Photochemistry and Photobiology, 20**, **: *–*
Electronic Interactions of Michler’s Ketone with DNA Bases in Synthetic Hairpins† Almaz S. Jalilov, Ryan M. Young, Samuel W. Eaton, Michael R. Wasielewski* and Frederick D. Lewis* Department of Chemistry and Argonne-Northwestern Solar Energy Research (ANSER) Center, Northwestern University, Evanston, IL Received 26 August 2014, accepted 30 September 2014, DOI: 10.1111/php.12360
ABSTRACT
undergo rapid intersystem crossing to yield triplet states that are capable of oxidizing purine nucleobases (9,10). The resulting triplet radical ion pairs have lifetimes which are substantially longer than those for the corresponding singlet radical ion pairs, as a consequence of the spin forbidden nature of triplet radical ion pair spin recombination (11). However, quinones are strong electron acceptors both in the ground and excited state. Thus, we turned our attention to other chromophores which are known to undergo efficient intersystem crossing that could be incorporated into synthetic DNA as a hairpin linker. Among the chromophores that we considered were the dihydroxyalkyl derivatives of the well-known triplet sensitizer Michler’s ketone (MK, Scheme 1) (12). In addition to undergoing fast intersystem crossing, MK derivatives are prone to undergo electron transfer reactions, serving as either electron donors or acceptors, as a consequence of the charge transfer character of their excited states (13,14). The ground state of DNA is also known to undergo oxidation and reduction reactions, the purine bases serving as electron donors and the pyrimidines as electron acceptors (15). To our knowledge, the interaction of electronically excited MK and its derivatives with DNA have not been investigated. We report here a study of the steady state and femtosecond time-resolved spectroscopies of two groups of MK-linked hairpins and the diol linker MK’ employed in their synthesis. The first group consisting of hairpins MK-1-3 possesses only a MK chromophore, whereas, the second group consisting of hairpins MK4-6 possess both MK and a perylenediimide (PDI) base surrogate, a strong ground state and singlet state oxidant (Scheme 1) (16).
The mechanism and dynamics of photoinduced electron transfer in two families of DNA hairpins possessing Michler’s ketone linkers have been investigated by means of steady state and time-resolved transient absorption and emission spectroscopies. The excited state behavior of the diol linker employed in hairpin synthesis is similar to that of Michler’s ketone in methanol solution. Hairpins possessing only a Michler’s ketone linker undergo fast singlet state charge separation and charge recombination with an adjacent purine base, attributed to well-stacked ground state conformations, and intersystem crossing to the triplet state, attributed to poorly stacked ground state conformations. The failure of the triplet to undergo electron transfer reactions on the 7 ns time scale of our measurements is attributed to the low triplet energy and reduction potential of the twisted triplet state. Hairpins possessing both a Michler’s ketone linker and a perylenediimide base surrogate separated by four base pairs undergo photoinduced hole transport from the diimide to Michler’s ketone upon excitation of the diimide. The efficiency of hole transport is dependent upon the sequence of the intervening purine bases.
INTRODUCTION The dynamics and efficiency of photoinduced hole injection and the ensuing hole transport in DNA have been extensively investigated using a wide variety of singlet and triplet electron acceptors (1–3). In contrast, studies of electron injection have been limited to a relatively small number of singlet electron donors and the dynamics and efficiency of electron transport beyond one or two base pairs remain poorly understood (4,5). In our continuing study of photoinduced electron transfer in DNA (2), we sought a chromophore with a high intersystem crossing quantum yield that could serve as both a linker for the formation of stable DNA hairpins and as a photoreductant for the formation of pyrimidine anion radicals. The N-hydroxypropyl derivative of anthraquinone-2-carboxamide (AQ, Scheme 1) has been widely used as a capping group for DNA duplexes and hairpins in studies of DNA photooxidation (6–8). Anthraquinone derivatives
MATERIALS AND METHODS Materials. N,N’-[Bis(methyl-3-hydroxypropylamino)-4,4’-benzophenone (MK’). 4,4’-difluorobenzo-phenone (1 g, 4.6 mmol), 3-methylaminopropanol (1 g, 11.2 mmol), anhydrous potassium carbonate (1 g, 7.3 mmol) and catalytic amount of 18-crown-6 ether in dry DMSO (4 mL) were heated at 100°C for 48 h. The resulting reaction mixture was further separated using column chromatography (using eluent hexane/ethylacetate/ triethylamine, 100/20/1 mixture by volume) to give a yellow product, 1.2 g (72%). 1H NMR (CDCl3, 500.137 MHz): d - 7.74 (d, 4 H, Ar-H), 6.71 (d, 4 H, Ar-H), 3.72 (t, 4H, N-CH2-C), 3.56 (t, 4H, O-CH2-C), 3.04 (s, 6H, N-CH3), 1.87 (p, 4H, C-CH2-C) ppm. N-[3-O-(dimethoxytrityl)propyl]-methylamine. The solution of 3-methylaminopropanol (0.5 g, 5.6 mmol) and dimethoxytrityl chloride (1.9 g, 5.6 mmol) in dry pyridine (20 mL) was stirred at room temperature for 24 h. After evaporation of the solvent, the reaction mixture was dissolved in water (50 mL), saturated with NaHCO3 (20 mL) and extracted with CHCl3 (3 9 50 mL). The combined organic layers were washed with H2O and dried over MgSO4. Evaporation of solvent resulted in crude
*Corresponding authors email: [email protected] (Michael R. Wasielewski); [email protected] (Frederick D. Lewis) †This manuscript is part of the Special Issue dedicated to the memory of Michael Kasha. © 2014 The American Society of Photobiology
1
2
Almaz S. Jalilov et al. Table 1. m/z values for MK-1-6 hairpins (determined by MALDI-TOF mass spectrometry) and TM values. Sequence MK-1 MK-2 MK-3 MK-4 MK-5 MK-6
Calculated, m/z
Measured, m/z
TM, °C*
4047.74 4049.73 4051.72 5509.05
4068.458 4065.464 4065.065 5514.407
5513.03
5448.336
54 67 85† 68 80 ~90†
*Obtained from the first derivative thermal dissociation profiles measured at 260 nm. †Extensive premelting observed.
Scheme 1. Structures for the duplex capping group AQ, hairpin linker MK’, base surrogate PDI and DNA hairpins MK-1-6. product, which was purified using column chromatography (using eluent hexane/ethylacetate/triethylamine, 100/20/1 mixture by volume) to give a yellow product, 2.5 g (72%). 1H NMR (CDCl3, 500.137 MHz): d - 7.45 (d, 2 H, Ar-H), 7.35 (d, 2 H, Ar-H), 7.29 (d, 2 H, Ar-H), 7.25 (t, 1H, Ar-H), 6.84 (d, 4 H, Ar-H), 3.82 (s, 6H, O-CH3), 3.16 (t, 2H, N-CH2C), 2.71 (t, 2H, O-CH2-C), 2.43 (s, 3H, N-CH3), 1.83 (p, 2H, C-CH2-C) ppm. 4-Fluoro-4’-N-(methyl-3-hydroxypropylamino)-benzophenone. 4,4’-difluorobenzophenone (3.67 g, 16.8 mmol), 3-methylaminopropanol (0.5 g, 5.6 mmol), anhydrous potassium carbonate (0.78 g, 5.6 mmol) and a catalytic amount of 18-crown-6 ether in dry DMSO (4 mL) were heated at 100°C for 48 h. The resulting reaction mixture was separated using column chromatography (using eluent hexane/ethylacetate/triethylamine, 100/20/1 mixture by volume) to give yellow product, 1.4 g (86%). 1H NMR (CDCl3, 500.137 MHz): d - 7.79 (d, 2 H, Ar-H), 7.77 (t, 2 H, ArH), 7.15 (t, 2 H, Ar-H), 6.75 (d, 2 H, Ar-H), 3.76 (q, 2H, N-CH2-C), 3.61 (t, 2H, O-CH2-C), 3.09 (s, 3H, N-CH3), 1.91 (p, 2H, C-CH2-C) ppm. 4-N-[3-O-(dimethoxytrityl)propyl]-methylamino-4’-N-(methyl-3-hydroxypropylamino)-benzophenone. 4-fluoro-4’-N-(methyl-3-hydroxypropylamino)-benzophenone (1.00 g, 3.5 mmol), N-[3-O-(dimethoxytrityl)propyl]-methylamine (1.40 g, 3.6 mmol), anhydrous potassium carbonate (0.76 g, 7.0 mmol) and catalytic amount of 18-crown-6 ether in dry DMSO (3 mL) were heated at 100°C for 48 h (1). The resulting mixture
was further separated using column chromatography (using eluent hexane/ethylacetate/triethylamine, 100/20/1 mixture by volume) to give yellow product, 1.73 g (76%). 1H NMR (CDCl3, 500.137 MHz): d - 7.76 (t, 4 H, Ar-H), 7.46 (d, 2 H, Ar-H), 7.34 (d, 2 H, Ar-H), 7.30 (t, 1 H, Ar-H), 7.23 (t, 1 H, Ar-H), 6.85 (d, 4 H, Ar-H), 6.75 (d, 2 H, Ar-H), 6.67 (d, 2 H, Ar-H), 3.82 (s, 6H, O-CH3), 3.76 (t, 2H, -CH2-OH), 3.59 (t, 4H, N-CH2-C), 3.16 (t, 2H, O-CH2-C), 3.07 (s, 3H, N-CH3), 2.96 (s, 3H, N-CH3), 1.91 (m, 4H, C-CH2-C) ppm. 4-N-[3-O-(dimethoxytrityl)propyl]-methylamino-4’-N-[3-[O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite]-methyl-3-propylamino)benzophenone. 2-cyano-ethyldidisopropylchlorophosphoramidite (29 lL, 0.15 mmol) was added dropwise to a solution of 1 (0.10 g, 0.15 mmol) and isopropylethylamiine (80 lL, 0.45 mmol) in dry acetonitrile (2 mL) at room temperature under N2 (2). The mixture was stirred for additional 1 h and then used without further purification for the solid phase synthesis of the oligonucleotides. Oligonucleotide synthesis. Phosphoramidite 2 was incorporated into oligonucleotide conjugates using phosphoramidite chemistry on an Expedite synthesizer following the standard automated synthetic procedures (17). The perylenediimide base surrogate PDI was prepared by the method of Wagner and Wagenknecht (18). DNA conjugates were purified by reverse phase HPLC and characterized by MALDI-TOF mass spectrometry. Mass spectral data and melting temperatures are reported in Table 1. Thermal dissociation profiles provided the hairpin melting temperatures (TM) are reported in Table 1. Values of TM increase, as expected, with increasing number of G-C base pairs. Methods. UV/Vis and CD spectra were measured in a 5 mm pathlength quartz cuvette at room temperature in Perkin–Elmer Lambda 2 and Jasco 815 spectrometers, respectively. Emission spectra were recorded using a Photon Technologies International fluorimeter. Transient absorption spectra were performed at room temperature in 2 mm glass cuvettes using the apparatus described previously (19). All samples were stirred during irradiation to minimize the effects of local heating and sample degradation. Time-resolved fluorescence measurements were performed using an instrument previously described (20).
