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J. Phys. Chem. B 2007, 111, 2322-2326
Hole Transfer in DNA and Photosensitized DNA Damage: Importance of Adenine Oxidation Kiyohiko Kawai,* Yasuko Osakada, Mamoru Fujitsuka, and Tetsuro Majima* The Institute of Scientific and Industrial Research (SANKEN), Osaka UniVersity, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan ReceiVed: September 21, 2006; In Final Form: December 22, 2006
Photosensitized DNA damage reactions were investigated for two well-known DNA-damaging photosensitizers (Sens), naphthalimide (NI) and napthaldiimide (NDI), which have similar photophysical properties but differ in their redox properties. NI and NDI derivatives (NIN, NDIN), which have cationic side chains and electrostatically binding to DNA due to favorable electrostatic interactions between the negatively charged phosphate groups of DNA and cationic groups, and NIP and NDIP, which possess phosphate groups and do not bind to DNA, were synthesized. NIN and NDIN can oxidize A and G via their singlet excited state, and NDIP oxidizes A and G via its triplet excited state, whereas NIP oxidizes only G. A combination of laser flash photolysis kinetic studies and quantitative HPLC analyses of photosensitized DNA damage was performed for several DNA sequences in the presence of Sens. NIN, NDIN, and NDIP, which oxidizes A, caused significant DNA damage upon photoirradiation, and DNA damage yield increased with the length of the consecutive A stretch. In contrast, NIP, which oxidizes only G, caused only moderate damage to DNA and showed no preference for the consecutive A sequences. These results clearly demonstrate the importance of A-oxidation, especially in consecutive A sequences, which triggers the rapid hole transfer between A’s.
Introduction The photosensitized reactions of endogenous or exogenous photosensitizers (Sens) in the vicinity of DNA have been extensively studied because this leads to the formation of oxidative lesions in DNA that cause carcinogenesis and aging. On the other hand, if this process could be efficiently controlled in a living cell, it may provide a new methodology for destroying malignant tissues during photodynamic therapy. To either suppress or to promote photosensitized DNA damage, it is important to understand the involved kinetic mechanisms.1-13 Photoirradiation of Sens triggers electron transfer from the nucleobases to the excited Sens to produce the radical anion of the Sens (Sens•-) and the radical cation of the nucleobase (hole). Among the four DNA bases, guanine (G) exhibits the lowest oxidation potential and is most subject to damage.14-21 For the photosensitized DNA damage by the one-electron oxidation mechanism to occur, the processes leading to the DNA damage, such as reaction of the G radical cation (G•+) with water, should take place faster than the charge recombination, a process which protects DNA from photochemical damage. It has been wellestablished that a hole can migrate through DNA by hopping between the G’s;22-34 however, the rate constants of hole transfer between G’s across A-T base pairs have been determined by Lewis et al. to be too slow to compete with charge recombination.35,36 In 2001, Giese et al. revealed that a hole can also be carried by the second most easily oxidized base adenine (A),37,38 and recently, we have demonstrated that hole transfer between adjacent A’s proceeds with a rate constant of ∼1010 s-1 and can compete with the charge recombination to produce a long* Corresponding authors. (K.K.) Phone: +81-6-6879-8496. Fax: +81-6-6879-8499. E-mail:
[email protected]. (T.M.) Phone: +81-6-6879-8495. Fax: +81-6-6879-8499. E-mail:
[email protected].
lived charge separated state.39-47 By using the DNA in which a photosensitizer is covalently attached to A-stretch sequences, it was clearly shown that, in the initial step of the photosensitized one-electron oxidation of DNA before a hole is trapped at G, the rapid hole transfer between A’s helps to separate the Sens•and a hole, causing DNA damage by providing the time for G•+ or Sens•-, or both, to react with water or O2.43 Consequently, consecutive A sequences were demonstrated to serve as a good target in photosensitized DNA damage, or G adjacent to such sequences may be a potential hot spot of oxidative DNA damage.44 However, photosensitizers are usually not covalently bound to DNA in the living cell. Therefore, it is important to describe the effect of adenine oxidation on the photosensitized DNA damage when photosensitizers are noncovalently bound or unbound to DNA. Here, to further investigate the importance of A oxidation and following rapid hole transfer between A’s on the photosensitized DNA damage, photosensitized DNA damage caused by the noncovalently bound or unbound Sens was investigated by a combination of laser flash photolysis transient absorption measurements and quantitative HPLC analyses of DNA damage. Materials and Methods Synthesis of Photosensitizers. N-(3-Dimethylaminopropyl)1,8-naphthalimide hydrochloride (NIN), N-propyl-1,8-naphthalimide, 3′-phosphoric acid (NIP), and N,N′-bis-(3-dimethylaminopropyl)-1,4,5,8-naphthaldiimide dihydrochloride (NDIN) were synthesized as previously reported.39,48 N,N′-Bis-propyl-1,4,5,8naphthaldiimide 3′,3′′-diphosphoric acid (NDIP) was synthesized as follows. N,N′-(3-propanol)-1,4,5,8-naphthalimide (12.3 mg, 32 µmol), which was synthesized according to the reported procedure,49 was dried by coevaporation with CH3CN (three times) and redissolved in 30 mL of CH3CN. To this solution, 1 mL of 0.3 M 1H-tetrazole and Chemical Phosphorylation
