A SITE-SPECIFIC TETRAFUNCTIONAL REAGENT FOR PROTEIN MODIFICATION - CROSS-LINKED HEMOGLOBIN WITH 2 SITES FOR FURTHER REACTION

A tetrafunctional site-directed reagent for protein modification has t he potential for introducing specific cross-links by reaction at two o f its four reactive sites. The remaining reactive groups on the link w ithin the protein an available for further reaction with added reagent s. The first example of such a designed multifunctional crosslinker, 3 ,5,3',5'-biphenyltetracarbonyl tetrakis(3,5-dibromosalicylate), BTDS ( 1), was prepared by treatment of the tetra tert-butyl ester with trifl uoroacetic acid. The ester was from reaction of the acid chloride of b iphenyl 3,5,3',5'-tetracarboxylic acid with tert-butyl-3,5-dibromosali cylate. BTDS contains four anionic sites each adjacent to four electro philic sites, The reaction of BTDS with deoxy human hemoglobin A gener ates in high yield the biphenyl bis carboxamide at the epsilon-amino o f lysine-82 of each of the beta subunits (BBS-Hb). The remaining ester groups from the biphenyl cross-link are available iv react with other nucleophiles, This is demonstrated by efficient reaction with ethylen ediamine. The use of multifunctional cross-linkers presents opportunit ies for introduction of probes and bioactive materials.