Time-Resolved Resonance Raman Study of the ... - ACS Publications

5018

J. Phys. Chem. B 2001, 105, 5018-5031

Time-Resolved Resonance Raman Study of the Exciplex Formed between Excited Cu-Porphyrin and DNA Sergei G. Kruglik,†,‡ Peter Mojzes,§ Yasuhisa Mizutani,† Teizo Kitagawa,† and Pierre-Yves Turpin*,# Institute for Molecular Science, Okazaki National Research Institutes, Nishigonaka 38, Myodaiji, Okazaki, Aichi 444-8585, Japan, B. I. StepanoV Institute of Physics, National Academy of Sciences of Belarus, F. Skaryna AVe. 70, Minsk 220072, Belarus, Institute of Physics, Charles UniVersity, Ke KarloVu 5, CS-12116 Prague 2, Czech Republic, and LPBC, CNRS ESA 7033, UniVersite´ Pierre et Marie Curie, Case Courrier 138, 4 Place Jussieu, 75252 Paris Cedex 05, France ReceiVed: NoVember 14, 2000; In Final Form: February 6, 2001

The photoinduced reversible process of exciplex formation and decay between the water-soluble cationic metalloporphyrin 5,10,15,20-tetrakis[4-(N-methylpyridyl)]porphyrin (Cu(T4MPyP)) and calf-thymus DNA has been studied by a picosecond time-resolved resonance Raman (ps-TR3) technique. For a detailed analysis of the exciplex properties, the following model compounds have also been investigated: double-stranded polynucleotides poly(dA-dT)2, poly(dG-dC)2, and poly(dA-dC)‚poly(dG-dT), single-stranded poly(dT), and the 32-mer d[(GC)7ATAT(GC)7]2. Additional Raman measurements have also been done in using cw and 20-ns laser sources. It is shown that this reversible exciplex is formed, with a yield depending on the nucleic base sequence, in less than 2 ps after photoexcitation, between photoexcited Cu(T4MPyP) and CdO groups of thymine residues in all thymine-containing sequences of nucleic acids. Such a rapid exciplex building process implies that it involves porphyrin molecules initially located, in the steady state of this interaction, at AT sites of the nucleic acids. This has two main consequences, which contradict previously reported assumptions (Strahan et al., J. Phys. Chem. 1992, 96, 6450): (i) although the binding mode of the porphyrin actually depends on the base sequence, there is no preferential binding of Cu(T4MPyP) to the various sites of DNA, and (ii) there is no photoinduced ultrafast porphyrin translocation from GC to AT sites of DNA. In addition, it is shown that with surrounding water molecules an exciplex can also be formed in ∼1 ps, whose spectral characteristics are not distinguishable from those formed with thymine residues. However, these two exciplex species can be distinguished from each other by their relaxation kinetics: the lifetime of the exciplex formed with water lies in the 3-12 ps range, while that of the exciplex formed with nucleic acids lies in the nanosecond time domain (1-3 ns). A set of possible routes is discussed for each of the exciplex building/ decay processes.

1. Introduction Since the discovery of Fiel et al.1 that water-soluble porphyrins can intercalate into B-form DNA, numerous studies on porphyrin complexes with DNA, RNA, and their model compounds have been performed,2-21 with the aim to exploit the great potential of medical, biological, and photophysical applications of porphyrins, on the basis of a good knowledge of their physicochemical properties. Cationic meso-substituted (metallo)porphyrins are proved to be of interest in many areas, for example, as probes of the local nucleic acid structure and dynamics, as artificial nucleases, and as possible DNA photosensitizers to be used in photodynamic therapy (see reviews, refs 2-5). It is now commonly accepted that there are three different porphyrin binding modes to nucleic acids,2-4,6 depending on many parameters, such as the base pairs sequence, the structural * To whom correspondence should be addressed. E-mail: turpin@ lpbc.jussieu.fr. † Okazaki National Research Institutes. ‡ National Academy of Sciences of Belarus. § Charles University. # Universite ´ Pierre et Marie Curie.

features of the proper (metallo)porphyrin, and the aqueous buffer properties: (i) intercalation between nucleic acids base pairs, (ii) various outside groove bindings (in the major and/or minor grooves of the double helix), and (iii) outside self-stacking along the nucleic acids surface. Concerning the influence of the base pairs sequence, intercalating drugs seek out GC-rich regions, while outside groove binders show a preference for AT-rich regions.2-7 It is possible that AT-rich structures, having only two connecting hydrogen bonds, are more flexible than GCrich ones, thus allowing a geometry optimization to make contacts with the porphyrin molecule easier. It is worth noting that the formation of self-stacked porphyrin assemblies is a quite peculiar type of interaction that only occurs for specific porphyrins under precise conditions of concentration and ionic strength.8 The Cu(II) derivative of 5,10,15,20-meso-tetrakis(4-N-methylpyridyl)porphyrin (in short Cu(T4MPyP) or CuP), which has no axial ligand in the ground electronic state, is known to be a good intercalator into B-form DNA.2-7,9-14 Since even a single GC base pair may determine a site of intercalation,5,13 the porphyrin can intercalate between both GC/CG and GC/AT steps. At the same time, it can also be groove bound at ATrich regions of the DNA.4,5,7,12-14

10.1021/jp004207q CCC: $20.00 © 2001 American Chemical Society Published on Web 05/03/2001

Ps-TR3 Study of CuP-DNA Exciplex In the recent past, Cu(T4MPyP) attracted a particular interest because, in addition to the steady-state interaction with DNA, a specific photoinduced reaction involving the porphyrin excited states had been discovered15 and then further studied.16-21 In resonance Raman (RR) under cw irradiation, Cu(T4MPyP) mixed with nucleic acids yields prominent vibrational bands around 1570 (ν2) and 1366 (ν4) cm-1 which originate from the ground electronic state.22 Under pulsed, high power excitation, extra Raman bands appear around 1550 (ν2*) and 1346 (ν4*) cm-1, pairing their ground-state counterparts and corresponding to a porphyrin exciplex state, i.e., a CuP-DNA complex formed in the porphyrin excited state.15 This transient exciplex is formed at AT sites of flexible helical structures in poly- and oligonucleotides,17,18 according to the following possible mechanism: an electronically excited Cu(T4MPyP) binds a carbonyl oxygen atom from a neighboring thymine residue as a fifth (axial) ligand to the central Cu2+ ion, thus producing a transient species in the excited 2(d,d) state of the metalloporphyrin.18,19 The puzzling aspect of this photoreaction in DNA is that Cu(T4MPyP) was usually considered as preferentially intercalated at GC sites of the duplex,9-11,17 while groove binding at AT site was thought to be required for the exciplex formation.15-17 Having in mind this logical discrepancy, an ultrafast photoinduced translocation of Cu(T4MPyP) from GC to AT sites has been proposed on the basis of very elegant nanosecond RR experiments.17 However, some ∼10 Å move of such a large molecule as CuP in less than 35 ns17 can be considered as surprisingly fast, and this hypothesis brought about further investigations. Recent RR studies21 of CuP-DNA made using cw radiation, as well as 10-ns and ∼50-ps laser pulses, have shown that neither a photoinduced release of Cu(T4MPyP) in the medium nor a change in the stationary distribution over its binding modes can occur under high power of irradiation. Consequently, the previous hypothesis15-18 that the exciplex is formed during the action of a laser pulse has been confirmed. Moreover, the estimated time range of exciplex formation has decreased from less than 8 ns17 to less than 50 ps.21 In using a rate equations approach, simulations of the measured Raman saturation dependencies have been performed for two models.21 The first one, assuming a preferential CuP intercalation followed by a photoinduced translocation, failed to describe the saturation Raman dependencies. The second one, assuming an initial population of Cu(T4MPyP) groove-bound at AT-sites, well described the ground-state saturation but failed to describe the exciplex behavior. A plausible reason for this failure lies in the fact that the models were too simple to describe such a complicated intermolecular complex as CuP-DNA. On the other hand, a better but more complicated model would involve an extended number of parameters making calculations too arbitrary. The purpose of the present study was to obtain direct dynamic information on the process of exciplex formation between Cu(T4MPyP) and DNA, DNA-model polynucleotides, and possibly water molecules in phosphate buffer, by using a picosecond time-resolved resonance Raman (ps-TR3) technique, which had been revealed very powerful to study ultrafast processes in complex molecular systems.23-25 2. Experimental Section 2.1. Samples. Copper(II) derivative of 5,10,15,20-mesotetrakis(4-N-methylpyridyl)porphyrin (chloride salt) was a generous gift from Professor K. Nakamoto (Marquette University,

