Practical preparation of UDP-apiose and its applications for studying apiosyltransferase.

UDP-apiose, a donor substrate of apiosyltransferases, is labile because of its intramolecular self-cyclization ability, resulting in the formation of apiofuranosyl-1,2-cyclic phosphate. Therefore, stabilization of UDP-apiose is indispensable for its availability and identifying and characterizing the apiosyltransferases involved in the biosynthesis of apiosylated sugar chains and glycosides. Here, we established a method for stabilizing UDP-apiose using bulky cations as counter ions. Bulky cations such as triethylamine effectively suppressed the degradation of UDP-apiose in solution. The half-life of UDP-apiose was increased to 48.1 ±â€¯2.4 h at pH 6.0 and 25 °C using triethylamine as a counter cation. UDP-apiose coordinated with a counter cation enabled long-term storage under freezing conditions. UDP-apiose was utilized as a donor substrate for apigenin 7-O-ß-D-glucoside apiosyltransferase to produce the apiosylated glycoside apiin. This apiosyltransferase assay will be useful for identifying genes encoding apiosyltransferases.

Efficient chemoenzymatic synthesis of UDP-α-6-N3-glucose.

A novel chemo-enzymatic synthetic method for UDP-α-6-N3-glucose was developed by combining the versatility of chemical synthesis and natural enzyme stereo-selectivity of Bifidobacterium longum (BLUSP). This flexible and efficient platform expanded the substrate scope for UDP-sugars on an improved scale, particularly for UDP-sugar substrates containing bioorthogonal functional groups.

Efficient enzymatic synthesis of guanosine 5'-diphosphate-sugars and derivatives.

An N-acetylhexosamine 1-kinase from Bifidobacterium infantis (NahK_15697), a guanosine 5'-diphosphate (GDP)-mannose pyrophosphorylase from Pyrococcus furiosus (PFManC), and an Escherichia coli inorganic pyrophosphatase (EcPpA) were used efficiently for a one-pot three-enzyme synthesis of GDP-mannose, GDP-glucose, their derivatives, and GDP-talose. This study represents the first facile and efficient enzymatic synthesis of GDP-sugars and derivatives starting from monosaccharides and derivatives.

Synthesis of 2-deoxy-hexopyranosyl derivatives of uridine as donor substrate analogues for glycosyltransferases.

A series of 2-deoxy-hexopyranosyl derivatives of uridine have been synthesized as analogues of UDP-sugar. These compounds were tested as inhibitors against bovine beta-1,4-galactosyltransferase I in fluorescent assays and showed no significant inhibition.

Probing replacement of pyrophosphate via click chemistry; synthesis of UDP-sugar analogues as potential glycosyl transferase inhibitors.

A series of potential UDP-sugar mimics were readily synthesised by copper(I) catalysed modified Huisgen cycloaddition of the corresponding alpha-propargyl glycosides with 5-azido uridine in aqueous solution. None of the compounds accessed displayed significant inhibitory activity at concentrations of up to 4.5mM in an assay against bovine milk beta-1,4-galactosyltransferase.

Synthesis of uridine 5'-diphosphoiduronic acid: a potential substrate for the chemoenzymatic synthesis of heparin.

An improved understanding of the biological activities of heparin requires structurally defined heparin oligosaccharides. The chemoenzymatic synthesis of heparin oligosaccharides relies on glycosyltransferases that use UDP-sugar nucleotides as donors. Uridine 5'-diphosphoiduronic acid (UDP-IdoA) and uridine 5'-diphosphohexenuronic acid (UDP-HexUA) have been synthesized as potential analogues of uridine 5'-diphosphoglucuronic acid (UDP-GlcA) for enzymatic incorporation into heparin oligosaccharides. Non-natural UDP-IdoA and UDP-HexUA were tested as substrates for various glucuronosyltransferases to better understand enzyme specificity.

Synthesis and photolytic activation of 6''-O-2-nitrobenzyl uridine-5'-diphosphogalactose: a 'caged' UDP-Gal derivative.

