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Characterization of Peroxy-A2E and Furan-A2E Photooxidation Products and Detection in Human and Mouse Retinal Pigment Epithelial Cell Lipofuscin

2005; Elsevier BV; Volume: 280; Issue: 48 Linguagem: Inglês

10.1074/jbc.m504933200

ISSN

1083-351X

Autores

Young Pyo Jang, Hiroko Matsuda, Yasuhiro Itagaki, Koji Nakanishi, Janet R. Sparrow,

Tópico(s)

Glaucoma and retinal disorders

Resumo

The nondegradable pigments that accumulate in retinal pigment epithelial (RPE) cells as lipofuscin constituents are considered to be responsible for the loss of RPE cells in recessive Stargardt disease, a blindness macular disorder of juvenile onset. This autofluorescent material may also contribute to the etiology of age-related macular degeneration. The best characterized of these fluorophores is A2E, a compound consisting of two retinoid-derived side arms extending from a pyridinium ring. Evidence indicates that photochemical mechanisms initiated by excitation from the blue region of the spectrum may contribute to the adverse effects of A2E accumulation, with the A2E photooxidation products being damaging intermediates. By studying the oxidation products (oxo-A2E) generated using oxidizing agents that add one or two oxygens at a time, together with structural analysis by heteronuclear single quantum correlation-NMR spectroscopy, we demonstrated that the oxygen-containing moieties generated within photooxidized A2E include a 5,8-monofuranoid and a cyclic 5,8-monoperoxide. We have shown that the oxidation sites can be assigned to the shorter arm of A2E, to the longer arm, or to both arms by analyzing changes in the UV-visible spectrum of A2E, and we have observed a preference for oxidation on the shorter arm. By liquid chromatography-mass spectrometry, we have also detected both monofuran-A2E and monoperoxy-A2E in aged human RPE and in eye cups of Abca4/Abcr–/– mice, a model of Stargardt disease. Because the cytotoxicity of endoperoxide moieties is well known, the production of endoperoxide-containing oxo-A2E may account, at least in part, for cellular damage ensuing from A2E photooxidation. The nondegradable pigments that accumulate in retinal pigment epithelial (RPE) cells as lipofuscin constituents are considered to be responsible for the loss of RPE cells in recessive Stargardt disease, a blindness macular disorder of juvenile onset. This autofluorescent material may also contribute to the etiology of age-related macular degeneration. The best characterized of these fluorophores is A2E, a compound consisting of two retinoid-derived side arms extending from a pyridinium ring. Evidence indicates that photochemical mechanisms initiated by excitation from the blue region of the spectrum may contribute to the adverse effects of A2E accumulation, with the A2E photooxidation products being damaging intermediates. By studying the oxidation products (oxo-A2E) generated using oxidizing agents that add one or two oxygens at a time, together with structural analysis by heteronuclear single quantum correlation-NMR spectroscopy, we demonstrated that the oxygen-containing moieties generated within photooxidized A2E include a 5,8-monofuranoid and a cyclic 5,8-monoperoxide. We have shown that the oxidation sites can be assigned to the shorter arm of A2E, to the longer arm, or to both arms by analyzing changes in the UV-visible spectrum of A2E, and we have observed a preference for oxidation on the shorter arm. By liquid chromatography-mass spectrometry, we have also detected both monofuran-A2E and monoperoxy-A2E in aged human RPE and in eye cups of Abca4/Abcr–/– mice, a model of Stargardt disease. Because the cytotoxicity of endoperoxide moieties is well known, the production of endoperoxide-containing