17948
J. Phys. Chem. 1995,99, 17948-17955
Direct Observation of Photoinduced Electron Transfer in Pyrene-Labeled dU Nucleosides and Evidence for Protonated 2'-Deoxyuridine Anion, dU(H)', as a Primary Electron Transfer Product Thomas L. Netzel*g+ and Kambiz Nafisi Department of Chemistry, Georgia State University, Atlanta, Georgia 30303
Jeb Headrick and Bruce E. Eaton* Department of Chemistry, Washington State University, Pullman, Washington 99164-4630 Received: July 14, 1995; In Final Form: September 18, 1995@
This paper examines the electron transfer (ET) photophysics of two pyrene-labeled nucleosides, 5-(1carboxypyrenyl)-2'-deoxyuridine (1) and 5-( 1-pyrenyl)-2'-deoxyundine (2) by a variety of spectroscopies in several different solvents. For 1 in methanol (MeOH), a maximal change-in-absorbance (AA) increase at 460 nm characteristic of pyrene" occurs during the time of photoexcitation ( 1 3 0 ps). (A control experiment ensures that pyrene'+ is formed from the singlet excited state of pyrene and not as result of multiphoton ionization of pyrene by the laser pulse.) The pyrene'+ signal decays in 20-70 ps slightly more slowly than does the S I state's positive AA signal at 510 nm. These results prove that the SI state of pyrene is quenched due to intramolecular ET. Similar results are also obtained for 1 in acetonitrile (MeCN) where the pyrenee+ absorbance at 460 nm decays in ca. 100 ps. For 1 and 2, changing solvent from MeOH to MeCN increases both emission lifetimes and quantum yields. For 2 the emission yield increases 13-fold, while for 1 it increases 3-fold. Even though the dielectric constants of MeOH and MeCN are similar, 33.6 and 37.5, respectively, both the large emission yield increase and the accompanying striking change in the emission spectrum for 2 on switching from MeOH to MeCN are consistent with lessened ET quenching due to raising the free energy of the pyrene'+/dU'- charge transfer product relative to the n,n*state of pyrene, where dU = 2'-deoxyuridine. Since dU(H)' requires less energy to form than does dU'-, ET quenching should be more favorable in MeOH, where dU(H)' can be formed, than in MeCN, where only dU'- can be produced. If this model is correct, the time of protonation of dU'- in MeOH is 1 3 0 ps for 1 based on transient absorbance measurements of the appearance of pyrene'+.
Introduction
phore/nucleic acid complexes. However, these latter studies are of systems which contain a large number of chromophore In this paper we lay the groundwork for studying electron microenvironments and thus are difficult to interpret without transfer (ET) reactions within DNA duplexes by examining the ambiguity. photophysics of the pyrene-labeled nucleosides 1,5-(1-pyrenoyl)Pyrene-DNA conjugates have been used previously because dU, and 2, 5-(l-pyrenyl)-dU, where dU = 2'-deoxyuridine, by pyrene is a stable chromophore with a reasonably long a variety of spectroscopies in different types of solvents. We fluorescence lifetime (ca. 200-400 ns) depending upon the type have previously reported the observation of pyrene-to-uridine of substitution and solvent.s-12 Its long emission lifetime is a charge transfer (CT) emission for 2 in methanol (MeOH).' consequence of the fact that n,n*absorption to its lowest energy Importantly, the CT emission in MeOH is replaced by n,n* singlet electronic excited state (SI) is spin-allowed but orbitally emission when 2 is dissolved in the low-dielectric solvent f ~ r b i d d e n . ' ~ .Since '~ it is conveniently derivatized at the tetrahydrofuran (THF). This finding is consistent with expecta1-position, it has frequently been employed as a fluorescent tions that the free energy of excited state ET in 1 and 2 should label, especially in biological s t ~ d i e s . ' ~ ,It~is~also - ~ ~true that be lower (more negative) in MeOH than in THF.2-4 However, pyrene can be reversibly oxidized and reduced in both its ground to date direct observation of the ET products following and lowest energy excited state^.^^-^^ Recently, its photophysics photoexcitation of either of 1 or 2 has not been reported. has been extensively studied as a carcinogenic and mutagenic Labeled nucleosides with photoredox-active labels attached benzo[a]pyrenediol epoxide (BPDE) derivative bound to the directly to ribose or to a base as in 1 and 2 are highly desirable exocyclic amino group of 2'-deoxyguanosine (dG) nucleosides synthons for creating labeled DNA oligonucleotides, because in native DNA.'7.18~2',25.29.30 A recent picosecond kinetics study the labels in duplexes formed from these oligonucleotides will was the first to provide unambiguous evidence of ET between have only a limited number of conformations making biophysiphotoexcited pyrene (pyrene*) and a covalently attached nucleic cal, NMR, and molecular modeling studies more t r a ~ t a b l e . ~ - ~ acid base by directly observing the pyrenyl anion following At present few studies of covalently substituted bases in wellphotoexcitation of the BPDE-dG n ~ c l e o s i d e . ~ ~ defined nucleic acid environments have been published. In Pyrene was chosen as a label for uridine in this and earlier contrast, there are many studies of the photophysical properties work, because (1) it can have a long-lived singlet excited state, of polyaromatic hydrocarbon (PAH)/DNA and other chromo(2) it is a reversible electron donor and acceptor in its ground and excited states, and (3) ET from pyrene to uridine is estimated Phone 404-651-3129; FAX 404-651-1416. to be very exergonic from pyrene's SI state, ca. -0.52 eV.' @Abstractpublished in Advance ACS Absrrucrs, November 15, 1995. +
0022-365419512099-17948$09.00/0 0 1995 American Chemical Society
Electron Transfer in Pyrene-Labeled Nucleosides
J. Phys. Chem., Vol. 99, No. 51, 1995 17949
