Efficient conversion of hemoglobin to a non-vasoactive oxygen carrier by site-specific cross-linking with azido acyl methyl phosphates followed by bio-orthogonal CuAAC with a Bis-Alkyne

While cross-linked hemoglobin tetramers are functional acellular oxygen carriers, their ability to scavenge endogenous nitric oxide (NO) by endothelial pore penetration results in adverse cardiovascular effects. Animal studies established that cross-linked human hemoglobins, chemically joined into a double protein, avoid NO scavenging, presumably due to their larger size preventing penetration into endothelial regions that produce NO. In the present report, we utilize azide-containing acyl phosphate reagents to form cross-linked hemoglobins then bio-orthogonally click-couple them with a bis-alkyne (CuAAC). The production of these larger oxygen-carrying hemoglobin conjugates is obtained in high yields through subunit-specific cross-linking between each βLys82 ε-amino group. The methyl phosphate leaving groups provide electrostatically induced β-subunit site-selectivity, producing azido-cross-linked hemoglobin that undergoes highly efficient CuAAC compared with previous cross-linkers. The acyl phosphates also efficiently cross-link both T-state and R-state hemoglobin. The resulting bis- and tris-tetrameric hemoglobin conjugates exhibit oxygen affinity and cooperativity that are comparable to those of the native protein. The hemoglobin derivatives from the process we describe can function as sources of oxygen in biomedical applications, such as in ex-vivo donor organ perfusion.