Phthalide Compounds Modulate Hemoglobin Oxygen Affinity

The patent introduces 2,3-BPG (2,3-bisphosphoglycerate) as an intermediary substance that binds to hemoglobin to modulate its oxygen affinity. This mediator enables the blood substitute to achieve both high oxygen transport capacity and efficient oxygen release by facilitating the transition between high-affinity (R-state) and low-affinity (T-state) conformational states of hemoglobin.

The patent employs chemical modification of hemoglobin by introducing allosteric effectors (2,3-BPG) that change the physical-chemical parameters of hemoglobin, specifically its oxygen affinity. By controlling the binding of 2,3-BPG to specific sites on hemoglobin subunits, the patent adjusts the P50 value (oxygen pressure at 50% saturation) to optimize both oxygen loading in lungs and unloading in tissues.

The patent applies local quality modification by introducing specific point mutations at targeted sites on the fetal hemoglobin β-subunits (positions 63, 64, 92, 93, 103, 104, 143, 146, 147, 148) while preserving the overall structural stability of the HbF tetramer. These localized changes create new binding sites or enhance existing sites for 2,3-BPG interaction without disrupting the global fold and stability of the hemoglobin molecule.

The patent creates a composite hemoglobin molecule that combines the stable tetrameric structure of fetal hemoglobin (α2γ2) with engineered binding sites that mimic adult hemoglobin's (α2β2) interaction characteristics with 2,3-BPG. This composite approach merges the structural advantages of HbF with the allosteric regulation capabilities of HbA.

The patent introduces dynamic allosteric regulation to the blood substitute by incorporating 2,3-BPG binding sites that allow hemoglobin to transition between different conformational states. In the lungs, high oxygen concentration promotes the R-state (high affinity) for efficient loading, while in peripheral tissues, lower oxygen concentration and the presence of 2,3-BPG promote the T-state (low affinity) for efficient unloading. This dynamic response to local conditions optimizes both loading and unloading efficiency.

The patent implements a feedback mechanism where 2,3-BPG binding to hemoglobin is influenced by local oxygen concentration, pH, and other physiological parameters. In oxygen-rich environments (lungs), hemoglobin releases 2,3-BPG and adopts high-affinity conformation for oxygen loading. In oxygen-poor environments (tissues), hemoglobin binds 2,3-BPG and adopts low-affinity conformation for oxygen unloading, creating a self-regulating oxygen delivery system.