6-Deoxy-D-glucose

Chemical Synthesis of a Keto Sugar Nucleotide.[Pubmed:37126664]

J Org Chem. 2023 Jun 2;88(11):7580-7585.

Keto sugar nucleotides (KSNs) are common and versatile precursors to various deoxy sugar nucleotides, which are substrates for the corresponding glycosyltransferases involved in the biosynthesis of glycoproteins, glycolipids, and natural products. However, there has been no KSN synthesized chemically due to the inherent instability. Herein, the first chemical synthesis of the archetypal KSN TDP-4-keto-6-Deoxy-D-glucose (1) is achieved by an efficient and optimized route, providing feasible access to other KSNs and analogues, thereby opening a new avenue for new applications.

Molecular Cloning and Characteristics of a Lectin from the Bivalve Glycymeris yessoensis.[Pubmed:36827096]

Mar Drugs. 2023 Jan 17;21(2):55.

C-type lectins (CTLs) are a family of carbohydrate-binding proteins that mediate multiple biological events, including adhesion between cells, the turnover of serum glycoproteins, and innate immune system reactions to prospective invaders. Here, we describe the cDNA cloning of lectin from the bivalve Glycymeris yessoensis (GYL), which encodes 161 amino acids and the C-type carbohydrate recognition domain (CRD) with EPN and WND motifs. The deduced amino acid sequence showed similarity to other CTLs. GYL is a glycoprotein containing two N-glycosylation sites per subunit. N-glycans are made up of xylose, mannose, D-glucosamine, 3-O-methylated galactose, D-quinovoses, and 3-O-methylated 6-Deoxy-D-glucose. The potential CRD tertiary structure of the GYL adopted CTL-typical long-form double-loop structure and included three disulfide bridges at the bases of the loops. Additionally, when confirming the GYL sequence, eight isoforms of this lectin were identified. This fact indicates the presence of a multigene family of GYL-like C-type lectins in the bivalve G. yessoensis. Using the glycan microarray approach, natural carbohydrate ligands were established, and the glycotope for GYL was reconstructed as "Galbeta1-4GlcNAcbeta obligatory containing an additional fragment", like a sulfate group or a methyl group of fucose or N-acetylgalactosamine residues.

Visualization of Protein-Specific Glycation in Living Cells via Bioorthogonal Chemical Reporter.[Pubmed:35982548]

Angew Chem Int Ed Engl. 2022 Oct 10;61(41):e202210069.

Due to the lack of suitable chemical tools, probing the protein-specific glycation is highly challenging. Herein, we present a strategy based on glycation chemical reporter and proximity-induced FRET signal readout for visualizing protein-specific glycation in living cells. We first developed a bioorthogonal glucose analogue, 6-azido-6-Deoxy-D-glucose (6AzGlc), as a novel glycation chemical reporter. Two types of DNA probes, glycation conversion probe and protein targeting probe, were designed to attach to glycation adducts and target proteins, respectively. After the protein was glycated by 6AzGlc, two DNA probes were sequentially applied to the target protein, triggering proximity-induced FRET signal readout. This strategy was successfully used to visualize glucose glycation of several proteins, including PD-L1 and integrin. More importantly, this strategy allowed us to analyze corresponding biological functions of glycated protein in the native environment.

Two RmlC homologs catalyze dTDP-4-keto-6-deoxy-D-glucose epimerization in Pseudomonas putida KT2440.[Pubmed:34099824]

Sci Rep. 2021 Jun 7;11(1):11991.

L-Rhamnose is an important monosaccharide both as nutrient source and as building block in prokaryotic glycoproteins and glycolipids. Generation of those composite molecules requires activated precursors being provided e. g. in form of nucleotide sugars such as dTDP-beta-L-rhamnose (dTDP-L-Rha). dTDP-L-Rha is synthesized in a conserved 4-step reaction which is canonically catalyzed by the enzymes RmlABCD. An intact pathway is especially important for the fitness of pseudomonads, as dTDP-L-Rha is essential for the activation of the polyproline specific translation elongation factor EF-P in these bacteria. Within the scope of this study, we investigated the dTDP-L-Rha-biosynthesis route of Pseudomonas putida KT2440 with a focus on the last two steps. Bioinformatic analysis in combination with a screening approach revealed that epimerization of dTDP-4-keto-6-Deoxy-D-glucose to dTDP-4-keto-6-deoxy-L-mannose is catalyzed by the two paralogous proteins PP_1782 (RmlC1) and PP_0265 (RmlC2), whereas the reduction to the final product is solely mediated by PP_1784 (RmlD). Thus, we also exclude the distinct RmlD homolog PP_0500 and the genetically linked nucleoside diphosphate-sugar epimerase PP_0501 to be involved in dTDP-L-Rha formation, other than suggested by certain databases. Together our analysis contributes to the molecular understanding how this important nucleotide-sugar is synthesized in pseudomonads.

