Oligomerization of  -Cleft Cross-Linked Human Hemoglobin: Synthesis, Intramolecular Cross-linking, and Intermolecular Coupling Studies with Polyfunctional Organic Reagents Hongyi Cai, Timothy A. Roach and Ramachandra S. Hosmane. Laboratory for Drug Design and Synthesis, Department of Chemistry & Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD Abstract: Current efforts to develop blood substitutes based on cell-free hemoglobin are directed toward (a) tuning its oxygen affinity to afford adequate oxygen delivery from lung to tissues via covalent cross-linking with an appropriate reagent that mimics the hemoglobin's natural allosteric modifier 2,3- diphosphoglycerate (DPG), and (b) increasing the steric bulk of the cross- linked hemoglobin to allow its retention in circulation for prolonged periods of time as well as to prevent its facile seeping through the endothelium and the subsequent interaction with nitric oxide, which results in vasoconstriction, and hence, the elevated blood pressure. As part of a program to address the current problems facing the blood substitute research, we report here the results of our studies on sequential intramolecular cross-linking and intermolecular coupling of human hemoglobin, employing a variety of polyfunctional organic reagents. Introduction: Attempts to develop a red blood cell (RBC) substitute in transfusion date back well over half a century. Hemoglobin (Hb), the natural oxygen carrier inside the RBC has been the preferred choice for such a substitute. Unfortunately, when Hb is outside of the RBC, its affinity to oxygen increases to an extent that may impair oxygen delivery from lungs to tissues. Furthermore, it suffers from short circulation times in the blood stream due to its breakdown from a large tetrameric protein into two smaller dimeric units, α1β1 and α2β2. These drawbacks are due to the loss of the natural Hb allosteric effector, 2,3-Bisphosphoglycerate (BPG). BPG aligns itself between the two β subunits of the Hb tetramer, and is surrounded by several positively charged amino acid residues residing on the β subunits, thus forming an anionic sink that is referred to the β-cleft or the BPG pocket. Therefore, the covalent cross-linking of Hb subunits with a BPG mimic, preferably in the β-cleft site, is anticipated to alleviate these drawbacks of cell-free Hb. We designed and synthesized several organic reagents (BCCEP, BPPCEP & Bis-Mal-PEG2000) for hemoglobin cross-linking studies. These reagents have been shown to modify Hb between different amino-acid residues in β chains. We propose to obtain intramolecular and intermolecular cross-linkedβchains by sequential reactions with these reagents. Experimental Procedures Two cross-linking reagents previously designed in our lab - BCCEPE 1 and BPPCEP 2, reacting with Lys82 and Lys144 of β chains were synthesized in schemes I and II, respectively. Bis-Mal-PEG2000 was reported to cross-link at Cys-93 of β subunits 3, and was synthesized using scheme III 4. Then Hemoglobin was reacted with BCCEPE or BPPCEP and Bis-Mal-PEG2000 in a series of reaction conditions. In a summary, we have demonstrated the intramolecular cross-linking (32KDa) of Hb with these reagents. Although the expected 64 kDa Hb dimer was not seen, partial intermolecular cross-linking (48KDa) with the above reagents was observed. It is possible that Hb changes its conformation after reacting with an reagent, causing the activity site to another cross-linking reagent to be hindered. We are currently altering the reaction conditions and purifying the intermediate products to optimize reaction efficiency. Results and Discussion The structures of three reagents were as determined by 1 H NMR (Figure 2), Mass Spectroscopy and IR. The extent of the cross- linking globin chains was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 3) and C4 reverse phase High performance liquid chromatography (HPLC) (Figure 4). Acknowledgements: I would like to express my great appreciation to my advisor, Dr. Ramachandra Hosmane, who has supported me throughout this work. In addition, I want to thank Dr. Tim Roach for his instruction and encouragement on this project. Finally, I would like to thank my colleagues and friends for all of their helpful suggestions. References: 1.R. S. Hosmane, S. P. Peri, V. S. Bhadti and V. W. Macdonald. Bis[2-(4- carboxyphenoxy) carbonylethyl] phosphinic Acid (BCCEP): A Nevel Affinity Reagent for the β-Cleft Modification of Human Hemoglobin. Bioorg. Med. Chem., 1998, 6, T. A. Roach, V. W. Macdonald, and R. S. Hosmane. A Novel Site-Directed Affinity Reagent for Cross-Linking Human Hemoglobin: Bis[2-(4-phosphonooxy phenoxy)carbonylethyl]phosphinic Acid (BPPCEP). J. Med. Chem., 2004, 47, B. N. Manjula, A. Malavalli, P. K. Smith, N. Chan, A. Arnone, J. M. Friedman and A. S. Acharya. Cys-93- -Sucinimidophenyl Polyethylene Glycol 2000 Hemoglobin A Intramolecular Cross-bridging of Hemoglobin Outside The Central Cavity. J. Biol. Chem, 2000, 275, M. E. Annunziato, U. S. Patel, M. Ranade, and P. S. Palumbo. p-Maleimidophenyl Isocyanate: A Novel Heterobifunctional Linker for Hydroxyl to Thiol Coupling. Bioconjugate Chem. 1993, 4, (A)(B) (C) Figture 2. NMR analysis of BCCEP(A), BPPCEP(B) and Bis-Mal-PEG2000(C). (B) Figture 4. (A) C4 reverse phase chromatogram of stroma-free hemoglobin Monitored at 214nm. (B) C4 reverse phase chromatogram of stroma-free hemoglobin, modified by Bis-Mal-PEG2000, Monitored at 214nm. (A) (B) Figture 3. SDS-PAGE analysis of the cross-linked Hb. (A) Hb reacted with BCCEP (HY-20) and Bis-Mal-PEG2000 (HY-14), (B) Hb reacted with BPPCEP (HY-8) and Bis-Mal-PEG2000 (HY-14). Lane 1, MW standards; XL,cross-linking (A) Figure 2. Experimental Procedures 48KDa 32KDa 48KDa 32KDa 16KDa