Georgia State University has been described fully in previous report^.^^-^'-' For this work sample solutions were prepared in 0\ air to have an absorbance of 0.35-0.38 at 355 nm and were 0 I excited with circularly polarized third-harmonicpulses from the Nd:YAG laser (-25 ps pulse duration fwhm at 355 nm; -6 d k m 2 per pulse). The monitoring light in the sample and 1 reference beams was depolarized both by scattering from a OH lightly sand-blazed quartz diffuser disk and by passage through an achromatic depolarizer. In this way possible kinetics artifacts due to photoselection and molecular rotation were eliminated. The 100-mLportions of ca. 0.5 L volume stock sample solutions were loaded into the sample reservoir and continuously circuO I ' 0 , lated through a 1 cm path length optical cell during data acquisition. Aliquots of the 100 mL reservoir solutions were 2 checked during kinetics data acquisition, and whenever more on than a few percent loss of initial absorbance was noted the entire Figure 1. Structural drawings of pyrene-labeled nucleosides 1, 5-(1solution in the sample reservoir was changed. Each changepyrenoy1)-dU, and 2, 5-( 1-pyreny1)-dU. in-absorbance (AA) point in a kinetics or spectral plot resulted from averaging data from 1920 laser shots (firings), 960 sample The goals of this study are (1) to observe directly the photoexcitations and an equal number of reference measurephotoinduced formation and subsequent decay of pyrenee+in 1 ments without sample excitation. The 1920 laser shots used and (2) to see if ET quenching of n,n*emission in 2 is as facile for each AA point were taken in alternating groups of 60 shots in a polar nonprotic solvent such as acetonitrile (MeCN) as in with excitation and 60 shots without excitation. Finally, AA the polar protic solvent MeOH. Since the emission quantum points in kinetics and spectral experiments were taken in random yield in MeOH of 1 is 0.002 while that of 2 is 0.027,' 1 is the order. better candidate for picosecond transient absorbance studies, Absorbance and Fluorescence Spectra and Quantum because it affords a lower level of interfering emission. 2, Yield Measurements. Absorbance spectra were recorded on however, is the better candidate for solvent dependent n,n*a Perkin-Elmer High-Performance Lambda-6 spectrophotometer quenching studies, because it shows a change from CT to n,n* equipped with a double monochromator for reduced stray light. emission as the solvent is changed from MeOH to THF, while Fluorescence spectra were recorded on an SLM-8000C (SLM 1 does not.' Aminco, Inc.) spectrofluorometerand corrected for the spectral Studies of pyrene-labeled nucleosides and oligonucleotides response of the optical system. The correction factors were are of interest from at least three different perspectives. First, determined at Georgia State University by technical support there is interest in understanding and controlling ET dynamics personnel from SLM Aminco, Inc., using a standard lamp whose among DNA bases and DNA adducts in oligomers and energy output was traceable to NIST calibrations. The corrected Second, knowledge of the rates of DNA base emission spectra reported in this paper are plotted as relative ionizations and neutralizations is useful in unraveling the mechanisms of DNA damage caused by ionizing r a d i a t i ~ n . ~ - ~ ~detected intensity versus wavelength. Solutions for fluorescence quantum yield measurements of 1 and 2 typically contained, Third, pyrene-adduct lesions in DNA strands can be inserted respectively, (2-4) x and (2-5) x M sample into cellular hosts as sites of genetic mutations and thus are concentrations. The excitation wavelength for emission spectra useful as tools for learning about DNA repair and replication mechanisms. and quantum yield measurements was 341 nm. Also, for relative emission quantum yield measurements, the excitation bandwidth was 1 nm and the absorbances of the two samples Materials and Methods being compared were made nearly identical at an absorbance Preparation of 5-(1-Pyrenoy1)-dU (1) and 5-(l-Pyrenyl)for value of ca. 0.1. The fluorescence quantum yield (aem) dU (2). Assembly of the uridine analogues 1 and 2 (depicted in pyrenebutanoic acid (PBA; Molecular Probes, Inc., High-Purity Figure 1) was accomplished by modification of previously Grade, lot number 4721-1) in MeOH (EM Science, Omnisolv published procedure^.^^^^^ Palladium coupling reactions which HR-GC Grade) was measured to be 0.065 relative to 9,lOjoined pyrene to uridine were conducted in pressure-equalizing diphenylanthracene (Aldrich, 98%) in cyclohexane (Aldrich, glass coupling apparatus equipped with an addition funnel and Spectrochemical Grade) with Oem= l.00.5' The emission high-vacuum Teflon valves. The starting reagents were comquantum yields of pyrene-labeled uridine nucleosides were bined in a Vacuum Atmospheres, Inc., inert (argon) glovebox. subsequently measured relative to PBA in MeOH. ApReactions conducted outside the glovebox were performed under propriately oriented polarizers were used to eliminate the an argon atmosphere. THF was distilled from benzophenone possible effects of nonisotropic emission from the samples for NalK alloy. S / P Brand 60 A (230-400 mesh ASTM) silica both spectra and quantum yield measurementsSs2 Also, for was used for flash chromatography. NMR data were acquired samples with very weak emission, an indirect quantum yield on a Bruker AMX (300 MHz 'H) instrument. Infrared data method was used to increase a c ~ u r a c y . ~All ~ - ~samples ~ used were acquired on a Perkin-Elmer 1600 FTIR instrument. Mass for emission spectra and quantum yield determinations were spectral data were obtained from the departmental facilities at deaerated by bubbling with solvent-saturated argon for 20-30 Washington State University and the University of California min while being magnetically stirred. at Berkeley. Complete descriptions of the syntheses and THF,MeOH, and MeCN (Aldrich, HPLC Grade) were also analytical characterizations of 1 and 2 have been reported elsewhere.' used as solvents for both emission quantum yield and lifetime measurements. In addition to air-equilibrated and freezePicosecond Transient Absorbance Measurements. The pump-thaw (FPT) prepared solutions of MeCN, "dry" MeCN active-passive mode-locked Nd:YAG laser system (1= 1064 was also used for emission quantum yield and lifetime measurenm; 15 Hz repetition rate; -30 ps pulse duration fwhm) at
J&l
LN
I" %
Netzel et al.
