Synthesis Of Uridine Research Articles

Characterization of Meiothermus taiwanensis Galactokinase and its Use in the One‐Pot Enzymatic Synthesis of Uridine Diphosphate‐Galactose and the Chemoenzymatic Synthesis of the Carbohydrate Antigen Stage Specific Embryonic Antigen‐3

AbstractHerein, we report the first characterization of a novel galactokinase from Meiothermus taiwanensis sp. nov. WR‐220 (MtGalK), which is overexpressed in E. coli and exhibits a kcat/Km value of 168.47 mM−1 s−1 toward Gal at 75 °C. The thermophilic MtGalK shows specific substrate recognition toward the Gal configuration with limited tolerance for modifications at the C‐2 position to form products such as GalN‐1‐P, GalN3‐1‐P, and GalNAc‐1‐P. Due to its unique thermostability toward elevated reaction temperatures, MtGalK served as an ideal biocatalyst in the synthesis of useful sugar‐1‐phosphates and was further combined with glucose‐1‐phosphate thymidylyltransferase (RmlA) and α‐1,4‐galactosyltransferase (LgtC) to achieve the efficient preparative‐scale synthesis of globotriose analogs (Gb3) using a one‐pot three‐enzyme system. In combination with a chemical synthetic strategy, a carbohydrate antigen from breast cancer stem cells, stage specific embryonic antigen‐3 (SSEA‐3) pentasaccharide, was synthesized from Gb3 in ten steps with a 23% overall yield. This flexible chemoenzymatic strategy for the synthesis of SSEA‐3 also allowed us to further expand the inventory of valuable natural and non‐natural glycans.magnified image

The in silico screening and X-ray structure analysis of the inhibitor complex of Plasmodium falciparum orotidine 5′-monophosphate decarboxylase

Orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) catalyses the final step in the de novo synthesis of uridine 5'-monophosphate (UMP) from orotidine 5'-monophosphate (OMP). A defective PfOMPDC enzyme is lethal to the parasite. Novel in silico screening methods were performed to select 14 inhibitors against PfOMPDC, with a high hit rate of 9%. X-ray structure analysis of PfOMPDC in complex with one of the inhibitors, 4-(2-hydroxy-4-methoxyphenyl)-4-oxobutanoic acid, was carried out to at 2.1 Å resolution. The crystal structure revealed that the inhibitor molecule occupied a part of the active site that overlaps with the phosphate-binding region in the OMP- or UMP-bound complexes. Space occupied by the pyrimidine and ribose rings of OMP or UMP was not occupied by this inhibitor. The carboxyl group of the inhibitor caused a dramatic movement of the L1 and L2 loops that play a role in the recognition of the substrate and product molecules. Combining part of the inhibitor molecule with moieties of the pyrimidine and ribose rings of OMP and UMP represents a suitable avenue for further development of anti-malarial drugs.

Glucose 1‐Phosphate Thymidylyltransferase in the Synthesis of Uridine 5′‐Diphosphate Galactose and its Application in the Synthesis ofN‐Acetyllactosamine

AbstractIn this study, we describe the direct synthesis of uridine 5′‐diphosphate galactose (UDP‐Gal) by a wild‐type bacterial thymidylyltransferase (RmlA), which is used to synthesize thymidine 5′‐diphosphate glucose (TDP‐glucose) in nature. By using magnesium (Mg2+) as a cofactor and a reaction temperature of 55 °C, a one hundred milligram‐scale synthesis of UDP‐Gal was achieved by RmlA. In addition, RmlA was site‐specifically and covalently immobilized on magnetic nanoparticles (MNPa) The resulting RmlA‐MNP complex retained almost 95% of its activity after reuse in ten consecutive enzyme assays. Furthermore, β‐1,4‐galactosyltransferase (GalT) fromNeisseria meningitideswas successfully overexpressed and purified by using an intein‐mediated protein expression system. GalT was relatively stable at 25 °C, and its activity was enhanced in the presence of DTT and BSA. Thus, it was feasible to synthesizeN‐acetyllactosamine (LacNAc) using RmlA and GalT in a sequential addition of enzyme and adjustment of thereaction temperature. These results demonstrate the potential applications of bacterial RmlA in carbohydrate synthesis.

Chemoenzymatic Synthesis of Uridine 5′-Diphospho-2-acetonyl-2-deoxy-α-d-glucose as C2-Carbon Isostere of UDP-GlcNAc

2-ketoGlc, which is the C(2)-carbon isostere of GlcNAc, is a novel GlcNAc analogue with a ketone group. The corresponding glycosyltransferase donor substrate, UDP-2-ketoGlc, is necessary for synthesizing 2-ketoGlc-containing molecules and is thus highly important for metabolic polysaccharide remodeling and engineering. We report here the first chemoenzymatic synthesis of UDP-2-ketoGlc using our two-enzyme (NahK and GlmU) system in vitro.

