Poly-l -glutamic acid (molecular weight = 50,000–100,000 Da) and all other chemicals, biochemicals, and solvents were purchased from Sigma-Aldrich Chemical Co (St. Louis, Missouri, USA). Influenza viruses [A/Wuhan/359/95 (H3N2) and A/turkey/MN/833/80 (H4N2), as well as their drug-resistant mutants] were obtained from the Centers for Disease Control and Prevention (Atlanta, Georgia, USA). Madin-Darby canine kidney (MDCK) cells were purchased from the ATCC. Zanamivir (1 , Fig. 1 ) was obtained from BioDuro (Beijing, China).
Zanamivir derivative2 (Fig. 1 ) was synthesized as described previously.12 (link) To prepare polymer conjugates 3–5 (Fig. 1 ), 2 was reacted with the benzotriazole ester of poly-l -glutamic acid, followed by quenching with NH4OH (Fig. 2 )11 (link). Bare poly-l -glutamine was synthesized analogously.11 (link) Zanamivir content in 3–5 was quantified by 1H NMR.
Plaque assays were performed in 12-well plates using a modified literature procedure.12 (link),13 (link) First, 55-μL aliquots of virus preparation (~800 pfu/mL in PBS) were incubated with equal volumes of inhibitor solutions (serially 10-fold diluted in PBS) for 1 h at room temperature. After washing, MDCK cells grown to confluency were infected at room temperature for 1 h with 100 μL of the virus–inhibitor mixture. The inoculum was removed by aspiration, and the cells were overlaid with 1 mL of plaque medium12 (link) [2×F12 medium, 0.01% DEAE-dextran, 0.1% NaHCO3, 100 units/mL of penicillin G, 100 μg/mL of streptomycin, 4 μg/mL of trypsin, and 0.6% purified agar (L28; Oxoid Co., Basingstoke, UK)]. Plaques were counted after 3 to 4 days of incubation at 37°C.
Neuraminidase inhibition assays were performed using a modified literature procedure.14 (link) Briefly, 20 μL of whole virus and 15 μL of inhibitor dilutions were incubated at room temperature for 1 h. Following addition of the fluorogenic NA substrate 4-methylumbelliferyl-α-d -N-acetylneuraminic acid (whose final concentration was 5- to 10-fold lower than the Km of the enzyme), generation of 4-methylumbelliferone was monitored for 1 h. Values of Ki were determined with nonlinear regression in KaleidaGraph.15 (link)
Zanamivir derivative
Plaque assays were performed in 12-well plates using a modified literature procedure.12 (link),13 (link) First, 55-μL aliquots of virus preparation (~800 pfu/mL in PBS) were incubated with equal volumes of inhibitor solutions (serially 10-fold diluted in PBS) for 1 h at room temperature. After washing, MDCK cells grown to confluency were infected at room temperature for 1 h with 100 μL of the virus–inhibitor mixture. The inoculum was removed by aspiration, and the cells were overlaid with 1 mL of plaque medium12 (link) [2×F12 medium, 0.01% DEAE-dextran, 0.1% NaHCO3, 100 units/mL of penicillin G, 100 μg/mL of streptomycin, 4 μg/mL of trypsin, and 0.6% purified agar (L28; Oxoid Co., Basingstoke, UK)]. Plaques were counted after 3 to 4 days of incubation at 37°C.
Neuraminidase inhibition assays were performed using a modified literature procedure.14 (link) Briefly, 20 μL of whole virus and 15 μL of inhibitor dilutions were incubated at room temperature for 1 h. Following addition of the fluorogenic NA substrate 4-methylumbelliferyl-α-
