Dynamics and Couplings of N−H Stretching ... - ACS Publications

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Dynamics and Couplings of N-H Stretching Excitations of Guanosine-Cytidine Base Pairs in Solution Ming Yang,† yukasz Szyc,† Katharina R€ottger,‡ Henk Fidder,† Erik T. J. Nibbering,*,† Thomas Elsaesser,† and Friedrich Temps‡ † ‡

Max Born Institut f€ur Nichtlineare Optik und Kurzzeitspektroskopie, Max Born Strasse 2A, D-12489 Berlin, Germany Institut f€ur Physikalische Chemie, Christian-Albrechts-Universit€at zu Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany

bS Supporting Information ABSTRACT: N-H stretching vibrations of hydrogen-bonded guanosine-cytidine (G 3 C) base pairs in chloroform solution are studied with linear and ultrafast nonlinear infrared (IR) spectroscopy. Assignment of the IR-active bands in the linear spectrum is made possible by combining structural information on the hydrogen bonds in G 3 C base pairs with literature results of density functional theory calculations, and empirical relations connecting frequency shifts and intensity of the IR-active vibrations. A local mode representation of N-H stretching vibrations is adopted, consisting of νG(NH2)f and νC(NH2)f modes for free NH groups of G and C, and of νG(NH2)b, νG(NH), and νC(NH2)b modes associated with N-H stretching motions of hydrogen-bonded NH groups. The couplings and relaxation dynamics of the N-H stretching excitations are studied with femtosecond mid-infrared two-dimensional (2D) and pump-probe spectroscopy. The N-H stretching vibrations of the free NH groups of G and C have an average population lifetime of 2.4 ps. Besides a vibrational population lifetime shortening to subpicosecond values observed for the hydrogen-bonded N-H stretching vibrations, the 2D spectra reveal vibrational excitation transfer from the νG(NH2)b mode to the νG(NH) and/or νC(NH2)b modes. The underlying intermode vibrational couplings are on the order of 10 cm-1.

1. INTRODUCTION Nucleobase pairing is one of the defining elements in the structures of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).1 The sequence of nucleobases (guanine, cytosine, adenine, and thymine or uracil) encodes the genetic information.2 Hydrogen bonding is the underlying interaction through which the nucleobase pairing occurs. In thermal equilibrium, nucleobase pair geometries and interactions with other parts of DNA and/or their aqueous environment have been studied for different forms of DNA, combining methods of X-ray diffraction, nuclear magnetic resonance (NMR), and spectroscopy with theoretical calculations.1 This detailed structural information represents an important prerequisite for studying basic nonequilibrium properties of base pairs and DNA, among them the pathways and mechanisms of electronic relaxation after photoexcitation,3-7 the dissipation of excess energy,8 and the transport of excitations, charge, and energy along helices.9,10 All such processes involve transient vibrational excitations that occur in a wide frequency range and that are characterized by elementary steps taking place in the femto- to picosecond time domain. Two approaches to study nucleobase pairing in DNA have been described in the literature. A bottom-up approach starts with the nucleobases themselves as molecular systems,11-18 or r 2011 American Chemical Society

model compounds,19-21 to study the hydrogen bond complexation. Advantages are control of molecular design of nucleobase derivatives and choice of solvent that does not necessarily have to be water.22,23 Disadvantages include the possibility of multiple hydrogen-bonded structures13,20,21 and possible keto-enol tautomerization.12-15,24,25 The second, top-down approach implies the use of DNA itself or DNA oligomers. Advantages include a well-defined Watson-Crick base pairing within the double helix structure, including the full interactions of the ribose-phosphate backbone, and in the case of smaller DNA oligomer structures, control of nucleobase sequence. A disadvantage is that IR spectroscopy on DNA samples cannot be performed in H2O solutions, due to spectral overlap of vibrational fingerprint modes of DNA and the solvent water. This can be overcome by controlling the amount of water to about one solvent shell in the case of hydrated films of DNA.8,26-28 The work reported in the following addresses hydrogen bonded base pairs of chemically modified guanosine and cytidine Special Issue: Shaul Mukamel Festschrift Received: November 4, 2010 Revised: December 16, 2010 Published: January 18, 2011 5484

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In this article, we present a study of G 3 C base pairs in solution, combining linear IR spectroscopy with ultrafast IR pump-probe and 2D IR measurements. We determine the rates of ultrafast population dynamics, the nature of the couplings between the different NH stretching vibrations, and elucidate the contributions of these NH stretching vibrations to linear and nonlinear spectra. The paper is organized as follows. The experimental techniques applied are summarized in section 2, followed by the presentation of experimental results in section 3. A detailed discussion of the linear and nonlinear vibrational response, including an assignment of the different NH stretching bands and theoretical calculations of the NH coupling pattern is presented in section 4. Conclusions are given in section 5.