J. Phys. Chem. B, Vol. 105, No. 21, 2001 5019 Milwaukee, WI). Highly polymerized calf-thymus DNA was purchased from Sigma. DNA-model polynucleotides, i.e., poly(dA-dT)2, poly(dG-dC)2, poly(dA-dC)‚poly(dG-dT), and poly(dT), were purchased from Sigma and ICN Biochemicals. All compounds have been used as received. The DNA-model oligonucleotide d[(GC)7ATAT(GC)7]2 (32-mer) was purchased from Genosys Co (G.B.). All samples have been prepared by an 1:1 (v:v) mixing of the porphyrin stock solution and the adequate nucleic acid dissolved in a 0.01 M phosphate buffer (pH ) 6.8). The ionic strength was adjusted to µ ) 0.2 M (as in all of the previous Raman studies of CuP-nucleic acids complexes15-21) by adding an appropriate amount of NaCl. Final concentrations of CuP and nucleic acids (in base pairs) determined spectroscopically were ∼2.5 × 10-5 and ∼5 × 10-4 M, respectively, thus giving a base pair/CuP ratio of about 20. After dissolution in the phosphate buffer, the 32-mer was twice annealed by heating to 110 °C and slow cooling to ambient temperature. For ps-TR3 measurements, a reliable Raman intensity calibration was necessary: 0.1 M of (NH4)2SO4 salt was added to the µ ) 0.2 M samples, and the SO42- band located at ∼982 cm-1 has been used as an internal standard of intensity. 2.2. Picosecond Raman Measurements. Picosecond-TR3 experimental setup has been described elsewhere.24b-27 The light source was based on a regenerative Ti:Sapphire amplifier pumped by a Q-switched Nd3+:YLF laser (repetition rate 1 kHz), whose seed pulse was generated by a mode-locked Ti:Sapphire oscillator (working in ps regime) pumped by a cw Ar+ laser. The second harmonic tuned at 383.4 nm fed two different channels: (i) a pump channel based on an OPG-OPA device,26 providing a broadband light (3-4 nm) centered at 548 nm with a pulse energy of ∼20 µJ at the sample cell, and (ii) a probe channel with a H2 Raman shifter27 that provided a narrowband light at 456 nm with a pulse energy of ∼ 6 µJ at the sample cell. Pump and probe beams were collinearly adjusted by using a dichroic mirror and focused onto the sample cell with cylindrical lenses. Typically, one pump pulse at 548 nm transferred 3040% of the molecules to their excited state. The relative orientations of the pump and probe beam polarizations were set at the magic angle (54.7°) to avoid any contribution from the molecular reorientation to the Raman kinetics. The Raman light scattered in a near backscattering geometry was dispersed through a Chromex-500 spectrograph (f ) 500 mm, 2400 rules‚mm-1 grating) and detected by a N2-cooled CCD (Princeton Instruments CCD-1100 PB). The spectral resolution was limited by the line width of the ps probe beam (12-15 cm-1 fwhm). The optical time delay (∆t) between the pump and the probe pulses was generated (from -5 to 1200 ps) by using a stepmotor-driven stage. Typical total accumulation time in 4 passes for one ∆t value was 4 min, and the total duration of a ps-TR3 experiment was typically 2 h. The ps-TR3 kinetics has been fitted by using a LevenbergMarquardt nonlinear least squares optimization method. The instrumental time response function was found to have a Gaussian shape with a half-width of TG ) 1.4 ps at 1/e of its maximum, as evaluated from absorption changes of copper(II)tetraphenylporphyrin (CuTPP) in toluene. Accuracy in the determination of the ∆t ) 0 point was better than 0.5 ps. 2.3. Cw Raman Measurements. Cw-RR spectra have been recorded by using the 441.6 nm (∼10 mW) and 457.9 nm (∼25 mW) lines of He-Cd and Ar+ lasers, respectively. The Raman scattering has been measured in a 90° geometry, through a Ritsu

5020 J. Phys. Chem. B, Vol. 105, No. 21, 2001

Kruglik et al. TABLE 1: Absorption Data for Cu(T4MPyP) in Phosphate Buffer (µ ) 0.2, pH 6.8) and in Complexes with Calf-Thymus DNA and DNA-Model Compounds sample

λmax, nm

bandwidth,a nm

% Hb

CuP - water buffer CuP - poly(dA-dT)2 CuP - poly(dT) CuP - DNA CuP - 32mer CuP - poly(dA-dC)‚poly(dG-dT) CuP - poly(dG-dC)2

425 427 429 430 438 440 440

28 22 23 29 32 31 31

-8 11 15 35 32 36

a fwhm. b The % hypochromicity (%H) was determined from ( f b)/f × 100, where f represents free porphyrins, b represents bound porphyrins, and f and b were determined at the respective Soret maxima. A negative hypochromicity demonstrates hyperchromicity.

Figure 1. Absorption spectra in the Soret-band region of Cu(T4MPyP) in water buffer (A) and bound to poly(dA-dT)2 (B), poly(dT) (C), calfthymus DNA (D), 32-mer (E), poly(dA-dC)‚poly(dG-dT) (F), and poly(dG-dC)2 (G). Spectra were displaced for clarity.

Oyo Kogaku DG-1000 spectrograph (f ) 1000 mm, 1200 mm-1 grating) and recorded with a N2-cooled CCD (Astromed LNC/ 1815). The spectral slit width was ∼3 cm-1. 2.4. Nanosecond Raman Measurements. Nanosecond transient resonance Raman spectra (ns-RR) have been recorded with the Raman setup built in Paris, based on a Q-switched Nd:YAG laser pumping a Datachrom 5000 dye laser (Quantel). Excitation at 425 nm (repetition rate 10 Hz, pulse duration 20 ns) has been obtained by frequency mixing the Nd:YAG and dye (LD-700) fundamental outputs. The Raman scattering signal was dispersed in a near backscattering geometry by a triplemate equipped with a N2-cooled CCD detector (Jobin Yvon T64000, f ) 640 mm, 1800 mm-1 grating). The spectral slit width was ∼5 cm-1. In all Raman measurements, the wavenumber scale has been calibrated by using, as a reference, the Raman spectrum of toluene acquired under identical experimental conditions. Samples were contained in spectrophotometric 10-mm quartz cuvettes and stirred by a small magnet. The sample integrity was checked before and after every Raman measurement by absorption spectroscopy in the Soret region, in 2-mm quartz cells by using Hitachi U-3210 and Cary-1E spectrophotometers. No significant change has been found after cw-RR and ns-RR experiments. However, in ps-TR3 experiments, long lasting (∼2 h) irradiation of some samples resulted in a ∼5-10% decrease of the Soret absorbance. Therefore, every new ps-TR3 experiment has been made with a fresh sample. All measurements were performed at room temperature. 3. Results 3.1. Absorption Spectra. All typical spectral changes previously ascribed to intercalation and outside-binding of porphyrins3,6,7 can be recognized in the absorption spectra of Cu(T4MPyP) mixed with calf-thymus DNA and synthetic polynucleotides (Figure 1, Table 1). A small red shift, a bandwidth narrowing and a weak hyperchromicity of the Soret

