Ultrafast Energy Exchange via Water−Phosphate Interactions in

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Ultrafast Energy Exchange via Water-Phosphate Interactions in Hydrated DNA Łukasz Szyc, Ming Yang, and Thomas Elsaesser* Max Born Institut fu¨r Nichtlineare Optik und Kurzzeitspektroskopie, Max Born Strasse 2A, D-12489 Berlin, Germany ReceiVed: February 6, 2010; ReVised Manuscript ReceiVed: April 12, 2010

The ionic phosphate groups in the DNA backbone play a key role for DNA hydration. We study ultrafast vibrational dynamics and local interactions of phosphate groups and water by femtosecond two-color pump-probe spectroscopy. The asymmetric (PO2)- stretching vibration νAS(PO2)- of artificial DNA oligomers containing 23 alternating adenine-thymine base pairs displays a lifetime of 340 fs, independent of the hydration level. For DNA at zero relative humidity, excess energy from the decay of the phosphate excitation is transferred within DNA on a 20 ps time scale. For fully hydrated DNA, the water shells around the phosphates serve as a primary heat sink accepting vibrational excess energy from DNA on a femtosecond time scale. OH stretching excitation of water molecules around fully hydrated DNA induces an ultrafast νAS(PO2)- response which includes rearrangements of the hydration shell and a reduction of the average number of phosphate-water hydrogen bonds. 1. Introduction Interaction of DNA with water plays a key role for its structural conformation, its local hydrogen bond geometries and the electrostatic coupling of charged groups in its backbone and with counterions.1-3 Studies of the structure of DNA hydration shells under equilibrium conditions have shown that both the ionic phosphate groups in the DNA backbone and particular functional groups of nucleic bases are hydrogen bonding docking sites for water molecules. Experimental work based on X-ray diffraction, neutron scattering, nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations of equilibrium DNA structures have shown that phosphate groups are main hydration sites, each being hydrated by up to 6 water molecules that directly interact with the two charged oxygen atoms.4-12 In the fully hydrated B conformer of DNA, each phosphate moiety is surrounded by its own hydration shell, interacting only weakly with other water shells (cf. Figure 1a).4-6 Under equilibrium conditions, residence times of individual water molecules at DNA hydration sites cover a very broad range from 10 ps up to nanoseconds.7-12 The interaction of DNA and water is characterized by long-range Coulomb forces and local hydrogen bonds fluctuating, however, on a substantially shorter time scale. The breaking and (re)formation of DNAwater and water-water hydrogen bonds and the reorientation of water molecules occur in the femto- to picosecond time domain.10,13 MD simulations have suggested such fluctuations within the residence time of water molecules at particular binding sites.10 Fluctuating interactions should also play a central role for the nonequilibrium energy exchange between DNA and its environment, a key step in accommodating the large amounts of excess energy released in the decay of electronic14,15 and vibrational DNA excitations. Vibrational spectroscopy addresses functional units of DNA in a selective way and, thus, has the potential to probe local geometries and interactions.16-23 The stretching vibrations of the phosphate groups have been studied in detail by infrared * Corresponding author. Phone: +49 30 63921400. Fax: +49 30 63921409. E-mail: [email protected].