2. EXPERIMENTAL TECHNIQUES 2.1. Sample Preparation and Stationary Infrared Spectra. 20 ,30 ,50 -TBDMS protected guanosine (denoted as G) and

Figure 1. Linear infrared spectra of G 3 C base pairs and C and G monomers dissolved in CDCl3 (from top to bottom). The extinction coefficient is plotted as a function of wavenumber, the sample concentrations were 50 mM (G 3 C base pairs), 2.0 mM (C), and 2.0 mM (G). The bottom-most panel shows the difference spectrum GC - G - C, indicating the absence of significant NH stretching absorption below 3000 cm-1. Inset: structure of the G 3 C base pair in Watson-Crick geometry.

in their electronic ground state. The complexes consist of tertbutyldimethylsilyl (TBDMS) derivatives of guanosine and cytidine (see molecular structure in Figure 1), denoted in this paper as G and C, respectively. With the substitution of the hydroxy groups of the ribose units with TBDMS, high solubilities are reached in weakly polar solvents such as chloroform, while the presence of the ribose units with bulky side groups prevents possible keto-enol isomerization of the guanine and cytosine nucleic bases, and Watson-Crick base pairing is strongly favored. The same molecular system has been used to study ultrafast internal conversion dynamics of G 3 C base pairs upon UV excitation.23 We exploit the potential of linear and ultrafast multidimensional spectroscopy29-32 to study the hydrogen bonded structure and dynamics of G 3 C base pairs. In particular, nonlinear infrared spectroscopy with femtosecond time resolution has provided detailed insight into vibrational dynamics, microscopic couplings and transient structures of hydrogen bonded systems such as bulk33-40 and nanoconfined water,41 ionic solutions,42,43 and macromolecular structures.44 Apart from a few experiments on model systems,19-21 the application of femtosecond pumpprobe and two-dimensional (2D) correlation spectroscopy on base pairs and DNA structures has, however, remained limited. Early 2D work by Krummel et al. has addressed guaninecytosine base pairs and DNA oligomers containing G 3 C in solution and suggests a complex interplay of intra- and interbase pair interactions of carbonyl, CN, and CC ring stretching modes.45,46 Numerical studies of the linear and nonlinear IR response of G 3 C and A 3 T oligomers in the fingerprint region also indicate an interplay of intra- and interbase interactions.47 In a more recent series of pump-probe and 2D experiments, double stranded DNA oligomers containing 23 alternating adenine-thymine (A 3 T) base pairs in Watson-Crick geometry have been studied over a wide range of hydration levels.8,26,28 These experiments point to predominant intrabase pair couplings of the different NH stretching excitations of the A 3 T pairs.

30 ,50 -TBDMS protected 20 -deoxycytidine (denoted as C) were synthesized as described before.23 The FT-IR spectra of G or C or G 3 C in CHCl3 or CDCl3 solution were measured with a Varian 640 FT-IR spectrometer, applying a concentration range of 2-100 mM. CHCl3 (p.A.) was purchased from Merck and CDCl3 was supplied by Deutero GmbH. 2.2. Femtosecond Experiments. 2D spectra of the G 3 C pairs were measured in a three-pulse photon echo experiment. Details of the photon echo setup and the procedures used for data analysis have been described in detail before.38,40,48 In short, the mid-IR pulses were generated by a home-built parametric frequency converter driven by a 1 kHz regenerative amplified Tisapphire laser system. The output pulse was centered at 3300 cm-1, with a spectral bandwidth exceeding 300 cm-1, a pulse duration of 50 fs, and a pulse energy of 8 μJ. A diffractive optical setup was employed to generate two pairs of phase-locked pulses of equal energy, three of which interacted with the sample in a photon echo sequence. The energy of such pulses at the sample was about 600 nJ each. The fourth pulse was attenuated and served as local oscillator to interfere with the generated nonlinear signal. Both nonlinear signal and local oscillator were dispersed by a monochromator providing the detection frequency ν3. The resulting spectral interferograms were detected by a HgCdTe detector array (resolution 8 cm-1). Interferograms were recorded as a function of the pulse delay τ, the coherence time, between pulse 1 and 2, to derive the excitation frequency ν1. 2D spectra were recorded for different waiting (population) times T, the delay between the second and the third pulse interacting with the sample. Pump-probe experiments were performed using the same experimental setup, with two of the four pulses blocked. One pulse served as a pump, and the other pulse attenuated to 1% by a copper mesh served as a probe and was detected in a spectrally resolved way. In the time-resolved experiments, the sample solution was kept between two 1 mm thick BaF2 windows, separated by a Teflon spacer with a thickness of 100 μm. In the photon echo experiments the solutions were made by mixing equal volumes of 100 mM G and of 100 mM C solutions, both in CHCl3. In the pump-probe experiments the concentrations were 50 mM G and 50 mM C.

3. RESULTS Figure 1 shows the linear IR spectra of the G 3 C base pair and of G and C monomers in CDCl3 solution. The concentration of the G 3 C base pair solution was 50 mM, the G and C monomer samples had a concentration of 2.0 mM. The G and C 5485

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Figure 2. 2D-IR spectra recorded for different population waiting times T. The contour plots have been scaled for maximum signal strength for each value of T. For comparison with the location of the IR-active N-H stretching transitions, the linear IR spectrum of the sample (50 mM G 3 C in CHCl3), not corrected for solvent contributions, has been added to the left and the top of the figure.