band (in comparison with free Cu(T4MPyP)), i.e., the common attributes of outside-bound porphyrins, can be seen in the spectra of CuP-poly(dA-dT)2 and CuP-poly(dT) complexes. Nevertheless, a detailed comparison of these two spectra yields slight spectral differences: this likely reflects some understandable differences in the modes of binding of Cu(T4MPyP) to doublestranded polymer versus single-stranded one.5,6 A large red shift, a substantial hypochromicity and an asymmetrical bandwidth broadening of the Soret maximum (Figure 1, Table 1), i.e., features characteristic for intercalated Cu(T4MPyP), are the most apparent spectral changes observed for CuP-poly(dG-dC)2 and CuP-poly(dA-dC)‚poly(dG-dT) complexes. The close resemblance of the absorption spectra of these two complexes well correlates with previous reports5,13 that a site of porphyrin intercalation within a duplex can be defined by a single GC base pair stacked with any other base pair. The absorption spectrum of Cu(T4MPyP) complexed with the 32-mer bears features typical for intercalation (ref 17 and Figure 1E, Table 1). This is quite understandable since there is only one tetrameric site (ATAT)2 suitable for outside-binding (more precisely, 2 AT/TA and 1 TA/AT steps not suitable for intercalation) and 28 steps suitable for intercalation (14 GC/ CG, 12 CG/GC, 2 CG/AT). The absorption spectrum of Cu(T4MPyP) complexed with calf-thymus DNA (Figure 1D) yields features characteristic for both intercalation and outside-binding.6 On one hand, there are an important hypochromism and a weak broadening of the Soret band, on the other, a rather limited red shift of the Soret maximum (along with an asymmetrical band shape) that differs from those of purely intercalated complexes. Cu(T4MPyP) complexed with calf-thymus DNA likely is outside-bound to runs of three5 and/or more consecutive AT base pairs, and also intercalated at GC/CG and AT/CG steps. Cu(T4MPyP) distribution among these different binding sites was shown to be a rather complicated function of the ionic strength, of the porphyrin loading (phosphate-to-porphyrin ratio), and of the percentage of GC base pairs in DNA.5,12,13,28 In this respect, it is worth noting that calf-thymus DNA contains ca. 42% of GC base pairs, and the average occurrences of two GC, two AT, and one ATone GC dimeric steps are 16, 36, and 48%, respectively.29 Consequently, supposing an equal affinity of Cu(T4MPyP) for both types of intercalative sites, intercalation between ATGC steps is three times more frequent than between two GC steps. The influence of the addition of ∼0.1 M of (NH4)2SO4 to the sample solutions, to be used as a Raman intensity standard (see section 2.1), has also been monitored by absorption spectroscopy (not shown). Only very minor spectral changes can be detected for Cu(T4MPyP) complexes with poly(dA-dT)2,

Ps-TR3 Study of CuP-DNA Exciplex

J. Phys. Chem. B, Vol. 105, No. 21, 2001 5021

Figure 2. Resonance Raman spectra (with cw excitation at 441.6 nm) of Cu(T4MPyP) in water buffer (A) and bound to poly(dA-dT)2 (B), poly(dT) (C), calf-thymus DNA (D), 32-mer (E), poly(dA-dC)‚poly(dG-dT) (F), and poly(dG-dC)2 (G).

poly(dT), and DNA: they generally remain within the limits of the measurement accuracy. In the complexes with poly(dG-dC)2 and 32-mer, a very weak blue shift (∆λmax ∼ -1 nm) and a slight broadening (∆λ ∼ 0.4-0.8 nm) at the blue limb of the Soret maximum can be observed. These changes are in qualitative agreement with what was previously reported for the free-base H2(T4MPyP)28: they might involve some small relocation/release processes of the porphyrin from intercalation to the buffer solution or to outside-binding sites as the ionic strength increases. Although these changes are even more pronounced for the Cu(T4MPyP) complex with poly(dA-dC)‚ poly(dG-dT) (i.e., ∆λmax ∼ -1.5 nm, 2.3 nm broadening), the overall spectral contour shows that the major part of Cu(T4MPyP) remains intercalated on addition of 0.1 M (NH4)2SO4. 3.2. Cw Raman Spectra. Figure 2 shows the 441.6 nm cwRR spectra of Cu(T4MPyP) free in aqueous buffer and bound to DNA and DNA-model compounds. Qualitatively, the porphyrin binding mode influences the Raman spectral features in two manners. First, in agreement with results of Schneider et al.,30 the wavenumber position of the line around 1100 cm-1 (Cβ-H bending motion30) is sensitive to the porphyrin intercalation. It goes from 1100 cm-1 for free or outside-bound species (Figure 2A-C), to 1105-1106 cm-1 for intercalated complexes (Figure 2E-G), presumably owing to the rotation of the methylpyridyl rings that allows the porphyrin to intercalate between the base pairs. Second, the Raman intensity ratio of the ν4 and ν2 symmetrical stretching bands (ca. 1365 and 1570 cm-1, respectively) depends on the porphyrin binding mode: it goes from ∼1.05 for free CuP (Figure 2A) or CuP groove-bound to AT runs (Figure 2B) to ∼0.70 when CuP is intercalated at GC/CG (Figure 2G) or AT/CG sites (Figure 2F). The same tendency holds with Raman excitation at 457.9 nm (data not shown). In addition, it is worth noting that the cw Raman spectrum of the CuP-32mer complex (Figure 2E) bears the

same features as those of the intercalative complexes, as expected. Concerning the Raman spectrum of Cu(T4MPyP) bound to calf-thymus DNA (Figure 2D), it shows spectral features intermediate between those of the groove-bound and intercalated species. Indeed, the contour of the Cβ-H bending mode of CuPDNA is asymmetric with a maximum at ∼1103 cm-1 and a prominent low-frequency shoulder, and the amplitude of the ν4-band is slightly weaker than that of the ν2 band. To analyze these cw-Raman spectra more quantitatively, a global fit analysis has been performed: it is possible to approximate almost perfectly the CuP-DNA RR spectrum by a least-squares fitting procedure using those of the following three model compounds CuP-poly(dA-dT)2, CuP-poly(dG-dC)2, and CuP-poly(dAdC).poly(dG-dT): the CuP-poly(dA-dC).poly(dG-dT) spectral contribution is nearly three times greater than that of CuPpoly(dG-dC)2, and again this fits the ratio of the relative proportions of AT-CG and GC-CG intercalative sites mentioned above in calf-thymus DNA (see section 3.1). 3.3. Picosecond TR3 Spectra and Kinetics. Ps-TR3 spectra of the various CuP complexes are shown in Figure 3 in the 1300-1700 cm-1 range. As expected, the probe-only excitation (a) yields CuP ground-state RR features, whereas the two-color pump-probe spectra (b) (taken with ∆t ) 10 ps) contain contributions from both ground and excited electronic states of the porphyrin. For obtaining the neat contribution from the excited state, the following subtractive procedure has been applied, which also takes into account the buffer contribution (spectra (c)). First, probe-only (a) and pump-probe (b) spectra have been normalized by using the internal Raman intensity standard (i.e., the 982 cm-1 line of SO42-) to account for the different light reabsorption in the ground- and excited electronic states, and the different scattering volume (depth of penetration of the probe


Kruglik et al.

Figure 3. Picosecond TR3 spectra of Cu(T4MPyP) bound to poly(dA-dT)2 (A), calf-thymus DNA (B), poly(dT) (C), poly(dA-dC)‚ poly(dG-dT) (D), 32-mer (E), and poly(dG-dC)2 (F). Spectra (a) were measured in a probe-only experiment. Spectra (b) were recorded at time delay ∆t ) 10 ps between pump (548 nm) and probe (456 nm) pulses. Spectra (c) represent the scattering from water molecules. Spectra (d) are difference spectra at ∆t ) 10 ps obtained by weighted subtraction of (a) and (c) from (b). Spectra (e) were obtained by the same procedure as (d), but at ∆t ) -5 ps. See text for further details.

beam) in the presence of the pump beam. Then, difference spectra (d) have been obtained by a weighted subtraction of (a) and (c) from (b) according to the formula:

difference spectrum (d) ) spectrum (b) K1 × spectrum (a) - K2 × spectrum (c) (1) where K1 and K2 factors were adjusted to suppress the Raman features of the ground-state CuP and water, respectively. It should be noted that K1(∆t) reflects the time dependence of the ground-state RR intensity (proportional to the population) measured in the pump-probe experiment, normalized to the probe-only RR intensity. The accuracy of the subtractive procedure (1) is outlined by the spectra (e) obtained at ∆t ) -5 ps. The excited-state difference TR3 spectra (d) are well consistent with the exciplex features previously observed in transient ns-RR spectra of photoexcited CuP bound to AT or T residues containing nucleic acids.15-21 The intensities of the most prominent exciplex bands at ∼1345 cm-1 (ν4*) and ∼1551 cm-1 (ν2*) decrease in the following order: poly(dA-dT)2 > DNA ≈ poly(dT) > poly(dA-dC)‚poly(dG-dT) > 32-mer. No ν4* exciplex features can be detected for the CuPpoly(dG-dC)2 complex: this was expected according to the reported inability of the exciplex to be formed in this case.15,17,18

Figure 4. Probe-only (ν4) and difference pump-probe (ν4*) ps-TR3 spectra of Cu(T4MPyP)-DNA, obtained at various time delays ∆t between pump and probe pulses. ∆t values are indicated in the right side of the corresponding spectra.

