The Experts below are selected from a list of 270 Experts worldwide ranked by ideXlab platform
Chi-huey Wong - One of the best experts on this subject based on the ideXlab platform.
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Large-Scale Enzymatic Synthesis of Glycans with Cofactor Regeneration
Glycoscience: Biology and Medicine, 2021Co-Authors: Tsung-i Tsai, Chung-yi Wu, Chi-huey WongAbstract:The glycosyltransferases, which catalyze the transfer of a saccharide from a Sugar Nucleotide donor to an acceptor, have been used for the synthesis of complex glycoconjugates. Nevertheless, narrow substrate specificity, high cost of both the enzymes and the Sugar Nucleotides, and limited enzyme availability limit their application. Moreover, the progression of glycosylation can be plagued by feedback inhibition caused by the generated nucleoside phosphate. Regeneration of the Sugar Nucleotide intermediate in situ can reduce the concentration of the nucleoside phosphate by-product by the use of a small amount of the Sugar Nucleotide; thus, the expenses of the Sugar Nucleotide and product inhibition are reduced simultaneously.
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Effective Sugar Nucleotide Regeneration for the Large-Scale Enzymatic Synthesis of Globo H and SSEA4
Journal of the American Chemical Society, 2013Co-Authors: Tsung-i Tsai, Hsinyu Lee, Shih-huang Chang, Chia-hung Wang, Yu-chen Lin, Der-ren Hwang, Chi-huey WongAbstract:We report here the development of chemoenzymatic methods for the large-scale synthesis of cancer-associated antigens globopentaose (Gb5), fucosyl-Gb5 (Globo H), and sialyl-Gb5 (SSEA4) by using overexpressed glycosyltransferases coupled with effective regeneration of Sugar Nucleotides, including UDP-Gal, UDP-GalNAc, GDP-Fuc, and CMP-Neu5Ac. The enzymes used in the synthesis were first identified from different species through comparative studies and then overexpressed in E. coli and isolated for synthesis. These methods provide multigram quantities of products in high yield with only two or three purification steps and are suitable for the evaluation and development of cancer vaccines and therapeutics.
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Chemo-enzymatic synthesis of fluorinated Sugar Nucleotide: useful mechanistic probes for glycosyltransferases.
Bioorganic & medicinal chemistry, 2000Co-Authors: Michael D. Burkart, Stéphane P. Vincent, Arno Düffels, Brion W. Murray, Steven V. Ley, Chi-huey WongAbstract:An effective procedure for the synthesis of 2-deoxy-2-fluoro-Sugar Nucleotides via Selectfluor-mediated electrophilic fluorination of glycals with concurrent nucleophilic addition or chemo-enzymatic transformation has been developed, and the fluorinated Sugar Nucleotides have been used as probes for glycosyltransferases, including fucosyltransferase III, V, VI, and VII, and sialyl transferases. In general, these fluorinated Sugar Nucleotides act as competitive inhibitors versus Sugar Nucleotide substrates and form a tight complex with the glycosyltransferase.
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selective inhibition of β 1 4 and α 1 3 galactosyltransferases donor Sugar Nucleotide based approach
Bioorganic & Medicinal Chemistry, 1999Co-Authors: Shuichi Takayama, Sang J Chung, Yasuhiro Igarashi, Yoshitaka Ichikawa, Armin Sepp, Robert I Lechler, Takashi Hayashi, Gary Siuzdak, Chi-huey WongAbstract:A combined rational and library approach was used to identify bisphosphonates (IC50 = 20 microM) and galactose type 1-N-iminoSugar (IC50=45 microM) as novel motifs for selective inhibition of beta-1,4-galactosyltransferase (beta-1,4-GalT) and alpha-1,3-galactosyltransferase (alpha-1,3-GalT), respectively. Our results demonstrate that, though these two galactosyltransferases both utilize the same donor Sugar-Nucleotide (UDP-Gal), the difference in their mechanisms can be utilized to design donor Sugar or Nucleotide analogues with inhibitory activities selective for only one of the galactosyltransferases. Investigation of beta-1,4-GalT inhibition using UDP-2-deoxy-2-fluorogalactose (UDP-2-F-Gal), UDP, and bisphosphonates, also led to the observation of metal dependent inhibition of beta-1,4-GalT. These observations and the novel inhibitor motifs identified in this study pave the way for the design and identification of even more potent and selective galactosyltransferase inhibitors.
