J. Phys. Chem. 1991,95, 5754-5756
5754
Contributlon of Resonance Raman Excitation Spectroscopy for Probing Electronically Excited States: Nature of a Porphyrin-DNA Excipiex Laurent Chinsky,**+Pierre-Yves Turpin: Ala H . R. AI-Obaidi,*Steven E. J. Bell,$ and Ronald E. Hester* L.P.C.B. (CNRS UA 198) Instirut Curie and UniversitP Pierre et Marie Curie, 75231 Paris Cedex 05, France, and Department of Chemistry, University of York, Heslington, York YO1 5DD, England (Received: November 12, 1990; In Final Form: January 24, 1991) The resonance Raman spectra of a water-soluble metalloporphyrin Cu(TMpy-P4), complexed with a synthetic nucleic acid, poly(dA-dT), were measured by using excitation wavelengths located within the B (Soret) transition of the porphyrin (417-470 nm), while its excited state was synchronously pumped with 545-nm pulsed excitation corresponding to the Q transition. In the presence of pump pulses, the aqueous solution of the Cu(TMpy-P4).poly(dA-dT) complex exhibits resonance Raman bands at 1558 and 1353 cm-l that are not observed in the absence of pump pulses. These new features were previously assigned to electronically excited Cu(TMpy-P4), stabilized by forming an exciplex with the A-T sites of the nucleic acid. Here we present rexmance Raman excitation profiles (RREP) of both the excited and ground states of the complex, and we experimentally confirm the very short lifetime of the exciplex. To our knowledge this is the first time that a RREP of a very short lived (ca. 20 ps) intermediate excited state has been obtained with a two-color experiment. We use this to help to characterize the nature of the porphyrin-AT specific complex formed in the porphyrin excited state. Introduction The porphyrins belong to a class of molecules which find application in photodynamic therapy: some of them are photosensitizers and on exposure to light are antitumor active.l*2 Thus it is of interest to study the nature of the porphyrin-DNA interaction and, in particular, the factors which are responsible for the DNA affinity and the photoinduced DNA strand-breaking. The binding of a water-soluble porphyrin, M(TMpy-P4), to nucleic acids has been studied with a variety of spectroscopic methods: UV-visible and CD,3 flow d i ~ h r o i s m ,fl~orescence,~ ~ ESR? NMR,’+ and RR (resonance Raman).l*I2 The major conclusions obtained from these investigations are that planar (four-coordinated) porphyrins [M = H2, Cu(II), and Ni(II)] are selectively intercalated at G-C sites while porphyrins having axial ligands ( H 2 0 ) [ M = Zn(II), Co(III), Fe(III), and Mn(III)] form groove-bound complexes at A-T sites of DNA. In addition, an exciplex formation can occur when planar Cu(TMpy-P4) is interacting with poly(dA-dT);I2 this exciplex might play a role in the DNA strand-breaking mechanism. In this communication, we examine the nature of this exciplex trapped selectively at the A-T sites of DNA by considering resonance Raman excitation profiles and lifetime measurements. Experimental Procedure Compounds. The tosylate salt of 5,10,15,20-tetrakis(4-Nmethy1pyridyl)porphyrinato cation, [H2(TMpy-P4I4+,was purchased from Midcentury, Posen, IL, and the Cu(I1) derivative was prepared by published procedure^.^ Poly(dA-dT) was purchased from Pharmacia Biochemicals. Sample solutions were prepared by mixing an aqueous solution of Cu(TMpy-P4) with a phosphate buffer solution of the nucleic acid. The final solution thus obtained (pH 6.8 and ionic strength 0.2) contained Cu(TMpy-P4) and nucleic acid at ca. 3 X and 1 X IO4 M, respectively, as determined spectroph~tometrically.~ The molar ratio of base pairs/Cu(TMpy-P4) was ca. 30. Spectral Measurements. The Raman excitation was provided by two dye lasers (Lambda Physik Models FL3002 and 2002) which were pumped by two excimer lasers (Lumonix Model HE460; repetition rate 20 Hz, pulse duration ca. 10 ns). The Raman spectra dispersed by a Spex Triplemate spectrometer were measured by an intensified diode array multichannel detector (Spectroscopy Instruments OSMA). The integration time was ca. 10 min. Pump pulse power was 0.5-1 mJ and probe pulse power was set at 25 pJ such that no exciplex feature would appear hstitut Curie. *University of York.
