Figure : A) Chemoezymatic synthesis of azidopropyl Le x -N3: (i) trimethylsilyl trifluoromethanesulfonate (TMSOTf), CH2Cl2, r.t, 2.5 h, 90%; (ii) 3-azidopropan-1-ol, camphorsulfonic acid, DCE, 80 °C, overnight, 30%; (iii) sodium methoxide, MeOH, r.t., 3 h, 68%. B) Enzymatic synthesis of Gb3-N3. HA-g-propargyl 6 with the highest and lowest DS were conjugated to Lac-N3 4 using CuAAC in the presence of CuSO4, sodium ascorbate, and tris((1-hydroxy-propyl-1H-1,2,3-triazol-4-yl)methyl)amine (THPTA), to give glycopolymers HA-g-Lac L and HA-g-Lac , where the superscripts L and H denote their comparatively low and high DS, respectively (Figure ). H NMR spectroscopy showed no unreacted alkyne remaining, and comparison of the integrations of the HA acetamide signal, the triazole proton in the aromatic region of the spectrum and the anomeric proton from glucose at 5.11 ppm indicated the DS was 12% for HA-g-Lac L and 35% for HA-g-Lac H . HA-gpropargyl 6 with the highest DS was also coupled to Le x -N3 3 and Gb3-N3 5 under the same conditions to give glycopolymers with comparable DS (30% for HA-g-Gb3 and 32% for HA-g-Le x ). In these cases, a small alkyne signal was still visible in the 1 H NMR spectra of the final glycopolymers, but as each glycopolymer had similar densities of glycans to HA-g-Lac , we concluded that the incomplete cycloaddition reactions would have no impact on our subsequent experiments. We note that the DS values estimated for HAg-propargyl samples were consistently higher than those for the corresponding HA-g-glycans. However, as the products of CuAAC reactions presented more distinct 1 H NMR signals for comparison, their integration was more reliable than for HA-g-alkyne, thus giving better estimation of DS.