Efficient CuAAC click formation of functional hemoglobin bis-tetramers.

A cross-linked hemoglobin bis-tetramer with oxygenation properties appropriate for a red cell substitute is produced by CuAAC conjugation of a dialkyne and two equivalents of an azide of β,β-cross-linked hemoglobin. The latter is formed efficiently by the aminolysis of the 3,5-dibromosalicylate ester of the β-82-β'-82-bis trimesyl amide of human hemoglobin A with (4-aminomethyl)benzyl azide.