Placing an 2-nitrobenzyl group on O-6 of the galactosyl residue in uridine-5'-diphosphogalactose (UDP-Gal) gives 6''-O-2-nitrobenzyl-UDP-Gal that is shown to be inactive as a donor substrate for beta-(1-->4)-galactosyltransferase (GalT). On irradiation at 365 nm, the nitrobenzyl group is completely removed yielding native UDP-Gal that then transfers normally to produce the expected betaGal-(1-->4)-betaGlcNAc disaccharidic linkage. 6''-O-2-Nitrobenzyl-UDP-Gal thus fulfils the minimum requirements of a 'caged' UDP-Gal for application in time-resolved crystallographic studies of beta-(1-->4)-GalT.

Stereoselective chemical synthesis of sugar nucleotides via direct displacement of acylated glycosyl bromides.

[structure: see text]. The use of Leloir glycosyltransferases to prepare biologically relevant oligosaccharides and glycoconjugates requires access to sugar nucleoside diphosphates, which are notoriously difficult to efficiently synthesize and purify. We report a novel stereoselective route to UDP- and GDP-alpha-D-mannose as well as UDP- and GDP-beta-L-fucose via direct displacement of acylated glycosyl bromides with nucleoside 5'-diphosphates.

Direct oxidation of sugar nucleotides to the corresponding uronic acids: TEMPO and platinum-based procedures.

The direct oxidation of UDP-alpha-d-glucose and UDP-N-acetyl-alpha-d-glucosamine to the corresponding uronic acids was explored using either TEMPO or platinum-catalysed oxidation with molecular oxygen. Whilst TEMPO-based procedures gave rise to substantial over-oxidation and/or degradation of UDP-glucose, oxidation of UDP-N-acetyl-glucosamine to UDP-N-acetyl-glucosaminuronic acid was achieved with >90% conversion and ca. 65% isolated yield using a platinum-catalysed procedure.

A chemical synthesis of UDP-LacNAc and its regioisomer for finding 'oligosaccharide transferases'.

A chemical synthesis of uridine 5'-diphospho-N-acetyllactosamine (Galbeta(1-->4)GlcNAc-UDP; UDP-LacNAc) and Galbeta(1-->3)GlcNAc-UDP is described. Coupling of the disaccharide imidate derivatives with dibenzylphosphate gave the corresponding 1-phosphates, which were condensed with UMP-imidazolate to give the target UDP-oligosaccharides after purification by anion exchange HPLC and gel filtration column chromatography. Using this methodology a variety of oligosaccharide nucleotide analogues can be synthesized. These UDP-oligosaccharides may be useful for finding so-called ;oligosaccharide transferases', the glycosyltransferases which transfer the oligosaccharide moiety onto glycosyl acceptors.

Preparation of UDP-galacturonic acid using UDP-sugar pyrophosphorylase.

UDP-galacturonic acid, the activated form of galacturonic acid (GalUA), is synthesized both de novo and by salvage pathways. The UDP-GalUA pyrophosphorylase gene involved in the salvage pathway has not been identified. Here we show that UDP-sugar pyrophosphorylase from Pisum sativum with a broad specificity has UDP-GalUA pyrophosphorylase activity. The enzyme catalyzed the formation of UDP-GalUA and pyrophosphate from GalUA 1-phosphate and UTP with an equilibrium constant value of 0.24. The recombinant UDP-sugar pyrophosphorylase had optimal pH of 6.0, and the apparent K(m) values for GalUA 1-phosphate, UTP, UDP-GalUA, and pyrophosphate were 2.27, 1.15, 0.70, and 1.26 mM, respectively. In the presence of inorganic pyrophosphatase, the recombinant enzyme produced UDP-GalUA in an 84% yield (based on the GalUA 1-phosphate substrate) on a preparative scale. Thus, this UDP-sugar pyrophosphorylase is useful for the highly efficient production of UDP-GalUA for studies on pectin biosynthesis.

Rational design, synthesis, and characterization of novel inhibitors for human beta1,4-galactosyltransferase.