17950 J. Phys. Chem., Vol. 99,No. 51, 1995
ments. In this case ca. 450 mL of MeCN was loaded with a generous amount of CaHz(s) into a round bottom flask in a NZ glovebag and attached to a solvent still with a flowing NZ atmosphere (N2 gas from liquid NZboil-off) held at greater than atmospheric pressure with an oil bubbler pressure-relief valve. MeCN was refluxed over CaH2 for 4 h, and ca. 100 mL was distilled off and discarded. A 250 mL portion of the remaining solution was used for spectroscopic experiments. Unused solution was stored in the still under a NZatmosphere, and each day fresh aliquots of MeCN were redistilled. For spectroscopic studies oven-heated flasks, optical cells, and stainless-steel cannulas were used with MeCN-washed rubber septa. Standard Schlenk flowing-N? transfer techniques were used to prepare samples of 1 and 2 in dry MeCN. Although the original motivation was to ensure that MeCN would have no water (or at least only a minimal amount), it was also true that the above procedures removed dissolved oxygen very well. Fluorescence Lifetime Measurements. All fluorescence decays were recorded on a Tektronix SCDl00O transient digitizer (10.35 ns rise time calculated from the bandwidth, I120 ps rise time for a step input 0.5 times the vertical range) and wavelengths resolved with a 0.1 m double monochromator (Instruments SA, Inc., model DH10) in additive dispersion. The 2 mm slits were used producing an 8 nm band-pass. The 1200 grooves/mm holographic gratings were blazed at 450 nm. After passing through the monochromator, the emission was detected with a Hamamatsu 1564U microchannel plate (200 ps rise time). The excitation and emission beams were oriented at 90” with respect to each other with the Glan-Thompson emission polarizer set at 54.7” (“magic angle”) with respect to the vertical excitation-polarization to eliminate rotational diffusion artifacts in the emission lifetime measurement^.^^ Emission for all lifetime measurements was excited at 355 nm with the third harmonic of an active-passive mode-locked Nd3+NAG laser manufactured by Continuum, Inc. Typically 35 pJ excitation pulses of ca. 25 ps duration were collimated into a 3 mm diameter beam and passed through a second Glan-Thompson polarizer before entering the sample cuvette. Photon Technology Incorporated software was modified by the manufacturer to process 1000 data points per decay curve and was used to deconvolute the instrument response from the emission decay to yield exponential lifetime fits to the emission decay data. Emission lifetime tests were carried out on commercial samples of anthracene (Aldrich, 99+%) and 1-aminoanthracene (Aldrich, 99+%) which were dissolved in cyclohexane and degassed in 0-ring-sealed optical cells with three FPT cycles on a vacuum line (2 x lov4 Torr). Recorded emission decays for these samples were fit with single exponential lifetimes of 5.1 and 22.5 ns, respectively, for anthracene and 1-aminoanthracene. These lifetimes agreed well with their respective literature values of 4.9 and 22.8 The generally observed temporal resolution of the emission kinetics system for multiexponential emission decays was ca. 0.2 ns; however, in ideal circumstances it was as good as ca. 50 ps after deconvolution. All samples used for lifetime measurements were also degassed with three FPT cycles on a vacuum line as described above unless otherwise indicated. Solutions for fluorescence lifetime measurements of 1 and 2 typically contained the same sample concentrations as for quantum yield measurements. A full description of the lifetime-fitting procedure used here is presented in a recent paper by Netzel et al.;’ included there are nine sets of emission decays on four time scales (20, 50, 100, and 500 ns) along with the following information: the equations used, plots of residual differences between experimental emission decays and calculated multiexponentialcurves, linear and logarithmic plots of emission decays, lamp decays
and exponential curves, and as specific
x?
values (the reduced values for lifetime fits reported here generally ranged from ca. 1 to 8. Other less extensive descriptions of the emission decay analysis procedure have also been r e p ~ r t e d . ~ . ~ ’ Because the accuracy to which a lifetime component can be determined is proportional to the relative emission area of that component, relative area data are presented with the lifetime values. On the other hand, relative emission amplitudes are proportional to the number of emitting species with the corresponding lifetime; thus these data are also given. The combination of finite detector response time and small relative emission areas (1-3%) for a number of the subnanosecond lifetime components presented in this work causes such values to be highly uncertain. Emission lifetime components 1 1 ns generally also have significant relative emission areas and are consequently much more reliable. However, whenever two emission lifetimes are less than a factor of 2 different for either bi- or triexponential decay processes, relative amplitude errors of f 2 0 % and lifetime errors of f10-20% are Typical errors for emission lifetimes that can be fit with only a single exponential are 2-4% for lifetimes 5 10 ns and 1-2% for lifetimes greater than 10 ns. Each kinetics trace (or curve) recorded 1000 data points; all fits used all 1000 points; and all data curves that were fit were themselves the result of averaging 1000 photoexcitation events as well as 1000 background events and subtracting the latter from the former.
x2 statistic) for the plotted exponential curves. x?
Results and Discussion Bimolecular Photoproduction of F‘yrene’-: A Control Experiment. The goal of picosecond transient absorbance experiments on 1 in MeOH is to determine whether or not ET products (especially pyrene’+/dU-) are formed as a result of photoexcitation. In the case of the closely related nucleoside 2, broad CT emission is seen in MeOH with an emission maximum at 475 nm.’ However, for 1 in MeOH only n,n* emission is seen with an emission maximum at 395 nm. At this wavelength, 30% of the emission amplitude decays in ca. 0.3 ns (2% relative emission area) and 70% of the emission amplitude decays in 8.7 ns. An immediate concern is whether or not ET is occumng in 1, since only n,n*emission can be seen. It is clear that emission quenching is occumng, because the emission quantum yield is only 0.002.’ This can be compared to reported emission quantum yields for 1-pyrenecarboxaldehyde in MeOH of 0.07-0.15.59-61 Two observations support the hypothesis that ET may be the cause of the very low emission quantum yield of 1. First, the n,n*emission yield of 1 increases 14-fold to 0.028 by changing from MeOH to the low-dielectric solvent THF. Decreasing the solvent dielectric constant is expected to raise the energy of CT product states more than that of x,n*states and thus should reduce ET It is equally important that the emission quantum yield increase for 1 on going from MeOH to THF is not consistent with an emission yield variation due to changing the location of the lowest energy n,n* state.62 When n,x* states are very near or below n,n*states in substituted aromatic systems, n,n*emission is quenched.63@ For 1 the lowest energy n,x* state is expected to be higher in MeOH (due to solvent proton donation) than in THF, and yet the n,n* emission yield is lower in MeOH than in THF. Second, the emission of 1 in MeOH at 495 nm exhibits triexponentialdecay kinetics with the following lifetimes: ca. 0.4 ns (78% amplitude, 15% area), 1.8 ns (11% amplitude, 10% area), and 7.2 ns (21% amplitude, 75% area). The very large amplitude of the shortest lifetime component could be due to ultrafast decay of a CT photoproduct.
