Vibrational dynamics of azide-derivatized amino acids studied by nonlinear infrared spectroscopy.

Vibrational dynamics of azide-derivatized amino acids studied by nonlinear infrared spectroscopy Masaki Okuda, Kaoru Ohta, and Keisuke Tominaga Citation: The Journal of Chemical Physics 142, 212418 (2015); doi: 10.1063/1.4917032 View online: http://dx.doi.org/10.1063/1.4917032 View Table of Contents: http://scitation.aip.org/content/aip/journal/jcp/142/21?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Distinguishing gramicidin D conformers through two-dimensional infrared spectroscopy of vibrational excitons J. Chem. Phys. 142, 212424 (2015); 10.1063/1.4917321 Interactions of aqueous amino acids and proteins with the (110) surface of ZnS in molecular dynamics simulations J. Chem. Phys. 140, 095101 (2014); 10.1063/1.4866763 Vibrational self-trapping in beta-sheet structures observed with femtosecond nonlinear infrared spectroscopy J. Chem. Phys. 131, 124503 (2009); 10.1063/1.3229891 Phosphorylation effect on the GSSS peptide conformation in water: Infrared, vibrational circular dichroism, and circular dichroism experiments and comparisons with molecular dynamics simulations J. Chem. Phys. 126, 235102 (2007); 10.1063/1.2738472 2DIR spectroscopic studies on cholic acid AIP Conf. Proc. 503, 303 (2000); 10.1063/1.1302883

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THE JOURNAL OF CHEMICAL PHYSICS 142, 212418 (2015)

Vibrational dynamics of azide-derivatized amino acids studied by nonlinear infrared spectroscopy Masaki Okuda,1 Kaoru Ohta,2 and Keisuke Tominaga1,2,a)

1 2

Graduate School of Science, Kobe University, Rokkodai-cho 1-1, Nada. Kobe 657-8501, Japan Moleuclar Photoscience Research Center, Kobe University, Rokkodai-cho 1-1, Nada. Kobe 657-8501, Japan

(Received 4 January 2015; accepted 25 March 2015; published online 13 April 2015) Recently, biomolecules which are labeled by azide or thiocyanate groups in solutions and proteins have been studied to examine microscopic environment around a solute by nonlinear infrared (IR) spectroscopy. In this study, we have performed two-dimensional infrared (2D-IR) spectroscopy to investigate the vibrational frequency fluctuations of two different azide-derivatized amino acids, Ala (N3-Ala) and Pro (N3-Pro), and N3− in water. From the 2D-IR experiments, it was found that the frequency-frequency time correlation function (FFTCF) of solute can be modeled by a delta function plus an exponential function and constant. FFTCF for each probe molecule has a decay component of about 1 ps, and this result suggests that the stretching mode of the covalently bonded azide group is sensitive to the fluctuations of hydrogen bond network system, as found in previous studies of N3− in water. In contrast to FFTCF of N3−, FFTCF of the azide-derivatized amino acids contains static component. This static component may reflect dynamics of water affected by the solutes or the structural fluctuations of the solute itself. We also performed the IR pump-probe measurements for the probe molecules in water in order to investigate vibrational energy relaxation (VER) and reorientational relaxation. It was revealed that the charge fluctuations in the azide group are significant for the VER of this mode in water, reflecting that the VER rate of N3− is faster than those of the azide-derivatized amino acids. While the behaviors of the anisotropy decay of N3-Ala and N3− are similar to each other, the anisotropy decay of N3-Pro contains much slower decaying component. By considering the structural difference around the vibrational probe between N3-Ala and N3-Pro, it is suggested that the structural freedom of the probe molecules can affect the reorientational processes. C 2015 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4917032] I. INTRODUCTION

