Demonstration of thio-sugar addition and production of hemi-A S-link heparosan

An acceptor, an aldehyde-containing glycoside of heparosan trisaccharide (GlcA-GlcNAc-GlcA-C₂H₄-amido-benzaldehyde³²; 0.2 mg/ml, 0.27 mM), was incubated with: (i) UDP-hexosamine sugar (either natural UDP-GlcNAc or UDP-4-SH-GlcNAc analog) alone (for single sugar addition), (ii) UDP-hexosamine sugar and UDP-GlcA (for polymerization), or (iii) UDP-GlcA alone (negative control), at 5 molar excess over the acceptor concentration, with a recombinant heparosan synthase chimeric construct, PmHS-G²¹ (2 mg/ml, 18 µM), in 1 mM MnCl₂, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at pH 7.2, for 20 h at 30 °C. For the preparation of UDP-4-SH-GlcNAc monomer, the UDP-thio-sugar donor dimer 11 was first reduced by treatment with 5 mM dithiothreitol in water at 22 °C for 20 min. Reaction products were diluted 1:10 in 50% acetonitrile, 0.1% trifluoroacetic acid, and a portion was mixed in a 1:1 ratio with the matrix solution of 6-aza-2-thiothymine (5 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid) and dried with a stream of air. The spot was assessed by matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectrometry (reflector negative mode using an Ultraflex II instrument, Bruker Daltonics, Billerica, MA)²¹ (Supplementary Fig. ³⁸). A ladder of defined HA oligosaccharides was employed as the mass calibrants (Hyalose, LLC, Oklahoma City, OK).