* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download DNA/RNA nucleotides and nucleosides: direct measurement of
Heat transfer physics wikipedia , lookup
Photoacoustic effect wikipedia , lookup
X-ray photoelectron spectroscopy wikipedia , lookup
Chemical thermodynamics wikipedia , lookup
Physical organic chemistry wikipedia , lookup
Rotational spectroscopy wikipedia , lookup
Mössbauer spectroscopy wikipedia , lookup
Chemical imaging wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Rotational–vibrational spectroscopy wikipedia , lookup
Two-dimensional nuclear magnetic resonance spectroscopy wikipedia , lookup
Atomic absorption spectroscopy wikipedia , lookup
Magnetic circular dichroism wikipedia , lookup
X-ray fluorescence wikipedia , lookup
Franck–Condon principle wikipedia , lookup
Astronomical spectroscopy wikipedia , lookup
9 November 2001 Chemical Physics Letters 348 (2001) 255±262 www.elsevier.com/locate/cplett DNA/RNA nucleotides and nucleosides: direct measurement of excited-state lifetimes by femtosecond ¯uorescence up-conversion Jorge Peon, Ahmed H. Zewail * Laboratory of Molecular Sciences, Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena, CA 91125, USA Received 20 August 2001 Abstract Fluorescence decay times of the nucleosides: adenosine, guanosine, cytidine and thymidine, and of the corresponding nucleotides, were determined using the technique of ¯uorescence up-conversion with femtosecond time resolution. The excited-state lifetimes of these nucleic acid molecules all fall in the sub-picosecond time scale, con®rming the presence of an ultrafast internal conversion channel for both the nucleotides and the nucleosides; the nucleotides lifetimes are longer than those of the nucleosides by up to 20%. The ultrafast internal conversion is biologically relevant to the stability of DNA, and our results support the sub-picosecond repopulation of the ground state, consistent with transient absorption studies on the femtosecond time scale. Ó 2001 Published by Elsevier Science B.V. 1. Introduction The pathways of electronically excited nucleic acid molecules are of considerable interest in photochemistry and photobiology. Areas of potential relevance of these studies include: the understanding of DNA and RNA photodamage and repair [1,2], the use of intrinsic ¯uorescence as a probe of DNA's molecular dynamics [3,4], and the studies of energy transfer among the bases in polynucleotides [5,6]. Our interest in this subject arises from the on-going research on * Corresponding author. Fax: +1-626-792-8456. E-mail address: [email protected] (A.H. Zewail). DNA-mediated electron transfer and DNA's characteristics when recognized by other ligands [7±10]. Early steady-state measurements of DNA and RNA bases, nucleosides and nucleotides demonstrated that these nucleic acid molecules exhibit extremely low ¯uorescence quantum yields, on the order of 10 4 at room temperature in aqueous solutions (see Table 1) [11±15]. Also, several studies have shown that the weak emission from these compounds exhibits a large degree of anisotropy, even in the solution phase, indicating that the ¯uorescent-state lifetimes are shorter than their rotational reorientation times [11,16±18]. This set of observations strongly suggested that the electronically excited-state dynamics of these 0009-2614/01/$ - see front matter Ó 2001 Published by Elsevier Science B.V. PII: S 0 0 0 9 - 2 6 1 4 ( 0 1 ) 0 1 1 2 8 - 9 256 J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262 Table 1 Measured lifetimes together with some photophysical parameters of the nucleosides and nucleotides Nucleosides a Fluorescence maximum (nm) a Fluorescence quantum yield 10 4 b Lifetime from transient absorption (fs) c Lifetime from ¯uorescence up-conversion (fs) Nucleotides A G T C A G T C 310 346 327 324 312 340 330 330 0.5 ± 1.0 0.7 0.5 0.8 1.2 1.2 290 460 540 720 ± 700 ± ± 860 100 980 120 950 120 530 120 690 100 700 120 760 120 520 160 Reported values correspond to room temperature aqueous solutions. kmax and /f for AMP, GMP, TMP and CMP were taken from [14, Table 1]; for Ado, Cyd and Thd from [15, Table 1]. kmax for Guo was taken from [19] . b The lifetimes of the nucleosides from transient absorption are taken from [24,25]. For GMP, the value is taken from [26]. The uncertainty in these values was reported as 40 fs. a molecules are dominated by a non-radiative decay to a non-emitting