15839-70-0Relevant articles and documents
Biochemical characterization of an α1,2-colitosyltransferase from Escherichia coli O55:H7
Wu, Zhigang,Zhao, Guohui,Li, Tiehai,Qu, Jingyao,Guan, Wanyi,Wang, Jiajia,Ma, Cheng,Li, Xu,Zhao, Wei,Wang, Peng G.,Li, Lei
, p. 493 - 500 (2016)
Colitose, also known as 3,6-dideoxy-l-galactose or 3-deoxy-l-fucose, is one of only five naturally occurring 3,6-dideoxyhexoses. Colitose was found in lipopolysaccharide of a number of infectious bacteria, including Escherichia coli O55 & O111 and Vibrio cholera O22 & O139. To date, no colitosyltransferase (ColT) has been characterized, probably due to the inaccessibility of the sugar donor, GDP-colitose. In this study, starting with chemically prepared colitose, 94.6 mg of GDP-colitose was prepared via a facile and efficient one-pot two-enzyme system involving an l-fucokinase/GDP-l-Fuc pyrophosphorylase and an inorganic pyrophosphatase (EcPpA). WbgN, a putative ColT from E. coli O55:H5 was then cloned, overexpressed, purified and biochemically characterized by using GDP-colitose as a sugar donor. Activity assay and structural identification of the synthetic product clearly demonstrated that wbgN encodes an α1,2-ColT. Biophysical study showed that WbgN does not require metal ion, and is highly active at pH 7.5-9.0. In addition, acceptor specificity study indicated that WbgN exclusively recognizes lacto-N-biose (Galβ1,3-GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP-l-Fuc (kcat/Km = 9.2 min-1 mM-1) as that toward GDP-colitose (kcat/Km = 12 min-1 mM-1). Finally, taking advantage of this, type 1 H-antigen was successfully synthesized in preparative scale.
Gram-scale production of sugar nucleotides and their derivatives
Li, Shuang,Wang, Shuaishuai,Wang, Yaqian,Qu, Jingyao,Liu, Xian-Wei,Wang, Peng George,Fang, Junqiang
supporting information, p. 2628 - 2633 (2021/04/21)
Here, we report a practical sugar nucleotide production strategy that combined a high-concentrated multi-enzyme catalyzed reaction and a robust chromatography-free selective precipitation purification process. Twelve sugar nucleotides were synthesized on a gram scale with a purity up to 98%.
A High-Throughput Glycosyltransferase Inhibition Assay for Identifying Molecules Targeting Fucosylation in Cancer Cell-Surface Modification
Zhang, Xiaohua,Chen, Fei,Petrella, Alessandro,Chacón-Huete, Franklin,Covone, Jason,Tsai, Teng-Wei,Yu, Ching-Ching,Forgione, Pat,Kwan, David H.
, p. 715 - 724 (2019/03/26)
In cancers, increased fucosylation (attachment of fucose sugar residues) on cell-surface glycans, resulting from the abnormal upregulation of the expression of specific fucosyltransferase enzymes (FUTs), is one of the most important types of glycan modifications associated with malignancy. Fucosylated glycans on cell surfaces are involved in a multitude of cellular interactions and signal regulation in normal biological processes, as well as in disease. For example, sialyl LewisX is a fucosylated cell-surface glycan that is abnormally abundant in some cancers where it has been implicated in facilitating metastasis, allowing circulating tumor cells to bind to the epithelial tissue within blood vessels and invade into secondary sites by taking advantage of glycan-mediated interactions. To identify inhibitors of FUT enzymes as potential cancer therapeutics, we have developed a novel high-throughput assay that makes use of a fluorogenically labeled oligosaccharide as a probe of fucosylation. This probe, which consists of a 4-methylumbelliferyl glycoside, is recognized and hydrolyzed by specific glycoside hydrolase enzymes to release fluorescent 4-methylumbelliferone, yet when the probe is fucosylated prior to treatment with the glycoside hydrolases, hydrolysis does not occur and no fluorescent signal is produced. We have demonstrated that this assay can be used to measure the inhibition of FUT enzymes by small molecules, because blocking fucosylation will allow glycosidase-catalyzed hydrolysis of the labeled oligosaccharide to produce a fluorescent signal. Employing this assay, we have screened a focused library of small molecules for inhibitors of a human FUT enzyme involved in the synthesis of sialyl LewisX and demonstrated that our approach can be used to identify potent FUT inhibitors from compound libraries in microtiter plate format.
