A Site-Specific Tetrafunctional Reagent for Protein Modification:  Cross-Linked Hemoglobin with Two Sites for Further Reaction

Abstract

A tetrafunctional site-directed reagent for protein modification has the potential for introducing specific cross-links by reaction at two of its four reactive sites. The remaining reactive groups on the link within the protein are available for further reaction with added reagents. The first example of such a designed multifunctional cross-linker, 3,5,3‘,5‘-biphenyltetracarbonyl tetrakis(3,5-dibromosalicylate), BTDS (1), was prepared by treatment of the tetra tert-butyl ester with trifluoroacetic acid. The ester was from reaction of the acid chloride of biphenyl 3,5,3‘,5‘-tetracarboxylic acid with tert-butyl-3,5-dibromosalicylate. BTDS contains four anionic sites each adjacent to four electrophilic sites. The reaction of BTDS with deoxy human hemoglobin A generates in high yield the biphenyl bis carboxamide at the ε-amino of lysine-82 of each of the β subunits (BBS-Hb). The remaining ester groups from the biphenyl cross-link are available to react with other nucleophiles. This is demonstrated by efficient reaction with ethylenediamine. The use of multifunctional cross-linkers presents opportunities for introduction of probes and bioactive materials.

Connecting Proteins by Design. Cross-Linked Bis-Hemoglobin

“…On the basis of the known reaction patterns of efficient and selective cross-linking reagents, 17,18 we designed doubly connected bis (3,5-dibromosalicyl)isophthalates. These should react preferentially with the -amino group of each β-Lys-82 and/or the R-amino group of the N-terminal β-Val- tional reagent derived from biphenyl tetracarboxylic acid (1) meets these requirements but it reacts with hemoglobin at only two of its sites, 19 suggesting that a greater separation between reaction sites is needed to connect tetramers. The oligoether reagent (2) reacts primarily at all four of its reaction sites but mainly within a single tetramer.…”

Efficient generation of dendritic arrays of cross-linked hemoglobin: symmetry and redundancy

“…While tetrafunctional reagents can cross-link within two tetramers, 11-13 they are inefficient due to the competing hydrolysis of the reagent: if hydrolysis inactivates only one site of four, the reagent becomes incapable of producing species with two cross-linked hemoglobins. 15 With an additional set of reaction sites, as in 1, the chances of forming connected tetramers is increased by 50%, and hydrolysis of a site leaves the reagent still able to produce cross-linked bis-tetramers. Furthermore, where there are single modifications (no cross-link forms) due to one site being hydrolyzed, the resulting modified subunit will be connected to the bis-tetramer.…”

“…These built-in redundancies increase the probability of the proper reaction and also provide a statistical advantage in competing with hydrolysis. 14, 15 We now report the successful implementation of such a strategy based on the convenient synthesis of a multifunctional reagent N,N ,N -tris[bis(sodium methyl phosphate)isophthalyl]-1,3,5-benzenetricarboxamide (1). Our approach to the synthesis of 1 is based on using symmetrical multi-sited core materials.…”

Protein–protein coupling and its application to functional red cell substitutes

“…Although the ester is slowly hydrolyzed, introduction of functionalized amines yields covalent conjugates of cross-linked tetramers. [77][78][79] Extension of this method leads to formation of hemoglobin clusters via the introduction of a ''Starburst'' dendritic polyamine core. 80 Fig.…”