RESULTS AND DISCUSSION Spectroscopic Characterization of the MK’ Linker The normalized UV absorption spectra of MK’ in acetonitrile, methanol and water are shown in Fig. 1 along with the fluorescence spectra determined for solutions excited at their absorption maxima having similar absorbance. The absorption maxima reported in Table 2 are similar to those reported previously for the 2-hydroxyethyl analog of MK’ (21). The maxima of the weak fluorescence in methanol and water are similar to those for MK (which is insoluble in water) in methanol (445 nm) (22). The fluorescence maximum in acetonitrile is blueshifted when compared to that for MK (487 vs 515 nm), plausibly as a consequence of intramolecular hydrogen bonding from the hydroxypropyl group to the amine lone pair which would increase the energy of the charge-transfer transition. The femtosecond time-resolved transient absorption spectra of MK’ in methanol and in acetonitrile and water are shown in
Photochemistry and Photobiology
Figure 1. Steady-state absorption (solid) and fluorescence (dash-dot) spectra of diol linker MK’ in acetonitrile, methanol and water. Fluorescence data acquired with 390 nm excitation.
Figs. 2 and S1, respectively. The negative band around 500 nm and positive band around 700 nm in methanol are attributed to the stimulated emission and absorption, respectively, of the Franck–Condon singlet state, both of which are formed during the pump pulse and decay rapidly. The decay of the stimulated emission is accompanied by a time-dependent Stokes shift. Following the slower rise and decay of a broad band with a maximum around 600 nm, the spectrum evolves to a single broad band around 675 nm assigned to triplet MK’, which is long lived on the 7 ns time scale of our measurements. The transient absorption spectra in water and acetonitrile (Figure S1) display much weaker stimulated emission and thus their dynamic Stokes shifts are not as well resolved. The single wavelength 500 nm and 685 nm transient kinetics of MK’ in methanol, acetonitrile and water are shown in Figure S2 and the dominant fast components are reported in Table 2. The transient absorption spectra of MK on the sub-ps time scale have previously been reported by Mondal et al. (23). Their published spectra are reconstructed from single wavelength decays in several solvents including 1-propanol, but not in methanol or water and their spectra in 1-propanol do not closely resemble our spectra in methanol. The fastest decay components
3
Figure 2. Femtosecond transient absorption spectra for the diol linker MK’ in methanol following 400 nm excitation.
in methanol, ethanol and 1-propanol were reported as 0.6, 1.6 and 2.4 ps, respectively, taken from the single wavelength fit at 630 nm. These differences are attributed to the sensitivity of MK excited state single bond torsion to specific solvation. Further insight into the excited state behavior of MK in methanol is provided by the femtosecond fluorescence up-conversion studies of Vedhoven et al. (24). The weighted fluorescence decay time for MK in methanol is 2 ps, similar to the decay of MK’ 685 nm singlet–singlet absorption in methanol (Figs. 2 and S2). These authors conclude that phenyl group twisting as well as solvent dynamics determine the dynamics of MK radiative and nonradiative decay at short times in methanol (24). Spectroscopic Characterization of Hairpins MK-1-3 The steady-state absorption and fluorescence spectra of hairpin MK-1 in aqueous buffer are shown in Fig. 3 along with those of MK’ in water. The absorption and fluorescence maxima and fluorescence quantum yields (Φf) for MK-1-3 are reported in Table 2. Values for the three hairpins are similar. The maxima of the long-wavelength bands of MK-1-3 are redshifted by 2 nm vs that of MK’. The fluorescence of MK-1-3 is substantially more intense and is redshifted by 40 nm vs that of MK’ in water or methanol. However, their maxima are similar to that of MK’ in acetonitrile, suggesting the large Stokes shift for the fluores-
Table 2. Absorption and fluorescence maxima, fluorescence quantum yields and fluorescence and transient absorption decay times for diol linker MK’ and hairpins MK-1-3. Percentage of each time component is given in parentheses. Φf (350ex)
kabs, nm
kfl nm (350ex)
MK’(CH3CN) MK’(CH3OH) MK’(H2O) MK-1*
357 372 389 391
487 ~ ~ 482
0.0023
1.7 (30%) 16 (36%) 219 (34%)
MK-2*
389
484
0.0013
MK-3*
391
482
0.0027
1.7 (55%) 13 (33%) 124 (11%) 0.5 (38%) 4.6 (41%) 44 (21%)
sfl, ps†
s455, ps‡
0.5(+) (28%) 1.3(+) (72%) 33() (87%) ~6000() (13%) 0.9(+) (100%) 25() (82%) ~7000() (18%) 1.0(+) (100%) 21() (46%) 65() (40%), >1 9 104() (14%)
s500, ps‡,§ 2.2‡ 1.5‡ 0.6‡ 0.65 (50%) 6.9 (50%) 0.15 (67%) 3.0 (33%) 1.50 (62%) 8.80 (38%)
s667, ps‡
1.9() (70%) 78(+) (100%) > 1 9 104() (30%) 1.5() (50%) 35(+) (100%) >1 9 104() (50%) 2.0() (32%) 160(+) (100%) >1 9 104() (68%)
*Values determined in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl. †Values from fits to fluorescence decay at 485 nm following 395 nm excitation. ‡Values from fits to transient absorption determined following 400 nm excitation. §Values from fits to stimulated emission band near 500 nm following 400 nm excitation. (+) = rise, () = decay.
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Almaz S. Jalilov et al.
Figure 3. Steady-state absorption (solid) and fluorescence (dash-dot) spectra of diol linker MK’ in water and 3.0 lM hairpin MK-1 in aqueous solution (0.1 M NaCl, 10 mM sodium phosphate, pH 7.2). Fluorescence data acquired with 390 nm excitation.
cence of hairpins MK-1-3 may reflect rapid twisting of the Franck–Condon singlet state prior to emission. Fluorescence decay times (sfl) determined by fluorescence up-conversion (Figure S3) are best fit as triexponentials, the decay times of which are reported in Table 2. The shortest lived decay components for MK-1,2 are similar to that for the linker MK’. The longer lived decay components for MK-1-3 are tentatively attributed to hairpin conformations having reduced torsional mobility. The transient absorption spectra of hairpins MK-1-3 in aqueous buffer are shown in Fig. 4. All of the spectra display negative bands similar to that for MK’ in methanol (Fig. 2) attributed to stimulated emission. These bands are formed during the pump pulse and decay with a time-dependent redshift as shown in Figs. 5a and S4a,c. The band minimum as a function of delay time was fit to a biexponential to determine the depopulation times of the singlet excited state reported in Table 2 (s500). The shorter depopulation times for MK-1-3 are between 0.2 and 1.5 ps and the longer depopulation times between 3 and 9 ps. Transient absorption bands are also observed at longer wavelengths for MK-1-3. The longer wavelength bands are similar to those for MK’ in methanol or water and are attributed to absorption by the vertical singlet state. These bands have decay times of 1.5–2 ps, similar to that of MK’ in methanol or water or MK in methanol (23). Since the appearance of neither the fluorescence nor the simulated emission is dependent upon the adjacent base pair, they plausibly arise from conformations in which there is weak electronic coupling between MK’ and the adjacent bases. The hairpin depopulation times are in only fair agreement with their two shorter fluorescence decay times (Table 2). The lack of better agreement may result from the different excited state populations probed by the two transient techniques: fluorescence decay times sampling only the minority population of fluorescent excited states and transient absorption sampling the overwhelming majority of the nonfluorescent excited states. In addition to the broad absorption band at longer wavelengths, hairpins MK-1-3 display a narrower absorption band at 455 nm that is partially formed during the laser pump pulse and continues to rise during the first few ps thereafter (Figs. 5b and
Figure 4. Femtosecond transient absorption spectra of hairpins MK-1-3 in aqueous buffer following 400 nm excitation.