Stepwise Post-glycosylation Modification of Sugar Moieties in Kanamycin Biosynthesis.[Pubmed:33403742]

Chembiochem. 2021 May 4;22(9):1668-1675.

Kanamycin A is the major 2-deoxystreptamine (2DOS)-containing aminoglycoside antibiotic produced by Streptomyces kanamyceticus. The 2DOS moiety is linked with 6-amino-6-Deoxy-D-glucose (6ADG) at O-4 and 3-amino-3-deoxy-d-glucose at O-6. Because the 6ADG moiety is derived from d-glucosamine (GlcN), deamination at C-2 and introduction of C-6-NH(2) are required in the biosynthesis. A dehydrogenase, KanQ, and an aminotransferase, KanB, are presumed to be responsible for the introduction of C-6-NH(2) , although the substrates have not been identified. Here, we examined the substrate specificity of KanQ to better understand the biosynthetic pathway. It was found that KanQ oxidized kanamycin C more efficiently than the 3''-deamino derivative. Furthermore, the substrate specificity of an oxygenase, KanJ, that is responsible for deamination at C-2 of the GlcN moiety was examined, and the crystal structure of KanJ was determined. It was found that C-6-NH(2) is important for substrate recognition by KanJ. Thus, the modification of the GlcN moiety occurs after pseudo-trisaccharide formation, followed by the introduction of C-6-NH(2) by KanQ/KanB and deamination at C-2 by KanJ.

Access to Galectin-3 Inhibitors from Chemoenzymatic Synthons.[Pubmed:33200927]

J Org Chem. 2020 Dec 18;85(24):16099-16114.

Chemoenzymatic strategies are useful for providing both regio- and stereoselective access to bioactive oligosaccharides. We show herein that a glycosynthase mutant of a Thermus thermophilus alpha-glycosidase can react with unnatural glycosides such as 6-azido-6-Deoxy-D-glucose/glucosamine to lead to beta-d-galactopyranosyl-(1-->3)-d-glucopyranoside or beta-d-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-d-glucopyranoside derivatives bearing a unique azide function. Taking advantage of the orthogonality between the azide and the hydroxyl functional groups, the former was next selectively reacted to give rise to a library of galectin-3 inhibitors. Combining enzyme substrate promiscuity and bioorthogonality thus appears as a powerful strategy to rapidly access to sugar-based ligands.

Biosynthetic access to the rare antiarose sugar via an unusual reductase-epimerase.[Pubmed:34122866]

Chem Sci. 2020 Mar 27;11(15):3959-3964.

Rubrolones, isatropolones, and rubterolones are recently isolated glycosylated tropolonids with notable biological activity. They share similar aglycone skeletons but differ in their sugar moieties, and rubterolones in particular have a rare deoxysugar antiarose of unknown biosynthetic provenance. During our previously reported biosynthetic elucidation of the tropolone ring and pyridine moiety, gene inactivation experiments revealed that RubS3 is involved in sugar moiety biosynthesis. Here we report the in vitro characterization of RubS3 as a bifunctional reductase/epimerase catalyzing the formation of TDP-d-antiarose by epimerization at C3 and reduction at C4 of the key intermediate TDP-4-keto-6-Deoxy-D-glucose. These new findings not only explain the biosynthetic pathway of deoxysugars in rubrolone-like natural products, but also introduce RubS3 as a new family of reductase/epimerase enzymes with potential to supply the rare antiarose unit for expanding the chemical space of glycosylated natural products.

Biochemical characteristics of maltose phosphorylase MalE from Bacillus sp. AHU2001 and chemoenzymatic synthesis of oligosaccharides by the enzyme.[Pubmed:31262243]

Biosci Biotechnol Biochem. 2019 Nov;83(11):2097-2109.

Maltose phosphorylase (MP), a glycoside hydrolase family 65 enzyme, reversibly phosphorolyzes maltose. In this study, we characterized Bacillus sp. AHU2001 MP (MalE) that was produced in Escherichia coli. The enzyme exhibited phosphorolytic activity to maltose, but not to other alpha-linked glucobioses and maltotriose. The optimum pH and temperature of MalE for maltose-phosphorolysis were 8.1 and 45 degrees C, respectively. MalE was stable at a pH range of 4.5-10.4 and at 6-Deoxy-D-glucose, d-xylose, d-lyxose, l-fucose, and l-sorbose as acceptors. The k(cat(app))/K(m(app)) value for d-glucosamine and 6-Deoxy-D-glucose was comparable to that for d-glucose, and that for other acceptors was 0.23-12% of that for d-glucose. MalE synthesized alpha-(1-->3)-glucosides through reverse phosphorolysis with 2-deoxy-d-glucose and l-sorbose, and synthesized alpha-(1-->4)-glucosides in the reaction with other tested acceptors.