiapp 16 microM) and, upon UV irradiation, irreversibly inactivates the beta-glucan synthase. The analogue was iodinated with Na125I to give a radiolabeled, photoreactive compound, and was used in photoaffinity labeling of UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, and several putative UDP-Glc-binding proteins from L. multiforum. The radiolabeled analogue specifically labeled the 31-kDa polypeptide in the immunoprecipitated synthase complex. The photolabeling of this polypeptide is strictly dependent on UV irradiation, is blocked by uridine 5'-diphosphoglucose and uridine 5'-diphosphate, and reaches saturation at analogue concentrations above 300 microM. These results indicate that the 31-kDa polypeptide in the beta-glucan synthase complex bears a uridine 5'-diphosphoglucose-binding site and is involved in the catalysis of beta-glucan synthesis.

Enzymatic synthesis of UDP-GlcN by a two step hollow fiber enzyme reactor system.

UDP-GlcN was synthesized from GlcN and UTP by a two step hollow fiber enzyme reactor method. In step 1, GlcN was converted to GlcN 6-P and then to GlcN 1-P by hexokinase and phosphoglucomutase, respectively, and UTP was used as the phosphate donor. In step 2, GlcN 1-P was converted to UDP-GlcN by UDP glucose pyrophosphorylase. All the enzymes required for the synthesis of UDP-GlcN were enclosed in hollow fiber bundles which allow for the free diffusion of substrates and products across the membranes to and from the enzymes, allow for the reutilization of the enzymes, and simplify the isolation of the product, UDP-GlcN. We show that both UTP and GlcN 6-P are inhibitors of the yeast UDPG pyrophosphorylase and therefore their concentrations must be regulated to obtain maximum yields of UDP-GlcN. The UDP-GlcN produced can be N-acetylated with [14C]acetic anhydride to produce UDP-[14C]GlcNAc. This method can also be used to synthesize [32P]UDP-GlcN and [32P]UDP-GlcNAc from [alpha-32P]UTP and GlcN 1-P.

Synthesis and properties of 5-azido-UDP-glucose. Development of photoaffinity probes for nucleotide diphosphate sugar binding sites.

A new active site directed photoaffinity probe, which is a model compound for studying nucleotide diphosphate sugar binding proteins, has been synthesized by coupling 5-azido-UTP and [32P]Glc-1-P using yeast UDP-glucose pyrophosphorylase to produce [beta-32P]5-azidouridine 5'-diphosphoglucose (5N3UDP-Glc). This probe has photochemical properties similar to that of 5-azidoUTP (Evans, R. K., and Haley, B. E. (1987) Biochemistry 26, 269-276). The efficacy of 5N3UDP-Glc as an active site directed probe was demonstrated using yeast UDP-Glc pyrophosphorylase. Saturation effects of photoinsertion were observed with an apparent Kd of 51 microM and the natural substrate, UDP-Glc, prevented photoinsertion of [beta-32P]5N3UDP-Glc with an apparent Kd of 87 microM. Prevention of photoinsertion was also seen with UTP and pyrophosphate with apparent Kd values less than 200 microM. UMP, UDP, ATP, and GTP were much less effective competitors. Selective photoinsertion was observed with several partially purified enzymes including UDP-Glc dehydrogenase, UDP-Gal-4-epimerase, Gal-1-P uridyltransferase, and phosphorylase a. The absence of nonselective photoinsertion into bulk proteins was demonstrated with crude homogenates of rabbit liver as well as with several UDP-Glc binding proteins. Of the six purified enzymes tested, only phosphoglucomutase has been shown to incorporate radiolabel from the photoprobe in the absence of UV irradiation. These results and a discussion of the utility of 5N3UDP-Glc for detecting UDP-Glc binding proteins and isolating active site peptides are presented.

Non-enzymatic synthesis of the coenzymes, uridine diphosphate glucose and cytidine diphosphate choline, and other phosphorylated metabolic intermediates.

The synthesis of uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) has been accomplished under simulated prebiotic conditions using urea and cyanamide, two condensing agents considered to have been present on the primitive Earth. The synthesis of UDPG was carried out by reacting G1P and UTP at 70 degrees C for 24 hours in the presence of the condensing agents in an aqueous medium. CDP-choline was obtained under the same conditions by reacting choline phosphate and CTP X G1P and G6P were synthesized from glucose and inorganic phosphate at 70 degrees C for 16 hours. Separation and identification of the reaction products have been performed by paper chromatography, thin layer chromatography, enzymatic analysis and ion pair reverse phase high performance liquid chromatography. These results suggest that metabolic intermediates could have been synthesized on the primitive Earth from simple precursors by means of prebiotic condensing agents.

Uridine 5'-(5-thio-alpha-D-glucopyranosyl pyrophosphate): chemical synthesis and activation of rat liver glycogen synthetase.

Uridine 5'-(5-thio-alpha-D-glucopyranosyl pyrophosphate), UDPTG, appears to be a potent activator of rat liver glycogen synthetase a even though it is not a substrate. At 1.0 mM, UDPTG causes over 400% activation of glycogen synthetase a activity. Activation by UDPTG is accompanied by normalization of the otherwise sigmoidal kinetics for UDPG with glycogen synthetase a and a decrease in the apparent Km for UDPG from approximately 2.0 to 0.62 mM. UDPTG inhibits catalytic activity at higher concentrations. At the concentrations examined, UDPTG has no effect on glycogen synthetase b activity. The use of glycogen synthetase free from glycogen synthetase b phosphatase and the selective inhibition of glycogen synthetase b phosphatase by 100 mM NaF-indicate that conversion of synthetase b to a is not responsible for the activation. Moreover, the use of the colorimetric assay for glycogen synthetase activity precludes effects of UDPTG on glycogen phosphorylase activity. UDPTG, chemically synthesized in 60% yield, is characterized by chromatographic and electrophoretic procedures, by its uv spectra, and by analysis of products after chemical and enzymatic hydrolysis.

[Mechanism of the enzymatic reaction catalyzed by uridine diphosphate glucose dehydrogenase. The chemical synthesis of uridine diphosphate glucose-6-H3 and its oxidation by uridine diphosphate glucose dehydrogenase]. / O mekhanizme fermentativnoi reaktsii, kataliziruemoi uridindifosfatgliukoza-degidrogenazoi. Khimicheskii sintez uridindifosfatgliukozy-6-H3 i ee okislenie uridindifosfatgliukoza-degidrogenazoi

The synthesis of UDP-glucose-6-s-H was performed through condensation of alpha-D-glucopyranosyl phosphate-6-3-H and uridine 5'-phosphomorpholidate. Enzymic oxidation of UDP-glucose-6-3-H with calf liver UDP-glucose dehydrogenase was found to proceed with direct transfer of the hydrogen from C-6 of UDP-glucose onto NAD.