However, a very weak excited-state feature centered around ∼1550 cm-1 is visible in the spectrum of Figure 3F, trace d. This might correspond to a RR contribution from the CuP tripletstate. Indeed, two independent studies31 have reported that the transient triplet-state Raman spectrum of Cu(TPP) exhibits a very weak and broad Raman band around the position of ν2*, while no trace of the ν4* band can be found in the 1320-1400 cm-1 range. Therefore, both the exciplex (d,d) and the triplet (π,π*) states of copper(II)-tetraarylporphyrins may contribute to ns- and ps-RR spectra in the 1500-1600 cm-1 region, whereas only the (d,d) exciplex ν4* Raman band can show up in the 1320-1400 cm-1 region. Figure 4 shows, in the 1300-1400 cm-1 spectral region, a set of difference ps-TR3 spectra of CuP-DNA obtained at various time delays ∆t from -5 to 1200 ps. It clearly shows that the exciplex ν4* band at 1345 cm-1 holds the same position and bandwidth during the whole time range, within the spectral resolution of the ps Raman setup. Similar results have been


J. Phys. Chem. B, Vol. 105, No. 21, 2001 5023 poly(dG-dT) (Figure 5D), two rise and two decay components can be clearly identified in the kinetics. Experimental kinetics have been fitted by using a convolution of a Gaussian instrumental response function with two exponential rise-decay processes, by using the following equation:

IRam Fit (∆t) )

Figure 5. Kinetics of the exciplex ν4*-band in difference ps-TR3 spectra of Cu(T4MPyP) bound to poly(dA-dT)2 (A), calf-thymus DNA (B), poly(dT) (C), and poly(dA-dC)‚poly(dG-dT) (D). Markers denote experimental points, and solid curves represent the best-fit profiles. See Table 2 for the best-fit parameters. Note the abscissa is a log scale.

obtained for the CuP-poly(dA-dT)2 and CuP-poly(dT) complexes. This unique exciplex ν4* band observed in all Tcontaining complexes suggests that there exists either a single exciplex species or several exciplex species having identical spectral characteristics. Figure 5 displays, on a ∆t logarithmic scale, the Raman intensity kinetics of the ν4* band of CuP bound to nucleic acids. The ν4* band has been chosen for the following reasons: (i) it allows the triplet-state contribution, which may occur in the ν2* band region (see above), to be avoided, and (ii) it allows the removal of possible contribution from the nonplanar distortions which may introduce an additional complication in the exciplex Raman kinetics of the ν2* mode, as indeed observed in the case of the CuP-poly(dA-dT)2 complex (see Table 2 and discussion below). It has actually been reported32 that the spectral line shape of the ν2 band is sensitive to nonplanar distortions of the porphyrin macrocycle for metal-tetraarylporphyrins, while that of the ν4 band is not. Since the process of exciplex formation goes through a step of axial ligand binding,18,19 domed porphyrin structure is likely to be formed in the course of excitation relaxation, thus implying a complicated behavior of the ν2* band during the kinetics. Three components can be qualitatively recognized in the kinetics of the CuP exciplexes formed with poly(dA-dT)2, DNA, and poly(dT) (Figure 5A, B, and C, respectively): a fast rise at ∆t ∼ 0...4 ps, a slow rise at ∆t ∼ 5...200 ps, and then a slow decay at longer ∆t. Despite a lower accuracy (( 20%) in the determination of the exciplex intensity for CuP-poly(dA-dC)‚

∫0

∞

e-(t - ∆t/TG)

kri

2

2

Ai ∑ (k i)1

ri

- kdi)

(e-kdit - e-krit)dt (2)

where kri ) 1/Tri, kdi ) 1/Tdi, and Ai, Tri, and Tdi represent the normalized intensity, rise, and decay times of the ith exponential rise-decay process (i ) 1,2). Ai, Tri, and Tdi values have been found from a fitting procedure by using a nonlinear least squares optimization method. Three different fitting models have been used, whose parameters are presented in Table 2: Fit 1 used no constraint on the fitting parameters, Tr1 and Td1 being shorter than Tr2 and Td2, respectively. This corresponds to a model involving two different exciplex species (“shortlived” and “long-lived”) spectroscopically identical but yielding independent kinetics. In fit 2, identical decay times of two processes (Td ) Td1 ) Td2) are assumed. This corresponds to a model involving a single exciplex species with a lifetime Td, created with two different, fast (Tr1) and slow (Tr2), rates of formation. Fit 3 used the constraint that the decay time of a first process is equal to the rise time of a second one (Td1 ) Tr2). This corresponds to the formation of a single exciplex species after photoexcitation (Tr1), which undergoes some kinetic transformation (Td1 ) Tr2) and then relaxes to the ground state (Td2). A fairly good agreement between experimental data points and calculated kinetics provided by fit 3 can be seen in Figure 5 for poly(dA-dT)2, DNA, and poly(dT). However, it should be noted that all three models provide almost identical calculated curves, well within the accuracy of the experimental points. Thus it is not possible to choose one particular model only from the fitted excited-state kinetics. The physical relevance of each model will be discussed below. On the other hand, only the fit 1 yields a good correspondence with the experimental data points for the CuP-poly(dA-dC)‚ poly(dG-dT) complex (Figure 5D). However, owing to the lower accuracy of the ν4*-area estimation, the time constants obtained can only be regarded as qualitative (Table 2, row 15). For the CuP-32-mer (Figure 3E) the weakness of the ν4* band (although quite detectable throughout the whole time range) precludes reliable kinetics to be determined. Other information can be derived from the ps-TR3 spectra, about the ground-state depletion/recovery, from a plot of the K1 factor of eq 1 versus the time delay ∆t (Figure 6): it represents the time evolution of the ground-state Raman intensity, normalized to its probe-only value. The ν4 groundstate intensity decreases after the pump pulse for all complexes within the instrumental rate limit and then gradually restores as does the ground-state population during the excitation relaxation. For CuP-poly(dT) and CuP-poly(dG-dC)2, the ν4 groundstate recovery can be fitted by a monoexponential function with a time constant of 1.1 and 4.4 ns, respectively (Table 2, rows 14 and 18, respectively). However, since a luminescence lifetime of ∼22 ns had been previously reported for CuP-poly(dG-dC)2,12 a double-exponential fit has also been tested in imposing Td2 ) 22 ns, which also gives a second major component with Td1 ) 2.8 ns (Table 2, row 19). Both fits (Table 2, rows 18 and 19)


Kruglik et al.

