Development of crosslinking reagents, and intra- and intermolecular multi-linking of hemoglobin molecules as potential blood substitutes

Blood substitutes have been sought for several centuries and in many ways. Hemoglobin-based blood substitutes are the most interesting among those because of hemoglobin's unique structure and function to transport oxygen. The tetrameric hemoglobin in solution can dissociate into dimers which are readily removed by the kidney. To overcome the limitations to hemoglobin it can be modified by biotechnology and/or chemistry. In chemistry, molecular modification requires crosslinking of hemoglobin to prevent formation of dimers. One of the most interesting small regions of the hemoglobin molecule is the {dollar}beta{dollar} cleft of the BPG binding site. A series of different kinds of crosslinking reagents were synthesized and reacted only with Val1, Lys82 and Lys144 of the {dollar}beta{dollar}-chain of hemoglobin. The negative charge from the phosphate or carboxyl groups of the reagents has an electrostatic interaction with positive charges from the residues in the BPG cleft.; For development of blood substitutes, a series of novel multi-(3,5-dibromosalicyl) linkers were designed and synthesized for modification of hemoglobin in this dissertation. In addition to electrostatic effects, selectivity and efficiency can be enhanced by taking advantage of steric effects which play an important role in determining the regioselectivity of organic reagents. Computer modeling techniques were utilized to model 3-dimensional structures of hemoglobin A molecules and deoxyhemoglobin crystal unit cells, and design crosslinkers. In a manner similar to that used in a successful synthesis and purification of tert-butyl salicylate, tert-butyl 3,5-dibromosalicylate was prepared using DCC to couple tert-butyl alcohol and 3, 5-dibromosalicylic acid. The crosslinking reagents successfully synthesized include: Two bis-, two tris-and two tetrakis(3,5-dibromosalicyl) reagents, and one tetrakis(sodium methyl phosphate) reagent.; Both oxy and deoxy hemoglobin were crosslinked with DBSTC. The denaturation transition ({dollar}Tsb{lcub}rm m{rcub}{dollar}) of the oxy crosslinked hemoglobin increased 14.5{dollar}spcirc{dollar}C and that of the deoxy crosslinked hemoglobin, 13.0{dollar}spcirc{dollar}C. The apparent rate constant (k{dollar}sb {lcub}rm app{rcub}{dollar}) of autoxidation for the oxy crosslinked hemoglobin remained the same as native hemoglobin but that of the deoxy crosslinked hemoglobin increased by 34%. The higher oxygen affinity and lower cooperativity of the crosslinked proteins compared with native hemoglobin indicated that the crosslink shifted the conformation to the R state.; The major species from reaction of deoxyhemoglobin with DBCPA contained high yields of both bi- and tri-linked proteins. The denaturation transition ({dollar}Tsb {lcub}rm m{rcub}{dollar}) of both bi- and tri-linked hemoglobins increased 14.0{dollar}spcirc{dollar}C. The apparent rate constant (k{dollar}sb {lcub}rm app{rcub}{dollar}) of autoxidation for the crosslinked hemoglobins remained the same as native hemoglobin. The bi-linked species had a lower oxygen affinity and unchanged cooperativity compared with native hemoglobin, while the tri-linked proteins exhibited increased oxygen affinity and decreased cooperativity. SDS-PAGE analysis showed multi-bands in dimer range, indicating heterogeneous reaction of DBCPA with hemoglobin because of the asymmetrical structure of the reagent.; The elongated bi-linkers were designed to crosslink between Lys82{dollar}betaspprime{dollar} of two different hemoglobin tetramers. However, no octamers were isolated by gel filtration. The tetra-linkers were designed to crosslink between two hemoglobin tetramers with bi-linking of each hemoglobin between Lys82{dollar}betaspprime{dollar}s in one step. Besides dimeric bands, trimeric and tetrameric bands were shown by SDS-PAGE, corresponding to tri-linking and tetra-linking of hemoglobin with the tetra-linkers.