monomers display narrow N-H stretching bands reflecting non-hydrogen-bonded NH groups. In the case of the C monomer, the symmetric and asymmetric N-H stretching transitions of the NH2 group are found at 3418 and 3534 cm-1, respectively. For the G monomer, the symmetric and asymmetric N-H stretching vibrations of the NH2 group are located at 3411 and 3521 cm-1, whereas the secondary amino N-H stretching band, hidden under bands of G 3 G dimers, has been suggested to be located near 3340 cm-1.23 For the concentrations used for the linear spectra, C dimer formation to C 3 C is almost negligible. This is not the case for solutions of G, where several N-H stretching bands indicative of hydrogen-bonded NH groups can clearly be observed. It has been established that preferentially the G 3 C base pair in the Watson-Crick geometry contributes to the linear IR spectrum.23 Here a narrow band, slightly asymmetric in line shape, is observed at 3491 cm-1 with a line width of 27 cm-1. Red-shifted bands, located at 3303 and 3145 cm-1, with line widths of 84 and 174 cm-1, respectively, are indicative of N-H stretching bands of hydrogen bonded NH groups in the G 3 C base pair. Both of these bands exhibit asymmetric line shapes. Between 2850 and 3000 cm-1, the C-H stretching bands of the TBDMS side groups of the ribose units are located, potentially masking additional spectral density of the N-H stretching manifold of the G 3 C base pair. As the same C-H stretching pattern is observed for solutions containing only C or G, we performed a subtraction procedure on the IR spectrum of the G 3 C base pair. It turns out that after this subtraction procedure, under the assumption that the C-H stretching bands remain unaffected in spectral position, cross section and line shape, no significant IR absorption is found below 3000 cm-1 (bottom panel of Figure 1). We conclude that the N-H stretching manifold of the G 3 C base pair only contributes to the IR spectrum in the frequency range between 3000 and 3500 cm-1, with absorption maxima at 3145, 3303, and 3491 cm-1. In Figure 2 we present results of 2D-IR measurements on the G 3 C base pair. The 2D correlation spectra, i.e., the sum of the rephasing and nonrephasing 2D signals, are plotted as a function

of excitation (ν1) and detection (ν3) frequency for several (population) waiting times T. In this series of measurements, we used CHCl3 as the solvent, because CDCl3 has an overtone transition strongly overlapping with the 3145 cm-1 N-H stretching band of the G 3 C base pair, leading to major distortions of the observed signals. The choice of CHCl3 as solvent prevents the investigation though of 2D-IR nonlinear signals in the spectral region below 3000 cm-1, which would already have been difficult due to the absorption of the C-H stretching bands of the TBDMS side groups anyway. As a result, signal contributions involving excited state absorption pathways of the hydrogen-bonded N-H stretching bands were not investigated in full detail. In the 2D-IR spectra we observe diagonal peaks at (ν1 [cm-1], ν3 [cm-1]) = (3145, 3145), (3303, 3303), and (3491, 3491). Whereas for population waiting time T = 0 fs the (3145, 3145) and (3491, 3491) diagonal peaks are only slightly elongated along the diagonal, the (3303, 3303) peak is a clearly elongated toward higher frequencies. The shapes of the diagonal peaks do not change profoundly as a function of T within the accuracy of our 2D-IR measurement. Off-diagonal peaks are present at (ν1 [cm-1], ν3 [cm-1]) = (3145, 3303), (3303, 3145), (3145, 3491), (3491, 3145), and (3303, 3491). An additional cross peak with opposite sign at (3491, 3360) indicates an excited state absorption contribution in connection with the 3491 cm-1 fundamental transition. The (3303, 3145) off-diagonal peak appears to increase its relative magnitude compared to the (3145, 3303) peak for increasing values of T. In Figure 3a we present the temporal behavior of the 2D-IR signals along the diagonal as a function of T. For pulse delays up to T = 1.0 ps, the peaks at 3145 cm-1 and at 3303 cm-1 decay significantly, while the narrow band at 3491 cm-1 only decays slightly. For comparison we show the transient spectra obtained in the pump-probe experiment in Figure 3b. Here the bleach at 3145 cm-1 shows a fast recovery, whereas the bleach recovery at 3491 cm-1 is significantly slower. The spectral region between 3280 and 3450 cm-1 shows a much more complex time-dependent pattern, with a broad-band bleach recovering within 1 ps, and 5486

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Table 1. Relaxation Times and Signal Amplitudes Determined by a Numerical Analysis of the Transient Pump-Probe and 2D-IR Spectra ν(0-1) (cm-1) 3145

Figure 3. (a) Diagonal cuts of the 2D-IR spectra as function of the population waiting time T. (b) Transient pump-probe spectra for various pump-probe pulse delay times.

Figure 4. Temporal behavior of diagonal and off-diagonal intensities located at (ν1 [cm-1], ν3 [cm-1]) obtained by integration of the 2D-IR spectra of Figure 2. The solid lines are fitting results with parameter values given in Table 1. The diagonal peaks are shown in (a), and the off-diagonal peaks in (b) (with the (3491, 3360) excited state absorption multiplied by -1), both shown on a logarithmic scale. The ratio of the (3303, 3145) and the (3303, 3303) peaks is presented in (c) on a linear scale.

