Azidopropyl Le x (Le x -N3, 3) was synthesized in two stages, starting with chemical attachment of an azide group into N-acetylglucosamine (GlcNAc), followed by enzymatic synthesis of Le x (Figure ). Per-acetylated GlcNAc was converted to an oxazoline using TMSOTf, and used to glycosylate azidopropanol in the presence of camphorsulfonic acid to give the -glycoside product. Deprotection of the hydroxyl groups using sodium methoxide in methanol provided azidopropyl GlcNAc 1. Enzymatic synthesis of Le x -N3 was performed in a one pot, two-step process. First, 3-azidopropyl N-acetyllactosamine 2 was made using two enzymes: UDP-Glc-4-epimerase (Glc(4)ep) converted uridine diphosphate glucose (UDP-Glc) into uridine diphosphate galactose (UDP-Gal) in situ, for Homo sapiens β-1,4-galactosyltransferase (β(1-4)GalT1) to glycosylate azidopropyl GlcNAc acceptor 1. The crude reaction mixture was then used directly for the synthesis of 3. Guanosine 5′-diphospho-β-L-fucose (GDP-Fuc) was synthesized using L-fucose (Fuc), using adenosine triphosphate (ATP) and guanosine-5'-triphosphate (GTP) as substrates for Bacteroides fragilis GDP-fucose pyrophosphorylase (FKP). Finally, the reaction of GDP-Fuc with 2 was catalyzed by Helicobacter pylori α-1,3-fucosyltransferase (α(1-3)FucT HP) to achieve Le x -N3. Synthesis of azido Gb3 5 was performed as reported previously, with in situ generation of UDP-Gal, and Neisseria weaveri (1,4)galactosyltransferase (Nw GalT) to glycosylate azido lactose 4 (Figure ). Alkyne-substituted HA has been prepared previously in the presence of EDC and NHS as activators in slightly acidic media (MES buffer at pH 6). However, our initial attempts to follow this method led to products that displayed a variety of additional signals in their NMR spectra that indicated other moieties derived from the coupling agents had become attached to the HA backbone, and could not be removed after multiple rounds of purification (Figure ). 55-58 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) has been described as an alternative to EDC/NHS for the formation of amides in monosaccharides and polysaccharides. The conditions described by Yu et al. were adapted to attach propargylamine, and we obtained clean conversion to HA-g-propargyl 6 (Figure and Figure ). The number of equivalents of propargylamine and DMTMM were varied to provide HA-gpropargyl with different degrees of substitution (DS) (Table ).