ABSTRACT
UDP-sugar analogs are useful for the study of glycosyltransferases and the production of unnatural glycans. The preparation of five UDP-GlcNAc derivatives is reported with 6-deoxy, 6-azido, 6-amino, 6-mercapto, or 6-fluoro substitutions. A concise chemoenzymatic synthesis was developed using the kinase NahK (B. longum JCM1217) and the uridyl transferase GlmU (E. coli K12).
Subject(s)
Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Carbohydrate Conformation , Uridine Diphosphate N-Acetylglucosamine/chemistryABSTRACT
We have investigated the applicability of different chemical methods for pyrophosphate bond formation to the synthesis of 5-substituted UDP-galactose and UDP-N-acetylglucosamine derivatives. The use of phosphoromorpholidate chemistry, in conjunction with N-methyl imidazolium chloride as the promoter, was identified as the most reliable synthetic protocol for the preparation of these non-natural sugar-nucleotides. Under these conditions, the primary synthetic targets 5-iodo UDP-galactose and 5-iodo UDP-N-acetylglucosamine were consistently obtained in isolated yields of 40-43%. Both 5-iodo UDP-sugars were used successfully as substrates in the Suzuki-Miyaura cross-coupling with 5-formylthien-2-ylboronic acid under aqueous conditions. Importantly, 5-iodo UDP-GlcNAc and 5-(5-formylthien-2-yl) UDP-GlcNAc showed moderate inhibitory activity against the GlcNAc transferase GnT-V, providing the first examples for the inhibition of a GlcNAc transferase by a base-modified donor analogue.
Subject(s)
N-Acetylglucosaminyltransferases/antagonists & inhibitors , Uridine Diphosphate Galactose/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Uridine Diphosphate/chemical synthesis , Animals , CHO Cells , Cricetinae , Diphosphates/chemistry , Enzyme Activation , Enzyme Assays , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Galactosephosphates/chemistry , Magnetic Resonance Spectroscopy , N-Acetylglucosaminyltransferases/chemistry , Recombinant Proteins/chemistry , Solvents/chemistry , Tetrazoles/chemistry , Time Factors , Uridine Diphosphate/analogs & derivatives , Uridine Diphosphate Galactose/chemistry , Uridine Diphosphate Galactose/pharmacology , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate N-Acetylglucosamine/pharmacologyABSTRACT
Eight N-acetylglucosamine-1-phosphate and N-acetylgalactosamine-1-phosphate analogs have been synthesized chemically and were tested for their recognition by the GlmU uridyltransferase enzyme. Among these, only substrates that have an amide linkage to the C-2 nitrogen were transferred by GlmU to afford their corresponding uridine diphosphate(UDP)-sugar nucleotides. Resin-immobilized GlmU showed comparable activity to nonimmobilized GlmU and provides a more facile final step in the synthesis of an unnatural UDP-donor. The synthesized unnatural UDP-donors were tested for their activity as substrates for glycosyltransferases in the preparation of unnatural glycosaminoglycans in vitro. A subset of these analogs was useful as donors, increasing the synthetic repertoire for these medically important polysaccharides.
Subject(s)
Chemistry Techniques, Synthetic/methods , Escherichia coli Proteins/metabolism , Multienzyme Complexes/metabolism , Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , PolymerizationABSTRACT
A Pasteurella multocida N-acetylglucosamine 1-phosphate uridylyltransferase (PmGlmU) was cloned and used efficiently with an N-acetylhexosamine 1-kinase (NahK_ATCC55813) and an inorganic pyrophosphatase (PmPpA) for one-pot three-enzyme synthesis of UDP-GlcNAc derivatives with or without further chemical diversification.
Subject(s)
Acetylglucosamine/chemistry , Bacterial Proteins/metabolism , Inorganic Pyrophosphatase/metabolism , Nucleotidyltransferases/metabolism , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Acetylglucosamine/chemical synthesis , Bifidobacterium/enzymology , Pasteurella multocida/enzymology , Uridine Diphosphate N-Acetylglucosamine/chemistryABSTRACT
6''-Azido-6''-deoxy-UDP-N-acetylglucosamine (UDP-6Az-GlcNAc) is a potential alternate substrate for N-acetylglucosaminyltransferases. This compound could be used to generate various glycoconjugates bearing an azide functionality that could in turn be subjected to further modification using Staudinger ligation or Huisgen cycloaddition. UDP-6Az-GlcNAc is synthesized from α-benzyl-N-acetylglucosaminoside in seven-steps with an overall yield of 6%. It is demonstrated to serve as a substrate donor for the glycosyl transfer reaction catalyzed by the human UDP-GlcNAc:polypeptidyltransferase (OGT) to the acceptor protein nucleoporin 62 (nup62).