oxo-A2E may account, at least in part, for cellular damage ensuing from A2E photooxidation. The bisretinoid fluorophores that accumulate in retinal pigment epithelial (RPE) 3The abbreviations used are: RPEretinal pigment epitheliumD2Odeuterium oxideESIelectrospray ionizationMSmass spectrometryHPLChigh performance liquid chromatographyHSQCheteronuclear singlet-quantum coherence spectroscopyLC-MSliquid chromatography-mass spectrometryMCPBAmeta-chloroperoxybenzoic acidTMStetramethylsilane. cells as lipofuscin constituents are considered to be responsible for the loss of RPE cells in recessive Stargardt disease (1Lois N. Halfyard A.S. Bird A.C. Holder G.E. Fitzke F.W. Am. J. Ophthalmol. 2004; 138: 55-63Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 2Shroyer N.F. Lewis R.A. Allikmets R. Singh N. Dean M. Leppert M. Lupski J.R. Vision Res. 1999; 39: 2537-2544Crossref PubMed Scopus (110) Google Scholar, 3Weng J. Mata N.L. Azarian S.M. Tzekov R.T. Birch D.G. Travis G.H. Cell. 1999; 98: 13-23Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar), an early onset form of macular degeneration, and may also be involved in the etiology of age-related macular degeneration (4Sparrow J.R. Boulton M. Exp. Eye Res. 2005; 80: 595-606Crossref PubMed Scopus (522) Google Scholar). The RPE lipofuscin fluorophores isolated thus far include A2E (5Eldred G.E. Nature. 1993; 364: 396Crossref PubMed Scopus (54) Google Scholar, 6Sakai N. Decatur J. Nakanishi K. Eldred G.E. J. Am. Chem. Soc. 1996; 118: 1559-1560Crossref Scopus (187) Google Scholar, 7Parish C.A. Hashimoto M. Nakanishi K. Dillon J. Sparrow J.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14609-14613Crossref PubMed Scopus (423) Google Scholar), iso-A2E (7Parish C.A. Hashimoto M. Nakanishi K. Dillon J. Sparrow J.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14609-14613Crossref PubMed Scopus (423) Google Scholar), less abundant double bond isomers of A2E (8Ben-Shabat S. Parish C.A. Vollmer H.R. Itagaki Y. Fishkin N. Nakanishi K. Sparrow J.R. J. Biol. Chem. 2002; 277: 7183-7190Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), and all-trans-retinal dimer conjugates (9Fishkin N. Sparrow J.R. Allikmets R. Nakanishi K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7091-7096Crossref PubMed Scopus (138) Google Scholar, 10Fishkin N. Pescitelli G. Sparrow J.R. Nakanishi K. Berova N. Chirality. 2004; 16: 637-641Crossref PubMed Scopus (29) Google Scholar) (Fig. 1). The most intensely studied of the RPE lipofuscin constituents are A2E and related photoisomers, pigments that, when accumulated by RPE cells in culture, have been shown to bestow a sensitivity to light damage (11Sparrow J.R. Nakanishi K. Parish C.A. Investig. Ophthalmol. Vis. Sci. 2000; 41: 1981-1989PubMed Google Scholar, 12Schutt F. Davies S. Kopitz J. Holz F.G. Boulton M.E. Investig. Ophthalmol. Vis. Sci. 2000; 41: 2303-2308PubMed Google Scholar, 13Sparrow J.R. Zhou J. Ben-Shabat S. Vollmer H. Itagaki Y. Nakanishi K. Investig. Ophthalmol. Vis. Sci. 2002; 43: 1222-1227PubMed Google Scholar). Blue light produces the most pronounced effect (11Sparrow J.R. Nakanishi K. Parish C.A. Investig. Ophthalmol. Vis. Sci. 2000; 41: 1981-1989PubMed Google Scholar). The augmentation of cell death under conditions that prolong the lifetime of singlet oxygen together with the protection provided by quenchers and scavengers of singlet oxygen has implicated singlet oxygen as having a role in the events leading to the death of the cells (14Sparrow J.R. Zhou J. Cai B. Investig. Ophthalmol. Vis. Sci. 2003; 44: 2245-2251Crossref PubMed Scopus (133) Google Scholar). retinal pigment epithelium deuterium oxide electrospray ionization mass spectrometry high performance liquid chromatography heteronuclear singlet-quantum coherence