Electron Transfer in Pyrene-Labeled Nucleosides There are two chief concerns when looking for AA signals characteristic of pyrene'+ following photoexcitation of 1. (AA signals due to dU'- are significantly smaller in the 360-750 nm observation range of our picosecond laser system than are signals due to the excited states of pyrene or its cation.) The first concern is to identify AA features characteristic of the S I and first excited triplet (TI) states of pyrene as well as of the pyrene cation and anion. The second is to demonstrate that pyrene cation found in a laser excitation experiment is due to ET quenching of pyrene* and not to direct two-photon ionization by the laser pulse. One method of demonstrating this latter point is to run a bimolecular ET-quenching control experiment which forms pyrene'- without forming pyrene'+. Then using laser pulses of the same fluence and a sample of 1 with the same absorbance at 355 nm (ca. 0.35),record the AA spectra and kinetics of 1. A study by Shafiiovich et al.25reports the required AA spectra for the closely related compound 7,8,9,1O-tetrahydroxytetrahydrobenzo[a]pyrene (BPT). The SI state of BPT in dimethylformamide (DMF) has a transient absorbance maximum at 490 nm with a broad shoulder in the 500-520 nm region and tailing absorbance beyond 600 nm. The TI state in DMF has an absorbance maximum at 420 nm with weak and very broad absorbance from 450 to 550 nm. BPT'- in MeCN has a sharp, strong absorbance band at 495 nm and tailing absorbance beyond 600 nm. This accords with an extremely sharp absorbance band for pyrenee- in methyl tetrahydrofuran at 492 nm ( E 93 OOO M-' cm-I), a weaker absorbance band at 712 nm (e 5000 M-I cm-I), and tailing absorbance extending as far as 1000 nm.26 BPT'+ in MeCN has a strong absorbance band at 455 nm with a distinct shoulder at ca. 440 nm. Importantly, B I T + does not have much absorbance in the 500-520 nm region where the S I state does. This accords with a sharp absorbance band for pyrene" in formamide (FM)at 449 nm (E 60 000 M-' cm-I) with a distinct vibrational peak at 438 nm ( E 25 000 M-' cm-I). Additionally, pyrene'+ shows six weak absorbance bands ( E ca. 5000 M-' cm-I) in the 490-800 nm region.26 Comparison of the wavelengths and extinction coefficients of thestrongest absorbance bands of the cations and anions of BPT and pyrene shows that the additional chemical substitution in BPT versus pyrene has two general consequences. The first is that while these absorbance bands are still strong for BPT, they are less intense and much less sharp than for pyrene. The second is that they nearly unshifted in wavelength (varing in different solvents by only 3-6 nm). The carbonyl substitution of pyrene in 1 suggests that the absorbance spectrum of the postulated photooxidized donor, pyrenoyl'+, will be modeled better by the spectrum of BPT'+ than by that of pyrene". Our attempts to record the transient absorbance spectra of ET products resulting from bimolecular quenching of photoexcited 1 -pyrenecarboxaldehyde failed due to fluorescence interference. Finally, for convenience the photooxidzed chromophore in 1, pyrenoyl'+, is referred to below as pyrene'+. The control experiment adds 0.08 M N,"-dimethylaniline (DMA) to 1.8 x M PBA in MeOH. DMA quenches BPT* in acetonitrile with a bimolecular quenching rate of 1.6 x 1Olo M-' s-', forming DMA'+ and BPT-.25 If the quenching rate for PBA and DMA in MeOH is similar, one would expect to see a AA increase near 495 nm grow in with a lifetime of 780 ps. Importantly, prompt (530 ps) absorbance near 455 nm would show direct photoionization of PBA and is undesirable. It is, however, important to note that the other ET-quenching product, DMA'+, absorbs moderately ( E 5000 M-' cm-') at 473 nm in FM and at ca. 470 nm in MeCN.25,26 Figure 2 shows the AA spectra for the DMAPBA quenching experiment in the 400-750 nm region immediately after (t =
J. Phys. Chem., Vol. 99, No. 51, 1995
17951
0.18 -j
2 m
li\ I 1
-.-I
,i__,.,,, ..
0u ::::id
9
1
c
0.02
-t=3ns
.. ......
0.00 400 450 500 550 600 650 700 750 800
Wavelength (nm) Figure 2. Change-in-absorbance (AA) spectra for PBA (1.8 x M) and DMA (0.08 M) in MeOH immediately after ( t = 0 ps) and 3 ns after (t = 3 ns) photoexcitation at 355 nm. Each AA point results from averaging data for 1920 laser shots; error bars represent 1 standard deviation in the averaged data. Data points are connected with straight lines for viewing convenience.
0
Q)
-._1
Bimolecular Quenching of PBA by DMA in MeOH
.
-1000 2000
SO&
8000
l l k 1 4 h O 17000
Time (ps) Figure 3. Plot of AA versus time at 500 nm for PBA and DMA in MeOH (same concentrations as in Figure 2) following laser excitation at 355 nm. The solid line shows a fit to the data from the end of the excitation pulse ( t = 0 ps) to 15 ns using the following equation: a exp(-bt) c exp(-dr) e, where f is time, a, c, and e are AA amplitudes, and b and d are rates. The parameter values for the fit shown are a = -0.09, l/b = 750 ps, c = 0.18, l/d = 22 ns, and e = 0.0 with R = 0.986. However, a range of values also provides fits that are nearly as good (lowest R = 0.982): a = -(0.086-0.090), llb = (750-770) ps, c = (0.14-0.18), lld = (16-22) ns, and e = (0.040.0) with the sum of c and e ca. 0.18.
+
+
0 ps) and 3 ns after photoexcitation. At 3 ns the strong, sharp peak at 500 nm is consistent with pyrene'- formation. Importantly, no shoulder in the 500-520 nm region characteristic of the St state is seen. The moderate absorbance at 470 nm is consistent with formation of DMA'+. At t = 0 ps it is likely that three species are present: unquenched S1 states, pyrene'-, and DMA'+. The latter two ET products arise from static quenching during the ca. 25 ps excitation pulse. Direct photoproduction of pyrene'+ would be seen in this spectrum in the 455-460 nm region. Importantly,the absorbance of D W + at 470 nm is even more prominent than is 455-460 absorbance at this time. To the extent direct photoexcitation of PBA would produce pyrene'+, an equal number of solvated electrons would also be produced. In water, solvated electrons have an absorbance maximum at 700 nm ( E 19 000 M-' ~ m - ' ) . However, ~~ no solvated electron band in the 600-750 nm region is apparent. Thus direct photoionization of PBA is not apparent at the laser power and fluence used here. Figure 3 shows kinetics data for the DMAPBA quenching experiment at 500 nm in the -200 ps (before photoexcitation) to 15 ns time range. One-half of the initial AA increase occurs during photoexcitation (due to static quenching and formation of S I states). After excitation the data can be fit with a three-
Netzel et al.