S4b,d). The 455 nm absorption bands of MK-1-3 are assigned to the anion radicals of MK’, based on the similarity of their appearance to the absorption spectrum reported by Mondal et al.
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5
Figure 6. Steady-state absorption spectra of hairpins MK-4, MK-5 and MK-6 in aqueous solution (0.1 M NaCl, 10 mM sodium phosphate, pH 7.2).
Figure 5. (a) Amplitude of the stimulated emission minimum (black) and time-dependent shift of the stimulated emission minima (gray) and (b) single wavelength transient kinetics monitored at 455 nm (black) and 667 nm (gray) following 400 nm excitation.
1
(a)
(b)
*MK-A
1
*MK τ st
τcs 1 (MK-.-A+.)
τf
τcr
MK-A
3
*MK
τ ts
MK
Scheme 2. Scheme Mechanisms for charge separation and recombination in (a) well-stacked hairpins with adjacent A-T base pairs and (b) for intersystem crossing in nonstacked hairpins MK-1-3.
for MK.generated by pulse radiolysis (25). In the case of MK1 and -2, the decay of this band can be fit as a single exponential with decay times of 33 ps and 25 ps, respectively, with a longlived offset (ca. 7 ns). The decay of the 455 nm band of MK-3
Figure 7. Steady-state fluorescence spectra of hairpins MK-4, MK-5 and MK-6 in aqueous solution (0.1 M NaCl, 10 mM sodium phosphate, pH 7.2) excited at 390 nm (solid lines) and 506 nm (dash-dot lines).
is best fit as a dual exponential with decay times of 25 and 65 ps with a long-lived offset. Since these decays are not accompanied by the rise of a successor band, we conclude that they occur by fast singlet radical ion pair recombination, similar to that observed for the singlet radical ion pairs of anthraquinones with purine bases (7). After decay times >0.5 ns the transient spectra of MK-1-3 consist of broad featureless bands similar to that for MK’ in methanol or water. These bands do not decay on the 7 ns time scale of our measurements and are attributed to absorption of the MK triplet state, which is described as having weak T1-Tn absorption at 650 nm (13). Only in the case of MK-2 is a rising component for the triplet observed with a time constant similar to that for decay of the 455 nm band attributed to the MK. anion radical. In view of the long decay times for the triplet states of MK-1-3, it seems unlikely that triplet electron transfer occurs in these hairpins. The excited state behavior of hairpins MK-1-3 is summarized in Scheme 2. The rapid formation and decay of the 455 nm band attributed to the MK. anion radical requires a significant population of these hairpins, in which the MK’ and purine chromophores are closely associated so that upon electronic excitation they can form a singlet radical ion pair. The time constants for
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Almaz S. Jalilov et al.
charge separation and recombination (Scheme 2a, scs and scr) can be estimated from the rise and decay times for formation of MK. (Table 2, s455 nm). The observation of rapid charge separation and charge recombination for singlet MK with adjacent A-T and G-C base pairs is analogous to the behavior of AQcapped hairpins for which singlet charge separation competes with intersystem crossing (7). Charge recombination for the singlet radical ion pairs of both MK and AQ is much faster than for the stilbenediamide hairpin linker, presumably as a consequence of the smaller energy gap for charge recombination (2). In competition with charge separation, the singlet states of MK1-3 can undergo behavior similar to that of the MK chromophore in methanol or water: twisting and solvent relaxation followed by efficient intersystem crossing to form the triplet state (Scheme 2b). Rate constants for fluorescence in these hairpins (kf) can be estimated from the fluorescence quantum yields and stimulated fluorescence decay times (kf = Φfs5001). The nonrigid nature of the MK’ linker (e.g. freedom of rotation about the phenyl-N bonds and aminopropyl linkers) provides ample opportunity for the formation of hairpins with multiple ground state conformations. Unlike the triplet of AQ (11), there is no evidence for the efficient formation of triplet radical ion pairs with the MK hairpins. The apparent lack of reactivity of the triplet state of MK-1-3 indicates that it is unable to form a triplet radical ion pair with a neighboring base, either because of lack of proximity or insufficient driving force for electron transfer. Estimating the energetics of singlet and triplet electron transfer reactions for the MK’-linked hairpins is complicated both because of uncertainties in the geometry of the MK’ linker and in its singlet and triplet energies and redox potentials within the DNA hairpins. Using the singlet energy (3.45 eV in a rigid glass) and ground state reduction potential (1.87 V vs SCE) reported by Loutfy for MK provides an excited state oxidation potential (1.58 V vs SCE) sufficiently large to oxidize G, but questionable for A (Eox = 1.24 and 1.69 V, respectively) (15,26). Using the triplet energy reported by Timpe et al. (14), triplet oxidation of purines is endoergic and using the oxidation potential of MK reported by Jockusch et al. (13), reduction of the pyrimidine bases by either singlet or triplet MK is likewise endoergic. Our hopes that the triplet state of the MK hairpins might prove to be an effective reductant of pyrimidines unfortunately were not realized. Spectroscopic Characterization of Hairpins MK-4-6 The UV–visible absorption spectra of hairpins MK-4-6 in aqueous buffer are shown in Fig. 6. The structured long-wavelength band has a first vibronic band maximum at 545 nm attributed to
PDI and a structureless second band at 385 attributed to the absorption of MK which overlaps the weaker absorption of PDI in this region of the absorption spectrum (16). The fluorescence spectra of MK–4-6 obtained with excitation wavelengths of 506 nm and 390 nm, corresponding to the absorption bands of the PDI and MK chromophores, respectively, are shown in Fig. 7 and absorption and fluorescence maxima are reported in Table 3. Fluorescence quantum yields have not been determined for these hairpins; however, the values of Φf for 390 nm excitation are presumed to be similar to the values for hairpins MK-13 (Table 2) and hence < 1%. The weaker PDI fluorescence for MK-6 vs MK-4,5 observed for 506 nm excitation is attributed to more extensive electron transfer quenching of singlet PDI by guanine (G) than by adenine (A). More efficient electron transfer quenching of PDI by G vs A has been previously reported and is attributed to the lower oxidation potential of G (16). Excitation at 390 nm results in the observation both MK and PDI fluorescence (Fig. 7). Dual fluorescence is attributed to competitive absorption at 390 nm; however, a minor contribution from resonant energy transfer cannot be ruled out. The transient absorption spectra of MK-4-6 obtained with 545 nm excitation are shown in Fig. 8. All three spectra display negative bands near 500 and 550 nm assigned to ground state bleach of PDI. The spectra of MK-4,5 also display a negative band near 580 nm assigned to PDI stimulated emission, which is not detected in the case of MK-6. The transient spectra of MK4,5 also display a positive band near 700 nm which undergoes a time-dependent redshift to ca. 725 nm. We have observed similar transient spectra for hairpins possessing A-T base pairs on both sides of a PDI base surrogate and assigned the redshifted absorption to reduction of singlet PDI to its anion radical (16). The spectra of MK-6 lack the 700 nm band assigned to singlet PDI, as previously observed for a hairpin possessing a single G–C base pair on one side of the PDI base surrogate. More rapid quenching of singlet PDI by G vs A is responsible for the absent or very weak transient absorption, stimulated emission and fluorescence of the PDI chromophore in MK-6. The 600 nm and 720 nm single wavelength kinetics for MK4-6 are shown in Fig. 9 and the multiple exponential fits are reported in Table 3. The behavior of hairpins MK-4,5 upon excitation of the PDI chromophore at 545 nm is summarized in Scheme 3a and the behavior of MK-6 is shown in Scheme 3b. Only the two chromophores and the connecting poly(purines) are shown in these schemes. The fast rising components of the 600 nm stimulated emission are attributed to the formation of the PDI.A+. or PDI.G+. radical ion pairs and the slower rising components to decay of the singlet (Fig. 9a). The somewhat shorter 600 nm decay time for MK-5 vs MK-4 plausibly reflects
Table 3. Absorption and fluorescence maxima and decay times for hairpins MK-4-6.* Percentage of each time component is given in parentheses.† kabs, nm
kfl, nm (350ex)
kfl, nm (506ex)
s600, ps
s720, ps
MK-4
389
474
555
0.3(+) (31%), 3.9(+) (46%), 24.3(+) (23%), >1 9 104()
MK-5
388
473
555
1.9(+) (52%), 12(+) (48%), >1 9 104()
MK-6
385
476
555
0.3(+) (~100%) 1.4() (89%) 15() (4%) 1600() (7%)
2.54() (4%), 25.4() (75%), 300() (11%), >1 9 104() (10%) 1.6() (9%), 48() (12%), 530() (52%), >1 9 104() (27%) 1.5() (89%), 32() (8%), 1320() (3%)
*All values determined in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl. †Time constants determined following 545 nm excitation. (+) = rise, () = decay.