10.1021/jp0661847 CCC: $37.00 © 2007 American Chemical Society Published on Web 02/10/2007
Adenine Oxidation and DNA Damage ‘Reagent (100 µmol, Glen Research) was added. The mixture was stirred for 24 h at room temperature under Ar. A 6-mL portion of Oxidizing Solution (0.02 M I2 in THF/pyridine/H2O, Glen Research) was added to the reaction mixture, which was stirred for 25 h at room temperature under Ar and evaporated. A 10-mL portion of K2CO3/MeOH was added to the reaction mixture under Ar and stirred for 2 days. After neutralization with 6 mL of 80% AcOH, the reaction mixture was purified by HPLC using a 5C18-MS column (8.0 × 150 mm, elution with a solvent mixture of 50 mM ammonium formate, 5-35%/020 min at a flow rate of 2.0 mL/min) and monitored at 355 nm. NDIP was identified by ESI-MS (positive ion): m/e 565 (M + Na). Association Constant Measurements. The ground-state absorption spectral changes were measured for NDIN (20 µM) and NDIP (30 µM) in 20 mM sodium phosphate buffer (pH 7.0) upon addition of DNA-A1 (0-27 µM). The association constants (Ka) of NDI derivatives to DNA were determined by fitting the change of absorption to the McGhee and von Hippel equation as previously reported.48,50 Laser Flash Photolysis. Nanosecond transient absorption measurements were performed using the laser flash photolysis technique.13,40,42-46,51 Briefly, the third-harmonic oscillation (355 nm, fwhm of 4 ns, 20 mJ/pulse) from a Q-switched Nd:YAG laser (Continuum, Surelight) was used for the excitation light, which was expanded to a 1-cm diameter. The light from a xenon flash lamp (Osram, XBO-450) was focused into the sample solution for the transient absorption measurement. Time profiles of the transient absorption in the UV-visible region were measured with a monochromator (Nikon, G250) equipped with a photomultiplier (Hamamatsu Photonics, R928) and digital oscilloscope (Tektronics, TDS-580D). DNA Damage Quantification. Photoirradiation was carried out in an aqueous solution containing 25 µM DNA (strand concentration) and 20 mM pH 7.0 Na phosphate buffer in the presence of 20 µM Sens (total 50 µL). The solution mixture was photoirradiated with a transilluminator (365 nm, 1.2 mW/ cm2), and the reaction mixture was directly subjected to enzymatic digestion with P1 nuclease and alkaline phosphatase. The yield of DNA damage (consumption of G) was quantified by reversed phase HPLC using A as an internal standard. Results and Discussion In this study, naphthalimide (NI) and napthaldiimide (NDI) were used as Sens’s which have similar photophysical properties and are both well-known as harmful Sens that cause DNA damage by the one-electron oxidation mechanism but differ in their redox properties.14,49,50,52 Following the absorption of light, Sens is activated to its singlet excited state (1Sens*), which may convert to its triplet excited state (3Sens*) through intersystem crossing. As described by Kelly and her co-workers, photosensitized DNA damage by NI and NDI can be induced by oxidation through electron transfer from DNA to 1Sens* or to 3Sens*. When NI and NDI are bound to DNA, electron-transfer mainly occurs via their singlet excited-state because the electrontransfer rates from DNA to 1NI* and 1NDI* are much faster as compared to the rates of the intersystem crossing that leads to the formation of their triplets. In contrast, in the case that NI and NDI are not bound to DNA, the collisional reactions via 3NI* and 3NDI* become more significant because the lifetime of 1NI* and 1NDI* are too short for the collisional reaction to take place.48,50,53 Both NI and NDI can oxidize A in their singlet excited state. Although NI has a higher triplet energy than NDI, only 3NDI* can oxidize A in the triplet excited state because of
J. Phys. Chem. B, Vol. 111, No. 9, 2007 2323
Figure 1. Structures of NIN, NIP, NDIN, and NDIP and sequences of DNA.
Figure 2. Ground-state absorption spectral changes observed upon addition of ODN-G1 (0-27 µM, strand concentration) to 20 µM NDIN and 30 µM NDIP (inset) in 20 mM sodium phosphate buffer (pH 7.0).
TABLE 1: Association Constants (Ka) of NI and NDI Derivatives to DNA-A1 Sens
Ka (M-1)
NIN NIP NDIN NDIP
4.7 × 104 (ref 48)