superimposed on this a narrower feature at 3360 cm-1 that does not change as strongly on this time scale. To quantify the dynamical behavior of diagonal and offdiagonal peaks, we integrated the areas around the diagonal and off-diagonal peaks in the 2D-IR spectra. The resulting integrated area for these peaks is plotted as a function of T in Figure 4a,b. Whereas the (3145, 3145), (3303, 3303) peaks have significantly decayed in magnitude for pulse delays as long

v=1

type of

lifetime (ps)

measurement

0.3 (-0.1/þ0.2)

pump-probe

0.4 (-0.1/þ0.2)

2D diagonal peak (3145,3145)

3303

0.24 (-0.06/þ0.08)

2D diagonal peak (3303,3303)

3491

2.9 ((0.7)

pump-probe

(ν1, ν3) (cm-1, cm-1)

decay time (ps)

(3145, 3303)

0.22 (-0.05/þ0.06)

(3303, 3145)

0.8 (-0.2/þ0.3)

as T = 1 ps (Figure 4a, filled black squares and filled red dots, respectively), the (3491, 3491) diagonal peak (Figure 4a, filled green triangles) has only diminished to 37% of its initial value. Such behavior is also reflected in the decay of the negative peak at (3491, 3360) (Figure 4b, open brown triangles). It should be noted that the normalized integral of the entire (3303, 3145) peak shows nearly the same behavior as the integrals for the lower and upper halves of the ν1 integration interval. This illustrates that the tail of the diagonal peak (3145, 3145) does not significantly alter the decay kinetics of this cross peak. Some difference is seen, however, between the integrations for the lower and upper halves of the ν3 integration interval, with a 0.6 ps decay for the upper range, and 1.1 ps decay for the lower range. This indicates some influence of the negative signals at ν1 = 3303 cm-1 and ν3 < 3100 cm-1 on the kinetics of the (3303, 3145) peak. However, the influence does not exceed the error margin range assigned to this cross peak. Figure 4b shows that the off-diagonal cross peak at (3303, 3145) decays slower than the diagonal peak at (3303, 3303). Figure 4c shows that the decay of the diagonal peak at (3303, 3303) and of the off-diagonal peak at (3303, 3145) are not identical. For pulse delays longer than T = 100 fs, the ratio of the off-diagonal peak and the diagonal peak increases with T, shown as blue squares. In contrast, the off-diagonal cross peak at (3145, 3303) decays faster than the diagonal peak at (3145, 3145). These findings hint at more complex dynamics of the hydrogen-bonded N-H stretching modes. From the pump-probe measurements (Figure 5) we derive effective time constants of the different spectral components contributing to the signals using a multiexponential fitting/ spectral decomposition procedure (see Table 1). The bleach recovery around 3145 cm-1 (Figure 5a) occurs with a predominant ultrafast component (0.34 ( 0.15 ps) and a weak slow part (∼2.4 ( 0.8 ps). The narrow 3491 cm-1 bleach exhibits a recovery with a 2.9 ( 0.7 ps time constant, similar to the decay of the enhanced absorption at 3360 cm-1. In general, the dynamics in the spectral region between 3280 and 3400 cm-1 turns out to be difficult to disentangle into different components. These findings show the limitation of transient IR pump-probe spectroscopy, where substantial spectral overlap may hamper the interpretation of the experimental results. In contrast, with ultrafast 2D-IR spectroscopy contributions originating from different Liouville space pathways appear at different positions in the 2D spectra. We show in the next section that disentangling these spectral components, assigning them to particular vibrational modes of 5487

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Table 2. Comparison of Vibrational Mode Assignment for the G 3 C Base Pair to Experimental Frequencies and DFT Results

a

experimental

anharmonic

scaled harmonic

vibrational

absorption

frequencies from

frequencies from

mode

maximum (cm-1)

DFTa (cm-1)

DFTb (cm-1)

νC(NH2)b

3145

2905.3

3243

νG(NH)

3145

3055.3

3264

νG(NH2)b νC(NH2)f

3303 3491

3347.9 3515.0

3343 3526

νG(NH2)f

3491

3522.9

3538

Reference 18. DFT at the level of B3LYP/6-311þþG**, no scaling (Gaussian 03). b Reference 12. DFT Hartree-Fock calculations with 6-31G(d,p) basis set, and scaling factor 0.893 (Gaussian 98).

Figure 5. Frequency-resolved pump-probe data as a function of pump-probe pulse delay time for selected detection frequencies. Red curves are the (bi)exponential fitting results. (a) shows dynamics near the absorption maxima 3145 cm-1 (νG(NH) and νC(NH2)b) and 3303 cm-1 (νG(NH2)b), related to hydrogen-bonded N-H stretching vibrations. (b) shows the slower excited state relaxation of the free NH groups absorbing at 3491 cm-1 (νG(NH2)f and νC(NH2)f), on both the v = 0 f v = 1 and the v = 1 f v = 2 transitions.

the G 3 C base pair molecular system, and deriving anharmonic couplings and energy flow pathways for the N-H stretching manifold are possible using ultrafast 2D-IR spectroscopy.