Subject(s)
N-Acetylglucosaminyltransferases/chemistry , Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives , Azides/chemical synthesis , Azides/chemistry , Humans , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , N-Acetylglucosaminyltransferases/metabolism , Nuclear Pore Complex Proteins/chemistry , Nuclear Pore Complex Proteins/metabolism , Substrate Specificity , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/chemistrySubject(s)
Galactosyltransferases/metabolism , Recombinant Proteins/metabolism , Uridine Diphosphate N-Acetylgalactosamine/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Combinatorial Chemistry Techniques , Escherichia coli Proteins/metabolism , Galactosyltransferases/chemical synthesis , Galactosyltransferases/chemistry , Humans , Molecular Structure , Multienzyme Complexes/metabolism , Recombinant Proteins/chemical synthesis , Recombinant Proteins/chemistry , Substrate Specificity , Uridine Diphosphate N-Acetylgalactosamine/chemistry , Uridine Diphosphate N-Acetylgalactosamine/metabolism , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate N-Acetylglucosamine/metabolismABSTRACT
The C-1-phosphonate analogue of UDP-GlcNAc has been synthesized using an alpha-configured C-1-aldehyde as a key intermediate. Addition of the anion of diethyl phosphate to the aldehyde produced the hydroxyphosphonate. The configuration of this key intermediate was determined by X-ray crystallography. Deoxygenation, coupling of the resulting phosphonic acid with UMP and deprotection gave the target molecule as a di-sodium salt. This analogue had no detectable activity as an inhibitor of (OGT).
Subject(s)
N-Acetylglucosaminyltransferases/antagonists & inhibitors , Organophosphonates/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Aldehydes , Crystallography, X-Ray , Molecular Structure , Organophosphates , Organophosphonates/pharmacology , Uridine Diphosphate N-Acetylglucosamine/pharmacologyABSTRACT
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.
Subject(s)
Chitin/metabolism , Deuterium/metabolism , Oligosaccharides/metabolism , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Acetylation , Carbohydrate Sequence , Chitin Synthase/analysis , Chitin Synthase/metabolism , Chromatography, Thin Layer , Molecular Structure , Spectrometry, Mass, Electrospray Ionization , Uridine Diphosphate N-Acetylglucosamine/chemistryABSTRACT
We describe the first synthesis of the C1-phosphonate analog of UDP-GlcNAc, based on a new preparation of the corresponding glycosyl phosphonate. This C-glycosyl analog is shown to be a very weak inhibitor (Ki>10 mM) of fungal chitin synthase, indicating that at least in this case the replacement of the anomeric oxygen with a methylene group is not an innocent substitution.
Subject(s)
Organophosphonates/chemistry , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Chitin Synthase/antagonists & inhibitors , Chitin Synthase/metabolism , Glycosides/chemistry , Models, Chemical , Molecular Structure , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate N-Acetylglucosamine/pharmacologyABSTRACT
[structure: see text] Chitin synthase (CS) polymerizes UDP-GlcNAc to form chitin (poly-beta(1,4)-GlcNAc), a key component of fungal cell wall biosynthesis. Little is known about the substrate specificity of chitin synthase or the scope of substrate modification the enzyme will tolerate. Following a previous report suggesting that 6-O-dansyl GlcNAc is biosynthetically incorporated into chitin, we became interested in developing an assay for CS activity based on incorporation of a fluorescent substrate. We describe the synthesis of two fluorescent UDP-GlcNAc analogues and their evaluation as chitin synthase substrates.
Subject(s)
Chitin Synthase/metabolism , Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives , Uridine Diphosphate N-Acetylglucosamine/metabolism , Chitin/biosynthesis , Chitin/chemistry , Chitin/metabolism , Fluorescent Dyes/chemistry , Molecular Structure , Substrate Specificity , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/chemistryABSTRACT
The synthesis and biological evaluation of a new UDP-GlcNAc competitor (I), designed to mimic the transition state of the sugar donor in the enzymatic reaction catalysed by chitin synthetase, is described. Compound (I) was found to competitively inhibit chitin synthetase from Saccharomyces cerevisiae with respect to UDP-GlcNAc, but displayed minimal antifungal activity.