17952 J. Phys. Chem., Vol. 99, No. 51, 1995 0.10
5-(1-pyrenoy1)-dU in MeOH
-0.01
1
I
0.10.
0.0o-ER.. . . . , . . . . . ,.. .... .. . , 1 ' -500 3500 7500 11500 15500
300
.
400
500
600
700
r . .
.
,
800
Time (ps)
Wavelength (nm) M Figure 4. AA spectra for 5-(l-pyrenoyl)-dU (1) at 5.1 x concentration in MeOH at three times after photoexcitation: 0 ps (immediately after photoexcitation), 3.5 ns, and 15 ns. Data points are connected with straight lines for viewing convenience. Laser excitation pulse fluence and sample absorbance at 355 nm are the same as in Figures 2 and 3. Data-averaging procedures and error bar symbols are the same as in Figure 2 . Error bars are not plotted in the 15 ns spectrum for viewing clarity, but they are similar to those in the other two spectra.
state first-order serial kinetics model, corresponding to the SI, pyrene'+, and TI species.66 The form of this equation requires five parameters, three for the respective molar extinction coefficients and two for the rate constants for the formation and decay of pyrene'+, respectively, from SI and to TI. The lifetime found for the growth of the 500 nm AA increase is 750 ps, which agrees with the 780 ps lifetime for bimolecular quenching estimated above. Photoproductionof Pyrene'+ in 1-Pyrenoyl-dU(1): Proof of ET. Figure 4 shows three AA spectra resulting from photoexcitation of 1 in MeOH with the same laser pulse fluence and sample absorbance as in the DMAPBA experiment: 0 ps (immediately after excitation), 3.5 ns, and 15 ns. In contrast to the DMAPBA quenching experiment, photoexcitation of 1 produces a maximum AA increase at 460 nm (not at 500 nm) at t = 0 ps (not at 2 ns). These observations are consistent with formation of pyrene'+ during the photoexcitation pulse (530 ps). Further inspection of the t = 0 ps spectrum shows a characteristic pyrene'+ shoulder at 440 nm as noted above. The AA increase at 500-510 nm, which falls off gradually until 600 nm, is consistent with formation of some SI states during photoexcitation. In other words, not all of the SI states are ET quenched in 530 ps. Comparison of the 3.5 and 15 ns spectra shows that a small, gradual relaxation occurs in this time range. Presumably the AA spectrum at 15 ns is due to mainly T I excited states. For BPT in DMF, the TI state has an absorbance maximum at 420 nm with weak absorbance extending to 550 nm. 1 has bleached ground state absorbance (-AA) offsetting the TI state's absorbance increase in the 400-410 nm region, but the peak at 420 nm in the 15 ns spectrum still accords with a TI assignment for this spectrum. Greatly enhanced (3-10 times) production of triplet excited states (relative to yields expected from simple intersystem crossing) as a result of ET quenching of BPT* by added dG has been previously rep0rted.2~ A similar situation appears to occur here for 1 in that at 3.5 ns pyrene'+ absorbance is absent, and mainly TI absorbance arising from charge recombination remains. Figure 5 presents plots of kinetics data for 1 in MeOH at 460 nm from -300 ps to 15 ns (experimental conditions as for Figure 4). The data are fit (without convolution of instrument response) to a biexponential function with decay lifetimes of 67 f 3 ps and ca. 6 ns. The AA decays at 510 nm (data not shown) are also fit with two decay lifetimes of 43 f 3 ps and 1-5 ns. It is reasonable to conclude that the majority of SI states decay slightly faster than the pyrenee+ CT product.
Figure 5. Plots of AA kinetics for 5-(l-pyrenoyl)-dU (1) in MeOH at 460 nm in the -500 ps (before excitation) to 15.5 ns time range. Sample absorbance, laser fluence, data-averaging, and error bar symbols are as in Figures 4 and 2 . The inset is an expanded plot of the same data in the -300 to 500 ps time range. The solid lines in both the main plot and the inset show fits to the data from 0 ps (immediately after excitation) to 15.5 ns using the same equation as in Figure 3. The parameter values for the fit shown are a = 0.075, l/b = 67 rt 3 ps, c = 0.006, lld = ca. 6 ns, and e = 0.015 with R = 0.999.
Without accurate knowledge of the shape of our instrument response function and considering the signal-to-noise limitations of the data, a reasonable estimate for the lifetime of the pyrene'+ CT product at 460 nm is 20-70 ps. Femtosecond laser techniques are needed to determine this relaxation time more accurately. Interestingly, the ca. 6 ns AA relaxation seen thoughout the visible region agrees well (considering the small amplitude of the relaxing signal) with the previously reported 8-9 ns emission component in the 380-450 nm range.' An unavoidable conclusion is that both ultrafast ( 530 ps) and slower (8-9 ns) ET-quenching processes are present for 1 in MeOH. It also appears that the overwhelming majority of quenching processes are ultrafast. Perhaps the best argument for this is based on the relative amplitudes of the fast and slow AA signals at 510 nm. These data indicate that 88 f 5% of the SI states decay in 43 & 3 ps. An important assumption here, which is supported by the known spectra of BPT'+ and pyrene'+ (see a b o ~ e )is, that ~ ~ pyrenee+ ~ ~ ~ has negligible absorbance at 5 10
nm. Evidence for Protonated dU'-, dU(H)', as a Primary ET Product for 2. In addition to their dielectric properties, solvents can also be classed with respect to their proton-donating properties. Thus although it was reported earlier that a change in the type of emission spectrum from CT to n,n*occurs for 2 on switching solvent from MeOH to THF, it was also noted that it was not clear how much of this change was due to removal of proton-donating capability and how much to lowering the dielectric constant from 33.6 (MeOH) to 7.6 (THF). The state of protonation of dU, dT, and dC upon reduction in aqueous solution remains an open question, where dT = 2'deoxythymidine and dC = 2'-deoxycytidine. A widely held opinion is that in double strand (ds) DNA dC'- would be readily protonated by its base-paired partner dG, and thus in ds DNA dC(H)' would be ca. 200 mV easier to form than dT-, which is not thought to protonate as It is known that in aqueous solution dC and dT have the same reduction potentials of -1.45 V (versus a saturated calomel e l e c t r ~ d e ) . ~It~is, ~ ~ not clear whether or not they are also protonated when they are reduced. Contrary to the above opinion, bimolecular ET quenching of BFT* by dT and dC is found to require a polar protic solvent, because it does not occur in the polar organic solvent dimethyl sulfoxide (DMSO).'" If this is true, the reduction potentials of dT and dC in water and in ds DNA are
Electron Transfer in Pyrene-Labeled Nucleosides
J. Phys. Chem., Vol. 99, No. 51, 1995 17953
5-(l-pyrenyl)-dU 475 nm J
in MeOH
TABLE 1: Emission Quantum Yields (ae,,,) and Lifetime (ns) for 2 in Three Solvents @em
lifetimes
0.004 . . . . , . . ; c 300 350 400 450 500 550 600
in "dry" MeCN
0.15, 342nm
in THF
300
350
400
450
500
550
600
Wavelength (nm) Figure 6. Plots of absorbance and relative emission intensity versus wavelength for 5-(l-pyrenyl)-dU (2) in three solvents: (top) in MeOH, M; and (bottom) in 4.9 x M; (middle) in dry MeCN, 4.7 x THF, 4.7 x M.