spectroscopy liquid chromatography-mass spectrometry meta-chloroperoxybenzoic acid tetramethylsilane. The propensity for A2E to undergo photooxidation was initially revealed by a tendency for fluorescence quenching of intracellular A2E upon blue light illumination (13Sparrow J.R. Zhou J. Ben-Shabat S. Vollmer H. Itagaki Y. Nakanishi K. Investig. Ophthalmol. Vis. Sci. 2002; 43: 1222-1227PubMed Google Scholar). HPLC analysis later confirmed this observation, the absorbance of the A2E peak after 430 nm irradiation, exhibiting a corresponding reduction (13Sparrow J.R. Zhou J. Ben-Shabat S. Vollmer H. Itagaki Y. Nakanishi K. Investig. Ophthalmol. Vis. Sci. 2002; 43: 1222-1227PubMed Google Scholar). Subsequent analysis by mass spectrometry showed that 430 nm irradiation of A2E, either in an acellular or cellular environment, yielded products that, starting from the M+ m/z 592 peak attributable to A2E, presented as consecutive peaks differing in m/z by 16 (15Ben-Shabat S. Itagaki Y. Jockusch S. Sparrow J.R. Turro N.J. Nakanishi K. Angew. Chem. Int. Ed. 2002; 41: 814-817Crossref PubMed Scopus (185) Google Scholar). Several lines of investigation suggested that singlet oxygen is involved in the photooxidation, with A2E serving as a sensitizer for the generation of singlet oxygen from triplet oxygen (13Sparrow J.R. Zhou J. Ben-Shabat S. Vollmer H. Itagaki Y. Nakanishi K. Investig. Ophthalmol. Vis. Sci. 2002; 43: 1222-1227PubMed Google Scholar, 15Ben-Shabat S. Itagaki Y. Jockusch S. Sparrow J.R. Turro N.J. Nakanishi K. Angew. Chem. Int. Ed. 2002; 41: 814-817Crossref PubMed Scopus (185) Google Scholar, 16Sparrow J.R. Vollmer-Snarr H.R. Zhou J. Jang Y.P. Jockusch S. Itagaki Y. Nakanishi K. J. Biol. Chem. 2003; 278: 18207-18213Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). For instance, the extent of photooxidation was found to increase in deuterium oxide (D2O), a solvent that prolongs the lifetime of singlet oxygen. Irradiation (430 nm) of A2E in chloroform also produces a luminescence at ∼1270 nm, which is typical of the phosphorescence of singlet oxygen. In addition, experiments demonstrated that singlet oxygen generated by an endoperoxide of 1,4-dimethylnaphthalene could substitute for blue light in mediating A2E oxidation. Singlet oxygen quenchers were also found to be inhibitory. Nonetheless, oxidation by other reactive forms of oxygen may also occur (17Pawlak A. Wrona M. Rozanowska M. Zareba M. Lamb L.E. Roberts J.E. Simon J.D. Sarna T. Photochem. Photobiol. 2003; 77: 253-258Crossref PubMed Scopus (60) Google Scholar, 18Gaillard E.R. Avalle L.B. Keller L.M.M. Wang Z. Reszka K.J. Dillon J.P. Exp. Eye Res. 2004; 79: 313-319Crossref PubMed Scopus (34) Google Scholar). Given that A2E has nine double bonds besides the pyridinium ring, together with the observation that nine m/z peaks (592 + n(16)) culminating in the m/z 736 peak (592 + 9(16)) (Fig. 2) (15Ben-Shabat S. Itagaki Y. Jockusch S. Sparrow J.R. Turro N.J. Nakanishi K. Angew. Chem. Int. Ed. 2002; 41: 814-817Crossref PubMed Scopus (185) Google Scholar, 18Gaillard E.R. Avalle L.B. Keller L.M.M. Wang Z. Reszka K.J. Dillon J.P. Exp. Eye Res. 2004; 79: 313-319Crossref PubMed Scopus (34) Google Scholar, 19Radu R.A. Mata N.L. Bagla A. Travis G.