spectroscopy in the 1000 to 1300 cm-1 range. For an increasing level of hydration, the asymmetric (PO2)- stretching vibration νAS(PO2)- undergoes a characteristic shift toward lower frequencies whereas the symmetric (PO2)- stretching vibration displays mainly changes of its spectral envelope.16,17,22 In principle, the lineshapes of the two bands and their changes upon hydration reflect DNA-water interactions in a timeintegrated way. Specific information on microscopic dynamics and the role of the different couplings is, however, very difficult to extract. Nonlinear vibrational spectroscopy with a femtosecond time resolution provides access to structural dynamics and ultrafast energy exchange.24,25 Femtosecond photon-echo and pumpprobe methods together with theoretical calculations and MD simulations have provided detailed insight into the microscopic dynamics of neat water (H2O) and isotopically substituted water (HOD in H2O or D2O) in the bulk and different (nano)confined environments.26-39 The fastest structural fluctuations in the sub100 fs time range are connected with high-frequency librational motions.30-32 The average lifetime of water-water hydrogen bonds is of the order of 1 ps.13 The lifetime of the OH stretching and bending vibration of H2O has a value of 200 and 170 fs, respectively.28,32,33 The excess energy released in the OH stretch and/or bend decay is transferred to librational modes, leading to a weakening of hydrogen bonds at the excited sites, followed by a spreading of excess energy on a time scale of a few picoseconds.33 The latter processes establish a macroscopically heated ground state with a larger fraction of broken hydrogen bonds. In contrast to the large body of work on ultrafast water dynamics, femtosecond studies of DNA vibrations and/or model systems mimicking base pair geometries have remained quite limited. So far, coupling schemes of modes in the fingerprint range have been addressed by 2D spectroscopy40,41 and vibrational lifetimes have been measured in pump-probe experiments.42,43 To get insight into water dynamics and solvation processes around DNA, the kinetics and spectral shift of emission from chromophores incorporated into DNA structures have been investigated.44-46 Here, we introduce femtosecond

10.1021/jp101174q  2010 American Chemical Society Published on Web 05/19/2010

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Szyc et al. in section 3.1. The response of the νAS(PO2)- oscillator after OH stretching excitation of the surrounding water molecules is addressed in section 3.2. Conclusions and a brief outlook are presented in section 4.

Figure 1. (a) Molecular structure of adenine-thymine (A-T) base pairs in Watson-Crick geometry together with the sugar and phosphate groups of the DNA backbone. The shaded areas indicate hydration sites on phosphate groups. Other hydration sites not addressed here are marked by dotted circles. Inset: Scheme of a fully hydrated phosphate group with arbitrary angular orientations of water molecules. (b) Sequence of base pairs in the double stranded DNA oligomers. (c) Steady-state infrared absorption spectrum of a thin film sample (d ) 30 µm) of the double stranded DNA oligomers on a Si3N4 substrate. Data are shown for relative humidities (r.h.) of 0% (dash-dotted line), 75% (dashed line) and 92% (solid line). Lower panel: Spectra of femtosecond pump pulses centered at 1220 and 3500 cm-1.

infrared spectroscopy for studying phosphate-water interactions of hydrated DNA. The nonequilibrium dynamics of energy exchange between DNA and water and the resulting changes of hydrogen bonded structure are mapped locally by probing phosphate vibrations on an ultrafast time scale. The asymmetric stretching vibration νAS(PO2)- of the phosphate groups serves as a sensitive local probe. We either excite the phosphate oscillator and follow its time evolution in interaction with the hydration shell or excite the hydration shell and measure the response of the phosphate oscillator. Such studies are complemented by measurements at low hydration level where single water molecules interact with the phosphate groups. We demonstrate that the water shells around the fully hydrated phosphates serve as a primary heat sink accepting vibrational excess energy from DNA on a femtosecond time scale. Upon heating, the hydrogen bond pattern of a hydration shell undergoes subpicosecond rearrangements, reducing the average number of phosphate-water hydrogen bonds. The paper is organized as follows. In section 2, we describe the preparation of the DNA thin film samples and discuss their infrared absorption spectra and the techniques applied in the femtosecond experiments. Experimental results for resonant femtosecond excitation of the νAS(PO2)- vibration are presented