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selective inhibition of β 1 4 and α 1 3 galactosyltransferases donor Sugar Nucleotide based approach
Bioorganic & Medicinal Chemistry, 1999Co-Authors: Shuichi Takayama, Sang J Chung, Yasuhiro Igarashi, Yoshitaka Ichikawa, Armin Sepp, Robert I Lechler, Takashi Hayashi, Gary Siuzdak, Chi-huey WongAbstract:Abstract A combined rational and library approach was used to identify bisphosphonates (IC50=20 μM) and galactose type 1-N-iminoSugar (IC50=45 μM) as novel motifs for selective inhibition of β-1,4-galactosyltransferase (β-1,4-GalT) and α-1,3-galactosyltransferase (α-1,3-GalT), respectively. Our results demonstrate that, though these two galactosyltransferases both utilize the same donor Sugar-Nucleotide (UDP-Gal), the difference in their mechanisms can be utilized to design donor Sugar or Nucleotide analogues with inhibitory activities selective for only one of the galactosyltransferases. Investigation of β-1,4-GalT inhibition using UDP-2-deoxy-2-fluorogalactose (UDP-2-F-Gal), UDP, and bisphosphonates, also led to the observation of metal dependent inhibition of β-1,4-GalT. These observations and the novel inhibitor motifs identified in this study pave the way for the design and identification of even more potent and selective galactosyltransferase inhibitors. ©
Jon S Thorson - One of the best experts on this subject based on the ideXlab platform.
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A general NMR-based strategy for the in situ characterization of Sugar-Nucleotide-dependent biosynthetic pathways.
Organic letters, 2014Co-Authors: Shanteri Singh, Pauline Peltier-pain, Marco Tonelli, Jon S ThorsonAbstract:A simple method for the study of Sugar-Nucleotide-dependent multienzyme cascades is highlighted where the use of selectively 13C-labeled Sugar Nucleotides and inverse 13C detection NMR offers fast, direct detection and quantification of reactants and products and circumvents the need for chromatographic separation. The utility of the method has been demonstrated by characterizing four previously uncharacterized Sugar Nucleotide biosynthetic enzymes involved in calicheamicin biosynthesis.
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broadening the scope of glycosyltransferase catalyzed Sugar Nucleotide synthesis
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Richard W Gantt, Shanteri Singh, Pauline Peltierpain, Maoquan Zhou, Jon S ThorsonAbstract:We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial Sugar Nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in kcat/Km of >400-fold and >15-fold for formation of NDP–glucoses and UDP–Sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP–Sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex Sugar Nucleotides and the practical synthesis of a unique set of Sugar Nucleotides.
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Using simple donors to drive the equilibria of glycosyltransferase-catalyzed reactions
Nature Chemical Biology, 2011Co-Authors: Richard W Gantt, Pauline Peltier-pain, William J Cournoyer, Jon S ThorsonAbstract:We report that simple glycoside donors can drastically shift the equilibria of glycosyltransferase-catalyzed reactions, transforming NDP-Sugar formation from an endothermic to an exothermic process. To demonstrate the utility of this thermodynamic adaptability, we highlight the glycosyltransferase-catalyzed synthesis of 22 Sugar Nucleotides from simple aromatic Sugar donors, as well as the corresponding in situ formation of Sugar Nucleotides as a driving force in the context of glycosyltransferase-catalyzed reactions for small-molecule glycodiversification. These simple aromatic donors also enabled a general colorimetric assay for glycosyltransfer, applicable to drug discovery, protein engineering and other fundamental Sugar Nucleotide–dependent investigations. This study directly challenges the general notion that NDP-Sugars are 'high-energy' Sugar donors when taken out of their traditional biological context. A systematic analysis of possible substrates for reverse glycosyltransferase reactions reveals thermodynamically favored pathways to the traditional 'activated' Sugar donors, enabling high-yielding enzymatically coupled Sugar transfers and a general colorimetric assay for Sugar Nucleotide formation and utilization.
