Ultrafast Electron-Transfer Reactions between Thionine and Guanosine Bases Gavin D. Reid,* Douglas J. Whittaker, Mark A. Day, Caitriona M. Creely, ² Eimer M. Tuite, ‡ John M. Kelly, ² and Godfrey S. Beddard School of Chemistry, UniVersity of Leeds Leeds, LS2 9JT, UK ReceiVed January 25, 2001 ReVised Manuscript ReceiVed June 8, 2001 Femtosecond electron-transport processes in DNA have re- ceived renewed attention both experimentally 1-3 and theoretically. 4-8 From an experimental perspective, optical excitation of an intercalated dye is one method by which electrons or holes can be rapidly injected into DNA strands. 3 Moreover, it has been argued recently that DNA flexibility is the key to its functionality and intercalated dyes can be used as probes of these dynamics. 9,10 Fiebig et al. 11 have recently reported on the photophysics of ethidium complexes with mononucleotides and polynucleotides in water. In this study it was necessary to consider the interplay between orientational motion and electron transfer, since both occurred on a picosecond time scale. In contrast, we present data here with thionine, which illustrate that both the forward and backward processes can be extremely rapid on the time scale of orientational motion, provided that both the energetics and the geometry are favorable. The excited state of DNA itself is extremely short-lived 12 and it has been suggested that this may protect the molecule from photodamage. However, photodynamic degradation of DNA may also be induced by ultrafast redox reactions. 13 Here, the pheno- thiazine family of dyes has attracted considerable attention 13-18 because the excited states of the dyes are strongly quenched when they bind near guanine bases. This quenching is believed to be due to electron transfer from the guanine to the dye excited state. 19 Quenching by adenine is less favorable. 17,20 We report here, for the first time, that for thionine this process and the subsequent back reaction to reform the ground state (eq 1) both occur on a femtosecond time scale in the polynucleotide, [poly(dG-dC)] 2 , which adopts a B-DNA structure, 21 and slightly more slowly in a thionine-5′-guanosine monophosphate (GMP) complex and in thionine bound to DNA. Solutions of thionine (50 μM, Aldrich) in 5 mM phosphate buffer (pH ) 6.9) containing either [poly(dG-dC)] 2 (0.5 mM), GMP (100 mM), or calf-thymus DNA (ca. 1.2 mM) (Sigma) were studied. Approximately 98% of the thionine is bound, and 1:2 complexes are expected to predominate for GMP. 22 Binding to the polynucleotide and to DNA is intercalative under these conditions, supported by experiment 18 and force field-based energy calculations on methylene blue with a decamer of alternating G-C bases. 15 Samples were excited with 25 fs pulses of 600 nm light from a Ti:sapphire pumped, noncollinear, optical parametric amplifier. The transient species, so-formed, were monitored using a second parametric amplifier, also with 25 fs resolution, either by following the reformation of ground-state thionine at 600 nm, or by loss of its singlet state by observing the stimulated emission at 670 nm. At this latter wavelength neither the ground state (Figure 1) nor the excited state absorb significantly. The relative polarization of the pump and probe was set to 54.7° (“magic angle”) to remove any contributions from orientational effects. As shown in Figure 2a the stimulated emission signal indicates that the thionine excited state, when bound to the polynucleotide, reacts with guanine and is strongly quenched with a single- exponential lifetime of 260 fs. The lifetime of free thionine in the absence of the polynucleotide is 320 ps, 17,23 more than a factor of 1200 longer. Monitoring the transient bleaching at 600 nm allows one to follow reformation of the ground state as the reaction products recombine. The signal recovers with a single- exponential lifetime of 760 fs, and this decay will be a lower limit for the return electron-transfer rate, k r . We estimate the error on the measured lifetimes to be <10%. Similarly, Figure 3 shows that with the mononucleotide, GMP, the excited-state lifetime is * Author for correspondence. E-mail: g.d.reid@chem.leeds.ac.uk. ² Trinity College, University of Dublin, Dublin 2, Ireland. ‡ Department of Chemistry, University of Newcastle, Newcastle, UK. 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(18) Tuite, E.; Norde ´n, B. J. Am. Chem. Soc. 1994, 116, 7548-7556. (19) Beddard, G. S.; Kelly, J. M.; Van der Putten, W. J. M. J. Chem. Soc., Chem. Commun. 1990, 1346-1347. (20) Seidel, C. A. M.; Schultz, A.; Sauer, M. H. M. J. Phys. Chem. 1996, 100, 5541-5553. (21) Loprete, D. M.; Hartman, K. A. Biochemistry 1993, 32, 4077-4082. (22) Tuite, E.; Kelly, J. M. Biopolymers 1995, 35, 419-433. (23) Archer, M. D.; Ferreira, M. I. C.; Porter, G.; Tredwell, C. J. NouV. J. Chim. 1977, 1, 9-12; Yamazaki, I.; Tamai, N.; Kume, H.; Tsuchiya, H.; Oba, K. ReV. Sci. Instrum. 1985, 56, 1187-1194. Figure 1. The absorption spectrum of the thionine-[poly(dG-dC)]2 complex and the emission spectrum of free thionine in water. Pump and probe wavelengths are shown. (The absorption spectrum of free thionine is blue shifted by ca. 15 nm, but the shape remains almost unchanged.) Th + *,G 9 8 k f [Th • ,G •+ ] 9 8 k r Th + ,G (1) 6953 J. Am. Chem. Soc. 2001, 123, 6953-6954 10.1021/ja015584z CCC: $20.00 © 2001 American Chemical Society Published on Web 06/23/2001