2. Experimental Techniques 2.1. Preparation and Stationary Infrared Spectra of DNA Thin-Film Samples. We study artificial DNA oligomer duplexes containing 23 alternating adenine-thymine (A-T) base pairs (Figure 1a,b) in Watson-Crick geometry (Thermo Scientific, HPLC, desalted). The oligomers were cast into ∼30 µm thick films on 0.5 µm thick Si3N4 substrates and held at a controlled humidity level.47,48 To generate films of high optical quality, the sodium counterions of the oligomers were replaced by the surfactant cetylmethyl-ammonium chloride (CTMA) forming complexes with DNA, as has been described in detail in refs 47-51. From the size of the DNA/surfactant complexes, one estimates a DNA concentration in the film of 1.5 × 10-2 M. The film samples were integrated into a home-built humidity cell made from stainless steel. The sample chamber was connected to a reservoir containing various agents to control the relative humidity (r.h.) in the cell and the film sample.52 X-ray diffraction studies have shown that DNA complexed with surfactant counterions undergoes water-content-driven changes in its helix conformation very similar to DNA with standard Na+ counterions.49-51 DNA oligomers with alternating A-T pairs have been shown to exist in the D conformation3 at r.h. between 40 and 70% and in the B conformation3 at high humidity levels. X-ray diffraction from thin films demonstrates a well-defined helix structure also at 0% r.h.50 In Figure 1c, infrared spectra of a DNA/CTMA film are plotted for 0%, 75% and 92% r.h., corresponding to ≈2, ≈12 and more than 20 water molecules per base pair in the DNA structure. At 92% r.h., the DNA is fully hydrated with n ) 6 water molecules per phosphate group. With increasing hydration level, the spectra show a strong absorption increase between 3000 and 3700 cm-1 which originates from the OH stretching vibrations of the additional water molecules. Concomitantly, the libration-bend combination tone of water around 2150 cm-1 becomes stronger. Additional changes occur in the fingerprint range below 1800 cm-1, the most characteristic ones being changes of (PO2)- stretching absorption. In Figure 2a, the symmetric νS(PO2)- and asymmetric νAS(PO2)- bands are plotted for the 3 hydration levels. The observed reshaping of the νS(PO2)- envelope and the red shift of the νAS(PO2)- band are very close to the behavior of DNA with Na+ counterions. The frequency shift of the νAS(PO2)- band relative to its position at 0% r.h. is identical for CTMA and Na+ counterions (Figure 2b),17,22 demonstrating a full hydration of the phosphate groups in the DNA/CTMA film samples at 92% r.h. Gravimetric studies of the DNA/CTMA samples at 92% r.h. give a number n g 20 water molecules per base pair, in agreement with measurements for DNA with Na+ counterions. The water concentration in the film samples is c ) 0.57 M and c g 5.7 M for 0% and 92% r.h., respectively. 2.2. Femtosecond Pump-Probe Experiments. Femtosecond pump and probe pulses which are independently tunable in the range from 800 to 3700 cm-1 (wavelength range 12.5 to 2.7 µm) were generated in two parametric frequency converters driven by amplified pulses from a Ti:sapphire laser (wavelength 800 nm, repetition rate 1 kHz).53 The pump spectra for exciting the νAS(PO2)- mode around 1230 cm-1 and the OH stretching mode of water around 3500 cm-1 are plotted in the bottom panel of Figure 1c. The energy of the pump pulses at the sample was

Ultrafast Energy Exchange in Hydrated DNA

Figure 2. (a) Infrared absorption bands of the phosphate stretching vibrations for relative humidities of 0% (dash-dotted line), 75% (dashed line), and 92% (solid line). The symmetric νS(PO2)- and the asymmetric νAS(PO2)- stretching band is located around 1090 and 1250 cm-1, respectively. (b) Spectral shift of the νAS(PO2)- band as a function of relative humidity for the DNA/CTMA samples studied here (squares) and DNA/Na+ samples (ref 17, circles, ref 22, triangles).

