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Synthetic hexynyl α-D-mannopyranoside and its α-1,6-linked disaccharide counterpart were fluorescently labelled through CuAAC click chemistry with 3-azido-7-hydroxycoumarin. The resulting triazolyl-coumarin adducts, which were amenable to analysis by TLC, HPLC and mass spectrometry, proved to be acceptor substrates for α-1,6-ManT activities in mycobacterial membranes, as well as α- and β-GalT activities in trypanosomal membranes, benchmarking the potential of the fluorescent acceptor approach against earlier radiochemical assays. Following on to explore the glycobiology of the benign protozoan alga Euglena gracilis, α-1,3- and α-1,2-ManT activities were detected in membrane preparations, along with GlcT, Glc-P-T and GlcNAc-P-T activities. These studies serve to demonstrate the potential of readily accessible fluorescent glycans as substrates for exploring carbohydrate active enzymes., Graphical abstract Image 1, Highlights • Assays for the analysis of carbohydrate-active enzymes that rely upon fluorescent acceptor substrates are set out. • New assays are validated by benchmarking against radiochemical work with known glycosyltransferase activities. • The installation of a fluorophore on acceptor substrates was easily achieved through click chemistry. • Fluorescence assays are used to discover GTs activities in Euglena gracilis microsomal membranes.

Graphical abstract, Glycosyltransferase MurG catalyses the transfer of N-acetyl-d-glucosamine to lipid intermediate I on the bacterial peptidoglycan biosynthesis pathway, and is a target for development of new antibacterial agents. A transition state mimic was designed for MurG, containing a functionalised proline, linked through the α-carboxylic acid, via a spacer, to a uridine nucleoside. A set of 15 functionalised prolines were synthesised, using a convergent dipolar cycloaddition reaction, which were coupled via either a glycine, proline, sarcosine, or diester linkage to the 5′-position of uridine. The library of 18 final compounds were tested as inhibitors of Escherichia coli glycosyltransferase MurG. Ten compounds showed inhibition of MurG at 1 mM concentration, the most active with IC50 400 μM. The library was also tested against Mycobacterium tuberculosis galactosyltransferase GlfT2, and one compound showed effective inhibition at 1 mM concentration.

Arabinosyltransferases (AraTs) play a critical role in mycobacterial cell wall biosynthesis and are potential drug targets for the treatment of tuberculosis, especially multi-drug resistant forms of M. tuberculosis (MTB). Herein, we report the synthesis and acceptor/inhibitory activity of Araf alpha(1-->5) Araf disaccharides possessing deoxygenation at the reducing sugar of the disaccharide. Deoxygenation at either the C-2 or C-3 position of Araf was achieved via a free radical procedure using xanthate derivatives of the hydroxyl group. The alpha(1-->5)-linked disaccharides were produced by coupling n-octyl alpha-Araf 2-/3-deoxy, 2-fluoro glycosyl acceptors with an Araf thioglycosyl donor. The target disaccharides were tested in a cell free mycobacterial AraTs assay as well as an in vitro assay against MTB H(37)Ra and M. avium complex strains.

Efficient syntheses are reported of the α- l -rhamnosidase inhibitors l -swainsonine [(1 R ,2 S ,8 S ,8a S )-octahydroindolizine-1,2,8-triol], (6 R )- C -methyl- l -swainsonine (1 R ,2 S ,6 R ,8 S ,8a S )-6-methyloctahydro-indolizine-1,2,8-triol, (6 S )- C -methyl- l -swainsonine (1 R ,2 S ,6 S ,8 S ,8a S )-6-methyloctahydro-indolizine-1,2,8-triol and related 6- C -methyl hydroxycastanospermines [(1 R ,2 S ,6 R ,7 R ,8 R ,8a R )-6-methyl-octahydroindolizine-1,2,6,7,8-pentaol and (1 R ,2 S ,6 S ,8 S ,8a S )-6-methyloctahydro-indolizine-1,2,8-triol]. (6 R )- C -Methyl- l -swainsonine [ K i = 0.032 μM] is a significantly more potent naringinase inhibitor than l -swainsonine [ K i = 0.45 μM].

Glycosyltransferases (GTs) play a crucial role in mycobacterial cell wall biosynthesis and are necessary for the survival of mycobacteria. Hence, these enzymes are potential new drug targets for the treatment of tuberculosis (TB), especially multiple drug-resistant TB (MDR-TB). Herein, we report the efficient syntheses of Araf(α 1 → 5)Araf, Galf(β 1 → 5)Galf, and Galf(β 1 → 6)Galf disaccharides possessing a 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl (dansyl) unit that were prepared as fluorescent disaccharide acceptors for arabinosyl- and galactosyl-transferases, respectively. Such analogs may offer advantages relative to radiolabeled acceptors or donors for studying the enzymes and for assay development and compound screening. Additionally, analogs possessing a 5-azidonaphthalene-1-sulfonamidoethyl unit were prepared as photoaffinity probes for their potential utility in studying active site labeling of the GTs (arabinosyl and galactosyl) in Mycobacterium tuberculosis (MTB). Beyond their preparation, initial biological testing and kinetic analysis of these disaccharides as acceptors toward glycosyltransferases are also presented.