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5928-5933Crossref PubMed Scopus (136) Google Scholar) appear following 430 nm irradiation of A2E, we previously suggested that A2E undergoes oxidation at all nine double bonds to form an unprecedented nonaoxirane structure. Because of the likelihood that numerous stereoisomers of the nonaoxirane species were present, together with its instability and the small amount of compound that was available, structural studies were not performed. As part of our effort to elucidate mechanisms involved in A2E oxidation, we also oxidized A2E with meta-chloroperoxybenzoic acid (MCPBA) (15Ben-Shabat S. Itagaki Y. Jockusch S. Sparrow J.R. Turro N.J. Nakanishi K. Angew. Chem. Int. Ed. 2002; 41: 814-817Crossref PubMed Scopus (185) Google Scholar). This approach led to the appearance of an m/z 624 peak (parent peak), to which was assigned a 7,8,7′,8′-bisoxido structure based on the nonaoxirane and mass spectrometry and 1H NMR data (15Ben-Shabat S. Itagaki Y. Jockusch S. Sparrow J.R. Turro N.J. Nakanishi K. Angew. Chem. Int. Ed. 2002; 41: 814-817Crossref PubMed Scopus (185) Google Scholar). More recently, it has been reported that photooxidation products of A2E can include a mono-furanoid oxide, a bisfuranoid oxide (Fig. 3B), and a mono-furanoid oxide with a second oxygen attached to the cyclohexenyl ring (20Dillon J. Wang Z. Avalle L.B. Gaillard E.R. Exp. Eye Res. 2004; 79: 537-542Crossref PubMed Scopus (67) Google Scholar). However, this conclusion was based on the fragmentation patterns generated by collision-induced dissociation and tandem mass spectrometry, an approach that cannot discriminate between a 5,8-furanoid and a 7,8-epoxide. Nor can the use of 1H NMR spectra distinguish a furanoid from an epoxide moiety because the chemical shift of the 7,8-protons in 1H NMR does not reveal the resonance of the 7,8-carbon. Here we provide definitive evidence from HSQC-NMR spectroscopy that the complex mixture of oxidized species resulting from A2E photooxidation includes a 5,8-monofuran-A2E. Further investigation has also uncovered a bisoxygenated photoproduct that has been structurally characterized as 5,8-monoperoxy-A2E. More importantly, through an analysis of chromatographic properties and UV-visible spectra together with mass spectrometry to provide molecular weight information, we have also detected a monofuran-A2E and monoperoxy-A2E in aged human RPE and in the eyecups of mice with null mutations in Abca4/Abcr, the gene responsible for recessive Stargardt disease.FIGURE 3Candidates for the structure of the oxygen-containing moiety generated by MCPBA oxidation of A2E. In each case, the moiety on only one retinoid arm of oxo-A2E is represented. A, 5,6-epoxide; B, 5,8-furanoid; C, 7,8-epoxideView Large Image Figure ViewerDownload Hi-res image Download (PPT) Reagents—MCPBA, ethanolamine, and trifluoroacetic acid were purchased from Aldrich; HEPES was obtained from Sigma; acetonitrile was purchased from Fisher; and Dulbecco's phosphate-buffered saline was from Invitrogen. All of the other chemicals were from Sigma. A2E was synthesized as described previously (7Parish C.A. Hashimoto M. Nakanishi K. Dillon J. Sparrow J.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14609-14613Crossref PubMed Scopus (423) Google Scholar). MCPBA Oxidation of A2E—To a solution of A2E (20.0 mg) in methanol (1.0 ml) was added MCPBA (33.0 mg, 2 eq), and the mixture was stirred for 12 h at room temperature in the dark. After concentration in vacuo, the reaction mixture was subjected to HPLC analysis. A2E mono- and bisfuranoid (Fig. 4) were eluted at 31 and 21 min, respectively, using a Vydac C18 column (22 mm × 250 mm) under the following conditions: solvent A, CH3CN, 0.1% trifluoroacetic acid; solvent B, H2O, 0.1% trifluoroacetic acid; gradient mode (A/B) 0 min, 85:15; 30 min, 90:10; 50 min, 100:0; flow rate 4.0 ml/min. UV-visible spectra were monitored at 430 nm. Endoperoxide of 1,4-Dimethylnaphthalene and A2E Oxidation—1,4-Dimethylnaphthalene endoperoxide was synthesized as described previously (21Turro N.J. Chow M.