In aqueous solutions, water molecules form three-dimensional hydrogen-bonding network which evolves continuously with time. These fluctuations induce a change of solute-solvent interactions and solvation structures.1 Because the solutesolvent interactions play important roles in various chemical reactions and relaxation processes in the condensed phase,2 it is crucial to understand how hydrogen bonding network structure around solute molecules changes in aqueous solution. It is known that vibrational transition of a solute molecule is a sensitive probe to explore the solute-solvent interaction and structural fluctuation. For these two decades, there have been considerable efforts to study vibrational dynamics of solute in water by using ultrafast spectroscopy in the infrared (IR) region.3–5 In these studies, vibrational dynamics such as frequency fluctuations, vibrational energy relaxations, and orientational relaxations have been rigorously investigated. The vibrational frequencies at each instant reflect the solvation structure around a solute molecule, which fluctuate as a function of time. This vibrational frequency fluctuation is expressed as the deviation from the mean frequency at time T, ∆ω(T) = ω(T) − ⟨ω⟩ , a)Email: [email protected]

0021-9606/2015/142(21)/212418/10/$30.00

(1)

where ω(T) is the time-dependent vibrational frequency at time T and ⟨ω⟩ is its average value. An experimental observable of the vibrational frequency fluctuations is its time-correlation function (TCF), C(T) = ⟨∆ω(T)∆ω(0)⟩ .

(2)

This function reflects the amplitude of the solute-solvent interaction and the time scale of the structural changes around the solute molecule. By using nonlinear IR spectroscopy, such as IR photon echo experiment and two-dimensional (2D) IR spectroscopy, we can obtain information on TCF of vibrational frequency fluctuations. In previous studies, the vibrational dynamics of small ions, such as N3− and SCN−, in water were studied by nonlinear IR spectroscopy such as IR pump-probe and photon echo methods. These are good vibrational probes because the N3 anti-symmetric stretching mode of these molecules has large IR activities and the frequencies of the stretching mode are in the range from 2000 to 2100 cm−1, which is called as a “transparent window” in aqueous solutions. In the early 1990s, Hochstrasser and co-workers studied the vibrational population relaxation and orientational relaxation of these ions.6–8 Hamm et al. have investigated the vibrational frequency fluctuations of the N3 anti-symmetric stretching mode of N3− in D2O by three-pulse photon echo experiments and found that the correlation function decays with time constants of 80 fs

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and 1.3 ps.9 We have reported the temperature dependence of vibrational dynamics of N3− in water and the vibrational dynamics of OCN− and SCN− in methanol.10–13 Recently, artificial amino acids which are labeled by azide or thiocyanate groups have been shown to be excellent vibrational probes to study the dynamics in water and biological environments. Bloem et al. incorporated an azide probe into peptide and studied the spectral diffusion process of the N3 anti-symmetric stretching mode in protein.14 Thielges et al. observed the vibrational dynamics of azidophenylalanine genetically incorporated into myoglobin at position at Phe43.15 Cho and co-workers reported vibrational energy relaxation and internal rotational mode of the proline derivatives which have azide or thiocyanate probes in D2O and CHCl3.16–18 The Bredenbeck group monitored vibrational energy transfer process between two non-native amino acids, β-(1-azulenyl)-alanine and -azidohomoalanine, in a model peptide.19 There are various kinds of unnatural amino acids bearing vibrational probes which can be incorporated into peptides and proteins by a peptide synthesis or genetic procedure.20–22 Their spectroscopic properties are affected by the molecular structures of their residues. Therefore, it is expected that the vibrational dynamics of azide labels depends on the properties of the side chain of amino acid such as structural flexibility, hydrophilicity, and hydrophobicity. However, there are still a few studies on the vibrational dynamics of the artificial amino acids which contain an azide group in water. In this study, we chose two unnatural amino acids, Boc3-azide-Ala-OH (dicyclohexylammonium) salt (abbreviated as N3-Ala) and N-Boc-cis-4-azide--proline (dicyclohexylammonium) salt (abbreviated as N3-Pro) as shown in Fig. 1. We examined the vibrational dynamics of the N3 anti-symmetric stretching of these molecules in water by nonlinear IR spectroscopy. For comparison, we also investigated vibrational dynamics of azide ion (N3−) in water. In this study, we performed the 2D-IR measurements to determine the TCFs of vibrational frequency fluctuations of N3-Ala, N3-Pro, and N3− in water. Moreover, we performed the polarization-controlled IR pumpprobe measurements in order to observe vibrational energy relaxation (VER) and anisotropy decay of them in water. The motivation to choose these molecules as a probe is following; so far, we chose ionic probes for measurements of the frequency fluctuations. Although both N3-Ala and N3-Pro have an ionic part (—COO−) in their structures, a vibrational probe part (—N3) is electronically neutral. Therefore, dependence of the frequency fluctuations on the electrical properties of the probe may be investigated. Furthermore, because the structures around the azide group of N3-Ala and N3-Pro are different, we

