Independent synthesis, solution behavior, and studies on the mechanism of formation of a primary amine-derived fluorophore representing cross-linkingof proteins by (E)-4-hydroxy-2-nonenal

Lipid peroxidation in aging and degenerative disease results in the product ion of 4-hydroxy-2-alkenals that modify proteins and give rise to both prot ein cross-linking and fluorophore generation. Recent model studies demonstr ated that the major ex/em 360/430 fluorophore formed from (E)-4-hydroxy-2-n onenal (HNE) or (E)-4-hydroxy-2-hexenal (HHE) and protein lysine-based amin e is a 2-alkyl-2-hydroxy-1,2-dihydropyrrol-3-one iminium 1:2 cross-link (1) , a structure that is further confirmed here using N-15-labeling, and which has pH stability characteristics the same as those of lipofuscin pigments isolated from human tissues. Fluorophore generation represents an overall f our-electron oxidation, requires dioxygen, and is enhanced by the presence of Cu(II). The HNE-propylamine-derived fluorophore 1a was independently syn thesized from either 3,4-dioxononanal (8) or (E)-4-oxo-2-nonenal (13), prov iding further evidence for its assigned structure and clues to how it forms from HNE. Mechanistic studies on HNE-derived fluorophore formation permit ruling out the initial reversible HNE-derived Schiff base Michael adduct (1 7) as an intermediate. In addition, the structurally related non-cross-link 2-pentyl-2-hydroxy-1,2-dihydropyrrol-3-one 9a that forms along with 1a fro m 8 does not form from HNE and does: not serve as a precursor to la in the HNE-amine reaction system. A mechanism involving two 2e oxidations followin g initial Schiff base formation is proposed that is consistent with interme diates independently accessed from 8 and 13.