Origins of specificity in protein modification, reaction patterns of tris-trimesates with human hemoglobin A, water, and n-propylamine

grantor: University of TorontoA series of anionic trifunctional reagents with different leaving group substituent patterns (carboxylphenolate derivatives) has been prepared to elucidate the mechanism of hemoglobin cross-linking. The reactivity of these reagents toward human hemoglobin A, water, and 'n'-propylamine has been evaluated. The substitution pattern of the reagent has critical effects upon reactivity with hemoglobin that are not reflected in reactions with the small nucleophiles. Specifically, the type and number of halogens and the number and position of the carboxylates on each leaving group affect the ability of the reagents to cross-link hemoglobin. Furthermore, the carboxylates have two functions on the reagents: (1) they direct the anionic reagents into the cationic site of hemoglobin; and (2) they acquire protons from protonated amino groups on the protein. The reagent that is most effective at cross-linking hemoglobin has two bromines, and one carboxylate 'ortho' to the esters on each leaving group. In this study, trimesoyl tris(3,5-dibromosalicylate) (TTDS, 1) is the most effective trifunctional cross-linking reagent. As a result of elucidating the mechanism of hemoglobin cross-linking, this information was used to design an asymmetric cross-linker (13). This reagent has four reactive ester sites: two should selectively react with hemoglobin, while the other two should selectively react with small nucleophiles. This asymmetric reagent will allow better efficiency in converting deoxyhemoglobin into a bioconjugative protein.Ph.D