-F. Rigaudyo J. J. Am. Chem. Soc. 1981; 103: 7218-7222Crossref Scopus (205) Google Scholar). Subsequently, endoperoxide of 1,4-dimethylnaphthalene (48.0 mg, 10 eq of A2E) was added to a solution of A2E (15.0 mg) in CD3OD (1.0 ml), and the mixture was stirred overnight at room temperature in the dark. After removal of solvent, the oxidation products of A2E were isolated and purified by HPLC using a YMC C18 column (10 × 250 mm) with the following solvent system: solvent A, CH3CN, 0.1% trifluoroacetic acid; solvent B, H2O, 0.1% trifluoroacetic acid; gradient mode (A/B), 0 min, 75:25; 40 min, 100:0; flow rate 2.5 ml/min. A2E mono- and bisperoxide (Fig. 5) were eluted at 21 and 12.5 min, respectively. NMR—1H NMR and heteronuclear singlet-quantum coherence spectroscopy (HSQC) spectra of 5,8,5′,8′-bisfuran-A2E were obtained at 500 MHz using a Bruker DMX-500 spectrometer. 1H NMR and HSQC spectra of 5,8,5′,8′-bisperoxy-A2E were recorded at 400 MHz (Bruker DRX-400). All data were recorded in CD3OD, and TMS was used as internal standard. NMR of Bisfuran—A2E: 1H NMR (500 MHz, CD3OD, 25 °C, TMS): δ = 1.14 (s, 6H), 1.41 (s, 3H), 1.42 (s, 3H), 1.77 (s, 3H), 1.91 (s, 3H), 2.07 (s, 3H), 3.87 (s, 2H), 4.50 (s, 2H), 5.15 (s, 1H; H-8), 5.19 (s, 1H; H-8′), 5.22 (s, 1H; H-7), 5.26 (s, 1H; H-7′), 6.26 (d, J = 11.3 Hz, 1H), 6.43 (d, J = 11.2 Hz, 1H), 6.56 (d, J = 15.0 Hz, 1H), 6.66 (s, 1H), 6.73 (d, J = 15.3 Hz, 1H), 6.93 (dd, J = 11.3, 15.0 Hz, 1H), 7.81 (dd, J = 11.2, 15.3 Hz, 1H), 7.86 (s, 1H), 7.91 (d, J = 7.0 Hz, 1H), 8.53 (d, J = 7.0 Hz, 1H). HSQC (500 MHz, CD3OD): 86.7 (C-8), 86.8 (C-8′), 117.6 (C-7), 117.6 (C-7′). NMR of Bisperoxy—A2E: 1H NMR (400 MHz, CD3OD, 25 °C, TMS): δ = 1.16 (s, 6H), 1.18 (s, 6H), 1.28 (s, 3H), 1.59 (s, 3H), 1.60 (s, 3H), 2.08 (s, 3H), 2.11 (s, 3H), 3.90 (t, J = 4.8 Hz, 2H), 4.54 (t, J = 4.8 Hz, 2H), 4.71 (d, J = 4.0 Hz, 1H, H-8), 4.77 (d, J = 4.0 Hz, 1H, H-8′), 5.63 (d, J = 4.0 Hz, 1H, H-7), 5.67 (d, J = 4.0 Hz, 1H, H-7′), 6.23 (d, J = 11.2 Hz, 1H), 6.41(d, J = 11.2 Hz, 1H), 6.58 (d, J = 15.2 Hz, 1H), 6.71 (s, 1H), 6.77 (d, J = 15.5 Hz, 1H), 6.98 (dd, J = 11.2, 15.2 Hz, 1H), 7.85 (dd, J = 11.2, 15.5 Hz, 1H), 7.91 (d, J = 2.0 Hz, 1H), 7.96 (dd, J = 2.0, 6.6 Hz, 1H), 8.57 (d, J = 6.6 Hz, 1H). HSQC (400 MHz, CD3OD): 82.9 (C-8), 83.6 (C-8′), 115.2 (C-7), 116.7 (C-7′). Cell Culture and Illumination—Human adult RPE cells (ARPE-19, American Type Culture Collection, Manassas, VA) lacking endogenous A2E (22Sparrow J.R. Parish C.A. Hashimoto M. Nakanishi K. Investig. Ophthalmol. Vis. Sci. 1999; 40: 2988-2995PubMed Google Scholar) were grown as described previously (22Sparrow J.R. Parish C.A. Hashimoto M. Nakanishi K. Investig. Ophthalmol. Vis. Sci. 1999; 40: 2988-2995PubMed Google Scholar). To generate A2E-laden RPE, nonconfluent cultures were allowed to accumulate A2E from a 20 μm concentration in medium. Cells were subsequently exposed to 430 nm illumination (0.36 milliwatt/mm2) as described previously (13Sparrow J.R. Zhou J. Ben-Shabat S. Vollmer H. Itagaki Y. Nakanishi K. Investig. Ophthalmol. Vis. Sci. 2002; 43: 1222-1227PubMed Google Scholar). Illuminated cells were harvested and added to a solution of chloroform and methanol at a 2:1 ratio. This mixture were homogenized in a glass tissue homogenizer and centrifuged at 10,000 × g for 10 min. The supernatant was dried under argon, redissolved in methanol, and subjected to liquid chromatography-mass spectrometry (LC-MS) study. Human and Mouse Tissue—Human donor eyes were obtained from the National Disease Research Interchange (Philadelphia). Abca4/Abcr null mutant mice (Rpe65 450Leu, pigmented, 129/SV × C57BL/6J) were generated as described previously (23Kim S.R. Fishkin N. Kong J. Nakanishi K. Allikmets R. Sparrow J.