TABLE 2: Best-Fit Parameters of Raman Intensity Kinetics of Cu(T4MPyP) Complexes with Calf-Thymus DNA, DNA-Model Nucleotides, and in Water Buffer N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

complex with poly(dA-dT)2

DNA

poly(dT) poly(dA-dC)‚poly(dG-dT) 32-mer poly(dG-dC)2 water buffer

modea

fitb

A1d

Tr1 (ps)

ν4* ν4* ν4* ν2* ν2* ν2* ν4 ν4* ν4* ν4* ν4 ν4* ν4* ν4 ν4* ν4 ν4 ν4 ν4 ν4* ν4* ν4

1 2 3 1 2 3 4c 1 2 3 4c 2 3 4c 1 4c 4c 4c 4c 3 4 4c

0.37 0.59 0.37 0.40 0.67 0.40

1.3 1.3 1.3 1.3 1.3 1.3

0.41 0.67 0.40 0.16 0.56 0.36

1.2 1.2 1.2

0.54 0.15 0.06

0.7

0.61 0.77 0.65 0.78

1.7 1.7

1.0 1.0

Td1 (ps) 48 Td2 Tr2 68 Td2 Tr2 e 93 Td2 Tr2 6.9 Td2 Tr2 22 5.7 53 2.8 × 103 3.3 3.3 3.0

A2

Tr2 (ps)

Td2 (ps)

0.63 0.41 0.63 0.60 0.33 0.60 1 0.59 0.33 0.60 0.84 0.44 0.64 1 0.46 0.85 0.94 1 0.39 0.23 0.35 0.22

48 48 48 68 68 68

1.9 × 103 1.9 × 103 1.9 × 103 1.8 × 103 1.8 × 103 1.8 × 103 1.8 × 103 2.7 × 103 2.5 × 103 2.5 × 103 2.4 × 103 1.1 × 103 1.1 × 103 1.1 × 103 3.1 × 103 5.5 × 103 3.9 × 103 4.4 × 103 22 × 103 12 12 25

71 44 44 64 64 60

Td1 Tr1

a Kinetics of the modes ν * and ν * represent the formation/decay of the excited-state species. Kinetics of the mode ν represents the ground4 2 4 state depletion/recovery. b Calculations have been performed according to eq 2. The estimated inaccuracy of time constants is ( 20%. Fit 1 was calculated without constrains on fitting parameters. Fit 2 was calculated with the constrain Td1 ) Td2. Fit 3 was calculated with the constrain Td1 ) Tr2. Fit 4 was calculated with the constrain Tr1 ) Tr2. c Fitting of the ground-state ν4-band kinetics was performed using a modified eq 2 with the constrain Tr1 ) Tr2 ) 0. d A1 and A2 were normalized on unity. e Plateau until ∼100 ps.

provide kinetic curves very close to each other, well within the experimental accuracy. The ν4 ground-state recovery of the CuP-32-mer complex is also close to a monoexponential curve with a 3.9 ns lifetime (Figure 6E, Table 2, row 17): the very weak component (amplitude 6%), with Td1 ) 53 ps, may not have any physical meaning. For the other complexes, the kinetics of ground-state recovery is not that straightforward. For CuP-poly(dA-dT)2, a plateau until ca. 100 ps precedes an monoexponential recovery with Td ) 1.8 ns (Figure 6A, Table 2, row 7). CuP-poly(dA-dC)‚ poly(dG-dT) clearly exhibits fast (Td1 ) 5.7 ps) and slow (Td2 ) 5.5 ns) recovery processes (Figure 6D, Table 2, row 16). Somewhat similar kinetics has been observed for CuP-DNA (Figure 6B, Table 2, row 11), although a rather complicated kinetic behavior can be observed within the time range ∆t < 20 ps. The behavior of CuP free in aqueous solution has been investigated in the same manner as in its complexes with nucleic acids. A ν4* exciplex band also appears at ∼1345 cm-1 (Figure 7). However, at ∆t ) 2 ps, this band is asymmetrical, slightly broader and ∼1 cm-1 downshifted as compared to its shape and position at ∆t g 5 ps. Nevertheless, except for these minor spectral peculiarities, the respective positions of the ν4* and ν2* bands are, within the accuracy of our measurements, the same for CuP free in aqueous solution and bound to nucleic acids. The fitting procedure applied to the kinetics of the ν4* band (Figure 8A) yields a rise time-constant of about 1 ps followed by a double-exponential decay with 3.3 and 12 ps time-constants (Table 2, rows 20, 21). It should be noted that a monoexponential-decay fit (Td ) 6.1 ps) results in a noticeable discrepancy between the fitting curve and experimental data points, mainly for ∆tg 20 ps. At last, no hint of exciplex bands can be observed at ∆tg 60 ps. Concerning the kinetics of ground-state depletion/recovery (Figure 8B) the depletion follows the instrument rate limit and

the recovery can well be fitted by a double-exponential process with 3 and 25 ps time constants (Table 2, row 22). If the short time constant reasonably fits that of the exciplex decay (Table 2, rows 20, 21), however the longer-lived recovery is about 2-fold longer than expected from the Td2 exciplex lifetime (25 vs 12 ps): this discrepancy is well outside the error margin of ps-TR3 measurements. 3.4. Nanosecond Transient RR Spectra. To confirm the existence of exciplex features in the ps-TR3 spectra of the CuP32mer complex, and to relate the current ps data to ns RR spectra published previously,17 a ns saturation RR experiment has been carried out. Figure 9 shows, in the 1300-1400 cm-1 region, the spectra of Cu(T4MPyP) bound to calf-thymus DNA (A), poly(dA-dC)‚poly(dG-dT) (B), 32-mer (C), and poly(dG-dC)2 (D), obtained with the same high-power excitation at 425 nm (the ν4* exciplex band is marked by a vertical dashed line). The exciplex intensity follows the same tendency as in ps-TR3 spectra (Figure 3), i.e., DNA > AC‚GT > 32-mer: it is clearly seen for the CuP-32-mer, thus confirming the previous results.17 It is also worth noting that for Cu(T4MPyP) intercalative complexes, the decrease of the ground-state ν4 mode intensity, under ns high-power of irradiation, is not balanced by a corresponding increase of the exciplex ν4* band (Figure 9BD). In particular this is evident for the CuP-poly(dG-dC)2 complex, where the ν4* exciplex band is not detected at all. The photoinduced depletion of the ground-state population, along with a storage of excited CuP molecules in the tripletstate manifold whose Raman cross-section is too small to be observed, is a plausible reason for this effect. 4. Discussion Before discussing the photoinduced interactions of Cu(T4MPyP) with nucleic acids, brief general consideration of the exciplex properties and then a discussion of the CuP specific reaction with water are worth making. 4.1. General Consideration about the Exciplex Properties. Cu-porphyrin photophysics has already been thoroughly re-



Figure 7. Probe-only (ν4) and difference pump-probe (ν4*) ps-TR3 spectra of Cu(T4MPyP) in aqueous buffer obtained at various time delays ∆t between pump and probe pulses. ∆t values are indicated in the left side of corresponding spectra. All spectra were baseline corrected.

Figure 6. Kinetics of the ground-state band ν4 in ps-TR3 spectra of Cu(T4MPyP) bound to poly(dA-dT)2 (A), calf-thymus DNA (B), poly(dT) (C), poly(dA-dC)‚poly(dG-dT) (D), 32-mer (E) and poly(dGdC)2 (F). The intensity of the ν4-band has been normalized on its value in the probe-only spectrum. All designations are as in Figure 5.

viewed (for example, see ref 5). Water-soluble CuP intercalated between the nucleic acids base pairs (e.g., in poly(dG-dC)2) exhibits a strong luminescence accompanying the 2T1 f 2S0 deactivation with a time constant of ca. 20 ns.5,12 In contrast, CuP free in buffer solution or outside-bound to nucleic acids (e.g., in poly(dA-dT)2) is nonluminescent: this results from a quenching process by a photoattached axial ligand to the central Cu2+ ion.12 Such a reaction had been previously discovered33 and then studied in details34-37 for non-water-soluble CuPs dissolved in oxygen-containing solvents such as tetrahydrofuran (THF) or 1,4-dioxane. On the other hand, water-soluble Cu(T4MPyP) gives rise to a photoinduced exciplex species15,16 when bound to T- or U-containing nucleic acids but not when the Cu2+ axial coordination sites are blocked: instead of water molecules, the CdO groups of thymine (uracil) residues can

Figure 8. Kinetics of the exciplex ν4*-band (panel A) and of the ground-state ν4-band (panel B) intensities in ps-TR3 spectra of Cu(T4MPyP) in aqueous buffer. The intensity of the ν4-band has been normalized on its value in the probe-only spectrum. Markers denote experimental points, and solid curves represent the best-fit profiles. See Table 2 for the best-fit parameters. Dashed line in panel A represents the instrument-limited response function.