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Figure 9. Normalized single wavelength kinetics for MK-4-6 in aqueous buffer determined at (a) 600 nm and (b) 720 nm following 545 nm excitation.
Figure 8. Femtosecond transient absorption spectra of hairpins MK-4-6 following 545 nm excitation.
confinement of the PDI.A+. radical ion pair within a AA dyad, whereas, the PDI.A+. radical ion pair in MK-4 can delocalize over a AAAA tetrad.
The 720 nm single wavelength decays for MK-4-6 display a long-lived component with band maximum at 725 nm assigned to the formation of the PDI./MK+. charge-separated state (Fig. 9b). Unfortunately, MK+ does not have a characteristic transient absorption band in 450–800 nm spectral window of our measurements that would permit direct determination of the hole transit times for the charge separation process, as implemented for stilbene donor–acceptor hairpins (27). The efficiency of formation of the charge-separated radical ion pair is dependent upon the purine base sequence, following the same order, A2G2 > A4 > G4, as previously observed for hole transport in stilbene donor–acceptor capped hairpins (28). Inefficient hole transport for Gn purines in the stilbene hairpins was attributed to fast charge recombination in the Sa–/G+. contact radical ion pair, as a consequence of a smaller energy gap than for Sa/A+..(28) More efficient charge separation for the A2G2 diblock sequence vs the A4 homopurine sequence was attributed to slow charge return once the hole exits A2 to form the Sa.AAG+ charge-separated radical ion pair (Scheme 3a) (28). The role of MK in hairpins MK-4-6 is to serve as a ground state electron donor or hole trap for the reduction of a A+ or G+. cation radical. The oxidation potential of MK (0.95 V vs SCE in acetonitrile) (26) is well below that of either of the nucleosides G or A (1.24 and 1.69 V, respectively, vs SCE) (15). While the potentials of MK, G and A within the hairpins MK-1-6 in water
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Almaz S. Jalilov et al. separation are also available from the amplitudes of the formation of long-lived charge-separated states (Fig. 9b). The values parallel those reported previously of the stilbenediether–stilbenediamide donor–acceptor capped hairpins (28), suggesting that it is the base sequence rather than the chromophores which, to a first approximation, control the dynamics and efficiency of charge transport in DNA. Acknowledgements—This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Chemical Sciences, Geosciences, and Biosciences Division under Award DE-FG02-96ER14604 (F.D.L.) and DE-FG02-99ER14999 (M.R.W.). R.M.Y gratefully acknowledges the Camille and Henry Dreyfus Foundation Postdoctoral Fellowship in Environmental Chemistry for funding. We thank Dr. Taiga Fujii for the preparation of the phosphoramidite of the diol linker.
SUPPORTING INFORMATION
Scheme 3. Mechanism for charge separation and charge recombination in (a) hairpins MK-4 and MK-5 and (b) hairpin MK-6.
are not known, it is unlikely that their order is different from that of MK and the nucleosides in acetonitrile.
CONCLUSIONS In summary, we find that the singlet excited state of MK but not its triplet state can serve as an electron acceptor for the oxidation of the purine bases G and A. The behavior of the hairpins MK1-3 is complicated by the presence of singlet conformers which undergo charge separation and those which do not. The difference in singlet and triplet state reactivities reflects not only the higher energy of the singlet state but also the difference in geometry and charge transfer character of the planar Franck–Condon singlet state and the relaxed twisted triplet state (23,24). Both excited state geometric relaxation and charge transfer may reduce the driving force for electron transfer processes and render the relaxed excited states less potent oxidants or reductants than the Franck–Condon excited states. The behavior of the MK-linked hairpins differs from that of the AQ-capped hairpins in several respects. The more rigid planar structure of AQ results in similar singlet and triplet energies (and presumably reduction potentials) and hence the observation of oxidation of neighboring A and G bases by both singlet and triplet AQ (7). In addition, more rapid intersystem crossing reduces the lifetimes of the singlet state and radical ion pairs of AQ in comparison with those of MK (10). The ground state of MK can also serve as an electron donor for the formation of PDI./MK+. charge-separated states in the hairpins MK-4-6. Hole transit times are in principle available from analysis of the time-dependent band shape changes of the 725–720 nm maxima for MK-4 and 5. Relative yields of charge
Additional Supporting Information may be found in the online version of this article: Figure S1. Transient absorption spectra of linker diol MK’ in (a) water and (b) in acetonitrile following 400 nm excitation. Figure S2. Kinetics for diol MK’ in various solvents following 400 nm excitation. Figure S3. Time-resolved fluorescence intensities for MK-1-3 in aqueous buffer monitored at 485 nm following 395 nm excitation. Figure S4. Amplitudes of the stimulated emission maximum (black) and time-dependent shift of the stimulated emission maxima (blue) and single wavelength transient kinetics monitored at 455 nm (black) and 667 nm (red) for (a, b) MK-2 and (c,d) MK-3 in aqueous buffer following 400 nm excitation. Figure S5. Kinetic fits at 600 and 720 nm for MK-4-6 in aqueous buffer following 545 nm excitation.
REFERENCES 1. Genereux, J. C. and J. K. Barton (2010) Mechanisms for DNA charge transport. Chem. Rev. 110, 1642–1662. 2. Lewis, F. D. (2013) Distance-dependent electronic interactions across DNA base pairs. Charge transport, exciton coupling, and energy transfer. Israel J. Chem. 53, 350–365. 3. Kawai, K. and T. Majima (2013) Hole transfer kinetics of DNA. Acc. Chem. Res. 46, 2616–2625. 4. Lewis, F. D., X. Y. Liu, S. E. Miller, R. T. Hayes and M. R. Wasielewski (2002) Dynamics of electron injection in DNA hairpins. J. Am. Chem. Soc. 124, 11280–11281. 5. Park, M. J., M. Fujitsuka, K. Kawai and T. Majima (2011) Direct measurement of the dynamics of excess electron transfer through consecutive thymine sequence in DNA. J. Am. Chem. Soc. 133, 15320–15323. 6. Armitage, B. (1998) Photocleavage of nucleic acids. Chem. Rev. 98, 1171–1200. 7. Lewis, F. D., A. K. Thazhathveetil, T. A. Zeidan, J. Vura-Weis and M. R. Wasielewski (2010) Dynamics of ultrafast singlet and triplet charge transfer in anthraquinoneDNA conjugates. J. Am. Chem. Soc. 132, 444–445. 8. Schuster, G. B. (2000) Long-range charge transfer in DNA: Transient structural distortions control the distance dependence. Acc. Chem. Res. 33, 253–260. 9. Armitage, B., C. J. Yu, C. Devadoss and G. B. Schuster (1994) Cationic anthraquinone derivatives as catalytic DNA photonucleases -
Photochemistry and Photobiology
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mechanisms for DNA-damage and quinone recycling. J. Am. Chem. Soc. 116, 9847–9859. Van Ramesdonk, H. J., B. H. Bakker, M. M. Groeneveld, J. W. Verhoeven, B. D. Allen, J. P. Rostron and A. Harriman (2006) Ultrafast intersystem crossing in 9,10-anthraquinones and intramolecular charge separation in an anthraquinone-based dyad. J. Phys. Chem. A 110, 13145–13150. Carmieli, R., A. L. Smeigh, S. M. Mickley Conron, A. K. Thazhathveetil, M. Fuki, Y. Kobori, F. D. Lewis and M. R. Wasielewski (2012) Structure and dynamics of photogenerated triplet radical ion pairs in DNA hairpin conjugates with anthraquinone end caps. J. Am. Chem. Soc. 134, 11251–11260. Murov, S. L. (1973) Handbook of Photochemistry. Marcel Dekker, New York. Jockusch, S., H. J. Timpe, W. Schnabel and N. J. Turro (1997) Photoinduced energy and electron transfer between ketone triplets and organic dyes. J. Phys. Chem. A 101, 440–445. Timpe, H. J., K. P. Kronfeld, U. Lammel, J. P. Fouassier and D. J. Lougnot (1990) Excited states of ketones as electron donors—ketone —iodonium salt systems as photoinitiators for radical polymerization. J. Photochem. Photobiol. A: Chem. 52, 111–122. Seidel, C. A. M., A. Schulz and M. H. M. Sauer (1996) Nucleobasespecific quenching of fluorescent dyes. 1 Nucleobase one-electron redox potentials and their correlation with static and dynamic quenching efficiencies. J. Phys. Chem., 100, 5541–5553. Zeidan, T. A., R. Carmieli, R. F. Kelley, T. M. Wilson, F. D. Lewis and M. R. Wasielewski (2008) Charge-transfer and spin dynamics in DNA hairpin conjugates with perylenediimide as a base-pair surrogate. J. Am. Chem. Soc. 130, 13945–13955. Letsinger, R. L. and T. Wu (1995) Use of a stilbenedicarboxamide bridge in stabilizing, monitoring, and photochemically altering folded conformations of oligonucleotides. J. Am. Chem. Soc. 117, 7323– 7328. Wagner, C. and H. A. Wagenknecht (2006) Perylene-3,4:9,10-tetracarboxylic acid bisimide dye as an artificial DNA base surrogate. Org. Lett. 8, 4191–4194.