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using simple donors to drive the equilibria of glycosyltransferase catalyzed reactions
Nature Chemical Biology, 2011Co-Authors: Richard W Gantt, Pauline Peltierpain, William J Cournoyer, Jon S ThorsonAbstract:Glycosyltransferases (GTs) constitute a predominant enzyme superfamily responsible for the attachment of carbohydrate moieties to a wide array of acceptors that include nucleic acids, polysaccharides, proteins, lipids, carbohydrates and medicinally relevant secondary metabolites (1, 2). The majority of GTs are LeLoir (Sugar Nucleotide-dependent) enzymes and utilize nucleoside diphosphate Sugars (NDP-Sugars) as donors for glycosidic bond formation (Fig. 1a). Recent studies have revealed certain GT-catalyzed reactions from bacterial secondary metabolism to be reversible, presenting new GT-catalyzed methods for NDP-Sugar synthesis as well as the GT-catalyzed exchange (Fig. 1b) or transfer (Fig. 1c) of Sugars attached to both simple glycoside Sugar donors(3, 4) and complex natural product glycosides including glycopeptides, enediynes(5), macrolides(6), macrolactams(7), (iso)flavonoids(8) and polyenes(9). Of the few examples which employed ‘activated’ Sugar donors (e.g., glycosyl halides or aromatic glycosides) for GT-catalyzed transglycosylation(3, 4), only indirect evidence for the intermediacy of Sugar Nucleotides was provided. Among GT-catalyzed ‘reverse’ reactions where NDP-Sugar formation has been confirmed, NDP-Sugar formation was thermodynamically disfavored (i.e., with NDP-Sugar as product, Keq < 1)(5, 10, 11) and typically required a 10- to 100-fold excess of NDP for Sugar Nucleotide production. In an effort to address the severe thermodynamic limitations of GT-catalyzed reactions run in reverse, herein we report the use of specific activated aromatic glycosides as donors in such reactions dramatically alters the equilibrium of GT-catalyzed reactions and thereby enables a variety of novel transformations (Fig. 1d) including: i) an unique platform for the efficient enzymatic syntheses of novel NDP-Sugars, ii) a coupled GT-catalyzed platform for the differential glycosylation of small molecules (including natural products and synthetic drugs/targets), and iii) a colorimetric readout upon glycosyltransfer amenable to high throughput formats for glycodiversification and glycoengineering which can be coupled to nearly any downstream Sugar Nucleotide-utilizing enzyme. Fig. 1 Representative GT-catalyzed reactions. a) Classical GT-catalyzed transformation wherein the Sugar, presented in the form of a Sugar Nucleotide donor, is conjugated to an acceptor target of interest to provide a thermodynamically-favored glycoside product. ...
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characterization of cale10 the n oxidase involved in calicheamicin hydroxyaminoSugar formation
Journal of the American Chemical Society, 2008Co-Authors: Heather D Johnson, Jon S ThorsonAbstract:As the first in vitro characterization of a Sugar N-oxidase, this study establishes CalE10 as the key oxidase involved in calicheamicin hydroxylamino glycoside formation. This study confirms that oxidation occurs at the Sugar Nucleotide stage prior to glycosyltransfer, and substrate specificity studies reveal CalE10-catalyzed oxidation to be regiospecific and to present trace amounts of the corresponding nitroSugar in vitro. This work also sets a precedent for the future study of other N-oxidases involved in hydroxylamino-, nitroso-, and/or nitroSugar formation.
Robert A Field - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of the GDP-d-Mannose Dehydrogenase from Pseudomonas aeruginosa Using Targeted Sugar Nucleotide Probes.