serve as axial ligands (i.e., luminescence quenchers) for CuP bound to nucleic acids.18,19 On the basis of ns-RR and picosecond transient absorption (ps-TA) data,18,19,34,35 the exciplex state has been assigned to a photoinduced five-coordinate complex between the excited 2(d,d) state of CuP, resulting from the promotion of a d(z2) inner metal electron to the half-filled d(x2-y2) orbital, and an oxygencontaining ligand (e.g., CdO group of thymine, THF, 1,4dioxane, etc.). The (d,d) assignment of this exciplex has been supported by the following signatures: (i) large Raman downshifts (≈20 cm-1) of core-size marker bands relative to their ground-state counterparts, (ii) characteristic Raman excitation


Figure 9. Nanosecond transient RR spectra of Cu(T4MPyP) bound to calf-thymus DNA (A), poly(dA-dC)‚poly(dG-dT) (B), 32-mer (C), and poly(dG-dC)2 (D). All spectra were obtained in using the same high-power excitation at 425 nm. Transient exciplex ν4* band is marked by a vertical dashed line.

profiles of these exciplex bands,16,19 and (iii) derivative-like redshifted ps-TA difference spectra.19,34,35 An alternative interpretation of this exciplex as arising from an excited-state having a (π,d) charge-transfer (CT) character,36-38 mainly based on the results of iterative extended Hu¨ckel molecular orbital calculations,39 has also been proposed. However, it should be noted that another calculation of the excited states of axially ligated CuP,40 using a model of intermediate neglect of differential overlap, showed that the exciplex state is mostly d(z2) f d(x2y2) but with an appreciable CT a2u(π) f d(x2-y2) character. Thus, there is no definite consensus in the literature yet about the assignment of CuP exciplex: some authors5,12,18,19,35 assigned it to the (d,d) state, while others36-38 are in favor of the CT state, although everybody agrees that the exciplex is a fivecoordinate complex. Since the present paper does not actually deal with this question, let us assume here the (d,d)-state interpretation that has been previously given and argued.19,35 4.2. Photophysics of Cu(T4MPyP) in Water. Photophysics of CuP in water has been extensively studied by luminescence,12 saturation38,41 and time-resolved42 RR, picosecond19,41 and femtosecond20,38,43 TA spectroscopic techniques. Although many features of the photoinduced processes in this system have already been revealed, the details of the exciplex formation (let us call it CuP*H2O) and decay are still under current debate. The present ps-TR3 kinetic data on Cu(T4MPyP) excited states in water are somewhat consistent with those reported from various fs-TA experiments.20,38,43 At the same time, careful examination of pure exciplex Raman spectra (Figure 7) and kinetics (Figure 8A), together with the ground-state recovery kinetics (Figure 8B), can provide a new insight into the photophysics of an investigated system (Scheme 1). First, although the Raman cross-section of the 2,4T1 triplet state of CuP (free or axially ligated) is so weak that practically no Raman feature of this state can be detected, in particular in the ν4- (or ν4*-) band region, Figure 7 shows that the exciplex ν4* Raman band nearly is at its maximum intensity at a 2 ps

Kruglik et al. delay. This means that after 2S0 f 2S1 photoexcitation, ultrafast relaxation into the triplet state first occurs (2S1 f 2T1 , < 0.1 ps), then the attachment of a water molecule to the central copper ion in the triplet state with the subsequent creation of the exciplex species in the Raman active 2(d,d) state of the molecule occurs with a time constant of ∼1 ps (2T1 + L f exciplex, Table 2, rows 20, 21), in excellent agreement with recent TA kinetic data.43 This diffusion-controlled process is quite plausible, since the energies of both the 2(d,d) and CT states are lowered in the process of axial ligation.40,44 Next, time evolution of the exciplex ν4* Raman band is instructive (Figure 7): at 2 ps it shows an asymmetrical lowfrequency broadening, while it becomes more symmetrical and its maximum undergoes a small upshift (∼1 cm-1) when time elapses. Both features can result from a vibrational heating/ cooling process:24,45 indeed, from a general point of view an ultrafast, nonradiative excitation relaxation from a high to a lower excited electronic state inevitably produces some vibrationally hot species having an extra energy equal to the energy gap between these two states. On the other hand, the CuP*H2O exciplex deactivation, as monitored by the ν4* band, is a biphasic process with time constants ∼3.3 and ∼12 ps (Figure 8A, Table 2, rows 20, 21), while the ground-state recovery, as monitored by the ν4 band, is dominated by an important component having a ∼3 ps lifetime and a much weaker component with a ∼25 ps lifetime (Figure 8B, Table 2, row 22). It should be noted, however, that the kinetics of ground-state recovery can also be accurately fitted by three decaying components, holding the intermediate time constant at 12 ps and allowing the longest one to vary in the 25-30 ps range (both with low amplitude, data not shown). The following explanation of the experimental data can be proposed (see Scheme 1). The vibrationally hot exciplex species can relax either through interaction with the solvent bath (let us call it “normal” cooling) or through direct axial ligand release. This ligand release, leading to the disruption of the exciplex, presumably induces the major part of excited Cu(T4MPyP) molecules to return to the ground state within ∼3 ps (hot exciplex f 2S0, path I in Scheme 1, major ground-state recovery component in Figure 8B). On the other hand, a minor part undergoes a “normal” cooling through collisional interactions with the bath (hot exciplex f cold exciplex, path II), and this is then followed by the exciplex deactivation (cold exciplex f 2S , 12 ps). It should be noted that, in the framework of this 0 consideration, the major exciplex decay component (τdecay ∼ 3.3 ps, Figure 8A) corresponds to some averaged relaxation via both pathways. Now remains another important observation to be interpreted, that the lifetime of the long-lived component of ground-state recovery (25 ps) is different from the longest exciplex lifetime (12 ps). It should be stressed that this kinetics of ground-sate recovery obtained in our ps-TR3 experiment (Figure 8B) is in agreement with the last fs-TA data.43 Therefore, we assume that another relaxation process, invisible in ps-TR3 and having a lifetime of ∼25 ps, occurs in parallel to the exciplex deactivation. Taking into account that (i) the absorption spectrum of the long-lived transient has been assigned to the triplet state of CuP,43 and (ii) the triplet-state Raman cross-section is very weak and actually displays no line in the ν4-band region,31 a relaxation route involving some hypothetical excited state (2X) having a “triplet-state character” is proposed to account for the ∼25 ps component of Raman kinetics of ground-state recovery (path III in Scheme 1). Such a very short triplet-state lifetime likely involves some quenching mechanism: a relevant analogy can