the same.69 Two studies also provide information on the rate of protonation of dT and dC in water at pH 7. Kinetics analysis of dynamic and static fluorescence quenching of BPT* by dT and dC finds lower limits of 0.6 and 6 ns, respectively, for ET to dT and dC.70 If ET is accompanied by proton transfer from water, these values are also lower limits for the rate of protonation of dT'- and dC'- in bimolecular complexes. Finally, a pulse radiolysis study of cytosine'-, dC'-, and 2'deoxycytidine-5'-monophosphate'- (dCMP-) finds that their rates of protonation are almost identical in the pH range of 5-12 with pseudo-first-order lifetimes of 400 n ~ . ~ ' Figure 6 presents absorbance and emission spectra for 2 in MeOH, dry MeCN, and THF. The results in MeOH and THF have been reported earlier' and are only shown here to provide a convenient comparison for the spectra in dry MeCN. Consistent with the above-referenced lack of fluorescence quenching for BPT* by dT in DMSO, the emission spectrum of 2 in the middle panel of Figure 6 shows that in MeCN most of the CT emission seen so clearly in MeOH (A, 475 nm) is replaced with n,n*emission as seen in THF (A, 395 nm). Switching solvent from MeOH to MeCN cleanly separates the effects of solvent proton donation and solvent dielectric response on ET quenching, because MeCN has a dielectric constant of 37.5, which is close to that of MeOH, 33.6. The emission data in Table 1 show that the emission quantum yield increases a factor of 13 on going from MeOH to MeCN
MeOHb
dry MeCN'
THFd
0.027 [0.691 0.05 (10) [0.2810.91 (70) [0.03] 2.2 (20)
0.35 [0.36] 0.19 (2) [ O H ] 5.8 (98)
L0.181 0.3 (1) [0.82] 6.7 (99)
0.42
[Fractional emission amplitude] emission lifetime (percent emission area). Lifetimes for MeOH and THF were measured in FPT degassed cells. Lifetimes measured at 475 nm. Data from ref 1. Quantum yield and lifetimes measured in cells prepared with Schlenk techniques, 4.7 x M sample concentrations for each measurement. Lifetimes measured at 430 nm. The quantum yield in HPLC grade acetonitrile prepared with argon bubbling was 0.26. Lifetimes measured at 395 nm. Data from ref 1.
and a factor of ca. 16 on going from MeOH to THF. The emission quantum yield and spectral shape changes as the solvent is varied are also reflected in the emission lifetime patterns of 2 in these solvents. As one changes solvent in the series MeOH, MeCN, and THF, there is a dramatic lessening of the amplitude of the shortest lifetime component accompanied by an equally dramatic increase in the amplitude of the longest lifetime component. Additionally, the overall lifetime patterns in MeCN and THF are very similar. These data indicate that for 2 switching from a polar protic to a polar nonprotic solvent lessens ET quenching of pyrene* almost as much as does switching from a polar protic solvent to one which is both nonpolar and nonprotic. The lessening of emission quenching on switching from MeOH to THF is greater than that found on switching from MeOH to MeCN, but the latter change combines both absence of proton donation and low dielectric response. Clearly, both factors affect the ET-quenching process. A straightforward mechanism by which each of them could lessen quenching is raising the free energy of the pyrene'+/dU'- CT state relative to the n,n*state of pyrene. Since dU(H)' requires less energy to form than does dU-, ET quenching should be more favorable in MeOH where dU(H)' can be formed than in MeCN, where only dU'- can be produced. This view is consistent with the ca. 1.0 V lower reduction potential of thymine derivatives in polar nonprotic organic solvents compared to that in aqueous s o l ~ t i o n s .However, ~ ~ ~ ~ the ~ ~ ~ ~ ~ ~ ~ increase in free energy of the CT state of 2 on switching from MeOH to MeCN does not appear to be this large. If it were, the free energy change for ET quenching would be ca. + O S eV in MeCN. The relative amplitude of the shortest lifetime component in this solvent, 0.36, appears too large for so unfavorable a reaction. Even in THF where the dielectric constant is low and proton donation is absent, 2 shows a relative emission amplitude of 0.18 for subnanosecond n,n*emission quenching. Photoinduced ET Quenching of 1 in MeCN. As the solvent is changed for 1 in the series MeOH, dry MeCN, and THF, the emission quantum yield varies, respectively, as 0.002, 0.005, and 0.028. The emission yield increase for 1 on going from MeOH to THF, 14-fold, is similar to the ca. 16-fold increase for 2. However, the ca. 3-fold emission increase for 1 on going from MeOH to MeCN is significantly less than 13-fold increase for 2. AA spectral and kinetics measurements for 1 in MeCN (5.5 x M sample concentration) made as for 1 in MeOH yield the following observations (data not shown). (1) A prominent 460 nm AA increase characteristic of pyrene'+ is formed within the laser pulse ( 530 ps). (2) This signal decays with biexponential kinetics with lifetimes of 103 f 9 ps and ca. 6 ns (assuming the AA spectrum at 15.5 ns is due almost entirely to the TI state). To the extent SIstates are not quenched at 15.5 ns, a longer second lifetime would result. (3) The 100 ps relaxation at 460 nm has a relative amplitude of 84 f 5%. (4) At 510 nm where SIstates have an absorbance maximum,
Netzel et al.