An affinity labeling reagent, uridine 5'-(6-amino-{2-[(7-bromomethyl-2-naphthyl)methoxycarbonylmethoxy]ethoxy}acetyl-6-deoxy-alpha-D-galactopyranosyl) diphosphate (1a), was designed on the basis of 3D docking simulation and synthesized to investigate the functional role of Trp310 residue located in the small loop near the active site of human recombinant galactosyltransferase (betaGalT-1). Mass spectrometric analysis revealed that the Trp310 residue of betaGalT1 can be selectively modified with the naphthylmethyl group of compound 1a at the C-3 position of the indole ring. This result motivated us to synthesize novel uridine-5'-diphosphogalactose (UDP-Gal) analogues as candidates for mechanism-based inhibitors for betaGalT-1. We found that uridine 5'-(6-O-[10-(2-naphthyl)-3,6,9-trioxadecanyl]-alpha-d-galactopyranosyl) diphosphate (2) is the strongest inhibitor (K(i) = 1.86 microM) against UDP-Gal (Km = 4.91 microM) among compounds reported previously. A cold spray ionization time-of-flight mass spectrometry study demonstrated that the complex of this inhibitor and betaGalT-1 cannot interact with an acceptor substrate in the presence of Mn2+.

Synthesis of unnatural sugar nucleotides and their evaluation as donor substrates in glycosyltransferase-catalyzed reactions.

New unnatural sugar nucleotides, UDP-Fuc and CDP-Fuc were synthesized from fucose-beta-1-phosphate and nucleotide monophosphates activated as morpholidates. Furthermore, a nucleotide analogue was prepared by phosphorylation of 1-(beta-D-ribofuranosyl)cyanuric acid, itself obtained as a protected derivative by condensation of the persilylated derivative of cyanuric acid with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose in 74% yield. This phosphate activated according to the same procedure was condensed with fucose-beta-1-phosphate, affording a new sugar nucleotide conjugate (NDP-Fuc) which was evaluated together with UDP-Fuc, CDP-Fuc and ADP-Fuc, as fucose donors in alpha-(1-->4/3)-fucosyltransferase (FucT-III) catalyzed reaction. Fucose transfer could be observed with each of the donors and kinetic parameters were determined using a fluorescent acceptor substrate. Efficiency of the four analogues towards FucT-III was in the following order: UDP-Fuc=ADP-Fuc>NDP-Fuc>CDP-Fuc. According to the same strategy ADP-GlcNAc was prepared from AMP-morpholidate and N-acetylglucosamine-alpha-1-phosphate; tested as a glucosaminyl donor towards Neisseria meningitidis N-acetylglucosaminyl transferase (LgtA), ADP-GlcNAc was recognized with 0.1% efficiency as compared with UDP-GlcNAc, the natural donor substrate.

Synthesis of thioglycoside-based UDP-sugar analogues.

Arbuzov reaction of O-acetyl-protected glycosylthiomethyl chlorides with triethyl phosphite and then phosphonate ethyl ester cleavage with trimethylsilyl bromide afforded glycosylthiomethyl phosphonates 13, 18, 22, and 26. These intermediates could be readily transformed into the O-deprotected phosphonates 7-10 and into title compounds 1-4. Similarly, sulfonomethyl phosphonate moieties containing UDP-sugar analogues 5 and 6 were obtained.

Chemical synthesis of UDP-4-O-methyl-GlcNAc, a potential chain terminator of chitin synthesis.

Chitin synthase converts uridine diphosphoryl-N-acetylglucosamine (UDP-GlcNAc) to chitin (poly-beta-(1-->4)-GlcNAc). During polymerization, elongation occurs at the 4-OH (nonreducing) terminus of the growing chitin chain. Blockage of the 4-OH via incorporation of UDP-N-acetyl-4-O-methylglucosamine (UDP-4-OMe-GlcNAc, 3) can potentially terminate chitin polymerization, and represents a novel strategy for chitin synthase inhibition. The chemical synthesis of 3 and preliminary evaluation of its possible incorporation by chitin synthase are reported herein.

