Optimized triazine-mediated amidation for efficient and controlled functionalization of hyaluronic acid

• Kinetics of the triazine-mediated amidation of hyaluronic acid were investigated.
• Degree of substitution decreases with increasing basicity of the amine precursor.
• The amine stoichiometry is the limiting factor for the degree of substitution.
• Several “clickable” hyaluronic acid derivatives were synthesized.
• Bioconjugates were prepared by azide-alkyne and maleimide-thiol click reactions.

Triazine-based coupling agents have the potential to replace carbodiimides in the functionalization of hyaluronic acid (HA) giving derivatives with high degrees of substitution (DS) under mild conditions with excellent efficiency. Kinetics of the triazine-mediated amidation of HA in aqueous solution were investigated to understand the reaction mechanism and the role of the amine reagent. The DS decreased with increasing basicity of the amine. The water soluble coupling agent was stable under the reaction conditions (t1/2 = 10 days) in the absence of amines. The activation of HA proceeded quantitatively. The stoichiometry of amine was the limiting factor in the substitution. Functional HA derivatives with DS up to 55% were obtained by the triazine-mediated amidation. They were used successfully to prepare well-defined HA conjugates via the maleimide-thiol and the azide-alkyne “click” reactions.