ACS chemical biology, 2020Co-Authors: Laura Beswick, Martin Rejzek, Eleni Dimitriou, Sanaz Ahmadipour, Ayesha Zafar, Jóhannes Reynisson, Robert A Field, Gavin J MillerAbstract:Sufferers of cystic fibrosis are at extremely high risk for contracting chronic lung infections. Over their lifetime, one bacterial strain in particular, Pseudomonas aeruginosa, becomes the dominant pathogen. Bacterial strains incur loss-of-function mutations in the mucA gene that lead to a mucoid conversion, resulting in copious secretion of the exopolysaccharide alginate. Strategies that stop the production of alginate in mucoid Pseudomonas aeruginosa infections are therefore of paramount importance. To aid in this, a series of Sugar Nucleotide tools to probe an enzyme critical to alginate biosynthesis, guanosine diphosphate mannose dehydrogenase (GMD), have been developed. GMD catalyzes the irreversible formation of the alginate building block, guanosine diphosphate mannuronic acid. Using a chemoenzymatic strategy, we accessed a series of modified Sugar Nucleotides, identifying a C6-amide derivative of guanosine diphosphate mannose as a micromolar inhibitor of GMD. This discovery provides a framework for wider inhibition strategies against GMD to be developed.
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discovery of an rmlc d fusion protein in the microalga prymnesium parvum and its implications for ndp β l rhamnose biosynthesis in microalgae
Journal of Biological Chemistry, 2019Co-Authors: Martin Rejzek, Lionel Hill, Sakonwan Kuhaudomlarp, Ben A. Wagstaff, Ilaria Mascia, Sergey A Nepogodiev, Helge C Dorfmueller, Robert A FieldAbstract:The 6-deoxy Sugar l-rhamnose (l-Rha) is found widely in plant and microbial polysaccharides and natural products. The importance of this and related compounds in host-pathogen interactions often means that l-Rha plays an essential role in many organisms. l-Rha is most commonly biosynthesized as the activated Sugar Nucleotide uridine 5'-diphospho-β-l-rhamnose (UDP-β-l-Rha) or thymidine 5'-diphospho-β-l-rhamnose (TDP-β-l-Rha). Enzymes involved in the biosynthesis of these Sugar Nucleotides have been studied in some detail in bacteria and plants, but the activated form of l-Rha and the corresponding biosynthetic enzymes have yet to be explored in algae. Here, using Sugar-Nucleotide profiling in two representative algae, Euglena gracilis and the toxin-producing microalga Prymnesium parvum, we show that levels of UDP- and TDP-activated l-Rha differ significantly between these two algal species. Using bioinformatics and biochemical methods, we identified and characterized a fusion of the RmlC and RmlD proteins, two bacteria-like enzymes involved in TDP-β-l-Rha biosynthesis, from P. parvum Using this new sequence and also others, we explored l-Rha biosynthesis among algae, finding that although most algae contain sequences orthologous to plant-like l-Rha biosynthesis machineries, instances of the RmlC-RmlD fusion protein identified here exist across the Haptophyta and Gymnodiniaceae families of microalgae. On the basis of these findings, we propose potential routes for the evolution of nucleoside diphosphate β-l-Rha (NDP-β-l-Rha) pathways among algae.
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Profiling of Sugar Nucleotides.
Methods in Enzymology, 2017Co-Authors: Martin Rejzek, Lionel Hill, Edward S. Hems, Sakonwan Kuhaudomlarp, Ben A. Wagstaff, Robert A FieldAbstract:Sugar Nucleotides are essential building blocks for the glycobiology of all living organisms. Detailed information on the types of Sugar Nucleotides present in a particular cell and how they change as a function of metabolic, developmental, or disease status is vital. The extraction, identification, and quantification of Sugar Nucleotides in a given sample present formidable challenges. In this chapter, currently used techniques for Sugar Nucleotide extraction from cells, separation from complex biological matrices, and detection by optical and mass spectrometry methods are discussed.
