Efficient chemical introduction of a disulfide cross-link and conjugation site into human hemoglobin at beta-lysine-82 utilizing a bifunctional aminoacyl phosphate.

The creation of a cross-link containing a disulfide into hemoglobin has been accomplished with a site-directed reagent, N,N'-bis(Cbz-cystinyl)bis(methyl phosphate) (1). This is prepared from the reaction of the bis acid chloride of N-protected cystine with dimethyl phosphate followed by O-demethylation with methyl iodide in acetone. Reaction with deoxyhemoglobin produces two main products: cross-linked hemoglobin as the bis(cystinyl amide) of the epsilon-amino group of the side chain of Lys-82 of the two beta subunits as well as material that has each of the same amino groups modified as the cysteinyl amide but not cross-linked. Addition of 2-mercaptoethanol cleaves the disulfide in the material that is not cross-linked while leaving the disulfide intact in the cross-linked species. Dithiothreitol reduces the disulfide in the cross-linked species as well as in the species that is not cross-linked. Spontaneous oxidation in air converts all of the reduced material to the cross-linked bis(cystinyl amide) of hemoglobin. The reagent permits controlled introduction of cystinyl groups at lysyl residues, leading to formation of sulfhydryl groups by reduction and the possibility of re-forming the cross-links or forming conjugates.