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 11668-11672Crossref PubMed Scopus (123) Google Scholar). The mice were raised under a 12-h on-off cycle lighting with an in-cage illuminance of ∼250 lux. Human RPE (from four eyes, ages 58–68) and murine posterior eye cups (six eye cups; age 17 months) were homogenized in phosphate-buffered saline using a tissue grinder. An equal volume of a mixture of chloroform/methanol (2:1) was added, and the sample was extracted three times. To remove insoluble material, extracts were filtered through cotton and passed through a reversed phase cartridge (C18 Sep-Pak, Millipore) with 0.1% trifluoroacetic acid in methanol. After removing solvent by evaporation under argon gas, the extract was dissolved in methanol containing 0.1% trifluoroacetic acid, for LC-MS analysis. HPLC—A Waters™ 600 equipped with photodiode array detector (model 996) and operating with Empower® software was used for HPLC analysis. A Cosmosil 5C18 column (4.6 × 150 mm; Nacalai Tesque, Japan) was used for analytical scale HPLC. Mass Spectrometry—Fast atom bombardment ionization-MS was performed on a JEOL JMS-HX110A/110A tandem MS (Akishima, Tokyo, Japan), using 10-kV acceleration voltage and fitted with a Xenon beam FAB gun (6 kV) on the MS-1 ion source. 3-Nitrobenzyl alcohol was used as matrix. HPLC separation combined with mass spectrometry for analysis of A2E oxidation products in cultured cells and tissues was performed on an Esquire 3000 (Bruker Daltonic Inc., Billerica, MA) with electrospray ionization (ESI) source. Samples were introduced with flow injection mode. A reversed phase column (Atlantis® dC18, 3 μm, 4.6 × 150 mm; Waters) was used under the following mobile phase conditions: solvent A, CH3CN, 0.1% trifluoroacetic acid; solvent B, H2O, 0.1% trifluoroacetic acid; gradient mode (% of A/B, v/v), 0 min, 10:90; 75 min, 100:0; flow rate, 0.3 ml/min. To generate a partially oxidized A2E (oxo-A2E) species, we began by using MCPBA as the oxidizing agent. Reaction of A2E with MCPBA (2 eq) in the dark followed by HPLC analysis revealed a bisoxo product with 1H NMR and mass spectral profiles that were identical to those reported previously for MCPBA-oxidized A2E (15Ben-Shabat S. Itagaki Y. Jockusch S. Sparrow J.R. Turro N.J. Nakanishi K. Angew. Chem. Int. Ed. 2002; 41: 814-817Crossref PubMed Scopus (185) Google Scholar). We surmised that the oxygen-containing moiety could exhibit one of three possible structures: 5,6-epoxide, 5,8-furanoid, or 7,8-epoxide (Fig. 3). The 5,6-epoxide and 5,8-furanoid were considered to be candidates because MCPBA oxidation of β-carotene with the same ring moiety yields a 5,6-epoxide; moreover, the 5,6-epoxide readily rearranges to the 5,8-furanoid structure even under mild acidic conditions (20Dillon J. Wang Z. Avalle L.B. Gaillard E.R. Exp. Eye Res. 2004; 79: 537-542Crossref PubMed Scopus (67) Google Scholar, 24Baldas J. Porter Q.N. Cholnoky L. Szabolos J. Weedon B.C.L. Chem. Commun. 1966; 23: 852-854Google Scholar). However, the 5,6-epoxide was eliminated as a possibility because NMR revealed that the 7,8,7′,8′ proton signals at 5.15–5.26 ppm are shifted upfield (see the NMR data under "Experimental Procedures") compared with those of A2E at 6.18 to 6.53 ppm (6Sakai N. Decatur J. Nakanishi K. Eldred G.E. J. Am. Chem. Soc. 1996; 118: 1559-1560Crossref Scopus (187) Google Scholar). Because fragment ions generated using collision-induced dissociation tandem mass spectral analysis for structural determination of oxo-A2E can form by rearrangement during ionization, we sought confirmatory evidence of a furanoid ring by NMR analysis. Accordingly, by using HSQC-NMR spectroscopy to reveal correlations between carbon atoms and directly attached protons (hydrogen), the structure was shown to be that of a 5,8-furanoid (Fig. 4). Specifically, HSQC analysis revealed that the two 87 ppm sp3 carbons are coupled to 5.15 (8-H)/5.19 (8′-H) ppm protons, whereas the two 117 ppm sp2 carbons are coupled to 5.22 (7-H)/5.26 (7′-H) ppm protons. In addition, because