Age-related macular degeneration, a major cause of blindness for which no satisfactory treatments exist, leads to a gradual decrease in central high acuity vision. The accumulation of f luorescent materials, called lipofuscin, in retinal pigment epithelial cells of the aging retina is most pronounced in the macula. One of the f luorophores of retinal pigment epithelial lipofuscin has been characterized as A2E, a pyridinium bis-retinoid, which is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine. An investigation aimed at optimizing the in vitro synthesis of A2E has resulted in the one-step biomimetic preparation of this pigment in 49% yield, readily producing more than 50 mg in one step. These results have allowed for the optimization of HPLC conditions so that nanogram quantities of A2E can be detected from extracts of tissue samples. By using 5% of the extract from individual aged human eyes, this protocol has led to the quantification of A2E and the characterization of iso-A2E, a new A2E double bond isomer; all-trans-retinol and 13-cis-retinol also have been identified in these HPLC chromatograms. Exposure of either A2E or iso-A2E to light gives rise to 4:1 A2E:iso-A2E equilibrium mixtures, similar to the composition of these two pigments in eye extracts. A2E and iso-A2E may exhibit surfactant properties arising from their unique wedge-shaped structures.Aging changes in retinal pigment epithelial (RPE) cells are generally assumed to contribute to the pathogenesis of agerelated macular degeneration (AMD) (1-3), the leading cause of acquired visual loss in persons older than 65 years of age (4-7) for which no cure exists. The most characteristic feature of aging in the RPE is the progressive cellular accumulation of lipofuscin (2,3,8). The deposition of this autofluorescent material is known to occur as a consequence of the role of RPE cells in phagocytosing packets of membrane that are shed by photoreceptor cells as part of the process by which photoreceptor outer segments undergo complete turnover every 2 weeks (9-11). In 1993, Eldred and coworkers (12, 13) proposed a bis-Schiff base structure (N-retinyl-N-retinylideneethanolamine) as one of the fluorescent pigments isolated from the pooled lipofuscin of a large number of aged human eyes. We later revised the proposed structure as the pyridinium bis-retinoid A2E (Fig. 1) (14) and succeeded in confirming this structure by a total chemical synthesis, which involved the coupling of the pyridine framework with the retinoid side chains (15).The genesis of A2E (Fig. 2), although speculative, could involve initial Schiff base formation between all-trans-retinal and ethanolamine or phosphatidylethanolamine to give (I), a [1,6]-proton tautomerization to enamine (II) followed by Schiff base generation with a second molecule of retinal to (III). This intermediate would undergo a [3,3]-sigmatropic rearrangement to (IV) and subsequent autooxidation to yield fluorescent pigment A2E or its phosphatidylethanolaminelinked adduct (which ...

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