4. DISCUSSION 4.1. Linear IR Spectrum. The G 3 C base pair is expected to adopt the Watson-Crick geometry, wherein the cytosine and guanine moieties adopt a planar structure.17,49,50 This picture is supported by the concentration dependent infrared spectra discussed in ref 23. In this geometry three of the five NH units form hydrogen bonds: one of the NH bonds of the NH2 groups for both G and C, and the secondary amine group of G (see Figure 1). Density functional theory (DFT) calculations at the BP86/TZ2P level of theory49 indicate that the N 3 3 3 O distance is much shorter for the C(NH2) 3 3 3 G(OdC) hydrogen bond than for the G(NH2) 3 3 3 C(OdC) hydrogen bond, i.e., 2.73 Å vs 2.87 Å, suggesting that the C(NH2) 3 3 3 G(OdC) hydrogen bond is significantly stronger. This is corroborated by DFT calculations by Wang et al.18 on guanine-cytosine in vacuo. A comparison of vibrational stretching transition frequencies from DFT calculations by Nir et al.12 and Wang et al.18 is presented in Table 2. In both publications the denotation symmetric and asymmetric N-H stretching is used for the NH2 groups. The three highest vibrational N-H stretching frequencies in these DFT calculations correspond well to the bands at 3303 and

3491 cm-1 (Figure 1), allowing for solvent induced frequency shifts of about 30-40 cm-1. On the other hand, the band at 3145 cm-1 falls between the values predicted by Nir et al. and Wang et al. As we will discuss next, the experimental spectra are well explained in a scheme of five local N-H stretching oscillators. Relevant for the Watson-Crick base pairing of G and C and the description of the spectral range between 3000 and 3500 cm-1 are mostly the five NH bonds, two in each NH2 groups and one in the guanosine NH group. First we focus on the C and G monomers, where the symmetric and asymmetric stretch vibrations of the NH2 groups can be considered linear “þ” and “-” combinations of two individual local N-H oscillators. In the experimental absorption spectra of our modified bases, ν(NH2)s and ν(NH2)a are found at 3418 and 3534 cm-1 for C, and at 3411 and 3521 cm-1 for G, respectively,23 slightly downshifted from the gas phase values for cytosine (3451.7 and 3572.7 cm-1 for ν(NH2)s and ν(NH2)a, respectively14) and guanine (3444.5 and 3544.5 cm-1 for ν(NH2)s and ν(NH2)a, respectively15,51). From this we can extract N-H stretching frequencies of 3476 and 3466 cm-1 for decoupled local N-H stretching modes in C and G, respectively, and (excitonic) couplings |V| of 58 and 55 cm-1 for C and G, respectively. Using the atomic coordinates of the structures of G, C, and G 3 C, and data on partial atomic charges (supplemental information in ref 49) as input, we have performed a preliminary analysis of the interactions in these systems, using an excitonic coupling model for the different vibrational transitions. On the basis of either dipolar coupling or Coulombic interactions, we find that all other couplings in G 3 C will be at least about 3 times smaller than the extracted monomeric NH2 couplings |V| of 58 and 55 cm-1. This implies that the positions of the bands at 3145 and 3303 cm-1, associated with the hydrogen-bonded N-H stretching oscillators, are scarcely influenced by couplings between the N-H stretching modes. Instead, they are dominated by diagonal frequency shifts caused by changes in the N-H stretching potential energy surface upon hydrogen bonding. Simultaneously, this indicates that the smallest diagonal shift is about -160 cm-1, or nearly 3 times larger than the couplings between the N-H stretching local modes in monomeric NH2 groups. A simple calculation shows that such a large detuning causes significant localization of the non-hydrogen-bonded N-H stretching mode, accompanied by a shift toward the decoupled frequency. This is observed when the G and C spectra are compared to the G 3 C base pair absorption spectrum, supporting the local mode approach. As a result, we use the adjective “f” for unbound NH groups, in line with ref 23, 5488