1.5 µJ for both excitation conditions. The fraction of phosphate oscillators excited by the pump pulses centered at 1220 cm-1 was of the order of 5%. The fraction of OH stretching oscillators excited by the pulses at 3500 cm-1 was less than 3%. The temporal width of the cross-correlation of pump and probe pulses was 200 fs (fwhm) for pump and probe pulses both around 1230 cm-1, and 150 fs (fwhm) with pump pulses at 3500 cm-1 and probe pulses around 1230 cm-1. Both pump and probe pulses were linearly polarized, and the change of absorbance of the DNA films ∆A ) -log(T/T0) (T, T0: sample transmission with and without excitation) was measured with parallel and perpendicular polarization of pump and probe. After interaction with the sample, the probe pulses were spectrally dispersed and detected with a 16-element HgCdTe detector array (resolution 2 cm-1). Measurements with uncoated Si3N4 substrates show that nonlinear signals from the substrate and the rear window of the cell are negligible. 3. Results and Discussion 3.1. Ultrafast Response of the Resonantly Excited νAS(PO2)- Mode. The femtosecond time evolution of νAS(PO2)excitations was studied at hydration levels of 0% and 92% r.h. Figure 3 summarizes results for 0% r.h. where typically a single water molecule interacts with a phosphate group. After excitation of the anharmonic νAS(PO2)- oscillator to its V ) 1 state (inset of Figure 3c), the transient infrared spectra (Figure 3a, symbols) exhibit an enhanced absorption on the 1-2 transition which is red-shifted compared to the absorption decrease on the 0-1 transition. The amplitudes of both contributions decrease within the first picosecond after excitation without changing their shapes, i.e., there is essentially no spectral diffusion of the νAS(PO2)- excitations during this period. Time traces (Figure 3b,c, symbols) recorded at fixed frequency positions (arrows in Figure 3a) show an initial fast decay, followed by a slow relaxation on a 20 ps time scale. The slow component is also present in the relaxation of other vibrations such as the NH stretching modes of the A-T base pairs (bottom

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Figure 3. (a) Transient spectra of νAS(PO2)- absorption at 0% r.h. (symbols) after femtosecond excitation centered at Eex ) 1220 cm-1. The absorbance change ∆A ) -log(T/T0) is plotted as a function of probe frequency for different delay times after excitation (T, T0: sample transmission with and without excitation, parallel linear polarizations of pump and probe). The spectra display a decrease of absorption (∆A < 0) on the fundamental 0-1 transition and an increase of absorption (∆A > 0) on the red-shifted 1-2 transition. Solid line: linear νAS(PO2)absorption at 0% r.h. Arrows: Frequency positions at which the transients in panels b and c were recorded. (b) Change of the νAS(PO2)absorption (symbols) at a fixed probe frequency Epr ) 1230 cm-1 on the 1-2 transition as a function of the pump-probe delay. Data are presented for a short time scale up to 2 ps and a longer time scale up to 20 ps. Solid line: numerical fit of the data with decay times of 340 fs and 5.5 ps. (c) Time dependent change of the νAS(PO2)- absorption (upper transient, symbols) at Epr ) 1268 cm-1 (0-1 transition). Solid line: numerical fit with the same time constants as in panel b. Lower transient: time-dependent change of NH stretching absorption of the DNA oligomers (symbols). For long delays, both the measured signal and a trace with the signal amplitude enlarged by a factor of 6 are shown. Solid lines: Numerical fit with a slow 5.5 ps decay. Inset: Schematic of the oscillator with pump (black arrow) and probe (gray arrows) transitions.

transient in Figure 3c, symbols). The transients in Figure 3b,c were analyzed by numerically fitting exponential kinetics to the data points. The calculated time evolution was convoluted with the cross correlation of pump and probe pulses (solid lines in Figure 3b,c). The femtosecond decays of the 1-2 absorption and the fast recovery of the 0-1 absorption are well reproduced by a single exponential component with a time constant of 340 fs. For the slower picosecond decay, one estimates a time constant of 5.5 ps. At 92% r.h., the νAS(PO2)- absorption of the fully hydrated phosphate groups occurs at lower frequency with modified spectral envelopes of both the linear and transient vibrational spectra (Figure 4a). During their femtosecond decay, the absorption changes undergo minor changes of their spectral envelopes. The V ) 1 lifetime derived from time-resolved data (Figures 4b,c) is again 340 fs. In contrast to the response at 0% r.h., however, the slow relaxation component is completely

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Figure 4. (a) Transient spectra of νAS(PO2)- absorption at 92% r.h. (symbols) after femtosecond excitation centered at Eex ) 1230 cm-1 (parallel polarization of pump and probe). Solid line: linear νAS(PO2)absorption at 92% r.h. (b, c) Time dependent changes of the νAS(PO2)absorption (symbols) at fixed probe frequencies Epr ) 1200 cm-1 (1-2 transition) and Epr ) 1255 cm-1 (0-1 transition). The measured transients follow a single-exponential decay with a time constant of 340 fs (solid lines: numerical fits). Note that the long-lived component found at low humidity is completely absent.