Chemical synthesis of uridine diphospho-D-xylose and UDP-L-arabinose.

Leloir transferases, like UDP-d-xylosyl transferase and arabinosyl transferase, utilize nucleoside diphosphate sugars to build up plant oligo- and polysaccharides. By the described, scalable three-step synthesis a simple route is described to arrive at the respective enzyme substrates, which are otherwise difficult to obtain.

The first total synthesis of lipid II: the final monomeric intermediate in bacterial cell wall biosynthesis.

Bacterial peptidoglycan is composed of a network of beta-[1,4]-linked glyan strands that are cross-linked through pendant peptide chains. The final product, the murein sacculus, is a single, covalently closed macromolecule that precisely defines the size and shape of the bacterial cell. The recent increase in bacterial resistance to cell wall active agents has led to a resurgence of activity directed toward improving our understanding of the resistance mechanisms at the molecular level. The biosynthetic enzymes and their natural substrates can be invaluable tools in this endeavor. While modern experimental techniques have led to isolation and purification of the biosynthetic enzymes utilized in peptidoglycan biosynthesis, securing useful quantities of their requisite substrates from natural substrates has remained problematic. In an effort to address this issue, we report the first total synthesis of lipid II (4), the final monomeric intermediate utilized by Gram positive bacteria for peptidoglycan biosynthesis.

Synthesis of nucleotide-activated oligosaccharides by beta-galactosidase from Bacillus circulans.

The enzymatic access to nucleotide-activated oligosaccharides by a glycosidase-catalyzed transglycosylation reaction was explored. The nucleotide sugars UDP-GlcNAc and UDP-Glc were tested as acceptor substrates for beta-galactosidase from Bacillus circulans using lactose as donor substrate. The UDP-disaccharides Gal(beta1-4)GlcNAc(alpha1-UDP) (UDP-LacNAc) and Gal(beta1-4)Glc(alpha1-UDP) (UDP-Lac) and the UDP-trisaccharides Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP and Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP) were formed stereo- and regioselectively. Their chemical structures were characterized by 1H and 13C NMR spectroscopy and fast atom bombardment mass spectrometry. The synthesis in frozen solution at -5 degrees C instead of 30 degrees C gave significantly higher product yields with respect to the acceptor substrates. This was due to a remarkably higher product stability in the small liquid phase of the frozen reaction mixture. Under optimized conditions, at -5 degrees C and pH 4.5 with 500 mM lactose and 100 mM UDP-GlcNAc, an overall yield of 8.2% (81.8 micromol, 62.8 mg with 100% purity) for Gal(beta1-4)GlcNAc(alpha1-UDP) and 3.6% (36.1 micromol, 35 mg with 96% purity) for Gal(beta1-4)Gal(beta1-4)GlcNAc(alpha1-UDP) was obtained. UDP-Glc as acceptor gave an overall yield of 5.0% (41.3 micromol, 32.3 mg with 93% purity) for Gal(beta1-4)Glc(alpha1-UDP) and 1.6% (13.0 micromol, 12.2 mg with 95% purity) for Gal(beta1-4)Gal(beta1-4)Glc(alpha1-UDP). The analysis of other nucleotide sugars revealed UDP-Gal, UDP-GalNAc, UDP-Xyl and dTDP-, CDP-, ADP- and GDP-Glc as further acceptor substrates for beta-galactosidase from Bacillus circulans.

Chemical synthesis of UDP-beta-L-arabinofuranose and its turnover to UDP-beta-L-arabinopyranose by UDP-galactopyranose mutase.

Uridine-5'-diphospho-beta-L-arabinofuranose, a possible donor of L-arabinofuranose residues in plants, was synthesized. This compound, in the presence of UDP-galactopyranose mutase, underwent interconversion with UDP-beta-L-arabinopyranose that is a likely precursor of L-arabinofuranose in vivo. This result provided a working model for the biogenesis of arabinofuranose in plants.