Subject(s)
Aminoglycosides , Chitin Synthase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Saccharomyces cerevisiae/enzymology , Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives , Uridine Diphosphate N-Acetylglucosamine/pharmacology , Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Binding Sites , Chitin Synthase/chemistry , Enzyme Inhibitors/chemistry , Kinetics , Pyrimidine Nucleosides/pharmacology , Structure-Activity Relationship , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/chemistryABSTRACT
For the enzymatic transfer of galactose, N-acetylglucosamine, and N-acetylgalactosamine, UDP-Gal (1), UDP-GlcNAc (2), and UDP-GalNAc (3) are employed, and UDP serves as a feedback inhibitor. In this paper the synthesis of the novel UDP-sugar analogues 4, 5, and 6 as potential transferase inhibitors is described. Compounds 4-6 feature C-glycosidic hydroxymethylene linkages between the sugar and nucleoside moieties in contrast to the anomeric oxygens in the natural derivatives 1-3.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Transferases/antagonists & inhibitors , Uridine Diphosphate Galactose/chemical synthesis , Uridine Diphosphate N-Acetylgalactosamine/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Magnetic Resonance Spectroscopy , Molecular Mimicry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Uridine Diphosphate Galactose/chemistry , Uridine Diphosphate Galactose/pharmacology , Uridine Diphosphate N-Acetylgalactosamine/chemistry , Uridine Diphosphate N-Acetylgalactosamine/pharmacology , Uridine Diphosphate N-Acetylglucosamine/chemistry , Uridine Diphosphate N-Acetylglucosamine/pharmacologySubject(s)
Escherichia coli/enzymology , Hexosyltransferases/analysis , N-Acetylglucosaminyltransferases , Carbohydrate Sequence , Disaccharides/biosynthesis , Glycolipids/biosynthesis , Glycolipids/isolation & purification , Molecular Sequence Data , Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives , Uridine Diphosphate N-Acetylglucosamine/chemical synthesisABSTRACT
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.
Subject(s)
Uridine Diphosphate Glucose/chemical synthesis , Uridine Diphosphate Sugars/chemical synthesis , Acetylglucosamine/metabolism , Chromatography, High Pressure Liquid , Glucosamine/analogs & derivatives , Hexokinase/metabolism , Phosphoglucomutase/metabolism , Substrate Specificity , UTP-Hexose-1-Phosphate Uridylyltransferase/antagonists & inhibitors , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Uridine Triphosphate/metabolismABSTRACT
Benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-alpha-D-glucopyranoside was converted into its 4-O-(methylsulfonyl) derivative (2) by treatment with methanesulfonyl chloride in pyridine. Displacement of the methylsulfonyloxy group of 2 with fluoride ion afforded benzyl 2-acetamido-3,6-di-O-benzyl-2,4-dideoxy-4-fluoro-alpha-D-galactopyranosi de, which on hydrogenolysis, followed by acetylation, furnished 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-4-fluoro-D-galactopyranose. Treatment of this and of 2-acetamido-1,3,4-tri-O-acetyl-2,6-dideoxy-6-fluoro-D-glucopyranose with trimethylsilyl trifluoromethanesulfonate in 1,2-dichloroethane at approximately 50 degrees afforded the 4-deoxy-4-fluoro- or the 6-deoxy-6-fluoro-oxazolines (5) and (11), respectively. Reaction of 5 and 11 with dibenzyl phosphate in 1,2-dichloroethane produced the alpha-linked dibenzyl phosphate derivatives 6 and 12, respectively. Catalytic hydrogenation of 6 provided 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-4-fluoro-alpha-D-galactopyranosy l phosphate (7), and that of 12 gave 2-acetamido-3,4-di-O-acetyl-2,6-dideoxy-6-fluoro-alpha-D-glucopyranosyl phosphate (13). Coupling of 7 and 13 with uridine 5'-monophosphomorpholidate in dry pyridine at approximately 37 degrees, followed by O-deacetylation, furnished the title compounds, respectively, isolated and characterized as their respective dilithium salts.