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The Journal of Physical Chemistry B instead of “a” for asymmetric as in ref 18, to indicate that in our description the two N-H oscillators of the NH2 groups become decoupled due to the hydrogen bonding, making the nonhydrogen bonded N-H oscillators nearly “free” local modes and therefore the designations symmetric and asymmetric stretch meaningless. Further corroboration of our assignment is obtained by investigating the relative intensities of the three absorption bands. First, we note that all three bands appear to be asymmetric in line shape. This can most reliably be stated for the 3145 and 3491 cm-1 bands, for which the half-width at half-maximum (HWHM) on the low frequency side is about 11/2 times that on the high frequency side. For the central band at 3303 cm-1 this determination is more difficult as the tail of the stronger 3145 cm-1 band extends underneath this band. Correcting for this it seems that for the 3303 cm-1 band the asymmetry may be in the opposite direction. These asymmetries may reflect a small variation in G 3 C base pair hydrogen bond geometry around the equilibrium structure. It is not uncommon for a symmetrical distribution around an equilibrium structure to result in an asymmetric distribution in interactions.52 As a rough indication of the relative integrated oscillator strengths contained in the three bands, we integrated the following regions in the absorption spectrum: 3000-3255, 3255-3431, and 3431-3600 cm-1, which gives the ratio 8:2:1 for the 3145, 3303, and 3491 cm-1 bands, respectively. Iogansen has investigated the influence of hydrogen bonding on the intensity of the absorption band of the hydrogen bonded species.53 He remarked that no general formula exists describing the correlation between the shift of the hydrogen-bonded mode and its absorption intensity change. However, Iogansen arrived at an empirical formula connecting the enthalpy change upon hydrogen bonding to the absorption intensity increase, which seems to present a fairly universal correlation for hydrogen bonding systems. Spencer et al.54 investigated for both N-H 3 3 3 N and N-H 3 3 3 OdC hydrogen-bonded systems the correlation between the enthalpy change and frequency shift upon hydrogen bonding. Combining these two investigations, we obtain for both types of hydrogen bonds in the G 3 C base pair an expression that relates the expected intensity increase to the observed red shift upon hydrogen bonding (see the Supporting Information). Recall that we concluded that the couplings in first approximation have little influence on the final positions. The analysis suggests that for the same red shift the absorption should increase more for N-H 3 3 3 OdC than for N-H 3 3 3 N hydrogen bonds. As the band at 3491 cm-1 is expected to correspond to two nonhydrogen-bonded (nearly) free N-H local modes (νG(NH2)f and νC(NH2)f), it can serve for normalizing the dependence for both types of hydrogen bonds. Using these expressions, we predict for a N-H 3 3 3 OdC bond at 3303 cm-1 (νG(NH2)b) an intensity enhancement to 3.45 times the intensity of a single free N-H oscillator and at 3145 cm-1 an enhancement factor of 6.65 for N-H 3 3 3 OdC (νC(NH2)b) and 5.15 for N-H 3 3 3 N hydrogen bonds (νG(NH)). Combined, this gives a ratio of 5.9:1.7:1 for the 3145, 3303, and 3491 cm-1 bands, which compares reasonably well to the experimental ratio of 8:2:1 and therefore supports the assignment of two hydrogen-bonded N-H stretching transitions to the 3145 cm-1 band. Thus far we have not considered the influence of any couplings in this intensity analysis. The most relevant couplings are those between the two local N-H stretching modes in the NH2 groups determined for the G and C monomers. Despite the fact that the diagonal shift of the hydrogen-bonded N-H oscillator of these

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NH2 groups localizes the free N-H stretching modes to 90% or more on a single NH bond, the residual amplitude on the hydrogen-bonded NH in combination with the enhancement of its transition dipole moment (intensity1/2) leads to reduction of the intensity of these “free” N-H modes to less than that of a real isolated free N-H mode. 4.2. Ultrafast IR Pump-Probe and 2D-IR Experiments. Having established the assignment, we now interpret the ultrafast pump-probe and 2D-IR results. It is well-known that hydrogen bonding often shortens the vibrational lifetimes of the involved N-H or O-H stretching modes.55 This shortening originates from enhanced anharmonic couplings with combination/overtone levels of fingerprint vibrations, often involving the N-H/ O-H bending mode, and a reduced energy mismatch between these combination/overtone levels and the v = 1 level of the N-H/O-H stretching vibration downshifted in frequency upon hydrogen bonding. Typical hydrogen-bonded N-H/ O-H stretching vibrational lifetimes are a few hundred femtoseconds. For hydrogen bonded complexes in liquid solution this ultrafast vibrational population decay is followed by intramolecular energy flow into other (low-frequency) modes. On longer time scales of picoseconds to tens of picoseconds dissipation takes place of excess vibrational energy into solvent modes. As a result, the time dependence of v = 1 f v = 2 excited state absorption signals of N-H/O-H stretching modes provide insight into the excited population kinetics, whereas the v = 0 f v = 1 bleach and v = 1 f v = 0 stimulated emission signals at the fundamental transition frequencies are governed by both population decay out of the v = 1 state, and vibrational cooling dynamics. The latter is a consequence of transient frequency shifts caused by anharmonic coupling of the hydrogen stretching oscillators with transiently highly excited low-frequency modes. In the case of G 3 C base pairs similar effects are observed as for other hydrogen-bonded complexes studied before.19,21,55 In the pump-probe measurements the bleach recovery at 3491 cm-1 (shown in Figure 5b) has an exponential dependence with a 2.9 ( 0.7 ps time constant. This indicates that the free N-H stretching vibrations νG(NH2)f and νC(NH2)f, associated with the 3491 cm-1 band have long vibrational lifetimes. From 3470 to 3500 cm-1 the recovery time increases fairly monotonously from 1.1 to 3.6 ps, resulting in an average lifetime of ∼2.4 ps for the 3491 cm-1 band. Whether the bleach recovery is governed by population decay only or involves vibrational cooling as well cannot be answered from only an analysis of the time dependence of the fundamental bleach. The asymmetry of the 3491 cm-1 band in linear absorption suggests that the νG(NH2)f and νC(NH2)f vibrations may absorb at slightly different frequencies. A possible difference in their vibrational lifetimes cannot conclusively be resolved. A positive absorption component at 3360 cm-1 (Figure 3b) is assigned to the v = 1 f v = 2 excited state absorption of the νG(NH2)f and νC(NH2)f vibrations, as the observed diagonal anharmonic frequency shift of the free N-H stretching signal of -130 cm-1 corresponds nicely with theoretical predictions18 and is of similar magnitude to experimentally determined values for free N-H stretching transitions of uracil22 and 7-azaindole.19 The 2.5 ps decay time of this signal (Figure 5b) represents the v = 1 lifetime and nicely matches the average decay time of the 3491 cm-1 band. For the bound νG(NH2)b, νG(NH), and νC(NH2)b stretching vibrations, femtosecond components in the bleach recoveries suggest a significant vibrational lifetime shortening. The negative bleach signal of the 3303 cm-1 band, assigned to the νG(NH2)b 5489

