Strain-promoted azide–alkyne cycloaddition for protein–protein coupling in the formation of a bis-hemoglobin as a copper-free oxygen carrier

Abstract

Conventional chemical approaches to protein–protein coupling present challenges due to the intrinsic competition between the desired interactions of reagents with groups of the protein as well as reactions with water. Biorthogonal Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC)-processes provide a basis to direct reactivity without functional group interference. However, the requirement for Cu(I) in CuAAC leads to complications that result from the metal ion's interactions with the protein. In principle, a similar but metal-free alternative approach to coupling could employ the reaction of an alkyne that is strained in combination with an azide (strain-promoted azide–alkyne cycloaddition, SPAAC). The method is exemplified by the combination of a cyclooctyne derivative of hemoglobin with an azide-modified hemoglobin. The bis-hemoglobin tetramer that is produced has properties consistent with those sought for use as a hemoglobin-based oxygen carrier (HBOC).