A Chemoenzymatic Approach to the Synthesis of Hemoglobin Oligomers

Polymerized hemoglobin (poly-Hb) molecules have been shown to have reduced toxicity compared to cell-free hemoglobins when transfused intravenously. Poly-Hbs are typically generated with non-specific crosslinking agents that yield a product that is polydisperse in molecular weight. We propose a chemoenzymatic approach to generate poly-Hb of defined molecular weight. The proposed method employs the site-specific ligation reaction of the sortase A enzyme from S. aureus. A Hb mutant previously developed in our lab has been 'sortagged' - modified by adding either the sortase recognition sequence (LPXTG) to the C-terminus of the α-subunit or a tetraglycine motif (GGGG) to the N-terminus. We show here that Hb subunits can be ligated directly by sortase A, using a mixture of substrate tagged (LPXTG) and nucleophile tagged (poly-G) Hbs. Additionally, using a novel approach for crosslinking Hb subunits using sortase A, we have generated a di-α construct that is functionalized with a strained cycloalkyne through an unnatural C-to-C fusion of sortagged α-subunits using a bifunctional sortase nucleophile peptide. We show that cycloalkyne-modified Hb molecules can be covalently linked to an azide functionalized fluorescein, using the well-established method of Huisgen cycloaddition. The work presented here establishes the feasibility of generating a monodisperse poly-Hb using azide decorated scaffolds.