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The Journal of Physical Chemistry B mode, unfortunately overlaps strongly with the 3360 cm-1 νG(NH2)f/νC(NH2)f excited state absorption signals, preventing a reliable determination of the kinetics of the band. However, the kinetic trace at 3304 cm-1 shown in Figure 5a indicates fast dynamics for this band, resulting in complete recovery in roughly 1 ps. The 3145 cm-1 band, associated with both νG(NH) and νC(NH2)b modes, shows mostly biexponential bleach recovery dynamics with the short component gradually increasing from ∼0.15 to ∼0.5 ps between 3110 and 3180 cm-1 and a second component of a few picoseconds, which is more prominent toward the lower end of this frequency range, which we ascribe to cooling dynamics. Below 3100 cm-1 strong absorption by the solvent CHCl3 and overlap with presumably the νG(NH) and νC(NH2)b excited state absorption hinder further analysis. With the 2D-IR spectra several of these ambiguities on the dynamics of the N-H stretching oscillators can be resolved, as the different spectral components overlapping in pump-probe are mapped into two dimensions in the photon echo experiment. For instance, the excited absorption connected with the νG(NH2)f and νC(NH2)f modes is now a cross peak located at (3491, 3360), clearly separated from the (3303, 3303) diagonal peak associated with the νG(NH2)b fundamental transition. Figure 4b shows that the integrated intensity decays of the (3491, 3491) diagonal peak and the off-diagonal (3491, 3360) excited state absorption peak perfectly match. The limited range of population times T up to 1 ps in our 2D-IR investigations prevents a reliable determination of time constants above 1 ps. Within the experimental accuracy, the intensity decay of the (3491, 3491) and (3491, 3360) peaks is consistent with the 2.5 ps decay time derived from the pump-probe data. The shapes of the diagonal peaks do not change significantly for population waiting times up to T = 1.0 ps, from which we conclude that spectral diffusion does not play a major role here. We conclude that the diagonal peaks of the hydrogen-bonded N-H stretching modes are governed by population decay dynamics on the subpicosecond time scale. Exponential fitting of the decay of the (3303, 3303) peak results in a 0.24 (-0.06/ þ0.08) ps time constant, a value that could not be determined from the pump-probe data. The (3145, 3145) diagonal peak exhibits slightly slower dynamics, as indicated by the 0.4 (-0.1/ þ0.2) ps time constant resulting from our fit. Within the experimental accuracy, this time constant agrees with the value of 0.3 ps derived from the pump-probe data. From our analysis of the steady state FT-IR spectrum we concluded that the 3145 cm-1 is actually composed of two hydrogen-bonded N-H stretching transitions. The 0.4 ps time constant may, thus, be a weighted average value for the population relaxation of these two N-H stretching modes. A comparison of the temporal characteristics of cross peaks and diagonal peaks reveals that the signal magnitude of the cross peaks is not only controlled by “instantaneous” anharmonic coupling between the hydrogen-bonded N-H stretching modes. If this were the case, the dynamics of the (3303, 3303) diagonal peak and the (3303, 3145) cross peak would be identical, as the decay of the signal strength would only be dictated by population dynamics of the 3303 cm-1 νG(NH2)b mode. The ratio of the magnitudes of the (3303, 3145) cross peak and the (3303, 3303) diagonal peak (Figure 4c) exhibits a pronounced increase with population time T, giving evidence of vibrational population transfer from the 3303 cm-1 νG(NH2)b mode to the 3145 cm-1 modes. As a result, the measured decay rate of the 3303 cm-1 mode of (1/0.24) ps-1 represents the sum of the population

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transfer rate and the relaxation rate back to the v = 0 state. The ratio plotted in Figure 4c rises on a time scale of approximately 1 ps. This time evolution is determined by the population transfer and the relaxation of the 3303 and 3145 cm-1 modes, the latter decaying with a 0.4 ps time constant. An extraction of the transfer rate would require independent information on the relaxation rate of the 3303 cm-1 mode, which is hard to acquire because of the strong spectral overlap of the different v = 0 f v = 1 and v = 1 f v = 2 transitions. As a result, we can derive only a lower limit of the transfer time of 240 fs and an upper limit of approximately 1.3 ps, which would be about 3 times the decay time of the 3145 cm-1 oscillator. Using a simple Fermi Golden rule approach to derive the corresponding vibrational coupling V, one estimates values of |V| ≈ 2.3-12 cm-1, again much smaller than the coupling of the N-H oscillators of the NH2 groups . If we assume a vibrational excitation transfer mechanism reminiscent of F€orster dipole-dipole coupling with an r-6 dependence on the dipole separation r for the population transfer rate,33 the likely candidate to which the transfer takes place is the νG(NH) mode, as this vibrational dipole is closer than that of the νC(NH2)b mode. The diagonal peaks are composed of two signal contributions having either ground or excited state populations during waiting time T, whereas the instantaneous positive contributions to the (3303, 3145) and (3145, 3303) cross peaks only involve Liouville pathways that reside in the (joint) ground state during T. Additional feeding occurs for the (3303, 3145) cross peak by population transfer from the v = 1 state of νG(NH2)b to the v = 1 state of either νG(NH) or νC(NH2)b (or both), depending on the coupling strengths, or the reverse for the (3145, 3303) peak. The slower 0.8 (-0.2/þ0.3) ps decay time of the (3303, 3145) cross peak, compared to the 0.24 ps decay of the (3303, 3303) diagonal peak could therefore reflect that the ground state recovery is slower than the excited state decay or, in case of population transfer from the 3303 cm-1 to the 3145 cm-1 band, reflect that the arrival state has a longer excited state lifetime than the departure state.

