Structural features required for the reactivity and intracellular transport of bis(3,5-dibromosalicyl)fumarate and related anti-sickling compounds that modify hemoglobin S at the 2,3-diphosphoglycerate binding site

Bis(3,5-dibromosalicyl)fumarate (I) reacts preferentially with oxyhemoglobin to cross-link the two beta 82 lysine residues within the 2,3-diphosphoglycerate (DPG) binding site and as a result markedly increases the solubility of deoxyhemoglobin S. The cross-link acts by perturbing the acceptor site for Val 6 within the sickle cell fiber (Chatterjee, R., Walder, R. Y., Arnone, A., and Walder, J. A. (1982) Biochemistry 21, 5901-5909). In the present studies we have compared a large number of analogs of I to determine the structural features of the reagent required for specificity and for transport into the red cell. Both electrostatic and hydrophobic interactions contribute to the binding of these compounds at the DPG site. The optimal position for the negatively charged groups on the cross-linking agent for productive binding is adjacent to the ester as in the original salicylic acid derivatives. There is a direct correlation between the reactivity toward hemoglobin and the hydrophobicity of the substituent attached at the para position. Phenyl and substituted phenyl derivatives as in the analgesic, antiinflammatory drug diflunisal are particularly effective. These groups probably interact with hydrophobic residues of the amino-terminal tripeptide and the EF corner of the beta chains adjacent to the DPG binding site. Although bis(3,5-dibromosalicyl)fumarate is very reactive toward hemoglobin in solution, it is much less effective in modifying hemoglobin within the red cell. The reaction with intracellular hemoglobin was shown to be limited by competing hydrolysis of the reagent catalyzed at the outer surface of the erythrocyte membrane. Inactivation of the red cell membrane acetylcholinesterase with phenylmethylsulfonyl fluoride did not inhibit this reaction. Introduction of a single methyl group onto the carbon-carbon double bond of the fumaryl moiety decreases the lability of the ester 10-fold, due to steric effects, and allows the reagent to be taken up by the red cell and modify intracellular hemoglobin. The kinetics of transport of the methylfumarate derivative, bis(3,5-dibromosalicyl)mesaconate, are first-order, consistent with passive diffusion. The attachment of larger alkyl groups onto the cross-link bridge further enhances the transport of the reagent into the red cell. The solubility of deoxyhemoglobin S cross-linked with the butylfumarate derivative was found to be increased by almost 10% compared to the original fumarate diester.(ABSTRACT TRUNCATED AT 400 WORDS)