Retinal age pigments, or lipofuscin granula, contain a number of fluorophores that accumulate as a consequence of light-related vitamin A recycling in association with aging and with some retinal disorders in retinal pigment epithelial (RPE) cells. 1 Lipofuscin is a mixture of incompletely degraded materials trapped in lysosomes of RPE cells and the fluorophores of lipofuscin granular are thought to represent a biomarker for cellular aging, since the byproducts in RPE cells are considered to be

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... a key contributor in the etiology of age-related macular degradation (AMD), a common cause of blindness in elderly people. 2,3 Although AMD is reported to be a leading cause of legal blindness in developed countries and several treatment options have been recently developed, 4-6 treatment of AMD still remains difficult because the molecular composition and pathogenic mechanisms of RPE lipofuscin have been poorly studied. This is mainly attributed to the heterogeneous nature of the most isolated lipofuscin preparations. Numerous fluorophores have been identified so far in RPE lipofuscin such as the pyridinium bisretinoid isomers A2E (N-retinyledin-N-retinylethanolamin), isoA2E, oxidized derivatives of A2E, and several conjugates ( Fig. 1) . 7 Among these fluorophores, A2E is known to play an important role in causing membrane permeabilization, 8 inhibition of cytochrome c oxygenase, 9 inhibition of the ATP-driven proton pump, 10 and partial mediation of light damage by acting as a photosensitizer. 11 A2E is also reported to perturb cholesterol metabolism in RPE cells without light exposure. 12 Of particular importance is the finding that the photo-damage to cells induced by A2E involves the formation of A2E photooxidation products. 1 The oxygen-containing moieties formed by the photoexcitation of A2E include epoxides, furanoid oxide structures and cyclic peroxides that are expected to be reactive. 13-15 Such bioactivities support a possible role for lipofuscin in AMD progression, followed by choroidal neovascularization (CNV) development, but the molecular mechanisms remain to be determined. Toward a better understanding of the pathogenesis caused by these potentially toxic lipofuscin fluorophores, we prepared a thiolated A2E derivative (A2S) in order to employ it for affinity chromatography as a first step to identify the target protein(s) of A2E in retina. As described in Experimental Section and shown in Scheme 1, the reaction time for A2S synthesis was increased to 72 h, compared to the reaction conditions for A2E synthesis. 11 A2S purification was performed by reverse-phase HPLC. The retention time of A2E as a control was found to be approximately 10 min under the HPLC conditions employed, and it was anticipated that the retention time of A2S would be delayed by the slightly higher hydrophobicity of the thiol functional group caused by the lower electronegativity of the sulfur atom. The fractionated solution at 15 min was analyzed via UV spectroscopy and MS for assigning the obtained fraction to A2S (Fig. 2) . Existence of the terminal sulfhydryl group in the fractionated solution was confirmed by using thiol and sulfide quantitation kit (Molecular Probes, OR) according to the manufacturer's manual (data not shown). UV data show λ max at two regions indicating that A2S is present in the fractionated solution. According to the previous reports, however, the absorbance intensity at 438 nm of A2E is approximately 2 times larger than that at 337 nm. Therefore, the inconsistency of the relative intensities in the 337 nm and 438 nm regions in Figure 2 (a) might result from a mixture of unknown compounds presumably including all trans-retinal, the thiolated derivatives of dihydro-A2E, or isoA2E. The presence of A2S in the fraction was also determined using mass spectrometry, as shown in Figure 2(b) . Since possible fragments during mass spectroscopy could be expected and molecular structure of A2S was confirmed considering the cleavable sites in the molecule, our obtained mass signals were accordingly assigned [m/z 608.43 (calculated), 608.46 (observed), for example]. Importantly, UV analysis implies that there are no trisretinoids or monoretinoids in the 15-min fraction and MS analysis confirmed the absence of the thiolated derivative of dihydro-A2E (i.e. no 606 m/z signal), which suggests that the 15-min fraction may be a mixture of A2S, retinal (showing a single λ max at the 337 nm region), and/or the thiolated derivative of isoA2E (or isoA2S that is interchangeable with