state. Using streak cameras [6,19,20], several studies have attempted the resolution of these lifetimes, but found that the decays were within the time resolution of the apparatus. The time resolution of the streak-camera method is limited to a few picoseconds, and latter studies have indicated that the reported lifetimes can only be considered an upper bound (vide infra). Pump-probe transient absorption studies have been made with sub-picosecond time resolution and have obtained excited-state lifetimes of the nucleobases on the order of 1 ps [21± 23]. Some of these studies were in¯uenced by multiphoton processes which lead to ionization in the solvent and/or solute. As pointed out elsewhere [24], for accurate results the experiments in the solution phase require careful consideration of the pulse intensities as in many cases ionization of the solvent can overwhelm the observed signal. Recently, Pecourt et al. [24±26] made the ®rst study of the excited-state dynamics of some nucleosides with femtosecond time resolution. In these transient absorption experiments, sub-picosecond decay signals in the visible region were observed and assigned to the lifetimes following the probing of the S1 ! Sn absorption. Also, when probing the UV spectral region near the red edge of the ground state absorption, signals characteristic of the vibrationally excited ground state were observed. These time dependent spectral features indicated that the ultrafast internal conversion gives rise to the electronic ground state with a large excess energy (up to about 34 000 cm 1 ) which is then transferred to the solvent on the time scale of about 2±4 ps (vibrational cooling). In order to obtain direct measurements of the lifetimes of the S1 state, without contribution from vibrational relaxation and solvation eects, direct resolution of the ¯uorescence is needed on the femtosecond time scale. In this Letter, we report a direct determination of the excited-state lifetimes of the eight nucleosides and nucleotides listed in Scheme 1. This was accomplished by resolving the emission decay using the technique of ¯uorescence up-conversion in a non-linear optical crystal. The sucient time resolution of this method provides accurate values of the decay and allows us to observe the dependence on base structure. The shortest lifetime measured in water solution is 520 160 fs and the longest is 980 120 fs. Clearly, in all the nucleosides and nucleotides studied, these molecules dissipate the energy very eciently. 2. Experimental 2.1. Up-conversion setup The third harmonic of a 1 kHz chirped pulse ampli®ed Ti:sapphire laser (k 270 nm, 0:5 lJ) was used to excite the samples. Emission was col- J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262 257 Scheme 1. Molecular structures of the nucleosides and nucleotides studied here. lected by a pair of parabolic focus mirrors and crossed at an angle of about 5° with the gate beam in the non-linear crystal. Fundamental pulses k 810 nm were used to gate the ¯uorescence in a 0.3 mm bBBO crystal (hcut 44°). The up-converted wavelength was in all cases near the maximum of the emission spectrum for each compound (see Fig. 1). The UV signal that results from the sum-frequency mixing was dispersed in a doublegrating monochromator and detected by a photomultiplier tube. The instrument response time of 360 fs was obtained from a dierence frequency-mixing scheme of the pump and gate pulses using the same crystal. The relative polarizations of the two beams were adjusted at the magic angle (54.7°) to remove the contribution from rotational reorientation. The crystal's acceptance axis for the type-I non-linear interaction was adjusted to be parallel to the gate beam's polarization axis. Solutions were studied in a 1 mm path-length rotating circular cell to avoid thermal and cumulative eects. The integrity of the samples was veri®ed by conventional absorption spectroscopy on the Fig. 1. Steady-state absorption and ¯uorescence spectra of the nucleotides studied here. The spectra were acquired in a 1 cm cell at the concentration of 10 4 M in a 50 mM, pH 7 phosphate buer. The excitation wavelength for the ¯uorescence was 260 nm. samples before and after the up-conversion experiments. 