Enzymatic synthesis and properties of uridine-5′-O-(2-thiodiphospho)-N-acetylglucosamine

Abstract

This paper describes an enzymatic approach to obtain a thio-containing UDP-GlcNAc analog. We use an assay based on capture of the carbohydrate and analysis by mass spectrometry to quantitatively characterize the activity of this unnatural sugar donor in a LgtA-mediated glycosylation reaction.

Effective one-pot multienzyme (OPME) synthesis of monotreme milk oligosaccharides and other sialosides containing 4-O-acetyl sialic acid

“…39 Briefly, Lc 3 trisaccharide GlcNAcβ3Galβ4GlcβProN 3 42 was synthesized from LacβProN 3 (17) and GlcNAc in a 94% yield using a one-pot four-enzyme (OP4E) GlcNAc activation and transfer system containing Bifidobacterium longum N-acetylhexosamine-1-kinase (BLNahK, NahK_ATCC55813), 43 Pasteurella multocida N-acetylglucosamine uridyltransferase (PmGlmU), 44 Pasteurella multocida inorganic pyrophosphatase (PmPpA), 40 and Neisseria meningitidis β1-3-N-acetylglucosaminyltransferase (NmLgtA). [45][46][47] LNnTβProN 3 (23) 42 was then produced from Lc 3 and galactose in an excellent (99%) yield using a OP4E galactosylation system 37,39 containing Escherichia coli galactokinase (EcGalK), 48 Bifidobacterium longum UDP-sugar pyrophosphorylase (BLUSP), 49 PmPpA, 40 and Neisseria meningitidis β1-4-galactosyltransferase (NmLgtB). 40,47 Synthesis of Neu4,5Ac 2 α3LNnTβProN 3 (34) from LNnTβProN 3 (23) and Neu4,5Ac 2 (1) was successfully achieved in a good 81% yield using NmCSS and PmST3 in a one-pot (Scheme 3).…”

“…Aspergillus oryzae β-galactosidase was from Sigma (St. Louis, MO). Recombinant enzymes Bifidobacterium longum strain ATCC55813 N -acetylhexosamine-1-kinase (BLNahK or NahK_ATCC55813), 43 Pasteurella multocida N -acetylglucosamine uridyltransferase (PmGlmU), 44 Pasteurella multocida inorganic pyrophosphatase (PmPpA), 40 Neisseria meningitidis β1–3- N -acetylglucosaminyltransferase (NmLgtA), 45, 46 Escherichia coli galactokinase (EcGalK), 48 Bifidobacterium longum UDP-sugar pyrophosphorylase (BLUSP), 49 Neisseria meningitidis β1–4-galactosyltransferase (NmLgtB), 40 Pasteurella multocida sialic acid aldolase (PmNanA), 25 Neisseria meningitidis CMP-sialic acid synthetase (NmCSS), 26 Pasteurella multocida CSS (PmCSS), Haemophilus ducreyi CSS (HdCSS), and two NmCSS mutants (NmCSS_S81R and NmCSS_Q163A), 27 Pasteurella multocida multifunctional α2–3-sialyltransferase 1 (PmST1) 29 and Pasteurella multocida α2–3-sialyltransferase 3 (PmST3) 31 were expressed and purified as described previously. Purified human influenza A viruses A/Puerto Rico/34/8 H1N1 (A/PR8), A/Philippines/2/82/X-79 H3N2 (A/Philips), A/Memphis/71 H3N1 (A/Mem71), and A/Udorn/307/72 H3N2 (A/Udorn72) were described previously.…”

“…45 (s, 3H), 1.37 (s, 3H);13 C NMR (200 MHz, D 2 O) δ 172.51, 169.48, 136.54, 134.94, 128.61(2C), 128.58(2C), 128.45, 128.27, 128.01(2C), 127.99(2C), 109.15, 95.39, 74.59, 73.41, 71.23, 69.56, 68.96, 67.61, 66.93, 66.76, 52.44, 38.98, 26.97, 25.28.…”

Mimicking Complex Biological Membranes and Their Programmable Glycan Ligands with Dendrimersomes and Glycodendrimersomes

“…Sugars are notoriously difficult to work with because of their sensitivity to a variety of common reaction conditions. Percec and co-workers employed the methods outlined in Schemes – to prepare sugars for conjugation to JD scaffoldings. , These procedures were chosen upon reviewing the literature. − In general terms, glycosylation was conducted such that the substituent attached to the anomeric carbon was trans to the vicinal hydroxyl group of the sugar rather than cis , as this stereochemistry afforded the most stable reaction intermediates and was thus most accessible. This stereochemistry is also employed by biological membranes .…”

One-pot three-enzyme synthesis of UDP-GlcNAc derivatives

“…A recombinant NahK cloned from another strain of Bifidobacterium longum (NahK_JCM1217) 19 was used in the synthesis of GlcNAc-1-phosphate, GalNAc-1-phosphate, and their derivatives. 20,21 The purified HexNAc-1-phosphates were then used in a one-pot two-enzyme system containing a commercially COMMUNICATION available inorganic pyrophosphatase (PpA) and a GlmU cloned from E. coli (EcGlmU) [22][23][24][25][26] or an AGX1 cloned from human 24 for the synthesis of UDP-GlcNAc, dNDP-GlcNAc, dNDP-Glc, UDP-GalNAc, and derivatives. Nevertheless, chemoenzymatic synthesis of UDP-GlcNAc derivatives using all three enzymes in one-pot has not been reported.…”