Enzymatic Synthesis of Human Milk Fucosides α1,2-Fucosyl para-Lacto-N-Hexaose and its Isomeric Derivatives
Fang, Jia-Lin,Tsai, Teng-Wei,Liang, Chin-Yu,Li, Jyun-Yi,Yu, Ching-Ching
supporting information, p. 3213 - 3219 (2018/08/06)
Enzymatic synthesis of para-lacto-N-hexaose and its isomeric structures as well as those α1,2-fucosylated variants naturally occurring in human milk oligosaccharide (HMOs) was achieved using a sequential one-pot enzymatic system. Three glycosylation routes comprising bacterial glycosyltransferases and corresponding sugar-nucleotide-generating enzymes were developed to facilitate efficient production of extended type-1 and type-2 N-acetyllactosamine (LacNAc) backbones and hybrid chains. Further fucosylation efficiency of two α1,2-fucosyltransferases on both type-1 and type-2 chains of the hexasaccharide was investigated to achieve practical synthesis of the fucosylated glycans. The availability of structurally defined HMOs offers a practical approach for investigating future biological applications. (Figure presented.).
Mechanism and active site residues of GDP-fucose synthase
Lau, Stephen T. B.,Tanner, Martin E.
experimental part, p. 17593 - 17602 (2009/07/19)
L-Fucose, 6-deoxy-L-galactose, is a key component of many important glycoconjugates including the blood group antigens and the Lewisx ligands. The biosynthesis of GDP-L-fucose begins with the action cof a dehydratase that converts GDP-D-mannose into GDP-4-keto-6-deoxy-mannose. The enzyme GDP-fucose synthase, GFS, (also known as GDP-4-keto-6-deoxy-D-mannose epimerase/reductase, GMER) then converts GDP-4-keto-6-deoxy-D-mannose into GDP-L-fucose. The GFS reaction involves epimerizations at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. This manuscript describes studies that elucidate the order of the epimerization steps and the roles of the active site acid/base residues responsible for the epimerizations. An active site mutant, Cys109Ser, produces GDP-6-deoxy-D-altrose as its major product indicating that C-3 epimerization occurs first and premature reduction of the GDP-4-keto-6-deoxy-D-altrose intermediate becomes competitive with GDP-L-fucose production. The same mutation results in the appearance of a kinetic isotope effect when [3 - 2H]-GDP-6-deoxy-4-keto- mannose is used as a substrate. This indicates that Cys109 is the base responsible for the deprotonation of the substrate at C-3. The Cys109Ser mutant also catalyzes a rapid wash-in of solvent derived deuterium into the C-5 position of GDP-fucose in the presence of NADP+. This confirms the order of epimerizations and the role of Cys109. Finally, the inactive His179Gln mutant readily catalyzes the wash-out of deuterium from the C-3 position of [3 - 2H]-GDP-6- deoxy-4-keto-mannose. Together these results strongly implicate an ordered sequence of epimerizations (C-3 followed by C-5 ) and suggest that Cys109 acts as a base and His179 acts as an acid in both epimerization steps.
Stereoselective chemical synthesis of sugar nucleotides via direct displacement of acylated glycosyl bromides
Timmons, Shannon C.,Jakeman, David L.
, p. 1227 - 1230 (2007/10/03)
Figure presented 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 GDPα-D-mannose as well as UDP- and GDP-β-L-fucose via direct displacement of acylated glycosyl bromides with nucleoside 5′- diphosphates.
Synthesis of the milk oligosaccharide 2′-fucosyllactose using recombinant bacterial enzymes
Albermann, Christoph,Piepersberg, Wolfgang,Wehmeier, Udo F
, p. 97 - 103 (2007/10/03)
The enzymatic synthesis of GDP-β-L-fucose and its enzymatic transfer reaction using recombinant enzymes from bacterial sources was examined. The GDP-D-mannose 4,6-dehydratase and the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase from Escherichia coli K-12, respectively, were used to catalyse the conversion of GDP-α-D-mannose to GDP-β-L-fucose with 78% yield. For the transfer of the L-fucose to an acceptor, we cloned and overproduced the α-(1 → 2)-fucosyltransferase (FucT2) protein from Helicobacter pylori. We were able to synthesise 2′-fucosyllactose using the overproduced FucT2 enzyme, enzymatically synthesised GDP-L-fucose and lactose. The isolation of 2′-fucosyllactose was accomplished by anion-exchange chromatography and gel filtration to give 65% yield.
An approach towards the synthesis of 1,2-trans glycosyl phosphates via iodonium ion assisted activation of thioglycosides
Veeneman,Broxterman,Van der Marel,Van Boom
, p. 6175 - 6178 (2007/10/02)
Phosphorylation of benzoylated ethyl 1,2-trans 1-thioglycosides with dibenzyl phosphate in the presence of NIS gave, after removal of all protecting groups, 1,2-trans glycosyl phosphates. The scope of the stereoselective method was demonstrated by the syn