2.2. Samples The following compounds were obtained from Sigma and used without further puri®cation; RNA nucleosides: adenosine (Ado), cytosine (Cyd) and guanosine (Guo). DNA nucleoside: thymidine 258 J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262 (Thd). RNA nucleotides: adenosine 50 -monophosphate (AMP), cytosine 50 -monophosphate (CMP) and guanosine 50 -monophosphate (GMP). DNA nucleotide: thymidine 50 -monophosphate (TMP). Unless indicated, aqueous solutions were prepared in a 50 mM, pH 7 phosphate buer using HPLC-spectrophotometric grade water (Omnisolv, EM Science). The concentration of the nucleosides and nucleotides in these studies was 3 mM with the exception of Guo; because of its reduced solubility in water, a concentration of 1.5 mM was used. Previous studies have veri®ed the absence of association eects at similar concentrations for several nucleic acid monomers [19,20,24]. Steady-state absorption measurements were made in a Cary 500 spectrophotometer (Varian). Static ¯uorescence spectra were taken in a Fluormax2 spectro¯uorometer (Instruments S.A.). All experiments were made at room temperature (294 1 K). 3. Results and discussion The steady-state absorption and ¯uorescence spectra of the nucleotides studied are presented in Fig. 1. The ¯uorescence spectra have been multiplied by a relative response function to correct for the wavelength dependence of the spectro¯uorometer. The spectra of the nucleosides are very similar to those of the corresponding nucleotides. The maximum emission wavelength and the overall shape of the spectra are consistent with the room temperature spectra available in the literature [4,6,11,13,14,19]. In all cases, the integrated ¯uorescence is very weak due to the extremely Fig. 2. Femtosecond ¯uorescence up-conversion transients obtained as a function of pump-gate delay time. Aqueous solutions of 3 mM concentration of the nucleosides or nucleotides were made in a 50 mM, pH 7 phosphate buer. J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262 259 Fig. 3. Femtosecond ¯uorescence up-conversion transients obtained as a function of pump-gate delay time. Aqueous solutions of 3 mM Thd (®lled circles) or TMP (open circles) were made in a 50 mM, pH 7 phosphate buer. small ¯uorescence quantum yield. This however is not necessarily a direct forecaster of the up-conversion signal magnitude since up-conversion resolves the emission in a very small time segment. Figs. 2 and 3 show ¯uorescence up-conversion signals from 3 mM solutions of the nucleotides and nucleosides in the pH 7 phosphate buer. The up-converted ¯uorescence wavelength was 330 nm for Cyd, CMP, Thd and TMP; 320 nm for Ado and AMP; and 340 nm for Guo and GMP. These wavelengths are near the maximum of the respective ¯uorescence spectra. In all cases, the signal corresponds to an instantaneous rise followed by a single exponential decay to the baseline. The decay times of these signals have been obtained from convoluted non-linear least-square ®ts to the data using the gaussian instrument response function. The results have been compiled with other relevant data in Table 1. It should be noticed, particularly for Ado and AMP, that the time resolution of these experiments is close to the sub-picosecond decay and the ®ts have a slight systematic deviation from the data on the decay side of the traces. We veri®ed that no up-conversion signal could be detected from the buered water free of the solute, under identical alignment conditions. Fig. 3 shows the ¯uorescence up-conversion scans for Thd and TMP on the same plot. The presence of the phosphate group in the deoxyribose sugar re- Fig. 4. Femtosecond ¯uorescence up-conversion transients obtained for a series of ¯uorescence wavelengths as a function of pump-gate delay time. The aqueous solution of 3 mM TMP was made in a 50 mM, pH 7 phosphate buer. 260 J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262 sults in a slight increase in the lifetime of the ¯uorescent state, which changes from 700 to 980 fs. Similarly, the lifetime increases from 690 to 860 fs in the Guo/GMP case and from 760 to 950 fs in the Cyd/CMP case. To test for contributions from solvation, Raman scattering and others, we made a series of experiments in which the up-converted ¯uorescence was varied from 310 to 350 nm. As shown for TMP, for all wavelengths, the same single exponential decay to the baseline was observed with only a small dierence in the decay times as indicated in Fig. 4; the scan for kfluor 310 nm where the S=N was only 4, is less reliable. The slightly shorter lifetimes observed in