absent. Measurements with parallel and perpendicular polarizations of pump and probe pulses give identical kinetics. The pump-probe anisotropies derived from such data have a constant value of 0.4 at both 0% and 92% r.h. The spectral shift between the 0-1 absorption decrease and the enhanced 1-2 absorption reflects the (diagonal) anharmonicity ∆ν ) ν(1-2) - ν(0-1) of the νAS(PO2)- oscillator. We performed a line shape analysis of the transient spectra to extract anharmonicities for the two hydration levels. As the lineshapes of the linear absorption spectra are not understood in detail, two limiting cases are considered: (i) For a homogeneously broadened phosphate absorption band, the bleaching spectrum of the 0-1 transition is given by the (inverted) linear absorption band (solid lines in Figure 5b,c). Adjusting the amplitude of this band to and subtracting it from the transient spectra (symbols in Figure 5b,c), one derives the 1-2 absorption spectra of the oscillator (solid lines). The shift of the maxima of the respective 1-2 band relative to the 0-1 band is taken as a measure of the anharmonicity ∆ν. (ii) For an inhomogeneously broadened phosphate absorption band, the bleaching spectrum of the 0-1 transition is given by the convolution of the (inverted) linear absorption band with the pump spectrum shown in Figure 5a (dash-dotted lines in Figure 5b,c). Following the same subtraction algorithm, one derives the corresponding 1-2 spectra (dash-dotted lines) and the anharmonicities. Both models give very similar values of ∆ν ) -12 ( 2 cm-1 at 0% r.h. and ∆ν ) -18 ( 2 cm-1 at 92% r.h. which represent anharmonicities averaged over the ensemble of oscillators. A more sophisticated treatment requires a detailed analysis of the line shape of the 0-1 and 1-2 bands by, e.g., two-dimensional spectroscopy.

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Figure 5. (a) Linear νAS(PO2)- absorption for 0% r.h. and 92% r.h. (solid lines). Dash-dotted line: Spectrum of the femtosecond pump pulse. (b) Transient spectrum of the νAS(PO2)- vibration at a delay time of 350 fs (symbols, 0% r.h.). Solid lines: Inverted linear absorption spectrum (0-1 transition, lower part) and calculated 1-2 absorption (upper part). Dash-dotted lines: Inverted linear absorption spectrum convoluted with the pump spectrum (0-1 transition, lower part) and calculated 1-2 absorption (upper part). (c) Same for a sample at 92% r.h.

We now discuss the mechanisms which underlie the ultrafast response of the νAS(PO2)- oscillators after resonant excitation on the 0-1 transition. The red shift of both the 0-1 and the 1-2 transition in the transient spectra and the increase of the diagonal anharmonicity from 12 to 18 cm-1 are in line with a more complete hydration of the phosphate groups at 92% r.h. Here, both the increased number of hydrogen bonds between the phosphate groups and water molecules16,17 and electronic structure effects54 are important, such as the relocation of electronic charge in the (PO2)- groups polarized by interaction with the water dipoles. The constant pump-probe anisotropy points to a minor role of resonant energy transfer between different νAS(PO2)- oscillators. Both the enhanced 0-1 absorption and the bleaching on the 0-1 transition display a fast decay with a time constant of 340 fs. This decay is due to the depopulation of the V ) 1 state and, thus, reflects its lifetime that is independent of the hydration level. The latter fact suggests that the V ) 1 population decay involves mainly couplings to DNA rather than water vibrations. The symmetric phosphate stretching vibration νS(PO2)- as well as combination tones involving the diester phosphate stretching and phosphate bending vibrations21 may serve as initial energy acceptors, an issue to be studied in future experiments. The νAS(PO2)- relaxation establishes a hot ground state of the phosphate group with the νAS(PO2)- mode being in its V ) 0 state and the released excess energy in other, mainly lowfrequency modes. Such modes are cooled by transferring energy to other DNA vibrations and to the water environment, in this way delocalizing the excess energy into a heated macroscopic volume. Population changes of low-frequency modes coupling to the νAS(PO2)- mode induce a reshaping of the νAS(PO2)-

