Synthesis and structural characterization of carboxyethylpyrrole-modified proteins: mediators of age-related macular degeneration

Protein modifications in which the ε-amino group of lysyl residues is incorporated into a 2-(ω-carboxyethyl)pyrrole (CEP) are mediators of age-related macular degeneration (AMD). They promote both angiogenesis into the retina (‘wet AMD’) and geographic retinal atrophy (‘dry AMD’). Blood levels of CEPs are biomarkers for clinical prognosis of the disease. To enable mechanistic studies of their role in promoting AMD, for example, through the activation of B- and T-cells, interaction with receptors, or binding with complement proteins, we developed an efficient synthesis of CEP derivatives, that is especially effective for proteins. The structures of tryptic peptides derived from CEP-modified proteins were also determined. A key finding is that 4,7-dioxoheptanoic acid 9-fluorenylmethyl ester reacts with primary amines to provide 9-fluorenylmethyl esters of CEP-modified proteins that can be deprotected in situ with 1,8-diazabicyclo[5.4.0]undec-7-ene without causing protein denaturation. The introduction of multiple CEP-modifications with a wide variety of CEP:protein ratios is readily achieved using this strategy.

Proteins in which the ε-amino groups of lysyl residues are incorporated into 2-(ω-carboxyethyl)-pyrroles are mediators of age-related macular degeneration. We report an efficient synthesis that accommodates a wide variety of CEP:protein ratios. Reaction of proteins with 4,7-dioxoheptanoic acid 9-fluorenylmethyl ester, and in situ deprotection with DBU provides CEP–proteins without causing denaturation. The structures of tryptic peptides derived from CEP–proteins were also determined.Figure optionsDownload as PowerPoint slide