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19. Brown, K. E., B. S. Veldkamp, D. T. Co and M. R. Wasielewski (2012) Vibrational dynamics of a perylene–perylenediimide donor– acceptor dyad probed with femtosecond stimulated raman spectroscopy. J. Phys. Chem. Lett. 3, 2362–2366. 20. Kim, C. H. and T. Joo (2008) Ultrafast time-resolved fluorescence by two photon absorption excitation. Opt. Express 16, 20742–20747. 21. El-Sayed, M., H. M€uller, G. Rheinwald, H. Lang and S. Spange (2001) Linear solvation energy (lse) correlations of the solvatochromic response and x-ray structure analysis of hydrophilically n-substituted Michler’s ketone derivatives. J. Phys. Org. Chem. 14, 247– 255. 22. Shoute, L. C. T. (1992) Dual fluorescence of 4,40 -bis(dimethylamino)benzophenone. Effects of specific and nonspecific interaction on the formation of twisted intramolecular charge transfer. Chem. Phys. Lett. 195, 255–261. 23. Mondal, J. A., H. N. Ghosh, T. K. Ghanty, T. Mukherjee and D. K. Palit (2006) Twisting dynamics in the excited singlet state of Michler’s ketone. J. Phys. Chem. A 110, 3432–3446. 24. Van Veldhoven, E., H. Zhang, W. Rettig, R. G. Brown, J. D. Hepworth and M. Glasbeek (2002) Femtosecond fluorescence studies of two-dimensional dynamics in photoexcited Michler’s ketones. Chem. Phys. Lett. 363, 189–197. 25. Samant, V., A. K. Singh, T. Mukerjee and D. K. Palit (2006) Spectroscopic properties of anion radicals studied using pulse radiolysis. Res. Chem. Intermed., 32, 767–776. 26. Loutfy, R. O. and R. O. Loutfy (1972) Correlations between the electrochemical and spectroscopic behavior of some benzophenones and thiobenzophenones. J. Phys. Chem. 76, 1650–1655. 27. Lewis, F. D., Y. S. Wu, L. G. Zhang, X. B. Zuo, R. T. Hayes and M. R. Wasielewski (2004) DNA-mediated exciton coupling and electron transfer between donor and acceptor stilbenes separated by a variable number of base pairs. J. Am. Chem. Soc. 126, 8206– 8215. 28. Vura-Weis, J., M. R. Wasielewski, A. K. Thazhathveetil and F. D. Lewis (2009) Efficient charge transport in DNA diblock oligomers. J. Am. Chem. Soc. 131, 9722–9727.
Electronic Interactions of Michler’s Ketone with DNA Bases in Synthetic Hairpins† Almaz S. Jalilov, Ryan M. Young, Samuel W. Eaton, Michael R. Wasielewski* and Frederick D. Lewis* Department of Chemistry and Argonne-Northwestern Solar Energy Research (ANSER) Center, Northwestern University, Evanston, IL Received 26 August 2014, accepted 30 September 2014, DOI: 10.1111/php.12360
ABSTRACT
undergo rapid intersystem crossing to yield triplet states that are capable of oxidizing purine nucleobases (9,10). The resulting triplet radical ion pairs have lifetimes which are substantially longer than those for the corresponding singlet radical ion pairs, as a consequence of the spin forbidden nature of triplet radical ion pair spin recombination (11). However, quinones are strong electron acceptors both in the ground and excited state. Thus, we turned our attention to other chromophores which are known to undergo efficient intersystem crossing that could be incorporated into synthetic DNA as a hairpin linker. Among the chromophores that we considered were the dihydroxyalkyl derivatives of the well-known triplet sensitizer Michler’s ketone (MK, Scheme 1) (12). In addition to undergoing fast intersystem crossing, MK derivatives are prone to undergo electron transfer reactions, serving as either electron donors or acceptors, as a consequence of the charge transfer character of their excited states (13,14). The ground state of DNA is also known to undergo oxidation and reduction reactions, the purine bases serving as electron donors and the pyrimidines as electron acceptors (15). To our knowledge, the interaction of electronically excited MK and its derivatives with DNA have not been investigated. We report here a study of the steady state and femtosecond time-resolved spectroscopies of two groups of MK-linked hairpins and the diol linker MK’ employed in their synthesis. The first group consisting of hairpins MK-1-3 possesses only a MK chromophore, whereas, the second group consisting of hairpins MK4-6 possess both MK and a perylenediimide (PDI) base surrogate, a strong ground state and singlet state oxidant (Scheme 1) (16).
The mechanism and dynamics of photoinduced electron transfer in two families of DNA hairpins possessing Michler’s ketone linkers have been investigated by means of steady state and time-resolved transient absorption and emission spectroscopies. The excited state behavior of the diol linker employed in hairpin synthesis is similar to that of Michler’s ketone in methanol solution. Hairpins possessing only a Michler’s ketone linker undergo fast singlet state charge separation and charge recombination with an adjacent purine base, attributed to well-stacked ground state conformations, and intersystem crossing to the triplet state, attributed to poorly stacked ground state conformations. The failure of the triplet to undergo electron transfer reactions on the 7 ns time scale of our measurements is attributed to the low triplet energy and reduction potential of the twisted triplet state. Hairpins possessing both a Michler’s ketone linker and a perylenediimide base surrogate separated by four base pairs undergo photoinduced hole transport from the diimide to Michler’s ketone upon excitation of the diimide. The efficiency of hole transport is dependent upon the sequence of the intervening purine bases.
INTRODUCTION The dynamics and efficiency of photoinduced hole injection and the ensuing hole transport in DNA have been extensively investigated using a wide variety of singlet and triplet electron acceptors (1–3). In contrast, studies of electron injection have been limited to a relatively small number of singlet electron donors and the dynamics and efficiency of electron transport beyond one or two base pairs remain poorly understood (4,5). In our continuing study of photoinduced electron transfer in DNA (2), we sought a chromophore with a high intersystem crossing quantum yield that could serve as both a linker for the formation of stable DNA hairpins and as a photoreductant for the formation of pyrimidine anion radicals. The N-hydroxypropyl derivative of anthraquinone-2-carboxamide (AQ, Scheme 1) has been widely used as a capping group for DNA duplexes and hairpins in studies of DNA photooxidation (6–8). Anthraquinone derivatives
MATERIALS AND METHODS Materials. N,N’-[Bis(methyl-3-hydroxypropylamino)-4,4’-benzophenone (MK’). 4,4’-difluorobenzo-phenone (1 g, 4.6 mmol), 3-methylaminopropanol (1 g, 11.2 mmol), anhydrous potassium carbonate (1 g, 7.3 mmol) and catalytic amount of 18-crown-6 ether in dry DMSO (4 mL) were heated at 100°C for 48 h. The resulting reaction mixture was further separated using column chromatography (using eluent hexane/ethylacetate/ triethylamine, 100/20/1 mixture by volume) to give a yellow product, 1.2 g (72%). 1H NMR (CDCl3, 500.137 MHz): d - 7.74 (d, 4 H, Ar-H), 6.71 (d, 4 H, Ar-H), 3.72 (t, 4H, N-CH2-C), 3.56 (t, 4H, O-CH2-C), 3.04 (s, 6H, N-CH3), 1.87 (p, 4H, C-CH2-C) ppm. N-[3-O-(dimethoxytrityl)propyl]-methylamine. The solution of 3-methylaminopropanol (0.5 g, 5.6 mmol) and dimethoxytrityl chloride (1.9 g, 5.6 mmol) in dry pyridine (20 mL) was stirred at room temperature for 24 h. After evaporation of the solvent, the reaction mixture was dissolved in water (50 mL), saturated with NaHCO3 (20 mL) and extracted with CHCl3 (3 9 50 mL). The combined organic layers were washed with H2O and dried over MgSO4. Evaporation of solvent resulted in crude
*Corresponding authors email: [email protected] (Michael R. Wasielewski); [email protected] (Frederick D. Lewis) †This manuscript is part of the Special Issue dedicated to the memory of Michael Kasha. © 2014 The American Society of Photobiology
1
2
Almaz S. Jalilov et al. Table 1. m/z values for MK-1-6 hairpins (determined by MALDI-TOF mass spectrometry) and TM values. Sequence MK-1 MK-2 MK-3 MK-4 MK-5 MK-6
Calculated, m/z
Measured, m/z
TM, °C*
4047.74 4049.73 4051.72 5509.05
4068.458 4065.464 4065.065 5514.407
5513.03
5448.336
54 67 85† 68 80 ~90†
*Obtained from the first derivative thermal dissociation profiles measured at 260 nm. †Extensive premelting observed.
