Nucleotide diphospho sugars

Nucleotide diphospho sugars (NDP-sugars) or sugar nucleotides are activated monosaccharide donors used by glycosyl transferases (GTs) for glycosylation of a variety of acceptors. The role of NDP sugars in Nature cannot be underestimated since most of polysaccharides and glycoconjugates are assembled from these highly energetic molecular building blocks. NDP-sugars originate from primary metabolism of common precursors, such as UDP-glucose, which are transformed to a diverse range of NDP-sugars by sugar nucleotide processing enzymes. Our long-standing interests in NDP-sugars cover various aspects of their chemical and enzymatic synthesis, chromatographic purification, biosynthesis, design and synthesis of NDP-sugar analogues as molecular probes, and metabolic profiling of these elusive but hugely important molecules.

Sugar Nucleotide Synthesis

In our studies of biochemical transformations of carbohydrates we use a small number of commercially available sugar nucleotides but most of all we rely on our ability to prepare these compounds. Depending on the structure of both sugar and nucleotide counterparts of a target molecule we use either chemical or enzymatic synthesis, or combination of both.

ndpsugar-synthesis

Purification of sugar nucleotides is another endeavour which requires a combination of strong anion exchange and reverse phase chromatography sometimes supplemented by gel filtration.  We routinely purify the synthetic NDP-sugars to homogeneity in quantities ranging from 100s  pg up to 100s mg.

Enzymatic synthesis of nucleobase-modified UDP-sugars

Unnatural auto-fluorescent derivatives of UDP-sugars can be very useful in assessing small molecule inhibitors of GTs (Pesnot, T.; Palcic, M. M.; Wagner, G. K. ChemBioChem 2010, 11, 1392-1398). Such derivatives should incorporate a fluorogenic substituent at 5 position of the uracil residue. A substituent can be introduced using palladium-mediated cross-coupling. The rest of the molecule is assembled by enzymatic synthesis as exemplified by preparation of 5-iodo-UDP-galactose.

Both chemical and enzymatic syntheses are used in our laboratory to access these unnatural UDP-sugars. UDP-glucose pyrophosphorylase in conjunction with inorganic pyrophosphatase was particularly effective at converting 5-substituted UTP derivatives, including 5-iodo-UTP, into a range of gluco-configured 5-substituted UDP-sugar derivatives in good yields. Erwinia amylovora UDP-glucose 4’’-epimerase effects 4’’-epimerization of 5-iodo-UDP-Glc to give 5-iodo-UDP-Gal. Given the established potential for Pd-mediated cross-coupling of 5-iodo-UDP-sugars, this provides convenient access to the galacto-configured 5-substituted-UDP-sugars from gluco-configured substrates and 5-iodo-UTP.

epimerisation

Strategies for enzymatic preparation of 5-iodo-modified UDP-glucose and UDP-galactose. GalPUT = galactose-1-phosphate uridylyltransferase; GalU = UDP-glucose pyrophosphorylase; GalE = UDP-Galactose 400-epimerase; IPP = inorganic pyrophosphatase. Arrows in black indicate a one pot reactions; arrows in grey indicate a separate reaction.

Sugar Nucleotide Profiling

Metabolic profiling of sugar nucleotides is the fingerprint-like measurement of these compounds in unicellular to multicellular biological systems. The metabolomics data thus obtained can be used to inform our transcriptome and genome sequencing efforts focused on unlocking glycomes of these organisms.  Our major technique for sugar nucleotides profiling is based on highly sensitive LC-MS protocols which rely upon authentic standards of NDP-sugars . If not commercially available, the standards required for this work are prepared in our laboratory.

martin-hplc-laila

Publications

1. Wagstaff, B. A.; Rejzek, M.; Pesnot, T.; Tedaldi, L. M.; Caputi, L.; O'Neill, E. C.; Benini, S.; Wagner, G. K.; Field, R. A., Enzymatic synthesis of nucleobase-modified UDP-sugars: scope and limitations. Carbohydr Res 2015, 404, 17-25.

2. Both, P.; Green, A. P.; Gray, C. J.; Sardzik, R.; Voglmeir, J.; Fontana, C.; Austeri, M.; Rejzek, M.; Richardson, D.; Field, R. A.; Widmalm, G.; Flitsch, S. L.; Eyers, C. E., Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing. Nat Chem 2014, 6 (1), 65-74.

3. Caputi, L.; Rejzek, M.; Louveau, T.; O'Neill, E. C.; Hill, L.; Osbourn, A.; Field, R. A., A one-pot enzymatic approach to the O-fluoroglucoside of N-methylanthranilate. Bioorgan Med Chem 2013, 21 (16), 4762-4767.

4. Alphey, M. S.; Pirrie, L.; Torrie, L. S.; Boulkeroua, W. A.; Gardiner, M.; Sarkar, A.; Maringer, M.; Oehlmann, W.; Brenk, R.; Scherman, M. S.; McNeil, M.; Rejzek, M.; Field, R. A.; Singh, M.; Gray, D.; Westwood, N. J.; Naismith, J. H., Allosteric Competitive Inhibitors of the Glucose-1-phosphate Thymidylyltransferase (RmlA) from Pseudomonas aeruginosa. Acs Chem Biol 2013, 8 (2), 387-396.

5. Rejzek, M.; Kannathasan, V. S.; Wing, C.; Preston, A.; Westman, E. L.; Lam, J. S.; Naismith, J. H.; Maskell, D. J.; Field, R. A., Chemical synthesis of UDP-Glc-2,3-diNAcA, a key intermediate in cell surface polysaccharide biosynthesis in the human respiratory pathogens B. pertussis and P. aeruginosa. Org Biomol Chem 2009, 7 (6), 1203-1210.

6. Tello, M.; Rejzek, M.; Wilkinson, B.; Lawson, D. M.; Field, R. A., Tyl 1a, a TDP-6-deoxy-D-xylo-4-hexulose 3,4-isomerase from Streptomyces fradiae: Structure prediction, mutagenesis and solvent isotope incorporation experiments to investigate reaction mechanism. Chembiochem 2008, 9 (8), 1295-1302.

7. Westman, E. L.; McNally, D. J.; Rejzek, M.; Miller, W. L.; Kannathasan, V. S.; Preston, A.; Maskell, D. J.; Field, R. A.; Brisson, J. R.; Lam, J. S., Identification and biochemical characterization of two novel UDP-2,3-dideoxy-alpha-D-glucuronic acid 2-epimerases from respiratory pathogens. Biochem J 2007, 405, 123-130.

8. Rejzek, M.; Mukhopadhyay, B.; Wenzel, C. Q.; Lam, J. S.; Field, R. A., Direct oxidation of sugar nucleotides to the corresponding uronic acids: TEMPO and platinum-based procedures. Carbohyd Res 2007, 342 (3-4), 460-466.

9. Errey, J. C.; Mukhopadhyay, B.; Kartha, K. P. R.; Field, R. A., Flexible enzymatic and chemo-enzymatic approaches to a broad range of uridine-diphospho-sugars. Chem Commun 2004, (23), 2706-2707.

zevo-lcms

Waters LC-MS Xevo TQ-S tandem mass spectrometer extensively used for NDP-sugars profiling in Rob Field lab.