Jeremy Koontz and Dr. Heidi Vollmer-Snarr, Chemistry and Biochemistry
Despite the enormous amounts of time and money spent to research effective cures for cancer—the second leading cause of death in the United States —little is known today about how to kill cancer cells without harming normal cells. Current traditional forms of cancer treatment are chemotherapy, radiotherapy, and invasive surgery. However, the challenge with such treatments is they do not discriminate between normal cells and cancerous cells; these therapies struggle to balance toxicity and efficiency. In comparison, photodynamic therapy (PDT) offers a more favorable alternative to traditional methods of cancer treatment. In PDT drugs, called photosensitizers, are activated through specific wavelengths of light in a localized region, resulting in an irreversible photodamage to tumor tissues.
A2-2,2’-(ethylenedioxy)-bis-ethylamine (A2NOON ) and A2-1,6-diaminohexane (A2DA) are compounds with similar structure and characteristics to A2-ethanolamine (A2E), a pyridinium bis-retinoid first isolated in lipofuscin from the eye. We have synthesized these compounds as potential novel photosensitizers for PDT. I have prepared both pyridinium bis-retinoids from all-trans-retinal and their respective amine linker. Additionally, I have linked A2DA to folic acid to improve the selective uptake in cancerous tissue. Ultimately, the aim of this study is to develop a more efficient photosensitizer, which will accumulate preferentially in tumor cells and leave peripheral cells unharmed, resulting in a highly selective cancer treatment.
In our lab we had hypothesized that A2NOON and A2DA would exhibit similar toxicity as A2E when irradiated. A2E, a biological by-product of the visual cycle often associated with age-related macular degeneration, is harmless to retinal pigment epithelial cells. However, blue light initiates the formation of oxidated species of A2E, which destroy irradiated cells but cause little harm to peripheral cells. The innocuous nature and significant photoactivated cytotoxicity of pyridinium bis-retinoids make them ideal candidates for PDT.
We also believe these potential drugs can be improved when tethered to folic acid. Numerous cancer cells are known to over-express the folate receptor and will preferentially uptake more folic acid bioconjugates than healthy cells. We can target cancer tissue through this preferential uptake by tethering both A2NOON and A2DA to folic acid. Rapidly growing cancer tissues will uptake our folate-linked pyridinium bis-retinoids at a higher rate due to a greater demand for folic acid.5
To accomplish this goal I combined one molar equivalent of amine—2,2’-(ethylenedioxy)-bis-ethylamine or diamino-1,6-hexane—with two molar equivalents of all-trans¬-retinal, ethanol, and one molar equivalent of acetic acid. This mixture was sealed in a round bottom flask and stirred in the absence of light for forty-eight hours. Afterwards I concentrated the mixture in vacuo, and purified each compound using silica gel column chromatography and a chemical gradient from methylene chloride (CH2Cl2) to elution with methanol and trifluoroacetic acid. The samples I collected were afterward subjected to analysis with a photodiode-array detector and a mass spectrometer equipped with both an electrospray ionization source and an atmospheric pressure chemical ionization source. To optimize the purity of the A2DA, I refined A2DA products on centrifugal thin-layer chromatography (TLC) and observed purity on through analytical high-pressure liquid chromatography (HPLC), H1-NMR, and C13-NMR.
To produce folate-linked pyridinium bis-retinoids I combined one molar equivalent of folic acid and one molar equivalent of pyridinium bis-retinoid in one molar equivalent of dicyclohexlcarbodiimide and catalytic amounts of dimethylaminopyridine. I stirred the mixture in dry dimethyl sulfoxide for twelve hours and then quenched the mixture with water. I filtered the solid product and dried it under high vacuum in preparation for purification and optimization via silica gel column chromatography, centrifugal TLC, and HPLC.
Through these procedures I had hypothesized I could produce a drug that could be subjected to cellular testing to evaluate cytotoxicity when irradiated. I was able to purify A2DA and a trace amount of purified folate-linked A2DA, but was unable to obtain anything past crude amounts of A2NOON. I was able to confirm the purity of collections of A2DA through mass spectrometry results, H1-NMR, and comparison of UV-visible spectra obtained from HPLC. From irradiation experiments I was able to assess the relative reactivity of A2DA and observe photoreactive similarities between A2DA and A2E.
Although purification procedures can be time consuming and frustrating, I have learned a great deal through this study. I hypothesize product yields are less than ten percent for three reasons. First, Parish, et al. reported the mechanism for pyridinium bis-retinoids as a four-step pathway in which all reactions are reversible and will drive products back to intermediates and even starting materials. Due to the large presence of all-trans-retinal and all-trans-retinal dimer, I believe the reaction works both ways even while being purified on a column. Next, pyridinium bis-retinoids are very delicate due to their all trans confirmation. Because of the great amount of impure compound eluted off the column I believe our target compound is being degraded in the harsh environment within the column. Finally, the photoreactivity of pyridinium bis-retinoids is a double-edged sword. The photoreactivity is the specific characteristic we plan on exploiting through PDT, however it is also a hindrance during purification. Any light contamination can result in isomerized pyridinium bis-retinoid product.
Regardless of the struggles I learned many lessons and have already started to formulate better approaches to purifying pyridinium bis-retinoid compounds. I look forward to cellular tests with A2DA as a cancer-fighting drug and hope to obtain larger amounts of folate-linked A2DA to evaluate preferential up-take in cancer cells. I still believe A2NOON holds potential as a drug because of the embedded oxygen, and look forward to purification through refined methods.
- American Cancer Society. Cancer Facts and Figures 2006 Atlanta: 2006. 1-64. Am. Canc. Soc.; 2006.
- Wilson, B.C. Photodynamic therapy for cancer: Principles Canadian J. of Gastroenter. 2002; 16(6): 393-396.
- Dolmans, D.; Fukumura, D.; Jain J.R. Photodynamic therapy for cancer Nature Reviews Canc. 2003; 3(5): 380-387.
- Sparrow, J.R.; Nakanishi, K.; Parish, C.A. The Lipofuscin Fluorophore A2E Mediates Blue Light-Induced Damage to Retinal Pigmented Epithelial Cells Invest. Ophthalmol. Vis. Sci. 2000, 41, 1981-1989.
- Parish, C.S.; Hashimoto, M.; Nakanishi, K.; Dillon, J.; Sparrow, J.R.; Isolation and one-step preparation of A2E and iso-A2E fluorophores from human retinal pigment epithelium Proc. Natl. Acad. Sci. 1998, 95, 14609-14613.