Subject(s)
Uridine Diphosphate N-Acetylgalactosamine/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/chemical synthesis , Uridine Diphosphate Sugars/chemical synthesis , Carbohydrate Conformation , Indicators and Reagents , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , Optical Rotation , Uridine Diphosphate N-Acetylgalactosamine/analogs & derivatives , Uridine Diphosphate N-Acetylglucosamine/analogs & derivativesABSTRACT
The lipid A disaccharide of the Escherichia coli envelope is synthesized from the two fatty acylated glucosamine derivatives UDP-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucosamine (UDP-2,3-diacyl-GlcN) and N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D-glucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) [Ray, B. L., Painter, G., & Raetz, C. R. H. (1984) J. Biol. Chem. 259, 4852-4859]. We have previously shown that UDP-2,3-diacyl-GlcN is generated in extracts of E. coli by fatty acylation of UDP-GlcNAc, giving UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc as the first intermediate, which is rapidly converted to UDP-2,3-diacyl-GlcN [Anderson, M. S., Bulawa, C. E., & Raetz, C. R. H. (1985) J. Biol. Chem. 260, 15536-15541; Anderson, M. S., & Raetz, C. R. H. (1987) J. Biol. Chem. 262, 5159-5169]. We now demonstrate a novel enzyme in the cytoplasmic fraction of E. coli, capable of deacetylating UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc to form UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine. The covalent structure of the previously undescribed UDP-3-O-[(R)-3-hydroxymyristoyl] glucosamine intermediate was established by 1H NMR spectroscopy and fast atom bombardment mass spectrometry. This material can be made to accumulate in E. coli extracts upon incubation of UDP-3-O-[(R)-3- hydroxymyristoyl]-GlcNAc in the absence of the fatty acyl donor [(R)-3-hydroxymyristoyl]-acyl carrier protein. However, addition of the isolated deacetylation product [UDP-3-O-[(R)-3-hydroxymyristoyl] glucosamine] back to membrane-free extracts of E. coli in the presence of [(R)-3-hydroxymyristoyl]-acyl carrier protein results in rapid conversion of this compound into the more hydrophobic products UDP-2,3-diacyl-GlcN, 2,3-diacyl-GlcN-1-P, and O-[2-amino-2-deoxy-N2,O3- bis[(R)-3-hydroxytetradecanoyl]-beta-D-glucopyranosyl]-(1----6)-2-amino- 2-deoxy-N2,O3-bis[(R)-3-hydroxytetradecanoyl]-alpha-D- glucopyranose 1-phosphate (tetra-acyldisaccharide-1-P), demonstrating its competency as a precursor. In vitro incubations using [acetyl-3H]UDP-3-O-[(R)-3-hydroxymyristoyl]-GlcNAc confirmed release of the acetyl moiety in this system as acetate, not as some other acetyl derivative. The deacetylation reaction was inhibited by 1 mM N-ethylmaleimide, while the subsequent N-acylation reaction was not. Our observations provide strong evidence that UDP-3-O-[(R)-3-hydroxymyristoyl]glucosamine is a true intermediate in the biosynthesis of UDP-2,3-diacyl-GlcN and lipid A.
Subject(s)
Escherichia coli/metabolism , Lipid A/biosynthesis , Myristic Acids/metabolism , Uridine Diphosphate N-Acetylglucosamine/metabolism , Uridine Diphosphate Sugars/metabolism , Acetic Anhydrides , Acyl Carrier Protein/metabolism , Adenosine Triphosphate/metabolism , Indicators and Reagents , Magnetic Resonance Spectroscopy , Myristic Acids/chemical synthesis , Uridine Diphosphate N-Acetylglucosamine/analogs & derivatives , Uridine Diphosphate N-Acetylglucosamine/chemical synthesisSubject(s)
Liver/enzymology , Phosphotransferases/isolation & purification , Transferases (Other Substituted Phosphate Groups) , Animals , Drug Stability , Female , Golgi Apparatus/enzymology , Humans , Intracellular Membranes/enzymology , Isotope Labeling/methods , Kinetics , Male , Organ Specificity , Phosphorus Radioisotopes , Phosphotransferases/metabolism , Rats , Rats, Inbred Strains , Species Specificity , Substrate Specificity , Tritium , Uridine Diphosphate N-Acetylglucosamine/chemical synthesisABSTRACT
Procedures for the preparation of UDP-N-[1-14C]acetyl-D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine with very high specific activities are described. The overall yield based on the amount of [1-14C]acetate used is greater than 80%. The N-acetyl-D-glucosamine-alpha-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-D-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-D-glucosamine-alpha-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. D-glucosamine-alpha-1-phosphate is N-acetylated with [14C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-D-glucosamine pyrophosphorylase. UDP-N-[1-14C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-14C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.