both of the two UV-visible absorbance maxima of A2E were blue-shifted (see below) in this bisoxo-A2E, it was established that the bisoxo-A2E is 5,8,5′,8′-bisfuran-A2E (Fig. 4). We were also able to identify the monooxo-A2E as 5,8-monofuran-A2E. Because A2E photooxidation is likely to occur, at least in part, via a singlet oxygen-mediated pathway (13Sparrow J.R. Zhou J. Ben-Shabat S. Vollmer H. Itagaki Y. Nakanishi K. Investig. Ophthalmol. Vis. Sci. 2002; 43: 1222-1227PubMed Google Scholar, 15Ben-Shabat S. Itagaki Y. Jockusch S. Sparrow J.R. Turro N.J. Nakanishi K. Angew. Chem. Int. Ed. 2002; 41: 814-817Crossref PubMed Scopus (185) Google Scholar), A2E was also reacted with 1,4-dimethylnaphthalene endoperoxide, an aromatic compound that decomposes to singlet oxygen and 1,4-dimethylnaphthalene with a convenient half-life of 5 h at 25°C. Analysis of the product by LC-MS revealed the presence of a peak that exhibited a UV-visible absorption spectrum suggestive of a bisoxo-A2E but a molecular mass (m/z 656) that was consistent with the addition of 4 oxygens. NMR studies, including 1H and HSQC NMR, showed upfield-shifted protons at the 7, 8, and 7′, 8′ positions, which together with the existence of sp2 carbons evidenced by HSQC data (115.2 (C-7), 116.7 (C-7′)) confirmed this tetraoxo-A2E as 5,8,5′,8′-bisperoxy-A2E (Fig. 5). The presence of the 1,2-dioxin moieties in this oxo-A2E species was corroborated by comparison with a previous investigation that demonstrated the production of a cyclic 5, 8-peroxide upon photolysis of A1E, a nonbiological single side arm counterpart to A2E (25Jockusch S. Ren R.X. Jang Y.P. Itagaki Y. Vollmer-Snarr H.R. Sparrow J.R. Nakanishi K. Turro N.J. J. Am. Chem. Soc. 2004; 126: 4646-4652Crossref PubMed Scopus (14) Google Scholar). We found that oxidation-associated changes in the UV-visible spectrum of A2E served as a means to determine the oxidation site (Fig. 6). This was possible because the absorption peaks of A2E at 337 nm (band S) and 439 nm (band L) could be assigned to the shorter and longer chains that extend from the pyridinium ring, respectively. Thus, whether the hypsochromic shift occurred in either band S or band L revealed the side arm on which the loss of conjugation had occurred. For instance, comparison of the UV-visible spectra of the mono-furanoid and mono-peroxide with that of A2E revealed that only band S was blue-shifted. In contrast, both bands were observed to undergo a hypsochromic shift in the bisfuranoid and bisperoxide. Both furanoid and peroxide formation resulted in the loss of two successive conjugation systems, hence the absorption maximum (either band S or L) of the affected side arm was shifted toward the blue region by ∼40 nm. HPLC analysis of products generated by photooxidation and chemical oxidation also revealed the presence of 335 and 400 nm absorbance peaks that corresponded to blue shifts in only the L band. This observation indicated oxidation exclusively on the longer arm. However, the minuscule amount of these compounds precluded further structural studies. Oxidation apparently occurs more readily on the shorter arm of A2E. Of the two polyenes extending ortho and para to the pyridinium nitrogen, electron delocalization is favored along the former ortho arm, which has one extra conjugation π bond; this leads to relatively lower density of electrons in each of the sp2 carbons, thus making it less nucleophilic and less susceptible to the MCPBA attack. In order to examine the formation of the oxidative products of A2E in a cellular environment, ARPE-19 cells were allowed to incorporate A2E, and the A2E-laden ARPE cells were irradiated at 430 nm. Chloroform/methanol extracts of the cells were analyzed by LC-MS. As shown in Fig. 7, two eluting components that