In contrast to most reported HA-NB syntheses, our EDC/NHS synthesis route is considerably less labour intensive and does not require the use of organic solvents like dimethyl sulfoxide or dimethylformamide. In one popular method, HA is first converted into a tetrabutylammonium salt and then reacted with either 5-norbornene-2-methylamine or 5norbornene-2-carboxylic acid in dimethyl sulfoxide, requiring up to 14 days of dialysis for purification. In a different approach, the NB is introduced in two steps by first attaching adipic acid dihydrazide to the carboxylic acid of HA, followed by purification and reaction with cis-5-norbonenen-endo-2,3-dicarboxylic acid. Our synthesis method only requires one synthesis step and uses water as solvent. A recently published protocol by Plaster et al. uses a similar approach to synthesize HA-NB using an aqueous buffer system and the water-soluble triazine derivative 4-(4,6-dimethoxy-1,3,5-triazin-2-yl) 4-methylmorpholinium chloride as coupling agent. HA-MA was synthesized as described before. In short, HA was dissolved at 2% w/v in PBS and reacted with methacrylic anhydride for 24 h at 5 °C (Scheme 1). For our studies we used HA-MA with Fene = 0.39-0.42.