the red side of the ¯uorescence spectrum of TMP, might be a signature of some solvation and vibrational relaxation processes occurring in the S1 state on this time scale. In fact, Callis has indicated that for the nucleobase thymine, vibrational relaxation most likely occurs on a time scale shorter than the excited-state lifetime. This idea was based on the observation that both the ¯uorescence spectrum and the anisotropy remain unchanged upon varying the excitation wavelength from near the 0±0 transition at 290 nm to an excess energy of 6000 cm 1 [15,16]. In Table 1, we also include the excited-state decay times obtained from pump-probe experiments by Pecourt et al. As can be seen, the lifetimes we report here are somewhat longer than those obtained from transient absorption studies. However, the ordering of the lifetimes reported here for the nucleosides is consistent with that from the transient absorption study: Ado < Guo < Thd < Cyd. The discrepancies between the transient absorption and the up-conversion measurements may be due to the fact that transient absorption, at certain wavelengths, could show a signi®cant contribution from solvation/vibrational relaxation. In ¯uorescence up-conversion, the observed decay at dierent wavelengths of the emission band separates the decay of unrelaxed and relaxed population. We further made studies of the eect of pH on the excited-state decay characteristics; these measurements were helpful in elucidating the power of up-conversion in resolving, with high sensitivity, the decay of protonated and unprotonated species. We made an up-conversion study of a solution of Guo at pH 3. Fujiwara et al. [19] performed streak-camera measurements of the ¯uorescence of these solutions and were able to isolate the weak, fast decaying ¯uorescence of neutral guanosine molecules from the more prominent, long lived ¯uorescence of guanosine molecules protonated at position 7 GuoH . The ¯uorescence spectrum of the protonated form is centered at 382 nm and overlaps the ¯uorescence from Guo, whose maximum occurs at 346 nm. As Fujiwara et al. showed, in the pH regime where protonated and un-protonated species coexist, the time resolved emission shows a fast decaying feature due to Guo and a much longer component from GuoH . The results of our up-conversion experiments are presented in Fig. 5. The ¯uorescence wavelength for this experiment was 340 nm; the pH of the solution was adjusted by adding a few milliliters of an HCl solution to the Guo solution. At this pH the Guo=GuoH ratio is approximately 6.3 (pKa GuoH 2:2) [19]. The traces in Fig. 5 are described by the sum of three exponential terms with s1 660 fs (67%), s2 3:4 ps (18%) and s3 209 ps (15%). The long lifetime we observed is consistent with the 197 ps component resolved in the streak-camera experiment and corresponds to the ¯uorescence lifetime of Fig. 5. Femtosecond ¯uorescence up-conversion transients obtained as a function of pump-gate delay time. Aqueous solutions of 3 mM Guo at pH 3 were used. J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262 GuoH . The sub-picosecond component we resolved for the pH 3 solution is the same as the ¯uorescence lifetime we measured at pH 7 for Guo. The 3.4 ps component is most likely due to relaxation processes in the S1 state of GuoH at the probing wavelength. In the streak-camera experiment, the ultrafast component was reported to have a 5 1 ps time constant. When we sum the contributions from our two observed ultrafast decays of 660 fs and 3.4 ps, a 85% total amplitude is obtained, in agreement with the relative amplitude of 90% of the 5 ps component measured with the streak-camera. Thus, with our up-conversion sensitivity we are able to resolve the true ultrafast dynamics of the species present. This case-study demonstrates the suitability and improved accuracy of the up-conversion experiments for the characterization of the ultrafast decay even in the presence of longer-lived ¯uorescent species. The mechanism for the rapid electronic deactivation of the nucleic acid molecules by internal conversion is clear, but the pathways for energy disposal need further elucidation. Theoretical studies have pointed out that the ultrafast quenching is probably related to the vibronic coupling between nearby np and pp states, which occurs in heterocyclic and aromatic-carbonyl molecules (proximity eect or pseudo Jahn±Teller distortion) [27±29]. Also, conical intersections have been proposed to be responsible for ultrafast internal conversion processes in several molecules [30±32], including nucleic acid components [24]. A complete understanding of the femtosecond deactivation mechanism should account for several observed phenomena, including the ordering of the excited-state lifetimes of the four nucleotides, the dramatic change upon modi®cations of the molecular structure, and the in¯uence of the phosphate binding (nucleosides vs nucleotides) on the observed lifetimes. 