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Sugar Nucleotide Recognition by Klebsiella pneumoniae UDP-d-Galactopyranose Mutase: Fluorinated Substrates, Kinetics and Equilibria
Organic & biomolecular chemistry, 2009Co-Authors: James C. Errey, Maretta C. Mann, Shirley A. Fairhurst, Lionel Hill, Michael R. Mcneil, James H. Naismith, Jonathan M. Percy, Chris Whitfield, Robert A FieldAbstract:A series of selectively fluorinated and other substituted UDP-D-galactose derivatives have been evaluated as substrates for Klebsiella pneumoniae UDP-D-galactopyranose mutase. This enzyme, which catalyses the interconversion of the pyranose and furanose forms of galactose as its UDP adduct, is a prospective drug target for a variety of microbial infections. We show that none of the 2''-, 3''- or 6''-hydroxyl groups of UDP-D-galactopyranose are essential for substrate binding and turnover. However, steric factors appear to play an important role in limiting the range of substitutions that can be accommodated at C-2'' and C-6'' of the Sugar Nucleotide substrate. Attempts to invert the C-2'' stereochemistry from equatorial to axial, changing D-galacto- to D-talo-configuration, in an attempt to exploit the higher percentage of furanose at equilibrium in the talo-series, met with no turnover of substrate.
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Structural and Mechanistic Basis of Bacterial Sugar Nucleotide-Modifying Enzymes†
Biochemistry, 2003Co-Authors: Robert A Field, James H. NaismithAbstract:Recently, carbohydrates have come to the fore because of their central role in many biological processes. One area of current interest concerns the enzymatic modification of Sugar Nucleotides, in relation to both secondary metabolite glycosylation and the formation of complex cell surface-associated glycoconjugates. Bacteria, in particular, have proven to be a rich field in which to study these transformations, because they are often unique to specific classes of organisms. This has led to the realization that such microbial biosynthetic pathways might be exploited in the generation of novel antibiotics, or indeed serve as targets for such compounds. This work illustrates the interplay between protein structure determination, chemistry, and molecular biology in providing insight into the mechanism of such biochemical transformations.
Xi Chen - One of the best experts on this subject based on the ideXlab platform.
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Recent progress in synthesis of carbohydrates with Sugar Nucleotide-dependent glycosyltransferases
Current opinion in chemical biology, 2020Co-Authors: Xi ChenAbstract:Abstract Sugar Nucleotide-dependent glycosyltransferases (GTs) are key enzymes that catalyze the formation of glycosidic bonds in nature. They have been increasingly applied in the synthesis of complex carbohydrates and glycoconjugates with or without in situ generation of Sugar Nucleotides. Human GTs are becoming more accessible and new bacterial GTs have been identified and characterized. An increasing number of crystal structures elucidated for GTs from mammalian and bacterial sources facilitate structure-based design of mutants as improved catalysts for synthesis. Automated platforms have also been developed for chemoenzymatic synthesis of carbohydrates. Recent progress in applying Sugar Nucleotide-dependent GTs in enzymatic and chemoenzymatic synthesis of mammalian glycans and glycoconjugates, bacterial surface glycans, and glycosylated natural products from bacteria and plants are reviewed.
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diversity oriented enzymatic modular assembly of abo histo blood group antigens
ACS Catalysis, 2016Co-Authors: Xianwei Liu, Xi Chen, Peng Peng, Fengshan Wang, Hongzhi CaoAbstract:Enzymatic synthesis of all 15 naturally occurring human ABH antigens was achieved using a diversity-oriented enzymatic modular assembly (EMA) strategy. Three enzyme modules were developed, each one-pot multienzyme module comprises a glycosyltransferase and one or two corresponding Sugar Nucleotide generating enzyme(s). These multienzyme cascade processes provide an efficient and convenient platform for collective synthesis of all 15 ABH antigens in three operationally simple steps from five readily available disaccharide acceptors and three simple free Sugars as donor precursors.