Scheme 1. Structures for the duplex capping group AQ, hairpin linker MK’, base surrogate PDI and DNA hairpins MK-1-6. product, which was purified using column chromatography (using eluent hexane/ethylacetate/triethylamine, 100/20/1 mixture by volume) to give a yellow product, 2.5 g (72%). 1H NMR (CDCl3, 500.137 MHz): d - 7.45 (d, 2 H, Ar-H), 7.35 (d, 2 H, Ar-H), 7.29 (d, 2 H, Ar-H), 7.25 (t, 1H, Ar-H), 6.84 (d, 4 H, Ar-H), 3.82 (s, 6H, O-CH3), 3.16 (t, 2H, N-CH2C), 2.71 (t, 2H, O-CH2-C), 2.43 (s, 3H, N-CH3), 1.83 (p, 2H, C-CH2-C) ppm. 4-Fluoro-4’-N-(methyl-3-hydroxypropylamino)-benzophenone. 4,4’-difluorobenzophenone (3.67 g, 16.8 mmol), 3-methylaminopropanol (0.5 g, 5.6 mmol), anhydrous potassium carbonate (0.78 g, 5.6 mmol) and a catalytic amount of 18-crown-6 ether in dry DMSO (4 mL) were heated at 100°C for 48 h. The resulting reaction mixture was separated using column chromatography (using eluent hexane/ethylacetate/triethylamine, 100/20/1 mixture by volume) to give yellow product, 1.4 g (86%). 1H NMR (CDCl3, 500.137 MHz): d - 7.79 (d, 2 H, Ar-H), 7.77 (t, 2 H, ArH), 7.15 (t, 2 H, Ar-H), 6.75 (d, 2 H, Ar-H), 3.76 (q, 2H, N-CH2-C), 3.61 (t, 2H, O-CH2-C), 3.09 (s, 3H, N-CH3), 1.91 (p, 2H, C-CH2-C) ppm. 4-N-[3-O-(dimethoxytrityl)propyl]-methylamino-4’-N-(methyl-3-hydroxypropylamino)-benzophenone. 4-fluoro-4’-N-(methyl-3-hydroxypropylamino)-benzophenone (1.00 g, 3.5 mmol), N-[3-O-(dimethoxytrityl)propyl]-methylamine (1.40 g, 3.6 mmol), anhydrous potassium carbonate (0.76 g, 7.0 mmol) and catalytic amount of 18-crown-6 ether in dry DMSO (3 mL) were heated at 100°C for 48 h (1). The resulting mixture
was further separated using column chromatography (using eluent hexane/ethylacetate/triethylamine, 100/20/1 mixture by volume) to give yellow product, 1.73 g (76%). 1H NMR (CDCl3, 500.137 MHz): d - 7.76 (t, 4 H, Ar-H), 7.46 (d, 2 H, Ar-H), 7.34 (d, 2 H, Ar-H), 7.30 (t, 1 H, Ar-H), 7.23 (t, 1 H, Ar-H), 6.85 (d, 4 H, Ar-H), 6.75 (d, 2 H, Ar-H), 6.67 (d, 2 H, Ar-H), 3.82 (s, 6H, O-CH3), 3.76 (t, 2H, -CH2-OH), 3.59 (t, 4H, N-CH2-C), 3.16 (t, 2H, O-CH2-C), 3.07 (s, 3H, N-CH3), 2.96 (s, 3H, N-CH3), 1.91 (m, 4H, C-CH2-C) ppm. 4-N-[3-O-(dimethoxytrityl)propyl]-methylamino-4’-N-[3-[O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite]-methyl-3-propylamino)benzophenone. 2-cyano-ethyldidisopropylchlorophosphoramidite (29 lL, 0.15 mmol) was added dropwise to a solution of 1 (0.10 g, 0.15 mmol) and isopropylethylamiine (80 lL, 0.45 mmol) in dry acetonitrile (2 mL) at room temperature under N2 (2). The mixture was stirred for additional 1 h and then used without further purification for the solid phase synthesis of the oligonucleotides. Oligonucleotide synthesis. Phosphoramidite 2 was incorporated into oligonucleotide conjugates using phosphoramidite chemistry on an Expedite synthesizer following the standard automated synthetic procedures (17). The perylenediimide base surrogate PDI was prepared by the method of Wagner and Wagenknecht (18). DNA conjugates were purified by reverse phase HPLC and characterized by MALDI-TOF mass spectrometry. Mass spectral data and melting temperatures are reported in Table 1. Thermal dissociation profiles provided the hairpin melting temperatures (TM) are reported in Table 1. Values of TM increase, as expected, with increasing number of G-C base pairs. Methods. UV/Vis and CD spectra were measured in a 5 mm pathlength quartz cuvette at room temperature in Perkin–Elmer Lambda 2 and Jasco 815 spectrometers, respectively. Emission spectra were recorded using a Photon Technologies International fluorimeter. Transient absorption spectra were performed at room temperature in 2 mm glass cuvettes using the apparatus described previously (19). All samples were stirred during irradiation to minimize the effects of local heating and sample degradation. Time-resolved fluorescence measurements were performed using an instrument previously described (20).
RESULTS AND DISCUSSION Spectroscopic Characterization of the MK’ Linker The normalized UV absorption spectra of MK’ in acetonitrile, methanol and water are shown in Fig. 1 along with the fluorescence spectra determined for solutions excited at their absorption maxima having similar absorbance. The absorption maxima reported in Table 2 are similar to those reported previously for the 2-hydroxyethyl analog of MK’ (21). The maxima of the weak fluorescence in methanol and water are similar to those for MK (which is insoluble in water) in methanol (445 nm) (22). The fluorescence maximum in acetonitrile is blueshifted when compared to that for MK (487 vs 515 nm), plausibly as a consequence of intramolecular hydrogen bonding from the hydroxypropyl group to the amine lone pair which would increase the energy of the charge-transfer transition. The femtosecond time-resolved transient absorption spectra of MK’ in methanol and in acetonitrile and water are shown in
Photochemistry and Photobiology
Figure 1. Steady-state absorption (solid) and fluorescence (dash-dot) spectra of diol linker MK’ in acetonitrile, methanol and water. Fluorescence data acquired with 390 nm excitation.
Figs. 2 and S1, respectively. The negative band around 500 nm and positive band around 700 nm in methanol are attributed to the stimulated emission and absorption, respectively, of the Franck–Condon singlet state, both of which are formed during the pump pulse and decay rapidly. The decay of the stimulated emission is accompanied by a time-dependent Stokes shift. Following the slower rise and decay of a broad band with a maximum around 600 nm, the spectrum evolves to a single broad band around 675 nm assigned to triplet MK’, which is long lived on the 7 ns time scale of our measurements. The transient absorption spectra in water and acetonitrile (Figure S1) display much weaker stimulated emission and thus their dynamic Stokes shifts are not as well resolved. The single wavelength 500 nm and 685 nm transient kinetics of MK’ in methanol, acetonitrile and water are shown in Figure S2 and the dominant fast components are reported in Table 2. The transient absorption spectra of MK on the sub-ps time scale have previously been reported by Mondal et al. (23). Their published spectra are reconstructed from single wavelength decays in several solvents including 1-propanol, but not in methanol or water and their spectra in 1-propanol do not closely resemble our spectra in methanol. The fastest decay components
3
Figure 2. Femtosecond transient absorption spectra for the diol linker MK’ in methanol following 400 nm excitation.