Mitochondrial DNA: An Up‐and‐coming Actor in White Adipose Tissue Pathophysiology

Mitochondrial DNA: An Up‐and‐coming Actor in White Adipose Tissue Pathophysiology

Optimization of the enzymatic one pot reaction for the synthesis of uridine 5′-diphosphogalactose

Five recombinant Escherichia coli extracts harboring overexpressed galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophophorylase, UMP kinase, and acetate kinase (AK) were utilized for the production of UDP-galactose (UDP-Gal). We analyzed the parameters which limit the yield of UDP-Gal in the reaction, and the reaction was optimized by increasing the concentration of AK. AK was used for the ATP regeneration as well as the conversion of UDP to UTP. The activities of four overexpressed enzymes were identically fixed, and then we increased the activity of AK to 20 times higher than others. The extracts catalyzed the production of UDP-Gal from UMP (10 mM), galactose (12 mM), ATP (1 mM), and acetyl phosphate (40 mM). As the result of the reaction, the conversion yield of UDP-Gal reached to 95% from 10 mMUMP.

Neocentromeres Come of Age

Neocentromeres Come of Age

Chemical synthesis of UDP-Glc-2,3-diNAcA, a key intermediate in cell surface polysaccharide biosynthesis in the human respiratory pathogens B. pertussis and P. aeruginosa

In connection with studies on lipopolysaccharide biosynthesis in respiratory pathogens we had a need to access potential biosynthetic intermediate sugar nucleotides. Herein we report the chemical synthesis of uridine 5'-diphospho 2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronic acid (UDP-Glc-2,3-diNAcA) (1) from N-acetyl-D-glucosamine in 17 steps and approximately 9% overall yield. This compound has proved invaluable in the elucidation of biosynthetic pathways leading to the formation of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid-containing polysaccharides.

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.

Structure–Activity Relationships of C6-Uridine Derivatives Targeting Plasmodia Orotidine Monophosphate Decarboxylase

Malaria, caused by Plasmodia parasites, has re-emerged as a major problem, imposing its fatal effects on human health, especially due to multidrug resistance. In Plasmodia, orotidine 5'-monophosphate decarboxylase (ODCase) is an essential enzyme for the de novo synthesis of uridine 5'-monophosphate. Impairing ODCase in these pathogens is a promising strategy to develop novel classes of therapeutics. Encouraged by our recent discovery that 6-iodo uridine is a potent inhibitor of P. falciparum, we investigated the structure-activity relationships of various C6 derivatives of UMP. 6-Cyano, 6-azido, 6-amino, 6-methyl, 6- N-methylamino, and 6- N, N-dimethylamino derivatives of uridine were evaluated against P. falciparum. The mononucleotides of 6-cyano, 6-azido, 6-amino, and 6-methyl uridine derivatives were studied as inhibitors of plasmodial ODCase. 6-Azidouridine 5'-monophosphate is a potent covalent inhibitor of P. falciparum ODCase. 6-Methyluridine exhibited weak antimalarial activity against P. falciparum 3D7 isolate. 6- N-Methylamino and 6- N, N-dimethylamino uridine derivatives exhibited moderate antimalarial activities.

Synthesis of Uridine 5′-diphosphoiduronic Acid: A Potential Substrate for the Chemoenzymatic Synthesis of Heparin

Synthesis of Uridine 5′-diphosphoiduronic Acid: A Potential Substrate for the Chemoenzymatic Synthesis of Heparin

Chemoenzymatic synthesis of stable isotope labeled UDP-N-[2H]-acetyl-glucosamine and [2H]-acetyl-chitooligosaccharides

Labeled UDP-GlcNAc and chitooligosaccharides should be powerful tools for studies of N-acetylglucosaminyltransferase such as chitin synthases. We describe here a rapid, inexpensive and a common strategie for the chemoenzymatic synthesis of uridine 5'-diphospho-N-[(2)H]-acetyl-glucosamine and the chemical preparation of N-[(2)H]-acetyl chitooligosaccharides (from 2 to 5 mers). Deuterated UDP-GlcNAc analogue was tested as chitin synthase substrate and it exhibited an incorporation level in chitin as the natural substrate. Deuterium labeling of carbohydrates present different advantages: it is a stable isotope and allows glycosyltransferase mechanism studies by a mass spectrometry approach.

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.

Crystallization and preliminary crystallographic analysis of orotidine 5'-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum.

Orotidine 5'-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5'-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 A resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 A. With a dimer in the asymmetric unit, the solvent content is 46% (V(M) = 2.3 A3 Da(-1)).

Palladium-catalyzed synthesis of uridines on polystyrene-based solid supports.

In this paper, the solid-phase synthesis of various substituted pyrimidine nucleosides is described starting from 2'-deoxyuridine, which has been attached through a base labile linker to polystyrene resins. The utility of the Pd(0) cross-coupling to functionalized pyrimidine nucleosides is expanded herein to include reactions of resin-supported 5-iodo-2'-deoxyuridine under Sonogashira, Stille, Heck, and Suzuki conditions. Upon cleavage with MeONa, a library of 5-substituted pyrimidine nucleosides was obtained in good (under Sonogashira and Stille conditions) to moderate (under Heck or Suzuki conditions) yields and high purity. Except the Suzuki-type reactions, the presented methods exhibit a significant improvement and facilitate the synthetic procedure with respect to purification and yields (determined after filtration over silica gel).