SCHEME 1: Proposed Relaxation Routes for Cu(T4MPyP) in Aqueous Buffer

be found in a ps-TA study of copper porphyrins in nitrogencontaining organic bases,44 where a spectroscopically unobservable CT state has been proposed to shorten the triplet-state lifetime to tens to hundreds of picoseconds. In the same way, the quenching influence of a CT state is suggested here, as one of the possible mechanisms, in the relaxation process of Cu(T4MPyP) in water, although no direct spectral evidence of this CT state has been obtained. Finally, equilibration of CuP triplet states 2T1 S 4T1 has been found to last hundreds of picoseconds at room temperature.46 Therefore, we presume that all of the relaxation processes in CuP-water complexes, which actually are very fast (3 Å). Since the length of an axial bond is ca. 2 Å, coordination of the porphyrin central Cu2+ ion to a CdO group of a thymine residue requires some changes in the doublehelix structure and/or a deformation of CuP. We propose that thermal motions of the double-helix and/or base pairs can provide a plausible mechanism for thymine CdO group axial coordination to CuP. Indeed, low-frequency intrahelical Raman modes of base pairs have been reported, for example at ∼85 cm-1 47 or ∼41 cm-1,48 which correspond to vibrational motions in the femtosecond time scale. Although these motions are of low amplitude, we speculate that a ∼1 ps duration is long enough for a superposition of many motions of helical fragments to occur in a direction and with an amplitude adequate for such a coordination to take place. Let us now consider the second rise component of the exciplex kinetics (Figure 5, Tr2 ∼ 40-70 ps). First, one can recall that water can be neglected as a main source of doublerise kinetic changes for all of the exciplexes observed in CuPnucleic acids complexes (see section 4.3). Second, porphyrin can bind in a face-on manner in the major groove but can also bind edgewise in the minor groove: one of those arrangements might be favorable for fast exciplex formation (Tr1), while the other might require some slow rearrangement during the time interval Tr2 (fitting model 2). However, although some heterogeneity in the CuP binding modes is certainly possible, i.e., CuP is attached in different manners to various polymers, the slow rise component (Tr2) is quite a common feature of the exciplex kinetics (Figure 5), even for the particular case of CuP bound to single-stranded poly(dT). Consequently, one should also consider the physical processes occurring within the metalloporphyrin moiety. As a possible candidate, structural relaxation of the whole exciplex system (namely, electronically excited CuP bound to polymer) can be suggested toward an energetically most favorable conformation. In the framework of this hypothesis, the “fast” exciplex formation is followed by a “slower” exciplex structural rearrangement (fitting model 3) which would be at the origin of the slow rise component of the exciplex kinetics. Let us develop this in more detail, in considering the relaxation route I shown in Scheme 2. Photoexcitation of CuP (2S0 f 2S1) is followed by a femtosecond excitation relaxation into the triplet state (2S1 f 2T1), in which the metalloporphyrin affinity for oxygencontaining neighbor molecules greatly increases.19,33-35 Provided that CuP is close enough to a free carbonyl group of a thymine residue, intrahelical low-frequency motions facilitate exciplex



SCHEME 2: Proposed Relaxation Routes for Cu(T4MPyP) in Interaction with Nucleic Acidsa

a Note that for different nucleic acids different combination of routes I-III may be realized. Route III has been suggested on the basis of data from ref 12.

formation (2,4T1 f constrained exciplex). The excited 2(d,d) state is populated in the exciplex building process through a CdO group attachment as an axial ligand to the central Cu(II), with a readily achieved porphyrin core expansion. Further structural evolution of the exciplex system toward a minimum-energy domed conformation (constrained exciplex f relaxed exciplex) is likely to occur in a picosecond time domain, as reported in a recent femtosecond TA study of photoinduced conformational changes in highly nonplanar metalloporphyrin.49 It should be noted that a 40-70 ps lasting process seems to be too slow for a structural rearrangement of a metalloporphyrin molecule.24,49,50 However, we would like to point out in this respect that the CuP exciplex under consideration is not free in solution, but bound to nucleic acids by both four methylpyridyl groups and axial attachment of a CO group of a thymine residue. Consequently, structural rearrangement in this case involves not only the metalloporphyrin, but also the attached nucleic acid neighborhood, and this appear to be a rather big molecular complex. Along with structural evolution of a nonplanar metalloporphyrin structure, a red shift of its absorption bands occurs, as reported recently.49 Since our Raman probing wavelength has been chosen at the red edge of the Soret maximum (λprobe ) 456 nm), such a red shift of the absorption profile and, therefore, of the Raman excitation profiles of the totally symmetrical modes ν2* and ν4* should lead to an enhancement of the exciplex Raman signal: this is what was actually observed. Comparison of the kinetics of the ν2* and ν4* modes observed for CuP-poly(dA-dT)2 somewhat supports this idea of “slow” structural rearrangement. As shown previously,32 ν2-band shape is sensitive to porphyrin nonplanar distortions while ν4 is not. Therefore, some differences in the exciplex Raman kinetics of these two modes can be expected, and that was indeed the case. The “fast” rise (Tr1) and slow decay (Td2) processes were found to be the same for both modes within the experimental error (Table 2, rows 1-3 and 4-6). However, the “slow” rise processes are different, i.e., longer for ν2* (Tr2 ∼ 68 ps) than for ν4* (Tr2 ∼ 48 ps). Now, let us consider the additional decay component (Td1 ∼ 22 ps) observed for CuP in interaction with poly(dA-dC)‚ poly(dG-dT) (Figure 5D, Table 2, row 15). Besides some possible contribution from the exciplex with water (see above), it might also correspond to vibrational cooling process of intercalated CuP at AT/GC steps. Indeed, in contrast to the outside-bound complex, intercalated CuP is screened from the collisional interactions with surrounding water molecules. Moreover, the details of static intermolecular interactions between CuP and nucleic acid backbone are different for intercalated and outside-bound complexes, and this difference may influence the vibrational cooling process of the photoexcited CuP*CdO species.

To further discuss the excitation relaxation pathways of CuP bound to nucleic acids, it is worth comparing the time constants of exciplex decay and ground-state recovery. For CuP in interaction with poly(dA-dT)2 and poly(dT), the situation is rather straightforward: exciplex decay and ground-state recovery occur with the same time constant Td2 (Table 2, rows 3 and 7, 13 and 14). This shows that the excitation relaxation of these outside-bound complexes is mainly linked to exciplex deactivation (route I in Scheme 2). Let us now consider the ground-state recovery of CuP intercalated in poly(dG-dC)2. The measured kinetics can be reasonably fitted by a single-exponential decay function with a time constant of 4.4 ns (Table 2, row 18). This value is noticeably shorter than the previously reported 22 ns luminescence lifetime.12 To overcome this discrepancy, a doubleexponential fit of our ps-TR3 data has been tried, in imposing a Td2 ) 22 ns value: the resulting best-fit gives a prominent fast-decay (Td1 ) 2.8 ns, A1 ) 0.61), the 22 ns component remaining minor (A2 ) 0.39, Table 2, row 19). It worth noting that the possibility of existence of several luminescent components has been admitted in ref 12. Thus ps-TR3 and luminescence data, considered altogether, allow (at least) two parallel relaxation processes to be suggested: the long-decay process related to the triplet state12 (Scheme 2, route III, 2,4T1 f 2S0) and the short-decay process involving some quenching mechanism from the environment (route II). An exciplex formation in CuP-poly(dG-dC)2 has been recently proposed, involving the binding of a nitrogen atom from cytosine residues.20 Our current ps-TR3 spectra show no trace of exciplex for this complex (Figure 3F); however, the idea about the quenching influence of nitrogen-containing guanine residues on the tripletstate deactivation seems plausible. It is also worth noting that a recent crystallographic study of intercalated CuP revealed that the porphyrin can bind both by normal intercalation and by extruding cytosine residues out of the DNA helical stack (socalled hemi-intercalation), depending on the nature of the binding site.51 Thus, it can be plausibly suggested that hemiintercalated photoexcited CuP interacts with nitrogen atoms of unpaired guanine residues, this resulting in an accelerated triplet state relaxation through an excited CT state (Scheme 2, route II, 2,4T1 f X f 2S0). However, more detailed time-resolved measurements at longer time delays are necessary to complete the picture of photoinduced processes of intercalated CuP complexes. Scheme 2 is a sketch summarizing all possible excitation relaxation pathways and available kinetic data for Cu(T4MPyP) bound to nucleic acids. The main idea is the following: depending on CuP location and base-pairs composition, excitation relaxation from the CuP triplet state can proceed through some combination of (at least) three possible relaxation routes.

