Anionic Acyl Methyl Phosphates for the Synthesis of Hemoglobin-Based Oxygen Therapeutics

Hemoglobin, contained within red blood cells (RBCs), is the oxygen-carrying unit within humans. Free of the immunogenic problems associated with red blood cells, acellular hemoglobin could be the basis for a universally accepted substitute for the oxygen-carrying function of RBCs. This inspired research on a class of molecules known as hemoglobin-based oxygen carriers (HBOCs) – an area of interest in our research group. Hemoglobin, however, does not function as intended when present outside of RBCs. Decades of research in the field has now established that a viable acellular HBOC must 1) include an intramolecular cross-link to retain function and prevent renal toxicity, 2) be enlarged to prevent adverse cardiovascular effects, notably hypertension, and 3) exhibit cooperativity and physiologically-relevant oxygen affinity. Based on these requirements, our group has developed hemoglobin bis-tetramers – a candidate oxygen carrier utilizing a combination of native protein modifications and bio-orthogonal “click” chemistry. Our method boasts highly selective protein modifications, and thus high homogeneity, in the resulting product. This also makes our method highly reproducible. Other HBOCs in development are often heterogeneous mixtures of compounds, resulting in potentially inconsistent oxygenation properties. The high homogeneity of our method, however, comes at the cost of low synthetic yields. In the present work I utilize anionic acyl phosphate-based reagents to optimize the synthesis of bis-tetramers. My efforts ultimately lead to a three-fold increase in overall yields and significant additional improvements towards scalability. I begin by optimizing the current leading bis-tetramer protocol using an acyl phosphate-based acetylating agent, methyl acetyl phosphate (MAP). Noticing critical shortcomings within this method, I then developed a novel procedure using an acyl phosphate-based azide cross-linker. The cross-linker, PacXL, is the first azide-containing cross-linker to react specifically at the target cross-linking site on hemoglobin. The new protocol also utilizes the R-state conformation of hemoglobin, which is significantly more advantageous towards scale-up compared with the previously used T-state conformation. Outside the context of bis-tetramers, my results also establish acyl phosphates as highly selective reagents for cationic sites and potentiates their broad application towards targeting proteins exhibiting strong cationic character.

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