Novel affinity cross-linking reagents for modification of human hemoglobin

The design and synthesis of bis[2-(3-carboxyphenoxy)carbonylethyl] phosphinic acid (m-BCCEP, HY-4) as a site-directed affinity reagent for cross-linking human hemoglobin (Hb) have been reported as part of our long-term goal to generate artificial blood for emergency transfusions. Molecular modeling techniques were used to design the reagent (HY-4 ), employing crystal coordinates of human hemoglobin A0 imported from the Protein Data Bank. The reagent was synthesized in four steps commencing from t-butyl 3-hydroxybenzoate. This reagent HY-4 was converted to its tri-sodium salt HY-4S to allow effective cross-linking in an aqueous medium. The reagent HY-4S was found to specifically cross-link stroma-free human hemoglobin in the beta--cleft under oxygenated reaction conditions at neutral pH. The SDS-PAGE analyses showed that the modified hemoglobin migrated at 32 kDa as anticipated. HPLC analyses of the modified hemoglobin suggested that the cross-link had formed between the beta1-beta2 subunits. Molecular dynamics simulation studies on the reagent-HbA0 complex suggested that the predominant amino acid residues involved in the cross-linking are N-terminus Val-1 or Lys-82 on one of the beta-subunits, and Lys-144 on the other. These predictions were confirmed by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of the peptide fragments obtained from tryptic digestion of the cross-linked product. The data also suggested the presence of a minor cross-link between Val-1 and Lys-82 on the opposing subunits. The oxygen equilibrium measurements of the m-BCCEP-Hb product at 37°C showed oxygen affinity (P50 = 25.8 Torr) comparable to that of the natural whole blood (P50 = 27.0 Torr) and significantly lower than that of stroma-free hemoglobin (P50 = 14.19 Torr) assayed under identical conditions. The measured Hill Coefficient value of 1.91 of the m-BCCEP-Hb product points to the reasonable retainment of oxygen-binding cooperativity after the cross-link formation.;Also reported herein is the synthesis of two potential intermolecular cross-linking reagents. The first one is a prototypical dendrimeric reagent (HY-28) that has four masked aldehydic groups as their respective acetals at the terminal positions. Upon unmasking, the reagent has the potential to effect covalent linkage of two, three or four Hb molecules using these aldehydic groups. Since the HY-4S-modified Hb is still relatively small as compared with an intact red blood cell, it suffers from facile filtration through the endothelium lining and subsequent reaction with vasorelaxing nitric oxide (NO), which results in elevating the blood pressure. Nitric oxide is essential for relaxing the muscular walls surrounding the blood vessels, and therefore, its depletion leads to elevated arterial pressure. The small size of modified Hb also suffers short circulation time in the blood stream as well as renal toxicity. The reagent HY-29, generated from HY-28 by unmasking, is anticipated to increase the size of modified Hb via oligomerization of the cross-linked Hb, thus alleviating the problem of filtration through endothelium, followed by reaction with nitric oxide. The increased size of modified Hb will also increase the retention time and reduce kidney dysfunction. The second potential intermolecular cross-linker is bis(maleidophenyl)-PEG2000 (HY-14). It is postulated to intermolecularly cross-link two intramolecular cross-linking Hb proteins together outside the central cavity to increase the molecular weight of the modified Hb. These modifications might solve the aforementioned problems associated with transfusions with cross-linked hemoglobins.