5. CONCLUSIONS We have investigated the N-H stretching vibrations of guanosine-cytidine base pairs in chloroform solution using linear and nonlinear IR spectroscopy. Comparison of the linear IR spectrum of G 3 C with those of the monomers of G or C shows that the G 3 C base pair has only IR-active transitions of N-H stretching vibrations located above 3000 cm-1. For the monomers of G and C the magnitude of the vibrational coupling between the two N-H stretching modes of the NH2 groups of 55-58 cm-1 justifies a description in terms of symmetric and asymmetric N-H stretching modes, ν(NH2)s and ν(NH2)a. In contrast, for G 3 C base pairs frequency downshifts of the N-H stretching vibrations of those NH groups forming hydrogen bonds (163 cm-1 for νG(NH2)b and 331 cm-1 for νC(NH2)b) are much larger than the vibrational coupling between the two N-H modes within the NH2 group. This fact makes a description based on a local mode basis for the N-H stretching vibrations more appropriate for the G 3 C base pair. Within this local mode representation we have analyzed the femtosecond 2D-IR photon echo spectra and transient pumpprobe data. The extensive spectral overlap between 3280 and 3400 cm-1 in the pump-probe data prevents an accurate determination of the population kinetics of in particular the νG(NH2)b mode. Ultrafast 2D-IR spectroscopy provides more insight into 5490

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The Journal of Physical Chemistry B the vibrational couplings and dynamics of the N-H stretching manifold of G 3 C base pair. Whereas the two N-H stretching modes involving motions of the free NH groups, νG(NH2)f and νC(NH2)f, show average vibrational population lifetimes of 2.4 ps, the vibrational population relaxation of the N-H stretching vibrations of the hydrogen-bonded NH groups is an order of magnitude faster. The νG(NH2)b mode has a population lifetime of ∼0.24 ps, whereas the νG(NH) and νC(NH2)b modes have an average lifetime of ∼0.4 ps. Comparison of the temporal behavior of the (ν1 [cm-1], ν3 [cm-1]) = (3303, 3303) diagonal peak with that of the (ν1 [cm-1], ν3 [cm-1]) = (3303, 3145) cross peak reveals that ultrafast vibrational excitation transfer occurs from the νG(NH2)b mode to the νG(NH) and/or νC(NH2)b modes.

’ ASSOCIATED CONTENT

bS

Supporting Information. Empirical relations connecting hydrogen bonding induced frequency shift to IR absorption intensity. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: þ49 30 63921477. Fax: þ49 30 63921409. E-mail: [email protected].

’ ACKNOWLEDGMENT The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013/ERC grant agreement no. 247051; T.E.) and the German Science Foundation (Deutsche Forschungsgemeinschaft; K.R. and F.T.). We cordially thank Dr. Nina K. Schwalb and Dr. Jason R. Dwyer for assistance in preliminary work. ’ REFERENCES (1) Saenger, W. Principles of Nucleic Acid Structure; Springer: New York, 1984. (2) Watson, J. D.; Crick, F. H. C. Nature 1953, 171, 737. (3) Crespo-Hernandez, C. E.; Cohen, B.; Kohler, B. Nature 2005, 436, 1141. (4) Middleton, C. T.; de La Harpe, K.; Su, C.; Law, Y. K.; CrespoHernandez, C. E.; Kohler, B. Annu. Rev. Phys. Chem. 2009, 60, 217. (5) Schwalb, N. K.; Temps, F. J. Am. Chem. Soc. 2007, 129, 9272. (6) Schwalb, N. K.; Temps, F. Science 2008, 322, 243. (7) Doorley, G. W.; McGovern, D. A.; George, M. W.; Towrie, M.; Parker, A. W.; Kelly, J. M.; Quinn, S. J. Angew. Chem., Int. Ed. 2009, 48, 123. (8) Szyc, y.; Yang, M.; Nibbering, E. T. J.; Elsaesser, T. Angew. Chem., Int. Ed. 2010, 49, 3598. (9) Boussicault, F.; Robert, M. Chem. Rev. 2008, 108, 2622. (10) Clegg, R. M.; Murchie, A. I. H.; Zechel, A.; Lilley, D. M. J. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 2994. (11) Nir, E.; Kleinermanns, K.; de Vries, M. S. Nature 2000, 408, 949. (12) Nir, E.; Janzen, C.; Imhof, P.; Kleinermanns, K.; de Vries, M. S. Phys. Chem. Chem. Phys. 2002, 4, 732. (13) Abo-Riziq, A.; Grace, L.; Nir, E.; Kabelac, M.; Hobza, P.; de Vries, M. S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 20. (14) Choi, M. Y.; Dong, F.; Miller, R. E. Philos. Trans. R. Soc. London A 2005, 363, 393.

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