Ultrafast Energy Exchange in Hydrated DNA absorption band, in this way mapping energy dissipation into the νAS(PO2)- kinetics.55 At 0% r.h., this mechanism gives rise to the longer-lived residual absorption changes that decay completely within 20 ps. A similar behavior is observed for NH stretching vibrations of the A-T base pairs in the DNA oligomers at 0% r.h. (cf. Figure 3c).47,48 The higher dynamic range of the latter measurements allows for a quantitative analysis of the slow decay giving a time constant of 5.5 ps. At 0% r.h., the single water molecule per phosphate group offers very few energy accepting modes only. Thus, DNA with its large manifold of low-frequency vibrations23 represents the main heat sink and the 5.5 ps decay reflects the time scale of energy transport within and along DNA. It is interesting to note that a very similar time scale has been found recently for the flow of vibrational excess energy along peptide structures56 and in longchain hydrocarbons.57 This scenario changes drastically for fully hydrated DNA at 92% r.h. (Figure 4). There are no slower kinetics in the νAS(PO2)- response. We conclude that the excess energy released on the decay of νAS(PO2)- is mainly transferred to the surrounding water shell and redistributed in the aqueous environment. The initial energy transfer from the phosphate group to water occurs on a time scale similar to or even faster than the 340 fs decay of νAS(PO2)-, in this way suppressing energy accumulation in the phosphate group. The absence of spectral diffusion points to an unchanged pattern of phosphate-water hydrogen bonds during energy transfer and, thus, a strong water-phosphate coupling. After the transfer to the phosphate hydration shell, the excess energy is rapidly delocalized in the aqueous environment. 3.2. Ultrafast Response of the νAS(PO2)- Mode to OH Stretching Excitations of Water Molecules. The ultrafast response of the νAS(PO2)- oscillator to an excitation of the hydration shell was studied at 92% r.h. with pump pulses centered at 3500 cm-1 (Figure 1c). The pump pulses mainly excite the OH stretching vibration of water andsto very minor extentsthe NH stretching modes of DNA. The transient spectra (Figure 6b,c) are dominated by the νAS(PO2)- response rather than by changes of the weak librational absorption of water (cf. Figure 6a).33 While the spectrum for a -100 fs delay displays a transient red shift, a strong bleaching at low frequencies and an enhancement of absorption at high frequencies develop on a 500 fs time scale and persist up to much longer time delays. The time evolution of such changes at two fixed probe frequencies is shown in Figure 7a. The pronounced signals at negative delay times are due to perturbed free induction decays,58 demonstrating the coupling of the OH stretching and νAS(PO2)modes. At positive delay times, the absorption changes reach their final long-lived values on a time scale of approximately 1 ps. In Figure 7b, time-resolved changes of the OH stretching absorption of the aqueous environment of DNA are presented. It is important to note that both transients exhibit a long-lived absorption change that develops on a very similar time scale as the changes of νAS(PO2)- absorption in Figure 7a. The longlived enhanced absorption observed at Epr ) 3550 cm-1 gives evidence of a vibrationally hot ground state of the aqueous environment (Figure 7b) that arises from the decay of the OH stretching excitations and the redistribution of the vibrational excess energy.48 In the experiments with OH stretching excitation, the νAS(PO2)- oscillator remains in its V ) 0 ground state. The measured changes of νAS(PO2)- absorption originate either from anharmonic vibrational couplings to other modes or from changes in the phosphate-water interaction induced by OH

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Figure 6. (a) Linear νAS(PO2)- absorption for 92% r.h. (solid line). Dash-dotted line: Linear absorption of bulk liquid H2O scaled for the water concentration at 92% r.h. (b, c) Transient spectra (symbols) of the νAS(PO2)- mode after excitation (Eex ) 3500 cm-1) of the OH stretching mode of water molecules in the sample at 92% r.h. Spectra are shown for 5 different pump-probe delays (parallel linear polarizations of pump and probe). The arrows in panel b mark the spectral positions at which the transients in Figure 7a have been recorded. Solid line in panel c: Difference δA ) A(0%) - A(92%) of the linear absorption spectra for 0% and 92% r.h. scaled for the fraction of excited water molecules.