17954 J. Phys. Chem., Vol. 99, No. 51, 1995
TABLE 2: Emission Lifetimes (ns)for 1 in Dry MeCN" 385 nm
400 nm
410 nm
[0.28] 0.44 (2) [0.61] 8.7 (59) [0.11] 40.3 (39)
[0.70] 0.37 (9) [O. 161 7.2 (40) [0.14] 38.5 (51)
[0.45] 0.3813) [0.50] 7.6 (71) [0.05] 39.4 (26)
420 nm
450 nm
495 nm
[0.50] 0.36 (4)
[0.71] 0.18 (23) [0.17] 4.3 (36) [0.10] 7.0 (13) [0.02] 40.1 (28)
[0.74] 0.28 (1 1) [0.24] 4.5 (55)
I0.441 6.8 (65) [0.06] 41.1 (31)
[0.02] 41.1 (34)
[Fractional emission amplitude] emission lifetime (percent emission area). Experimental conditions as in Table 1 for dry MeCN; 4.7 x M sample concentration. a
the AA signal decays with a single exponential lifetime of 95 k 5 ps. The 2-fold lower signal amplitude at 510 nm than at 460 nm causes nanosecond-lived relaxations at 5 10 nm to occur within the noise of the data. Emission lifetimes for 1 in dry MeCN are presented in Table 2. Except for the presence of 40-ns-lived components with small (0.02-0.14) relative emission amplitudes, the emission lifetime pattern as wavelength is varied is similar to that reported for 1 in Me0H.I In MeOH biexponential emission kinetics are found from 382-450 nm with subnanosecond and 8-9 ns lifetime components. Since AA measurements in MeOH show that ca. 90% of the SI states of 1 decay in 1 4 3 ps, it is clear that the amplitudes of the ultrashort emission decays in MeOH are significantly underestimated by the emission detection equipment used here. The ultrafast 510 nm AA relaxation (due to S I states) of 1 in MeCN is slightly longer than this, ca. 100 ps; thus, in MeCN, the emission amplitudes of the shortest lifetime components will not be as severely underrepresented as in MeOH. With the exception of the data at 400 nm, there are two significant trends in the lifetime data as wavelength is increased from 385 to 495 nm. First, the relative emission amplitudes of the shortest lifetime components progressively increase. Second, in concert with the first trend, the relative emission amplitudes of the two longest lifetime components progressively decrease. A similar pattern of variation of emission amplitudes with wavelength is seen for 1 in MeOH. In contrast, 1 in THF shows no variation of emission amplitudes with wavelength. Also, the longest emission lifetime of 1 in this solvent is 94 ns, significantly longer than in MeCN.' From the point of view of emission quantum yield and lifetime, and AA spectral and kinetics data, 1 behaves very similarly in MeCN and in MeOH. A natural question is, Why does switching solvent from MeOH to MeCN for 2 produce a dramatic change in ETquenching behavior while the same switch for 1 produces a much more modest change? One possibility is that the free energy of photoinduced ET quenching is more negative for 1 than for 2. Emission-quenching results for 1 and 2 in MeOH do not contradict this possibility, since both show predominantly ultrafast n,n*quenching kinetics. If this is true, it is possible in MeCN that ET to form the pyrene'+/dU- product may remain quite favorable for 1 while for 2 ET to form the same product may be only weakly favorable or even modestly unfavorable. However, for both 1 and 2, forming pyrene'+/dU(H)' in a protic solvent appears to be more favorable than forming pyrene'+/ dU'- in a nonprotic one.
Conclusions A control transient absorbance study of ET quenching of photoexcited PBA by added DMA in MeOH showed that the pyrene'- signal at 500 nm can be easily observed 2 ns after photoexcitation without photoproduction of pyrene'+ and sol-
vated electron. The same laser pulse fluence and sample absorbance were then used to study 1 in MeOH. In this case a maximal AA increase at 460 nm characteristic of pyrene'+ occurs during the time of photoexcitation (130 ps). The pyrene" signal decays in 20-70 ps slightly more slowly than the SI state's positive AA signal decreases at 510 nm. These results prove that the SI state of pyrene in 1 is quenched due to intramolecular ET. Similar results are also obtained for 1 in MeCN, where the pyrene'+ absorbance at 460 nm decays in ca. 100 ps. Additionally, biexponential AA relaxations are found in both solvents throughout the visible spectral region. These AA relaxations match the emission decay kinetics for 1 in both solvents and further establish that S I states in 1 are quenched in polar solvents on both ultrashort and ca. 6 ns time scales. For 1 in MeCN, a small amplitude emission decay component with a lifetime of 40 ns is also present, indicating additional ET-quenching complexity in this solvent as compared to MeOH. Presumably the strikingly different time scales for n,n*emission quenching reflect a distribution of relative orientations of pyrenoyl, uracyl, and ribosyl groups in 1 which is heterogeneous for tens of nanoseconds. The emission properties of both 1 and 2 were studied in the solvent series MeOH, MeCN, and THF. For both compounds changing from MeOH to MeCN increased emission lifetimes and quantum yields. For 2 the emission yield increase was 13fold, while for 1 it was 3-fold. A dramatic change in the emission spectrum of 2 upon changing solvent from MeOH to THF was previously noted.' In the former solvent, broad CT emission is present, while in the latter solvent exclusively n,n* emission is seen. This work reports the striking observation that changing solvent from MeOH to MeCN changes the emission spectrum of 2 nearly as much as does changing to THF. The main difference between emission in THF and in MeCN is that in MeCN a small amount of CT emission is also seen in combination with strong n,n*emission. The emission quantum yields in this solvent series for 2 are 0.027, 0.35, and 0.42, respectively. Since the dielectric constants of MeOH and MeCN are similar, 33.6 and 37.5, respectively, the large emission yield increase and striking change in the emission spectrum for 2 on switching from MeOH to MeCN is consistent with lessened ET quenching due to raising the free energy of the pyrene'+/dU'- CT product relative to the n,n* state of pyrene. Since dU(H)' requires less energy to form than does dU'-,67,70*72,73 ET quenching should be more favorable in MeOH, where dU(H)' can be formed, than in MeCN, where only d U can be produced. This conclusion agrees with recent results by Shafirovich et al.70in which quenching of emission from BFT* by dT and dC occurred in aqueous solutions but not in the polar nonprotic solvent DMSO. If this model is correct, the time of protonation of d U - in MeOH is 5 3 0 ps for 1 based on transient absorbance measurements of the appearance of pyrenee+. The observation that switching solvent from MeOH to MeCN increases the emission quantum yield only 3-fold for 1 but 13fold for 2 suggests that the free energy change for ET quenching of pyrene* might be more negative (more favorable) for 1 than for 2. It would be interesting to test this possibility with electrochemical experiments. Additionally, femtosecond transient absorbance experiments may be able to time resolve the primary ET steps in these compounds. If so, measurements of the solvent associated H/D kinetic isotope effect on the quenching steps may be possible.