in methanol, ethanol and 1-propanol were reported as 0.6, 1.6 and 2.4 ps, respectively, taken from the single wavelength fit at 630 nm. These differences are attributed to the sensitivity of MK excited state single bond torsion to specific solvation. Further insight into the excited state behavior of MK in methanol is provided by the femtosecond fluorescence up-conversion studies of Vedhoven et al. (24). The weighted fluorescence decay time for MK in methanol is 2 ps, similar to the decay of MK’ 685 nm singlet–singlet absorption in methanol (Figs. 2 and S2). These authors conclude that phenyl group twisting as well as solvent dynamics determine the dynamics of MK radiative and nonradiative decay at short times in methanol (24). Spectroscopic Characterization of Hairpins MK-1-3 The steady-state absorption and fluorescence spectra of hairpin MK-1 in aqueous buffer are shown in Fig. 3 along with those of MK’ in water. The absorption and fluorescence maxima and fluorescence quantum yields (Φf) for MK-1-3 are reported in Table 2. Values for the three hairpins are similar. The maxima of the long-wavelength bands of MK-1-3 are redshifted by 2 nm vs that of MK’. The fluorescence of MK-1-3 is substantially more intense and is redshifted by 40 nm vs that of MK’ in water or methanol. However, their maxima are similar to that of MK’ in acetonitrile, suggesting the large Stokes shift for the fluores-
Table 2. Absorption and fluorescence maxima, fluorescence quantum yields and fluorescence and transient absorption decay times for diol linker MK’ and hairpins MK-1-3. Percentage of each time component is given in parentheses. Φf (350ex)
kabs, nm
kfl nm (350ex)
MK’(CH3CN) MK’(CH3OH) MK’(H2O) MK-1*
357 372 389 391
487 ~ ~ 482
0.0023
1.7 (30%) 16 (36%) 219 (34%)
MK-2*
389
484
0.0013
MK-3*
391
482
0.0027
1.7 (55%) 13 (33%) 124 (11%) 0.5 (38%) 4.6 (41%) 44 (21%)
sfl, ps†
s455, ps‡
0.5(+) (28%) 1.3(+) (72%) 33() (87%) ~6000() (13%) 0.9(+) (100%) 25() (82%) ~7000() (18%) 1.0(+) (100%) 21() (46%) 65() (40%), >1 9 104() (14%)
s500, ps‡,§ 2.2‡ 1.5‡ 0.6‡ 0.65 (50%) 6.9 (50%) 0.15 (67%) 3.0 (33%) 1.50 (62%) 8.80 (38%)
s667, ps‡
1.9() (70%) 78(+) (100%) > 1 9 104() (30%) 1.5() (50%) 35(+) (100%) >1 9 104() (50%) 2.0() (32%) 160(+) (100%) >1 9 104() (68%)
*Values determined in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl. †Values from fits to fluorescence decay at 485 nm following 395 nm excitation. ‡Values from fits to transient absorption determined following 400 nm excitation. §Values from fits to stimulated emission band near 500 nm following 400 nm excitation. (+) = rise, () = decay.
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Almaz S. Jalilov et al.
Figure 3. Steady-state absorption (solid) and fluorescence (dash-dot) spectra of diol linker MK’ in water and 3.0 lM hairpin MK-1 in aqueous solution (0.1 M NaCl, 10 mM sodium phosphate, pH 7.2). Fluorescence data acquired with 390 nm excitation.
cence of hairpins MK-1-3 may reflect rapid twisting of the Franck–Condon singlet state prior to emission. Fluorescence decay times (sfl) determined by fluorescence up-conversion (Figure S3) are best fit as triexponentials, the decay times of which are reported in Table 2. The shortest lived decay components for MK-1,2 are similar to that for the linker MK’. The longer lived decay components for MK-1-3 are tentatively attributed to hairpin conformations having reduced torsional mobility. The transient absorption spectra of hairpins MK-1-3 in aqueous buffer are shown in Fig. 4. All of the spectra display negative bands similar to that for MK’ in methanol (Fig. 2) attributed to stimulated emission. These bands are formed during the pump pulse and decay with a time-dependent redshift as shown in Figs. 5a and S4a,c. The band minimum as a function of delay time was fit to a biexponential to determine the depopulation times of the singlet excited state reported in Table 2 (s500). The shorter depopulation times for MK-1-3 are between 0.2 and 1.5 ps and the longer depopulation times between 3 and 9 ps. Transient absorption bands are also observed at longer wavelengths for MK-1-3. The longer wavelength bands are similar to those for MK’ in methanol or water and are attributed to absorption by the vertical singlet state. These bands have decay times of 1.5–2 ps, similar to that of MK’ in methanol or water or MK in methanol (23). Since the appearance of neither the fluorescence nor the simulated emission is dependent upon the adjacent base pair, they plausibly arise from conformations in which there is weak electronic coupling between MK’ and the adjacent bases. The hairpin depopulation times are in only fair agreement with their two shorter fluorescence decay times (Table 2). The lack of better agreement may result from the different excited state populations probed by the two transient techniques: fluorescence decay times sampling only the minority population of fluorescent excited states and transient absorption sampling the overwhelming majority of the nonfluorescent excited states. In addition to the broad absorption band at longer wavelengths, hairpins MK-1-3 display a narrower absorption band at 455 nm that is partially formed during the laser pump pulse and continues to rise during the first few ps thereafter (Figs. 5b and
Figure 4. Femtosecond transient absorption spectra of hairpins MK-1-3 in aqueous buffer following 400 nm excitation.
S4b,d). The 455 nm absorption bands of MK-1-3 are assigned to the anion radicals of MK’, based on the similarity of their appearance to the absorption spectrum reported by Mondal et al.
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Figure 6. Steady-state absorption spectra of hairpins MK-4, MK-5 and MK-6 in aqueous solution (0.1 M NaCl, 10 mM sodium phosphate, pH 7.2).
Figure 5. (a) Amplitude of the stimulated emission minimum (black) and time-dependent shift of the stimulated emission minima (gray) and (b) single wavelength transient kinetics monitored at 455 nm (black) and 667 nm (gray) following 400 nm excitation.
1
(a)
(b)
*MK-A
1
*MK τ st
τcs 1 (MK-.-A+.)
τf
τcr
MK-A
3
*MK
τ ts
MK
Scheme 2. Scheme Mechanisms for charge separation and recombination in (a) well-stacked hairpins with adjacent A-T base pairs and (b) for intersystem crossing in nonstacked hairpins MK-1-3.
for MK.generated by pulse radiolysis (25). In the case of MK1 and -2, the decay of this band can be fit as a single exponential with decay times of 33 ps and 25 ps, respectively, with a longlived offset (ca. 7 ns). The decay of the 455 nm band of MK-3
Figure 7. Steady-state fluorescence spectra of hairpins MK-4, MK-5 and MK-6 in aqueous solution (0.1 M NaCl, 10 mM sodium phosphate, pH 7.2) excited at 390 nm (solid lines) and 506 nm (dash-dot lines).
is best fit as a dual exponential with decay times of 25 and 65 ps with a long-lived offset. Since these decays are not accompanied by the rise of a successor band, we conclude that they occur by fast singlet radical ion pair recombination, similar to that observed for the singlet radical ion pairs of anthraquinones with purine bases (7). After decay times >0.5 ns the transient spectra of MK-1-3 consist of broad featureless bands similar to that for MK’ in methanol or water. These bands do not decay on the 7 ns time scale of our measurements and are attributed to absorption of the MK triplet state, which is described as having weak T1-Tn absorption at 650 nm (13). Only in the case of MK-2 is a rising component for the triplet observed with a time constant similar to that for decay of the 455 nm band attributed to the MK. anion radical. In view of the long decay times for the triplet states of MK-1-3, it seems unlikely that triplet electron transfer occurs in these hairpins. The excited state behavior of hairpins MK-1-3 is summarized in Scheme 2. The rapid formation and decay of the 455 nm band attributed to the MK. anion radical requires a significant population of these hairpins, in which the MK’ and purine chromophores are closely associated so that upon electronic excitation they can form a singlet radical ion pair. The time constants for
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Almaz S. Jalilov et al.