5030 J. Phys. Chem. B, Vol. 105, No. 21, 2001 Experimentally, route I has been detected in ps-TR3 exciplex kinetics, route II in ps-TR3 kinetics of ground-state recovery, and route III in luminescence kinetics.12 The existence of one or more parallel relaxation channels can explain the different intensities of the exciplex Raman bands observed for CuP complexes with various nucleic acids. At last, for CuP-calf thymus DNA complexes, the excitation deactivation is expected to proceed through all of the three relaxation routes, resulting in a complex kinetic behavior, since CuP is nonpreferentially bound in this case, thus involving both intercalated and outside-bound species. The fact that the groundstate recovery in ps-TR3 kinetics (Td2 ) 2.4 ns, Table 2, row 11) is much quicker than the reported luminescence lifetime (∼18 ns12) can be explained in the same way as in the case of CuP-poly(dG-dC)2 where multiple relaxation components most likely participate in the nanosecond time domain. 5. Conclusions On the basis of the data obtained in absorption, steady-state, ns- and ps-TR3 spectroscopies, the following main conclusions can be formulated: (i) When a light pulse impinges Cu(T4MPyP)-DNA/polynucleotides complexes a reversible exciplex can be formed, in less than 2 ps after photoexcitation, between excited porphyrins and CdO groups of thymine residues in all AT-containing sequences of nucleic acids. The yield of exciplex formation depends on the nucleic base sequence, i.e., in decreasing order: poly(dA-dT)2 > calf thymus DNA > poly(dA-dC)‚poly(dGdT). (ii) Such a rapid exciplex building process implies that it is formed between CuP molecules initially located, in the steady state of this interaction, at AT sites of the nucleic acids. This also has two main consequences, which contradict conclusions previously reported in ref 17: (a) although the binding mode of the porphyrin actually depends on the base sequence, there is no preferential binding of Cu(T4MPyP) to the various sites of DNA, and (b) there is no photoinduced ultrafast porphyrin translocation from GC to AT sites of DNA. (iii) An exciplex is also formed in ∼1 ps for CuP free in aqueous buffer. CuP exciplexes with water and with CdO groups of thymine residues are spectroscopically identical. However, they can be distinguished from each other by their relaxation kinetics. The lifetime of the exciplex formed with water lies in the 3-12 ps range, while that of the exciplex formed with nucleic acid lies in the nanosecond time domain (1-3 ns). (iv) The preliminary step of exciplex formation involves an axial ligation of a O-containing ligand (water or CdO group of thymine) to the Cu-porphyrin in its excited triplet state. In water, the exciplex is likely formed as a vibrationally “hot” species, and its deactivation goes through (i) a direct axial water ligand release from the “hot” exciplex (τ ∼ 3 ps) and (ii) molecular collisions with the solvent bath leading to a “cold” exciplex species and then a ligand release (τ ∼ 12 ps). In parallel to the exciplex deactivation, a slower but noticeable relaxation (τ ∼ 25 ps) occurs probably involving the quenching influence of a CT state (invisible in Raman spectra) of water-ligated porphyrins in their triplet state. (v) In nucleic acids, excitation relaxation of the porphyrin can proceed through (at least) three possible channels, or some combination of them depending on the CuP location within various base sequences. Outside binding mode of CuP at AT/ TA steps provides the structurally most favorable geometry for exciplex formation with the CdO group of thymine residues

Kruglik et al. in less than 2 ps. A subsequent structural rearrangement of the Cu-porphyrin-polymer system is proposed to occur within a 40-70 ps time range after exciplex formation. Intercalation at GC/AT steps also allows exciplex to be formed with CdO groups of thymines, but with a much lower efficiency than in the case of groove-bound complexes, while intercalation at GC/ CG steps strictly prohibits exciplex formation. Acknowledgment. The authors warmly thank Dr. V. Chirvony for very useful discussions of the manuscript, and V. Galievsky for communication of transient absorption data prior to publication. S.G.K. acknowledges the postdoctoral fellowship grant P96309 from the Japanese Society for the Promotion of Science and personal visiting grants from the University Pierre and Marie Curie (Paris VI) and from the French Embassy in Belarus. P.M. acknowledges the Japanese Society for the Promotion of Science and Academy of Sciences of the Czech Republic for partially supporting this research in the framework of the Joint Research Project Kontakt ME271. This work has been partially supported by the INTAS-Belarus Grant 97-0428. References and Notes (1) Fiel, R. J.; Howard, J. C.; Mark, E. N.; Dattagupta, N. Nucleic Acids Res. 1979, 6, 3093. (2) Fiel, R. J. Biomol. Struct. Dyn. 1989, 6, 1259. (3) Pasternack, R. F.; Gibbs, E. J. In Metal-DNA Chemistry; Tullius, T. D., Ed.; ACS Symposium Series No. 402: New York, 1989; pp 59-73. (4) Marzilli, L. G. New J. Chem. 1990, 14, 409. (5) McMillin, D. R.; McNett, K. M. Chem. ReV. 1998, 98, 1201. (6) (a) Pasternack, R. F.; Gibbs, E. J.; Villafranca, J. J. Biochemistry 1983, 22, 2406. (b) Bustamante, C.; Gurrieri, S.; Pasternack, R. F.; Purrello, R.; Rizzarelli, E. Biopolymers 1994, 34, 1099. (7) Ward, B.; Skorobogaty, A.; Dabrowiak, J. C. Biochemistry 1986, 25, 7827. (8) Pasternack, R. F.; Bustamante, C.; Collings, P. J.; Giannetto, A.; Gibbs, E. J. J. Am. Chem. Soc. 1993, 115, 5393. (9) Dougherty, G.; Pilbrow, J. R.; Skorobogaty, A.; Smith, T. D. J. Chem. Soc., Faraday Trans. 2 1985, 81, 1739. (10) Feng, Y.; Pilbrow, J. R. Biophys. Chem. 1990, 36, 117. (11) Dougherty, G.; Pasternack, R. F. Biophys. Chem. 1992, 44, 11. (12) Hudson, B. P.; Sou, J.; Berger, D. J.; McMillin, D. R. J. Am. Chem. Soc. 1992, 114, 8997. (13) Eggleston, M. K.; Crites, D. K.; McMillin, D. R. J. Phys. Chem. A, 1998, 102, 5506. (14) Strickland, J. A.; Marzilli, L. G.; Wilson, W. D. Biopolymers 1990, 29, 1307. (15) Turpin, P.-Y.; Chinsky, L.; Laigle, A.; Tsuboi, M.; Kincaid, J. R.; Nakamoto, K. Photochem. Photobiol. 1990, 51, 519. (16) Chinsky, L.; Turpin, P.-Y.; Al-Obaidi, A. H. R.; Bell, S.; Hester, R. E. J. Phys. Chem. 1991, 95, 5754. (17) Strahan, G. D.; Lu, D.; Tsuboi, M.; Nakamoto, K. J. Phys. Chem. 1992, 96, 6450. (18) Mojzes, P.; Chinsky, L.; Turpin, P.-Y. J. Phys. Chem. 1993, 97, 4841. (19) Kruglik, S. G.; Galievsky, V. A.; Chirvony, V. S.; Apanasevich, P. A.; Ermolenkov, V. V.; Orlovich, V. A.; Chinsky, L.; Turpin, P.-Y. J. Phys. Chem. 1995, 99, 5732. (20) Jeoung, S. C.; Eom, H. S.; Kim, D.; Cho, D. W.; Yoon, M. J. Phys. Chem. A, 1997, 101, 5412. (21) Shvedko, A. G.; Kruglik, S. G.; Ermolenkov, V. V.; Orlovich, V. A.; Turpin, P.-Y.; Greve, J.; Otto, C. J. Raman Spectrosc., 1999, 30, 677. (22) Skeletal modes designation according to Li, X.-Y.; Czernuszewicz, R. S.; Kincaid, J. R.; Su, Y. O.; Spiro, T. G. J. Phys. Chem. 1990, 94, 31. (23) Mizutani, Y.; Kitagawa, T. Science, 1997 278, 443. (24) (a) Kruglik, S. G.; Mizutani, Y.; Kitagawa, T. Chem. Phys. Lett. 1997, 266, 283. (b) Mizutani, Y.; Uesugi, Y.; Kitagawa, T. J. Chem. Phys. 1999, 111, 8950. (25) Uesugi, Y.; Mizutani, Y.; Kitagawa, T. J. Phys. Chem. A, 1998, 102, 5809. (26) Uesugi, Y.; Mizutani, Y.; Kitagawa, T. ReV. Sci. Instrum. 1997, 68, 4001. (27) Uesugi, Y.; Mizutani, Y.; Kruglik, S. G.; Shvedko A. G.; Orlovich V. A.; Kitagawa, T. J. Raman Spectrosc. 2000, 31, 339. (28) Pasternack, R. F.; Garrity, P.; Ehrlich, B.; Davis, C. B.; Gibbs, E. J.; Orloff, G.; Giartosio, A.; Turano, C. Nucleic Acids Res. 1986, 14, 5919.

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