ARTICLE pubs.acs.org/JPCB
Excited-State Dynamics in 6-Thioguanosine from the Femtosecond to Microsecond Time Scale Christian Reichardt, Cao Guo, and Carlos E. Crespo-Hernandez* Department of Chemistry and the Center for Chemical Dynamics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, United States ABSTRACT: Patients treated with the immunosuppressant and anticancer drugs 6-thioguanine, azathioprine, or mercaptopurine can metabolize and incorporate them in DNA as 6-thioguanosine. The skin of these patients is sensitive to UVA radiation, and long-term treatment can result in extremely high incidence of sunlight-induced skin cancer. In this contribution the photophysics of 6-thioguanosine have been studied in aqueous buffer solution and in acetonitrile after excitation with UVA light to provide mechanistic insights about the origin of its phototoxicity. It is shown that most of the initial excited-state population in the S2(ππ*, La) state decays by ultrafast intersystem crossing to the triplet manifold. A triplet quantum yield of 0.8 ( 0.2 is determined in aqueous buffer solution. A minor fraction of the S2 population bifurcates on an ultrafast time scale to populate the S1(nSπ*) state, which decays back to the ground state in tens of picoseconds. Quantum-chemical calculations that include solvent effects support the experimental results. The high triplet yield of 6-thioguanosine, which we argue can result in photosensitization of molecular oxygen and photooxidative DNA damage, is proposed to explain the high phototoxicity exhibited by these pro-drugs in patients upon sunlight exposure. Finally, the experimental and computational results for 6-thioguanosine are compared with those reported for the DNA/RNA guanine monomers.
1. INTRODUCTION 6-Thioguanine (6tGua) and other purine derivatives are members of a family of biomolecules known as pro-drugs, which are widely prescribed for maintenance therapy of acute lymphoblastic leukemia, for inflammatory bowel disease that is unresponsive to steroids,1 and for gliomas.2,3 In particular, 6tGua is a cytotoxic agent of clinical relevance4-6 and has been used in cross-linking studies and as a site-specific optical probe.7,8 6-Thioguanosine (6tGuo), a metabolite of 6tGua,9,10 is a single-atom-substituted guanine base analogue that absorbs in the ultraviolet A region (UVA) of the electromagnetic spectrum (Figure 1). Previous works have shown that irradiation of DNA containing 6tGuo with UVA light can damage DNA causing cell death.11-14 Furthermore, the DNA of patients treated with immunosuppressant and anticancer drugs azathioprine or mercaptopurine incorporates 6tGuo after metabolization.9,10 The skin of these patients is sensitive to UVA radiation, and long-term treatment can result in extremely high incidences of sunlightinduced skin cancer.15 Studies have shown that radiation of 6tGuo with UVA light results in the formation of reactive oxygen species in cells treated with thiopurines.16-18 It is expected that the triplet excited state of 6tGuo plays a major role in its phototoxicity, but direct evidence of the population of the triplet state after UVA light absorption and its quantum yield is lacking. In this contribution, we investigated the excited-state dynamics of 6tGuo in phosphate buffer and in acetonitrile solutions. As recently shown for the DNA thymidine analogue, 4-thiothymidine (4tThd),19,20 replacement of the oxygen in the carbonyl group of guanosine by a sulfur atom results in the r 2011 American Chemical Society
ultrafast population of the triplet state in high yield due to enhanced spin-orbit and vibronic coupling between the singlet and triplet manifold. A triplet yield of 0.8 ( 0.2 is estimated in aqueous buffer solution with reference to that of 4tThd.19 Our results support the idea that when 6tGuo is incorporated in DNA it can act as an efficient UVA photosensitizer causing oxidative DNA damage and cell death. Finally, the steady-state photophysics and excited-state dynamics of 6tGuo are compared with those of the DNA/RNA guanine monomers.
2. EXPERIMENTAL METHODS 2.1. Chemicals and Steady-State Measurements. 2-Amino6-mercapto-9-(β-D-ribofuranosyl)purine hydrate (>98% purity), also known as 6-thioguanosine or 6-mercaptoguanosine, was obtained from Carbosynth Limited, Berkshire, UK, and used as received. Phosphate buffer solutions were freshly prepared using 0.24 g of sodium dihydrogen phosphate and 0.177 g of disodium hydrogen phosphate dissolved in 200 mL of ultrapure water and adjusted to pH 7.0 using a diluted solution of NaOH. The steadystate absorption spectrum was measured at room temperature using a Cary 100 spectrophotometer (Varian, Inc.). Fluorescence measurements were recorded using a Cary Eclipse spectrofluorimeter (Varian, Inc.). The absorbance of the solutions at the excitation wavelength of 310 nm was
