Bis[2-(3-carboxyphenoxy)carbonylethyl]phosphinic acid (m-BCCEP): A novel affinity cross-linking reagent for the β-cleft modification of human hemoglobin

The design and synthesis of bis[2-(3-carboxyphenoxy)carbonylethyl] phosphinic acid (m-BCCEP, 1) as a site-directed affinity reagent for cross-linking human hemoglobin 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, employing crystal coordinates of human hemoglobin A₀ imported from the Protein Data Bank. It was synthesized in four steps commencing from 3-hydroxybenzoic acid. The reagent 1 was converted to its trisodium salt to allow effective cross-linking in an aqueous medium. The reagent 1, as its trisodium salt, was found to specifically cross-link stroma-free human hemoglobin A₀ in the β-cleft under oxygenated reaction conditions at neutral pH. The SDS-PAGE analyses of the modified hemoglobin pointed to the molecular mass range of 32 kDa as anticipated. The HPLC analyses of the product suggested that the cross-link had formed between the β₁-β₂ 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 β-subunits and Lys-144 on the other. These predictions were borne out by MALDI-TOF MS analyses data 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-modified hemoglobin product at 37 °C showed oxygen affinity (P ₅₀ = 25.8 Torr) comparable to that of the natural whole blood (P ₅₀ = 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-modified Hb product points to the reasonable retainment of oxygen-binding cooperativity after the cross-link formation.