4. Conclusion Our results provide direct measurements of the femtosecond decay of the electronically excited states of eight nucleosides and nucleotides. 261 Because we monitor the population loss to the ground state, these measurements provide accurate description of the non-radiative decay by internal conversion. The range of lifetimes measured is from 520 to 980 fs. Vibrational relaxation and solvation occur on a time scale less than or comparable to those observed here. On such a time scale, the conversion of electronic energy to vibrational dissipation is biologically relevant as it ensures a highly photostable molecular structure in the DNA and RNA assemblies. Acknowledgements This work was supported by a grant from the National Science Foundation. We wish to thank Dr. Dongping Zhong for his help with the initial setup of the experiments. References [1] H. Mukhtar, C.A. Elmets, Photochem. Photobiol. 63 (1996) 355. [2] M.D. Sevilla, in: B. Pullman, N. Goldblum (Eds.), Excited States in Organic Chemistry and Biochemistry, Reidel, Dordrecht, 1977, p. 15. [3] S. Georghiou, T.D. Bradrick, A. Philippetis, J.M. Beechem, Biophys. J. 70 (1996) 1909. [4] S. Georghiou, L.S. Gerke, Photochem. Photobiol. 69 (1999) 646. [5] M. Gueron, J. Eisinger, R.G. Shulman, J. Chem. Phys. 47 (1967) 4077. [6] R. Plessow, A. Brockhinke, W. Eimer, K. Kohse-H oinghaus, J. Phys. Chem. B 104 (2000) 3695. [7] T. Fiebig, C. Wan, S.O. Kelley, J.K. Barton, A.H. Zewail, Proc. Natl. Acad. Sci., USA 96 (1999) 1187. [8] C. Wan, T. Fiebig, S.O. Kelley, C.R. Treadway, J.K. Barton, A.H. Zewail, Proc. Natl. Acad. Sci., USA 96 (1999) 6014. [9] B. Onfelt, P. Lincoln, B. Norden, J.S. Baskin, A.H. Zewail, Proc. Natl. Acad. Sci., USA 97 (2000) 5708. [10] C. Wan, T. Fiebig, O. Schiemann, J.K. Barton, A.H. Zewail, Proc. Natl. Acad. Sci., USA. 97 (2000) 14052. [11] J.P. Morgan, M. Daniels, Chem. Phys. Lett. 67 (1979) 533. [12] J.P. Morgan, M. Daniels, Photochem. Photobiol. 31 (1980) 207. [13] P. Vigny, M. Duquesne, in: J.B. Birks (Ed.), Organic Molecular Photophysics, Wiley, New York, 1976, p. 167. [14] P. Vigny, J.P. Ballini, in: B. Pullman, N. Goldblum (Eds.), Excited States in Organic Chemistry and Biochemistry, Reidel, Dordrecht, 1977, p. 1. 262 J. Peon, A.H. Zewail / Chemical Physics Letters 348 (2001) 255±262 [15] P.R. Callis, Ann. Rev. Phys. Chem. 34 (1983) 329. [16] P.R. Callis, Chem. Phys. Lett. 61 (1979) 563. [17] R.W. Wilson, J.P. Morgan, P.R. Callis, Chem. Phys. Lett. 36 (1975) 618. [18] J.P. Morgan, M. Daniels, Photochem. Photobiol. 27 (1978) 73. [19] T. Fujiwara, Y. Kamoshida, M. Ryuji, M. Yamashita, J. Photochem. Photobiol. B: Biol. 41 (1997) 114. [20] T. H aupl, C. Windolph, T. Jochum, O. Brede, R. Hermann, Chem. Phys. Lett. 280 (1997) 520. [21] D.N. Nikogosyan, D. Angelov, S. Beno^õt, L. Lindqvist, Chem. Phys. Lett. 252 (1996) 322. [22] A. Reuther, H. Iglev, R. Laenen, A. Laubereau, Chem. Phys. Lett. 325 (2000) 360. [23] A. Reuther, D.N. Nikogosyan, A. Laubereau, J. Phys. Chem. 100 (1996) 5570. [24] J.-M. Pecourt, J. Peon, B. Kohler, J. Am. Chem. Soc. 122 (2000) 9348. [25] J.-M. Pecourt, J. Peon, B. Kohler, Errata, J. Am. Chem. Soc. 123 (2001) 5166. [26] J.-M.L. Pecourt, Ph.D. Thesis, Femtosecond pump-probe investigation of excited state dynamics in liquids: intramolecular charge transfer in dimethylaminobenzonitrile and ultrafast internal conversion in nucleic acid components, The Ohio State University, Columbus, OH, 2000. [27] E.C. Lim (Ed.), Excited States, Academic Press, New York, 1977, p. 305. [28] E.C. Lim, J. Phys. Chem. 90 (1986) 6770. [29] A. Broo, J. Phys. Chem. A 102 (1998) 526. [30] M.J. Bearpark, F. Bernardi, S. Cliord, M. Olivucci, M.A. Robb, B.R. Smith, T. Vreven, J. Am. Chem. Soc. 118 (1996) 169. [31] M. Chachisvillis, A.H. Zewail, J. Phys. Chem. A 103 (1999) 7408. [32] A.L. Sobolewski, W. Domcke, Chem. Phys. Lett. 180 (1991) 381.