The assembly of the arabinan portions of cell wall polysaccharides in mycobacteria involves a family of arabinosyltransferases (AraT's) that promote the polymerization of decaprenolphosphoarabinose. Mycobacterial viability depends upon the ability of the organism to synthesize an intact arabinan and thus compounds that inhibit these AraT's are both useful biochemical tools as well as potential lead compounds for new anti-tuberculosis agents. We describe here the preparation of oligosaccharide fragments of mycobacterial arabinan that contain arabinofuranosyl residues modified at C-3 by the replacement of the hydroxyl group with an amino, azido or methoxy functionality. Subsequent testing of these oligosaccharides as inhibitors of mycobacterial AraT's revealed that all inhibited the enzymes, but to varying degrees. In further studies, each compound was shown to have only low activity as an inhibitor of mycobacterial growth.

Analogs of the α- d -Man p -(1→6)-α- d -Man p -O(CH 2 ) 7 CH 3 disaccharide 4 , a known substrate for a polyprenol monophosphomannose-dependent α-(1→6)-mannosyltransferase involved in mycobacterial LAM biosynthesis, have been synthesized and screened as potential substrates and inhibitors of the enzyme. In the disaccharides synthesized, the hydroxyl groups at C-2 and C-6 on the reducing end residue have been replaced by combinations of amino, fluoro, and methoxy functionalities 9 – 14 . In addition, a disaccharide in which the nonreducing mannopyranose residue was replaced with a 3,6-anhydromannopyranose residue 34 was synthesized from a byproduct formed during one of the reactions leading to 14 . When tested against the enzyme, none were active as substrates, as would be expected as all lack the C-6′ hydroxyl group to which an additional sugar residue would be transferred. Evaluation of these compounds as inhibitors of the enzyme revealed that only three, 11 , 12 , and 13 , all of which contain one or more amino groups, inhibited the enzyme. The most potent inhibitor was the diamino-disaccharide, 11 .

Mannosyltransferases play a crucial role in mycobacterial cell-wall biosynthesis and are potential new drug targets for the treatment of tuberculosis. Herein, we describe the synthesis of α-(1 → 2)- and α-(1 → 6)-linked mannopyranosyl disaccharides possessing a 5-azidonaphthlene-1-sulfonamidoethyl group as photoaffinity probes for active-site labeling studies of mannosyltransferases in Mycobacetrium tuberculosis .

Several octyl 5- O -(α- d -arabinofuranosyl)-α- d -arabinofuranoside disaccharide analogs substituted at the 5-position of the non-reducing end sugar were synthesized and tested in vitro against Mycobacterium tuberculosis ( M.tb. ), Mycobacterium avium complex (MAC) as well as in a cell free assay system for arabinosyltransferase acceptor/inhibitor activity. A few compounds showed interesting inhibitory activity in the cell free assay as well as against the whole microorganism in vitro.

Analogues of the natural antibiotic thiolactomycin, with acetylene-based side chains, have the highest recorded in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis β-ketoacyl-ACP synthase mtFabH condensing enzyme. In particular, 5-[3-(4-acetyl-phenyl)-prop-2-ynyl]-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited more than an 18-fold increased potency, compared to thiolactomycin, against this key condensing enzyme, involved in M. tuberculosis mycolic acid biosynthesis. Analogues of the antibiotic thiolactomycin, with acetylene-based side chains, have the highest recorded activity against cloned mtFabH condensing enzyme.

Dolichol phosphate-mannose (Dol-P-Man) is a mannose donor in various eukaryotic glycosylation processes. So far, two groups of Dol-P-Man synthases have been characterized based on the way they are stabilized in the endoplasmic reticulum membrane. Enzymes belonging to the first group, such as the yeast Dpm1, are typical integral membrane proteins harboring a transmembrane segment (TMS) at their C terminus. In contrast, mammalian Dpm1, enzymes of the second group, lack the typical TMS and require the association with the small hydrophobic proteins Dpm3 to be properly stabilized in the endoplasmic reticulum membrane. In Mycobacterium tuberculosis, the Polyprenol-P-Man synthase MtPpm1 is involved in the biosynthesis of the cell wall-associated glycolipid lipoarabinomannan. MtPpm1 is composed of two domains. The C-terminal catalytic domain is homologous to eukaryotic Dol-P-Man synthases. The N-terminal domain of MtPpm1 contains six TMS that anchor the enzyme in the cytoplasmic membrane. In contrast, in Mycobacterium smegmatis, orthologs of the two domains of MtPpm1 are encoded by two distinct open reading frames, Msppm1 and Msppm2, organized as an operon. No TMS are predicted in MsPpm1, and subcellular fractionation experiments indicate that this enzyme is cytosolic when produced in Escherichia coli. Computer-assisted topology predictions and alkaline phosphatase insertions showed that MsPpm2 is an integral membrane protein. Using a recently developed bacterial two-hybrid system, it was found that MsPpm2 interacts with MsPpm1 to stabilize the synthase MsPpm1 in the bacterial membrane. This interaction is reminiscent of that of mammalian Dpm1 with Dpm3 and mimics the structure of MtPpm1 as demonstrated by the capacity of the two domains of MtPpm1 to spontaneously interact when co-expressed in E. coli.

The appearance multi-drug resistant Mycobacterium tuberculosis (MTB) throughout the world has prompted a search for new, safer and more active agents against tuberculosis. Based on studies of the biosynthesis of mycobacterial cell wall polysaccharides, octyl 5- O -(α- d -arabinofuranosyl)-α- d -arabinofuranoside analogues were synthesized and evaluated as inhibitors for M. tuberculosis and Mycobacterium avium . A cell free assay system has been used for the evaluation of these disaccharides as substrates for mycobacterial arabinosyltransferase activity.