Characterization of dihydro-A2PE: an intermediate in the A2E biosynthetic pathway.

A number of observations over the years have shown that the deposition of lipofuscin fluorophores in RPE is dependent on the availability of vitamin A and/or vitamin A derivatives. For instance, Rpe65 null mutation (1), amino acid variants in Rpe65 (2), antagonists of RPE65 (3) and retinoids (13-cis retinoic acid) that inhibit 11-cis retinol dehydrogenase (4, 5), all of which slow the visual cycle and reduce the flux of all-trans-retinal, also limit the formation of RPE lipofuscin. In addition, RPE lipofuscin accumulation in normal rat retina can be reduced by dietary vitamin A deficiency (6, 7), while pharmacological agents that reduce serum vitamin A also decrease RPE lipofuscin in mice (8). The first of the vitamin A aldehyde derivatives to be identified in RPE lipofuscin was A2E (Fig. 1), a pyridinium bisretinoid conjugate (9-12) (C42H58NO, molecular weight 592), named because it can be synthesized biomimetically from vitamin A aldehyde and ethanolamine when combined in a 2:1 ratio. Later, a C13-C14 Z-isomer of A2E (isoA2E) (Fig. 1) was identified (12), along with minor isomers having cis-double bonds at other positions (13). Confirmation of the structure of A2E was obtained by extensive nuclear magnetic resonance (NMR) studies (12) and by total synthesis (14). Other constituents of RPE lipofuscin include pigments generated via the condensation of two all-trans-retinal (15, 16). These compounds include all-trans-retinal dimer (atRAL dimer) and the conjugates all-trans-retinal dimer-phosphatidylethanolamine (atRAL dimer-PE) and all-trans-retinal dimer-ethanolamine (atRAL dimer-E). Figure 1 Proposed biosynthetic pathway of A2E. All-trans-retinal that forms from photoisomerization of 11-cis-retinal reacts with phosphatidylethanolamine (PE) to generate the Schiff base N-retinylidene-phosphatidylethanolamine (NRPE). This adduct undergoes a ... We previously proposed an A2E biosynthetic cascade (12, 17) (Fig. 1) that is initiated with a reaction between the membrane phospholipid phosphatidylethanolamine (PE) and all-trans-retinal, the latter being generated upon photoisomerization of 11-cis-retinal (Fig. 1). We demonstrated by mass spectrometry analysis (17) that the compound formed by this reaction is the Schiff base conjugate, N-retinylidene-phosphatidyl-ethanolamine (NRPE) while others showed that NRPE is probably the ligand for ABCA4 (ABCR) (18-20), the photoreceptor-specific ATP-binding cassette transporter that is mutated in recessive Stargardt disease (21). We suggested that reaction with a second molecule of all-trans-retinal, would lead to the formation of a phosphatidyl dihydropyridinium molecule (dihydro-A2PE) that we envisioned would undergo automatic oxidative aromatization to yield A2PE, a phosphatidyl pyridinium bisretinoid. Although the formation of dihydro-A2PE was not, at that time, corroborated, mass spectrometry was utilized to confirm the structure of A2PE (17) and experiments demonstrating the release of A2E upon phospholipase D-mediated cleavage of A2PE established A2PE as the immediate precursor of A2E (13, 17). Since an understanding of A2E biosynthesis is essential to efforts aimed at limiting the formation of this lipofuscin pigment, we have revisited our A2E biogenesis scheme, with the aim of providing evidence for the formation of the dihydropyridinium intermediate, dihydro-A2PE. This transitional compound is of interest to us because it is likely the last intermediate before stable compound is generated. Dihydropyridinium compounds, such as dihydro-A2PE, are notoriously unstable and undergo automatic oxidative aromatization (22); thus to aid in the identification of dihydro-A2PE in our reaction mixture, we first obtained computer simulations of the UV-visible absorbance spectrum of the compound using TDDFT (23, 24). In subsequent experiments we sought for evidence of a compound with similar UV-visible spectra and identical mass.