+
during probe-only measurements. Ethanol was used as an internal intensity standard, by adding ca. 0.5% ethanol to the sample solution for 465-,457-, 450-, and 445-nm probe wavelengths and a.10% for 435-,427-, and 418-nm probe wavelengths, all of these being in the Soret region. In all experiments the samples were excited under a grazing incidence geometry; thus any possible effects of differential reabsorption of the Raman photons at various diffusion wavelengths can be neglected in estimating the band intensities. For all experiments the 0.5-1-mJ pump pulse was delivered at 545 nm (corresponding to the Q absorption band of Cu(TMpy-P4)); this wavelength was chosen because it creates a good yield of the exciplex state. intensity Measurements. In order to compare some characteristics of the ground and excited (exciplex) states of the Cu(TMpy-P4) molecule, we chose to determine the excitation profiles of the 1574- and 1370-cm-’ (ground state) and 1558- and 1353-cm-I (exciplex state) Raman bands; thus we must obtain the intensities of these bands along with that of the ethanol Raman band (at ca. 900 cm-’ for ethanol in water) needed for the normalization procedure. To make the individual band determinations under similar conditions, we used a computer program to build up five model spectra, each of them containing only one of the bands of interest (i.e., 1574,1370,1558, 1353 cm-’ and the ethanol band). Each model spectrum has the peak value taken as unit intensity. These five model spectra were used to decompose the experimental spectra into their component features, thus determining the individual band intensities in the regions of band overlap. ( I ) Fiel, R. J.; Datta-Gupta, N.; Mark, E. H.; Howard, J. C. Cancer Res. 1981, 41, 354. (2) Praseuth, D.; Gaudemer, A,; Verlhac, J. B.; Kraljic, 1.; Sissoeff, I.; Guille, E. Phorochem. Phorobiol. 1986, 44, 717. (3) Pasternack, R. F.; Gibbs, E. J.; Villafranca, J. J. Biochemistry 1983, 22, 2406. (4) Geacintov, N. E.; Ibanez, V.; Rougee, M.; Bensasson, R.V. Biochemistry 1987, 26, 3087. ( 5 ) Kelly, J. M.; Murphy, M. J.; McConnell, D. J.; OhUigin, D. Nucleic Acids Res. 1985, 13, 167. (6) Dougherty, G.;Pilbrow, J. R.;Skorobogaty, A.; Smith, T. D. J . Chem. Soc., Faraday Trans. 2 1985,81, 1739. (7) Banville, D. L.; Marzilli, L. G.; Strickland, J. A.; Wilson, W. D. Biopolymers 1986, 25, 1837. (8) Marzilli, L. G.; Banville, D. L.; Zon, G.;Wilson, W. D. J . Am. Chem. SOC.1986, 108, 4188. (9) Strickland, J. A.; Banville, D. L.; Wilson, W. D.; Mardlli, L. G. lnorg. Chem. 1987, 36, 3398. (10) Blom, N.; Odo, J.; Nakamoto, K.; Strommen, D. P. J . Phys. Chem. 1986, 90, 2847. ( 1 1) Schneider, J. H.; Odo, J.; Nakamoto, K. Nucleic Acids Res. 1988, 16, 10323. (12) Turpin, P. Y.; Chinsky, L.; Laigle, A.; Tsuboi, M.; Kincaid. J. R.; Nakamoto, K. Phorochem. Phorobiol. 1990, 51, 519.
0022-3654191 (2095-5754%02.50/0 0 1991 American Chemical Society
The Journal of Physical Chemistry, Vol. 95, No. 15, I991 5755
Nature of Porphyrin-DNA Exciplex I
L
lPQ0
1241
1500
U
I
I
B
I
/ crl
Yav&
Figure 1. Raman spectra of the Cu(TMpy-P4).poly(dA-dT) exciplex,
obtained with 0.5 mJ of 545 nm pump power and 25 pJ probe power: A, 457 nm; 9, 450 nm; C, 445 nm excitation.
475
450
425
Ywelength / nm
Figure 3. Raman excitation spectrum of ground state porphyrin: (-) 1369 cm-l; (---) 1574-cm-' band; (-.-.-) B (Soret) absorption band (au).
I
1241
1500 Ya&
1MM / crl
Figure 2. Raman spectra of Cu(TMpy-P4)-poly(dA-dT)exciplex, obtained with 1 mJ of pump power (545 nm)and 25 pJ probe power (450
nm): A, without time delay; B, with a 20-11s delay between pump and probe pulses.