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Systematic chemoenzymatic synthesis of O-sulfated sialyl Lewis x antigens
Chemical science, 2015Co-Authors: Abhishek Santra, Nova Tasnima, Musleh M. Muthana, Jie Zeng, Nicholas J. Kenyon, Angelique Y. Louie, Xi ChenAbstract:O-Sulfated sialyl Lewis x antigens play important roles in nature. However, due to their structural complexity, they are not readily accessible by either chemical or enzymatic synthetic processes. Taking advantage of a bacterial sialyltransferase mutant that can catalyze the transfer of different sialic acid forms from the corresponding Sugar Nucleotide donors to Lewis x antigens, which are fucosylated glycans, as well as an efficient one-pot multienzyme (OPME) sialylation system, O-sulfated sialyl Lewis x antigens containing different sialic acid forms and O-sulfation at different locations were systematically synthesized by chemoenzymatic methods.
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Fermenting next generation glycosylated therapeutics.
ACS chemical biology, 2011Co-Authors: Xi ChenAbstract:Mutants of glycosyltransferases and related Sugar Nucleotide biosynthetic enzymes have been essential for in vitro glycorandomization to create libraries of novel glycosylated natural products and derivatives. These diverse glycorandomized compounds can now be produced in vivo economically by fermenting engineered Escherichia coli cells that express enzyme mutants.
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Enzymatic Approaches to O-Glycoside Introduction: Glycosyltransferases
Comprehensive Glycoscience, 2007Co-Authors: Harshal A. Chokhawala, Xi ChenAbstract:Glycosyltransferases that catalyze glycosylation reactions in a highly regio- and stereo-selective manner have proven to be important tools for preparative and large-scale synthesis of biologically and medicinally important glycans and glycoconjugates. A number of glycosyltransferases from various sources (mammalian, bacterial, and viral) and their mutants with or without Sugar Nucleotide biosynthetic enzymes have been widely used in the synthesis. Different synthetic strategies have been developed, including one-pot multi-enzyme (OPME), Sugar Nucleotide recycling, immobilized carbohydrate biosynthetic enzymes, solid phase synthesis, and cell-based systems. The trans-glycosidase and revsersible glycosylation activities of glycosyltransferases have also been explored. Glycosylation catalyzed by glycosyltransferases and their mutants will continue to play important roles in producing diverse glycan structures for elucidating their biological functions and developing carbohydrate-based therapeutics.
David L. Jakeman - One of the best experts on this subject based on the ideXlab platform.
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Stereospecific synthesis of Sugar-1-phosphates and their conversion to Sugar Nucleotides.
Carbohydrate research, 2008Co-Authors: Shannon C. Timmons, David L. JakemanAbstract:As Leloir glycosyltransferases are increasingly being used to prepare oligosaccharides, glycoconjugates, and glycosylated natural products, efficient access to stereopure Sugar Nucleotide donor substrates is required. Herein, the rapid synthesis and purification of eight Sugar Nucleotides is described by a facile 30 min activation of nucleoside 5'-monophosphates bearing purine and pyrimidine bases with trifluoroacetic anhydride and N-methylimidazole, followed by a 2 h coupling with stereospecifically prepared Sugar-1-phosphates. Tributylammonium bicarbonate and tributylammonium acetate were the ion-pair reagents of choice for the C18 reversed-phase purification of 6-deoxySugar Nucleotides, and hexose or pentose-derived Sugar Nucleotides, respectively.
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Lipophilic Sugar Nucleotide synthesis by structure-based design of nucleotidylyltransferase substrates
Organic & biomolecular chemistry, 2007Co-Authors: Malcolm P. Huestis, Gaia A. Aish, Joseph P. M. Hui, Evelyn C. Soo, David L. JakemanAbstract:Structure-based design of alkyl Sugar-1-phosphates provides an efficient nucleotidylyltransferase-catalyzed synthesis of a series of new lipophilic Sugar Nucleotides possessing long or branched alkyl chains, thereby demonstrating the utility of nucleotidylyltransferases to catalyze the synthesis of Sugar Nucleotides with potential applications in lipopolysaccharide and lipoglycopeptide biosynthesis.
