Western CEDAR

A Chemoenzymatic Approach for Synthesizing Polymeric Hemoglobin

Miller Hall

Polymerized hemoglobin (Hb) molecules have been shown to decrease previously observed adverse events associated with the administration of cell-free hemoglobin. To create these polymers, a method will be developed which employs the site specific ligation reaction of the sortase A enzyme from S. aureus. An Hb mutant (“αcpβ“) previously developed in our lab has been further modified by adding either the sortase recognition sequence, LPXTG, to the C-terminus of the α-subunit (s-αcpβ), or a tetraglycine motif, GGGG, to the N-terminus (n-αcpβ). Three types of sortase mediated ligation (SML) will be employed in this study. First, we will attempt to ligate Hb subunits directly, using a mixture of s-αcpβ (substrate) and n-αcpβ (nucleophile). The second method will be two-part, first appending either an azide or strained cyclooctyne to the C- and N-termini of the subunits. Using the well-established method of Huisgen cycloaddition, the Hb molecules can then be covalently linked via these chemoenzymatically appended functional groups. Initially, we will be using doubly modified Hb, and we expect our products will be a mixture of polymeric states. If this is successful, we will then construct singly modified monomers to better control the polymeric states of our products.