stretching excitation. The ultrafast response of the νAS(PO2)mode to an OH stretching excitation of the hydration shell demonstrates the strong water-phosphate coupling. The decay of the OH stretching excitation on a subpicosecond time scale (Figure 7b) transfers the excess energy into low-frequency modes of water, forming a hot water ground state within 1 to 2 ps,48 very similar to the behavior of bulk water.33 During and after this process, the interaction of the excited hydration shell with the unexcited νAS(PO2)- oscillator modifies its V ) 0 to 1 absorption. The νAS(PO2)- transients in Figure 7a exhibit a time evolution very similar to the OH stretching vibration (Figure 7b), showing that the phosphate response is dictated by the behavior of the hydration shell. The transient spectra of Figure 6 reflect changes in the local interaction pattern of phosphate and water. Heating the hydration shell introduces defects in the hydrogen bond pattern, i.e., the fraction of geometrically distorted and/or broken hydrogen bonds increases.33 This mechanism also affects phosphate-water hydrogen bonds. With a decreasing number of phosphate-water hydrogen bonds, the νAS(PO2)- mode undergoes a transient shift to higher frequencies, the behavior found in the transient spectra taken at positive delay times. Under steady-state conditions, a similar shift occurs when the hydration level of the DNA films is reduced (Figure 2). The solid line in Figure 6c represents the difference δA ) A(0%r.h.) - A(92%r.h.) of the steady-state νAS(PO2)- spectra for 0% r.h and 92% r.h. which displays the same features, a decrease of absorption at low and an increase at high frequencies. This similarity of lineshapes in the transient and steady state difference spectra strongly suggests that a substantial number of water-phosphate hydrogen bonds are broken in the hot water ground state. In the hot ground state,

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Szyc et al. Acknowledgment. We would like to thank Erik T. J. Nibbering for valuable discussions. This work has been supported in part by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 450). References and Notes

Figure 7. (a) Time-resolved change of νAS(PO2)- absorption at fixed frequency positions Epr of the probe after excitation of the OH stretching band of water at Eex ) 3500 cm-1 (92% r.h., parallel linear polarization of pump and probe). (b) Time resolved change of OH stretching absorption of water in the DNA sample at 92% r.h. for two different excitation and probe positions (symbols, ref 48). The amplitude of the transient recorded at Epr ) 3500 cm-1 has been reduced by a factor of 3 to facilitate a comparison of the different time traces. The long-lived signals reflect the hot ground state of the aqueous environment.

the fraction of broken hydrogen bonds is enhanced at constant water concentration whereas a change of the hydration level also changes the number of water molecules interacting with the phosphate groups. As a result, the transient and the steady state difference spectra are not fully equivalent although showing the same type of spectral reshaping. 4. Conclusions We have studied the interaction of phosphate groups in the DNA backbone with their hydration shells to establish the ultrafast time scales of nonequilibrium energy dissipation and characterize the concomitant changes of the water-phosphate hydrogen bond pattern. While the transfer of excess energy from DNA into the hydration shell occurs on a femtosecond time scale, energy delocalization within DNA is a slower picosecond event. The phosphate hydration shells represent a highly efficient heat sink with an intrinsic dynamic behavior very close to bulk water. In a heated aqueous environment, the number of hydrogen bonds between the phosphate groups and their water shells is reduced. Future work will include the measurement of 2D vibrational spectra, in particular two-color 2D spectra, to address couplings between the relevant DNA modes and the phosphate-water interaction in a more quantitative way. In the present experiments, the total amount of energy deposited into DNA and/or exchanged with the hydration shell is limited to individual vibrational quanta. Much higher excess energies are released in the relaxation of electronically excited states of base pairs by internal conversion to the electronic ground state. Probing phosphate-water interactions during and after electronic relaxation will elucidate the pathways and time scales of energy flow and can map the resulting jumps of vibrational temperature. The analysis of such processes will complement and improve our understanding of the high photostability of DNA.

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