Results and Discussion Traces A, B, and C of Figure 1 show resonance Raman spectra from a mixture of Cu(TMpy-P4) with poly(dA-dT) obtained with excitation (at 25 rJ/pulse) in the Soret region at 457,450, and 445 nm, respectively, with simultaneous 545-nm pumping (0.5 mJ/pulse). These exhibit two bands, at 1558 and 1353 cm-I, which are not present in the spectrum of Cu(TMpyP4) alone excited under the same pump and probe conditions. These extra bands do not appear either when the resonance Raman spectra of the Cu(TMpy-P4)*poly(dA-dT)solution are obtained without pump pulses, or with a ca. 20-11s delay between pump and probe pulses (Figure 2B). Figure 3 presents the Raman excitation spectrum of the ground-state molecule characterized by the 1574- and 1370-cm-' bands, while Figure 4 presents that of the exciplex state characterized by the 1558- and 1353-cm-' bands. These profiles were obtained with the decomposition procedure described in the Experimental Section. Since the wavelength and energy of the pump laser were the same in all experiments, it follows that the population proportion of molecules in the ground state and in all the excited states remained constant. The intensities of the two Raman bands attributed to the exciplex species (at 1558 and 1353 cm-', see Figure 1) vary systematically relative to those of the ground-state species (1 574 and 1370 cm-I) as the probe wavelength is changed from 457 to 450
Yawlength / M
Figure 4. Raman excitation spectrum of exciplex: (-) 1350 cm-I; (---)
1558-~m-~ band.
and 445 nm. Since these relative band intensities are dependent only on the resonance enhancement factors for the two species, it is clear that they must have different excitation profiles. This is as expected. However, the fact that the Raman bands for the exciplex species are comparable in intensity with those for the ground state, for excitation in the wavelength region characteristic of the ground-state B (Soret) absorption band, is entirely unexpected. Excitation profiles, as plotted in Figures 3 (ground state) and 4 (exciplex state), generally mimic the vibronic absorption spectra. Consistent with this, the ground-state B-band peaks at ca. 426 nm and, correspondingly, we may predict the Occurrence of the exciplex transient absorption peak to be shifted only some 4-7 nm to the red (cf. Figures 3 and 4). This transient absorption has not been measured directly. Since the proportion of the porphyrin molecules which are in the exciplex state in our experiments is unknown, although constant, the intensities of the two excitation profiles cannot, of course, strictly be compared. However, it remains true that the transient exciplex species would appear to have an absorption spectrum which is only slightly red-shifted from that of the ground state. Spectra were also obtained with the lowest possible delay which could be used without overlap between the pump and probe pulses:
5756 The Journal of Physical Chemistry, Vol. 95, No. 15, 1991 since pump and probe pulse widths were ca. 10 ns, a delay between the pulses of 20 ns was chosen. In this case, Raman spectra are exactly the same with and without pump pulses (see Figure 2). This is consistent with the estimate of the lifetime of the exciplex state (ca. 10 ps) that previously was made by varying the power in a pump-probe single-pulse experiment.12 To give a coherent explanation of these different observations, it is necessary to consider the energy level structures of porphyrins, which have been studied in detail by several authors."-I6 The following facts have been established: 1. Pumping at the B (Soret) absorption band wavelength of ca. 430 nm yields an excited state with very short lifetime ( < I PSI. 2. Pumping at the Q absorption band wavelength of ca. 550 nm yields an excited state which also has a very short lifetime. 3. Pumping at the T absorption band wavelength of ca. 740 nm yields 'tripmultiplet" states with much longer lifetimes ( > l o ns at room temperature). Before considering the process of exciplex formation, it is worthwhile to recall the nature of the Cu(TMpy-P4).poly(dA-dT) interaction in the ground state of the porphyrin. Evidence has previously been presented"*" that Cu(TMpyP4) is bound to the periphery of the double helix via Coulombic interaction between the N+-CH3 group of the porphyrin and the PO, group of poly(dA-dT). On so binding, the Cu(TMpy-P4) gave a ground-state RR spectrum, with Kr ion laser excitation (CW, 10-20 mW), showing only very small downward shifts of 0.1 cm-' for the 1571-cm-l band and 0.5 cm-' for the 1366-cm-' band, consistent with only very slight perturbation of the porphyrin ring. Thus, for the Cu(TMpy-P4).poly(dA-dT) interaction in the porphyrin excited state (i.e., the exciplex), the much larger spectral perturbation suggests that the role of poly(dA-dT) here is to trap the porphyrin molecule in a modified excited state whose lifetime is much longer than that of the unperturbed Q-excited state. The porphyrin ring evidently is significantly distorted in this state, as is shown by the additional Raman features. Let us now consider the true nature of the so-called exciplex state. It is helpful first to gather together the tentative conclusions which may be drawn from the experimental results here and in the literature: 1. The exciplex lifetime is probably of the order of a few tens of picoseconds.I2 This is similar to the lifetime of the [Cu(TPP)] T-T* excited state in the strongly coordinating solvent piperidine, attributed to a transient charge-transfer (CT) state involving 5-coordinate Cu(I1). This CT state serves to quench the longer lived tripmultiplet manifold of excited states.'*J9 The unperturbed B- or Q-excited singlet states would have still shorter lifetimes (e.g.