were more polar than A2E and with m/z of 624 and 608 were resolved. The UV-visible spectra of these peaks had similar profiles; specifically, absorbance maxima occurred at 296 nm (band S) and ∼435 nm (band L). Because only band S exhibited a hypsochromic shift, relative to A2E, it was apparent that in both cases two successive conjugate systems on the shorter arm of A2E were lost by oxidation. On the basis of the presence of two peaks, which by LC-MS exhibited molecular sizes that corresponded to a mono-oxo (m/z 608) and bisoxo-A2E (m/z 624), together with our prior NMR analysis that established the structures of these products, it was evident that in this cellular system the mono-furanoid and mono-peroxide formed. We also detected oxo-A2E in chloroform/methanol extracts of RPE cells isolated from human donor eyes and in extracts of posterior eye cups of Abca4/Abcr–/– mice. The latter mice are considered to be a model of Stargardt macular degeneration. Again we used LC-MS for this analysis. It is our experience that the use of MS/MS fragmentation analysis with collision-induced dissociation is not a reliable approach to the assignment of oxo-A2E structure because the fragmentation patterns vary, probably because the oxygen-containing fragment ions can undergo complex intramolecular rearrangements. As shown in Fig. 8, examination of the human and mouse samples by LC-MS revealed the presence of reversed phase HPLC eluates with extracted ion signals indicative of m/z 592, 608, and 624. The corresponding compounds could be identified as A2E (m/z 592), monofuran-A2E (m/z 608), and monoperoxy-A2E (m/z 624) because they had the same retention time and UV-visible absorbance spectra as the species detected in A2E-laden ARPE-19 cells following 430 nm irradiation (Fig. 7). To determine whether these partially oxidized forms of A2E could serve as intermediates on a pathway to further oxidation of A2E, we incubated A2E with MCPBA and isolated the product 5,8-monofuran-A2E by HPLC. The monofuranoid in phosphate-buffered saline with 0.5% Me2SO was subsequently submitted to blue light irradiation (430 nm), and unirradiated and irradiated samples were analyzed by MS. As shown in Fig. 9, the 5,8-monofuran-A2E (m/z 608) underwent further oxidation to generate higher molecular species representing the incorporation of additional oxygens (i.e. m/z 640, 672, and 720 peaks corresponding respectively to addition of 2, 4, and 7 oxygens). Most interestingly, however, if irradiation of 5,8-monofuran-A2E was carried out with methanol as solvent, no additional oxidation occurred. We also proceeded to revisit the issue of solvent effects on A2E photooxidation. To this end, we performed HPLC analysis of unirradiated samples of A2E and samples in which A2E was irradiated in an environment of Me2SO/H2O and samples irradiated in Me2SO/D2O (Fig. 10). Consistent with the ability of deuterium solvent to extend the lifetime of singlet oxygen (26Rodgers M.A.J. Snowden P.T. J. Am. Chem. Soc. 1982; 104: 5541-5543Crossref Scopus (452) Google Scholar), irradiation in D2O resulted in more pronounced photooxidation, the latter reflected in the greater diminution in the A2E peak relative to the sample irradiated in H2O. Specifically, quantitation of the peak area revealed that with irradiation in Me2SO/H2O A2E was decreased by 75%, whereas in Me2SO/D2O under the same conditions of irradiation the reduction was 90%. In these experiments, only the singlet oxygen that escaped to the medium would be affected by the deuterated solvent. Based on the foregoing results, we propose that A2E oxidation occurs by means of multiple independent mechanisms. For instance, the addition of one atom of oxygen can occur at the 5,6 p

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