Biocatalytic synthesis of uridine 5′-diphosphate N-acetylglucosamine by multiple enzymes co-immobilized on agarose beads

Recombinant N-acetylglucosamine kinase, pyruvate kinase, N-acetylglucosamine phosphate mutase, uridine 5′-diphosphate N-acetylglucosamine pyrophosphorylase, and inorganic pyrophosphatase were overexpressed in E. coli and co-immobilized on agarose beads for the practical synthesis of uridine 5′-diphosphate N-acetylglucosamine.

Synthesis and separation of diastereomers of uridine 2′,3′-cyclic boranophosphate

The first boron-containing 2′,3′-cyclic phosphate-modified analogue, uridine 2′,3′-cyclic boranophosphate (2′,3′-cyclic-UMPB), was synthesized. 5′- O-Protected uridine was cyclophosphorylated by diphenyl H-phosphonate to yield uridine 2′,3′-cyclic H-phosphonate, which upon silylation followed by boronation and subsequent acid treatment gave 2′,3′-cyclic-UMPB in high yield. The two diastereomers of 2′,3′-cyclic-UMPB were separated by HPLC. An alternative method for synthesis of uridine 2′,3′-cyclic phosphorothioate (2′,3′-cyclic-UMPS) via H-phosphonate was also described.

A novel synthesis of N-acetyl-α-d-fucosamine 1-phosphate and uridine 5"-diphospho-N-acetyl-α-d-fucosamine

In connection with studies on the biosynthesis of capsular polysaccharides from Staphylococcus aureus, a new synthesis of uridine 5"-(2-acetamido-2,6-dideoxy-α-d-galactopyranosyl diphosphate) (uridine 5"-diphospho-N-acetyl-α-d-fucosamine) using 2-azido-3,4-di-O-acetyl-2,6-dideoxy-α-d-galactopyranosyl nitrate as the key intermediate was carried out. The reaction of this product with cesium dibenzyl phosphate smoothly affords the corresponding β-glycosyl dibenzyl phosphate, which undergoes anomerization on treatment with BF3·Et2O and 2-bromopyridine to give α-glycosyl dibenzyl phosphate in high yield. This product was then transformed into 2-amino-3,4-di-O-acetyl-2,6-dideoxy-α-d-galactopyranosyl phosphate, subsequently converted into 2-acetamido-2,6-dideoxy-α-d-galactopyranosyl phosphate and the target nucleoside diphosphate sugar.

Structural basis for the catalytic mechanism of a proficient enzyme: orotidine 5'-monophosphate decarboxylase.

Orotidine 5'-monophosphate decarboxylase (ODCase) catalyzes the decarboxylation of orotidine 5'-monophosphate, the last step in the de novo synthesis of uridine 5'-monophosphate. ODCase is a very proficient enzyme [Radzicka, A., and Wolfenden, R. (1995) Science 267, 90-93], enhancing the reaction rate by a factor of 10(17). This proficiency has been enigmatic, since it is achieved without metal ions or cofactors. Here we present a 2.5 A resolution structure of ODCase complexed with the inhibitor 1-(5'-phospho-beta-D-ribofuranosyl)barbituric acid. It shows a closely packed dimer composed of two alpha/beta-barrels with two shared active sites. The orientation of the orotate moiety of the substrate is unambiguously deduced from the structure, and previously proposed catalytic mechanisms involving protonation of O2 or O4 can be ruled out. The proximity of the OMP carboxylate group with Asp71 appears to be instrumental for the decarboxylation of OMP, either through charge repulsion or through the formation of a very short O.H.O hydrogen bond between the two carboxylate groups.

Synthesis of uridine 5′-monophosphate glucose as an inhibitor of UDP-glucose pyrophosphorylase

Uridine 5′-monophosphate α- d-glucose (UMPG) was evaluated as a novel and potent inhibitor of the enzymatic reaction involved in sugar nucleotide metabolism. UMPG was synthesized by chemical coupling of 2,3,4,6-tetra- O-acetyl-α- d-glucopyranosyl bromide with uridine 5′-monophosphate (UMP) to give uridine 5′-monophosphate 2″,3″,4″,6″-tetra- O-acetyl-α- d-glucose (UMPTAG), followed by deacetylation of UMPTAG with sodium methoxide. In addition to UMPG, UMPTAG showed potent inhibitory activity toward yeast UDPG pyrophosphorylase (UDPG synthetase). UMPG and UMPTAG were competitive with UDPG in the pyrophosphorolytic reaction, with inhibition constants ( K i) of 4.8 and 20.7 μM, respectively, but non-competitive with inorganic pyrophosphate. UMPG and UMPTAG also inhibited the enzyme non-competitively in the reverse reaction to synthesize UDPG from UTP and glucose 1-phosphate (G1P). The acetyl group of UMPTAG was thought to enhance its hydrophobic interaction, possibly with an active site region of the enzyme functional for binding with UDPG.