Matthew Sparks and Dr. Heidi Vollmer-Snarr, BYU Chemistry & Biochemistry Department

Age-related macular degeneration (AMD) is an ocular disease that currently afflicts approximately 25–30 million people around the globe.1,2 The disease is characterized by the destruction of the macula, the central region of the retina. As the disease progresses, central vision becomes distorted, clouded, and eventually lost entirely.3 Although currently receiving much attention from researchers, the chemical pathway(s) causing the disease have remained elusive. Much research has focused on A2E, the most commonly known photo-toxic compound implicated in AMD pathogenesis.

A2E consists of one equivalent ethanolamine and two equivalents all-trans-retinal. By reacting two equivalents all-trans-retinal with one equivalent of various other biogenic amines, the Vollmer-Snarr research group has developed a library of A2E-similar molecules, known as pyridinium bis-retinoids (PBR). Because A2E exhibits cytotoxicity through a photo-initiated mechanism, it is conceivable that other PBRs such as A2-Cadaverine (A2C), A2-Dopamine (A2D), and A2-Phenylethanolamine (A2-PEA) would have similar photo-toxic properties. If so, these compounds might participate in the degenerative pathways of AMD and could potentially also serve as novel photodynamic therapy drugs for use against cancer. To explore this possibility, I worked with graduate student MacKenzie Pew to synthesize A2C, A2D, and A2-PEA and subject them to light experiments to determine their photo-reactivity.

PBR synthesis is a time-consuming process resulting in disappointingly low yields. To form PBRs, the necessary equivalents of all-trans-retinal, biogenic amine, and acid catalyst are placed in a round bottom flask with ethanol as the solvent. The reaction mixture is stirred at room temperature for an optimal amount of time (ex. A2C is stirred for 48 hours). Purification requires slow chromatographic (reverse-phase column and chromatotron) techniques. The low yields (<15%) made multiple reactions necessary to obtain enough material to run further experiments. I successfully synthesized A2C and A2D, but was unable to fully characterize the A2-PEA product.

To test the photo-reactivity of A2C & A2D, light experiments were performed using a high intensity light source and filter that selects only blue light (λ = 430nm). A fiber optic cable was attached to the source to keep the sample at a distance and minimize heat-related errors. A box was placed over the source to minimize error due to unwanted light. The entire experiment was performed in a closed hood with aluminum foil covering the hood windows to block any other interfering light. The PBR was dissolved in a DMSO/H2O mixture and transferred to a cuvette prior to irradiation. Small samples were taken for analysis from the cuvette at various time intervals during irradiation.

Mass spectrometry and UV-Visible detection (HPLC) were chosen as the analytical methods for studying the chemical changes resulting from blue light irradiation. It would have been interesting to use nuclear magnetic resonance imaging to identify the oxidation species created by the irradiation, but this method was impractical due to the large number of possible species produced and their short life-spans. Because PBRs are highly conjugated molecules (successive double bonds), they absorb in the UV region of the electromagnetic spectrum and give distinct UV-Vis spectra. The molecule’s conjugation is broken up during irradiation and results in a different UV-Vis spectrum. UV-Visible spectra helped me obtain relative quantitative information about chemical species populations as a function of time and gave a general idea of when the reaction had reached completion. Mass spectrometry showed the molecular mass of A2C increasing stepwise by 16, corresponding to suspected oxidation species. This evidence suggests that A2C and A2D both exhibit similar photo-reactivity to A2E, as hypothesized.

Photodynamic therapy involves irradiating a photosensitizer to activate its cytotoxic properties. The photo-toxic properties of PBRs make them ideal as photosensitizers. Due to the length of time required to synthesize the PBRs, I was unable to perform cellular assays to explore the cytotoxicity of A2C or A2D. Other researchers in the Vollmer-Snarr research group were able to run some cellular assays using A2C and A2D, with varying degrees of success. It was decided that to increase the effective cytotoxicity of A2C, it should be tethered to folic acid. Cancer cells over-express the folate receptor and therefore have a higher level of folic acid uptake than healthy cells. By tethering folic acid to A2C, the compound should be easier for cancer cells to uptake. After entering the cancer cell, the PBR would be activated by irradiation and initiate cell apoptosis. Folic-acid A2C (FA-A2C) synthesis is complicated and results in even lower yields than PBR synthesis. MacKenzie Pew developed a synthetic procedure and I worked to produce FA-A2C for further experimentation.

To determine the photodynamic properties of the pyridinium bis-retinoids A2C, A2D, and A2-PEA, I performed and purified 50 A2C reactions, four A2D reactions, and eight A2-PEA reactions. With the purified PBRs, I performed light experiments that give evidence supporting the production of oxidative species. These oxidative species could be part of the pathogenesis of AMD and may also lead to a new cancer treatment.

Further research must be completed to accurately assess the effectiveness of PBRs as cancer therapy drugs. More cell assays must be run to compare cytotoxicity of different types of PBRs and to determine what variables increase the cytotoxicity of these compounds. Before such research can be done on a large scale, the synthetic product yields must be optimized.

References

• Ambati, J et al., An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice, Nat. Med., 2003, 9, 1390–1397.
• Bulletin World Health Organization, 1995, 73, 115–121.
• Ben-Shabat, S. et al, Formation of a nonaoxirane from A2E, a lipofuscin fluorophore related to macular degeneration, and evidence of singlet oxygen involvement, Angew. Chem. Int. Ed., 2002, 41 (5), 814¬–817.