charge separation and recombination (Scheme 2a, scs and scr) can be estimated from the rise and decay times for formation of MK. (Table 2, s455 nm). The observation of rapid charge separation and charge recombination for singlet MK with adjacent A-T and G-C base pairs is analogous to the behavior of AQcapped hairpins for which singlet charge separation competes with intersystem crossing (7). Charge recombination for the singlet radical ion pairs of both MK and AQ is much faster than for the stilbenediamide hairpin linker, presumably as a consequence of the smaller energy gap for charge recombination (2). In competition with charge separation, the singlet states of MK1-3 can undergo behavior similar to that of the MK chromophore in methanol or water: twisting and solvent relaxation followed by efficient intersystem crossing to form the triplet state (Scheme 2b). Rate constants for fluorescence in these hairpins (kf) can be estimated from the fluorescence quantum yields and stimulated fluorescence decay times (kf = Φfs5001). The nonrigid nature of the MK’ linker (e.g. freedom of rotation about the phenyl-N bonds and aminopropyl linkers) provides ample opportunity for the formation of hairpins with multiple ground state conformations. Unlike the triplet of AQ (11), there is no evidence for the efficient formation of triplet radical ion pairs with the MK hairpins. The apparent lack of reactivity of the triplet state of MK-1-3 indicates that it is unable to form a triplet radical ion pair with a neighboring base, either because of lack of proximity or insufficient driving force for electron transfer. Estimating the energetics of singlet and triplet electron transfer reactions for the MK’-linked hairpins is complicated both because of uncertainties in the geometry of the MK’ linker and in its singlet and triplet energies and redox potentials within the DNA hairpins. Using the singlet energy (3.45 eV in a rigid glass) and ground state reduction potential (1.87 V vs SCE) reported by Loutfy for MK provides an excited state oxidation potential (1.58 V vs SCE) sufficiently large to oxidize G, but questionable for A (Eox = 1.24 and 1.69 V, respectively) (15,26). Using the triplet energy reported by Timpe et al. (14), triplet oxidation of purines is endoergic and using the oxidation potential of MK reported by Jockusch et al. (13), reduction of the pyrimidine bases by either singlet or triplet MK is likewise endoergic. Our hopes that the triplet state of the MK hairpins might prove to be an effective reductant of pyrimidines unfortunately were not realized. Spectroscopic Characterization of Hairpins MK-4-6 The UV–visible absorption spectra of hairpins MK-4-6 in aqueous buffer are shown in Fig. 6. The structured long-wavelength band has a first vibronic band maximum at 545 nm attributed to
PDI and a structureless second band at 385 attributed to the absorption of MK which overlaps the weaker absorption of PDI in this region of the absorption spectrum (16). The fluorescence spectra of MK–4-6 obtained with excitation wavelengths of 506 nm and 390 nm, corresponding to the absorption bands of the PDI and MK chromophores, respectively, are shown in Fig. 7 and absorption and fluorescence maxima are reported in Table 3. Fluorescence quantum yields have not been determined for these hairpins; however, the values of Φf for 390 nm excitation are presumed to be similar to the values for hairpins MK-13 (Table 2) and hence < 1%. The weaker PDI fluorescence for MK-6 vs MK-4,5 observed for 506 nm excitation is attributed to more extensive electron transfer quenching of singlet PDI by guanine (G) than by adenine (A). More efficient electron transfer quenching of PDI by G vs A has been previously reported and is attributed to the lower oxidation potential of G (16). Excitation at 390 nm results in the observation both MK and PDI fluorescence (Fig. 7). Dual fluorescence is attributed to competitive absorption at 390 nm; however, a minor contribution from resonant energy transfer cannot be ruled out. The transient absorption spectra of MK-4-6 obtained with 545 nm excitation are shown in Fig. 8. All three spectra display negative bands near 500 and 550 nm assigned to ground state bleach of PDI. The spectra of MK-4,5 also display a negative band near 580 nm assigned to PDI stimulated emission, which is not detected in the case of MK-6. The transient spectra of MK4,5 also display a positive band near 700 nm which undergoes a time-dependent redshift to ca. 725 nm. We have observed similar transient spectra for hairpins possessing A-T base pairs on both sides of a PDI base surrogate and assigned the redshifted absorption to reduction of singlet PDI to its anion radical (16). The spectra of MK-6 lack the 700 nm band assigned to singlet PDI, as previously observed for a hairpin possessing a single G–C base pair on one side of the PDI base surrogate. More rapid quenching of singlet PDI by G vs A is responsible for the absent or very weak transient absorption, stimulated emission and fluorescence of the PDI chromophore in MK-6. The 600 nm and 720 nm single wavelength kinetics for MK4-6 are shown in Fig. 9 and the multiple exponential fits are reported in Table 3. The behavior of hairpins MK-4,5 upon excitation of the PDI chromophore at 545 nm is summarized in Scheme 3a and the behavior of MK-6 is shown in Scheme 3b. Only the two chromophores and the connecting poly(purines) are shown in these schemes. The fast rising components of the 600 nm stimulated emission are attributed to the formation of the PDI.A+. or PDI.G+. radical ion pairs and the slower rising components to decay of the singlet (Fig. 9a). The somewhat shorter 600 nm decay time for MK-5 vs MK-4 plausibly reflects
Table 3. Absorption and fluorescence maxima and decay times for hairpins MK-4-6.* Percentage of each time component is given in parentheses.† kabs, nm
kfl, nm (350ex)
kfl, nm (506ex)
s600, ps
s720, ps
MK-4
389
474
555
0.3(+) (31%), 3.9(+) (46%), 24.3(+) (23%), >1 9 104()
MK-5
388
473
555
1.9(+) (52%), 12(+) (48%), >1 9 104()
MK-6
385
476
555
0.3(+) (~100%) 1.4() (89%) 15() (4%) 1600() (7%)
2.54() (4%), 25.4() (75%), 300() (11%), >1 9 104() (10%) 1.6() (9%), 48() (12%), 530() (52%), >1 9 104() (27%) 1.5() (89%), 32() (8%), 1320() (3%)
*All values determined in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl. †Time constants determined following 545 nm excitation. (+) = rise, () = decay.
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Figure 9. Normalized single wavelength kinetics for MK-4-6 in aqueous buffer determined at (a) 600 nm and (b) 720 nm following 545 nm excitation.
Figure 8. Femtosecond transient absorption spectra of hairpins MK-4-6 following 545 nm excitation.
confinement of the PDI.A+. radical ion pair within a AA dyad, whereas, the PDI.A+. radical ion pair in MK-4 can delocalize over a AAAA tetrad.
The 720 nm single wavelength decays for MK-4-6 display a long-lived component with band maximum at 725 nm assigned to the formation of the PDI./MK+. charge-separated state (Fig. 9b). Unfortunately, MK+ does not have a characteristic transient absorption band in 450–800 nm spectral window of our measurements that would permit direct determination of the hole transit times for the charge separation process, as implemented for stilbene donor–acceptor hairpins (27). The efficiency of formation of the charge-separated radical ion pair is dependent upon the purine base sequence, following the same order, A2G2 > A4 > G4, as previously observed for hole transport in stilbene donor–acceptor capped hairpins (28). Inefficient hole transport for Gn purines in the stilbene hairpins was attributed to fast charge recombination in the Sa–/G+. contact radical ion pair, as a consequence of a smaller energy gap than for Sa/A+..(28) More efficient charge separation for the A2G2 diblock sequence vs the A4 homopurine sequence was attributed to slow charge return once the hole exits A2 to form the Sa.AAG+ charge-separated radical ion pair (Scheme 3a) (28). The role of MK in hairpins MK-4-6 is to serve as a ground state electron donor or hole trap for the reduction of a A+ or G+. cation radical. The oxidation potential of MK (0.95 V vs SCE in acetonitrile) (26) is well below that of either of the nucleosides G or A (1.24 and 1.69 V, respectively, vs SCE) (15). While the potentials of MK, G and A within the hairpins MK-1-6 in water
8
Almaz S. Jalilov et al. separation are also available from the amplitudes of the formation of long-lived charge-separated states (Fig. 9b). The values parallel those reported previously of the stilbenediether–stilbenediamide donor–acceptor capped hairpins (28), suggesting that it is the base sequence rather than the chromophores which, to a first approximation, control the dynamics and efficiency of charge transport in DNA. Acknowledgements—This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Chemical Sciences, Geosciences, and Biosciences Division under Award DE-FG02-96ER14604 (F.D.L.) and DE-FG02-99ER14999 (M.R.W.). R.M.Y gratefully acknowledges the Camille and Henry Dreyfus Foundation Postdoctoral Fellowship in Environmental Chemistry for funding. We thank Dr. Taiga Fujii for the preparation of the phosphoramidite of the diol linker.
SUPPORTING INFORMATION
Scheme 3. Mechanism for charge separation and charge recombination in (a) hairpins MK-4 and MK-5 and (b) hairpin MK-6.
are not known, it is unlikely that their order is different from that of MK and the nucleosides in acetonitrile.
CONCLUSIONS In summary, we find that the singlet excited state of MK but not its triplet state can serve as an electron acceptor for the oxidation of the purine bases G and A. The behavior of the hairpins MK1-3 is complicated by the presence of singlet conformers which undergo charge separation and those which do not. The difference in singlet and triplet state reactivities reflects not only the higher energy of the singlet state but also the difference in geometry and charge transfer character of the planar Franck–Condon singlet state and the relaxed twisted triplet state (23,24). Both excited state geometric relaxation and charge transfer may reduce the driving force for electron transfer processes and render the relaxed excited states less potent oxidants or reductants than the Franck–Condon excited states. The behavior of the MK-linked hairpins differs from that of the AQ-capped hairpins in several respects. The more rigid planar structure of AQ results in similar singlet and triplet energies (and presumably reduction potentials) and hence the observation of oxidation of neighboring A and G bases by both singlet and triplet AQ (7). In addition, more rapid intersystem crossing reduces the lifetimes of the singlet state and radical ion pairs of AQ in comparison with those of MK (10). The ground state of MK can also serve as an electron donor for the formation of PDI./MK+. charge-separated states in the hairpins MK-4-6. Hole transit times are in principle available from analysis of the time-dependent band shape changes of the 725–720 nm maxima for MK-4 and 5. Relative yields of charge
Additional Supporting Information may be found in the online version of this article: Figure S1. Transient absorption spectra of linker diol MK’ in (a) water and (b) in acetonitrile following 400 nm excitation. Figure S2. Kinetics for diol MK’ in various solvents following 400 nm excitation. Figure S3. Time-resolved fluorescence intensities for MK-1-3 in aqueous buffer monitored at 485 nm following 395 nm excitation. Figure S4. Amplitudes of the stimulated emission maximum (black) and time-dependent shift of the stimulated emission maxima (blue) and single wavelength transient kinetics monitored at 455 nm (black) and 667 nm (red) for (a, b) MK-2 and (c,d) MK-3 in aqueous buffer following 400 nm excitation. Figure S5. Kinetic fits at 600 and 720 nm for MK-4-6 in aqueous buffer following 545 nm excitation.
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