Summary of Abbreviations ET = electron transfer; CT = charge transfer; MeOH = methanol; MeCN = acetonitrile; DMA = N,"-dimethylaniline;
Electron Transfer in Pyrene-Labeled Nucleosides
FM = formamide; DMSO = dimethyl sulfoxide; DMF = dimethylformamide; THF = tetrahydrofuran; dU = 2’-deoxyuridine; dT = 2’-deoxythymidine; dG = 2’-deoxyguanosine; dC = 2’-deoxycytidine; dCMP = 2’-deoxycytidine 5’-monophosphate; DNA = 2’-deoxyribonucleic acid; CaH2 = calcium hydride; BIT= 7,8,9,1O-tetrahydroxybenzo[a]pyrene;BPDE = benzo[a]pyrenediol epoxide; PBA = pyrenebutanoic acid; NZ = dinitrogen; PAH = polyaromatic hydrocarbon; A4 = change in absorbance; NIST = National Institute of Standards and Technology; FTIR = founier transform infrared; N M R = nuclear magnetic resonsance; fwhm = full width at one-half of maximum; FPT = fieeze-pump-thaw; ds = double stranded; E = molar extinction coefficient; @‘em = emission quantum yield; SI= lowest energy singlet electronic excited state; T I = lowest energy triplet electronic excited state; X? = the reduced chisquare statistic. Acknowledgment. This work was supported at Georgia State University by a grant to T.L.N. from the United States Department of Energy, Office of Health and Environment, Radiological and Chemical Physics Research Division (Grant No. DE-FG05-03ER61604). T.L.N. is pleased to acknowledge helpful conversations with Dr. Nicholas Geacintov. References and Notes (1) Netzel, T. L.; Zhao, M.; Nafisi, K.; Headrick, J.; Sigman, M. S.; Eaton, B. E. J. Am. Chem. SOC.1995, 117, 9119. (2) Brunschwig, B. S.; Ehrenson, S.; Sutin, N. J. Phys. Chem. 1986, 90, 3657. (3) Sutin, N.; Brunschwig, B. S.; Creutz, C.; Winkler, J. R. Pure Appl. Chem. 1988, 60, 1817. (4) Marcus, R. A,; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265. (5) Kierzek, R.; Li, Y.; Tumer, D. H.; Bevilacqua, P. C. J. Am. Chem. SOC.1993, 115, 4985. ( 6 ) Veal, J. M.; Wilson, W. D. J. Biomol. Struct. Dyn. 1991, 8, 1119. (7) Kollman, P. In Protein Des. Dev. New Ther. Vaccines; Hook, J. B., Poste, G., Eds.; Plenum: New York, 1990; p 229. (8) Manoharan, M.; Tivel, K.; Zhao, M.; Nafisi, K.; Netzel, T. L. J. Phys. Chem., in press. (9) Cundall, R. B. Photochemistry 1992, 23, 3. (10) Ahuja, R. C.; Moebius, D. Langmuir 1992, 8, 1136. (1 1) Kalyanasundaram, K.; Thomas, J. K. J. Am. Chem. SOC.1977,99, 2039. (12) Lianos, P.; Cremel, G.Photochem. Photobiol. 1980, 31, 429. (13) Lianos, P. C. Photophysical Properties of Pyrene of Biophysical Importance; University of Tennessee: Knoxville, TN, 1978; Avail. Univ. Microfilms Int., Order No. 7903440; From: Diss. Abstr. Int. B 1979, 39 (8), 3656. (14) Turro, N. J. Modem Molecular Photochemistry; BenjamidCummings Publishing Co., Inc.: Menlo Park, CA, 1978. (15) Lianos, P.; Duportail, G. Eur. Biophys. J. 1992, 21, 29. (16) Bevilacqua, P. C.; Kierzek, R.; Johnson, K. A,; Tumer, D. H. Science (Washington D.C.) 1992, 258, 1355. (17) Eriksson, M.; Kim, S. K.; Sen, S.;Graslund, A.; Jemstrom, B.; Norden, B. J. Am. Chem. SOC.1993, 115, 1639. (18) Geacintov, N.; Prusik, T.; Khosrofian, J. J. Am. Chem. SOC. 1976, 98, 6444. (19) Graslund, A.; Kim, S. K.; Eriksson, S.; Norden, B.; Jemstroem, B. Biophys. Chem. 1992, 44, 21. (20) Kano, K.; Matsumoto, H.; Hashimoto, S.;Sisido, M.; Imanishi, Y. J. Am. Chem. SOC.1985, 107, 6117. (21) Weston, A.; Bowman, E. D. Carcinogenesis 1991, 12, 1445. (22) Telser, J.; Cruickshank, K. A,; Momson, L. E.; Netzel, T. L. J. Am. Chem. SOC.1989, 111, 6966. (23) Telser, J.; Cruickshank, K. A.; Momson, L. E.; Chan, C.-K.; Netzel, T.L. J. Am. Chem. SOC.1989, 111, 7226. (24) Kubota, T.; Kano, J.; Uno, B.; Konse, T. Bull. Chem. SOC.Jpn. 1987, 60, 3865. (25) Shafirovich, R. Y.; Levin, P. P.; Kuzmin, V. A.; Thorgeirsson, T. E.; Kliger, D. S.; Geacintov, N. E. J. Am. Chem. SOC.1994, 116, 63. (26) Shida, T. Electronic Absorption Spectra of Radical Ions; Elsevier: New York, 1988. (27) Weinstein, Y. A.; Sadovskii, N. A.; Kuz’min, M. G.High Energy Chem. 1994, 28, 211. (28) Pysh, E. S.;Yang, N. G. J. Am. Chem. SOC.1963, 85, 2124. (29) Geacintov, N. E.; Zhao, R.; Kuzmin, V. A.; Seog, K. K.; Pecora, L. J. Photochem. Photobiol. 1993, 58, 185. (30) Vahakangas, K.; Yrjanheikki, E. IARC Sci. Publ. 1990, 104, 199.
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