FIG. 1. The molecular structures of (a) Boc-3-azide-Ala-OH (dicyclohexylammonium) salt and (b) N -Boc-cis-4-azide-L-proline (dicyclohexylammonium) salt. Azide groups are highlighted in red circles.

J. Chem. Phys. 142, 212418 (2015)

may expect to observe different vibrational dynamics such as orientational relaxation and vibrational energy relaxation.

II. MATERIALS AND METHODS

The details of the experimental setup for the IR pumpprobe experiments have been described elsewhere.23 Briefly, a mid-IR pulse was generated by difference frequency generation on a AgGaS2 crystal between the two near-IR outputs, signal and idler pulses of an optical parametric amplifier. A mid-IR pulse has a pulse width of about 100 fs with a repetition rate of 1 kHz, a bandwidth of about 100 cm−1, and pulse energy of about ∼3 µJ. Peak frequency of the IR pulse was tuned to around 2150 cm−1 for N3-Ala and N3-Pro, and 2000 cm−1 for N3 − . For the IR pump-probe measurements, the mid-IR pulse was split into pump and probe pulses with a ZnSe wedged window. The time delay between the pump and probe pulses was controlled by motorized delay stages. The pump and probe pulses were focused on the sample with a parabolic mirror. The polarizations of the probe pulses were tilted by 45◦ with respect to the pump pulses. Pump-probe signals parallel and perpendicular to the pump pulses were detected independently by liquid N2-cooled 64-channel mercury cadmium telluride array (MCT) detectors after passing through a monochromator. The properties of mid-IR pulses for the 2D-IR measurement were the same as those for the IR pump-probe experiments. We adopted the so-called collinear pump-probe geometry for measuring 2D-IR spectra. The mid-IR pulses were split into three components: the two are for pump pulses (k1 and k2) and the other is for probe pulse (k3), where ki is the wavevector of each IR pulse and was focused on the sample. The delay time between first and second pump pulses is referred as coherence time τ, and that between second pump pulse and probe pulse is referred as population time T (Fig. 2). The phase matching condition of third-order nonlinear optical effects is expressed as follows: ksig = −k1 + k2 + k3,

(3)

where ksig is the wavevector of the vibrational echo signal. In the pump-probe geometry, because the two pump pulses are collinear (k1 = k2), the wavevector of the vibrational echo signals ksig was in the same direction as that of the probe pulse, ksig = k3.24–26 Therefore, the vibrational echo signals were automatically overlapped with the probe pulse, and the probe pulse worked as a local oscillator, which interferes with the vibrational echo signals for heterodyne detection. The echo signal and local oscillator were passed through a

FIG. 2. Pulse geometry around the sample for 2D-IR measurements and the definitions of coherence time τ and population time T .


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monochromator and detected by liquid N2-cooled 64ch-MCT detectors in order to measure slices of the 2D-IR spectra along the ω3 frequency axis (vertical axis). For a fixed T, we scanned τ to observe interferograms at each ω3 frequency, and these interferograms were Fourier-transformed to obtain the 2D-IR spectra along the ω1 frequency axis (horizontal axis). To obtain a series of 2D-IR spectra, we repeated 2D-IR measurements over a range of T. N3-Ala, N3-Pro, and tetrabutylammonium azide were purchased from Sigma-Aldrich and used without further purification. As a solvent, we used distilled H2O. The sample was contained in a cell with an optical path length of 25 µm and CaF2 windows. The samples were passed through a 0.20 µm filter, and the concentrations of the samples used for the IR pump-probe and 2D-IR experiments were around 50 mM for N3− and 250 mM for N3-Ala and N3-Pro. In order to dissolve N3-Ala and N3-Pro into water, we added a small amount of NaOH to the solutions and controlled the pH of the solutions at around 9.0.

III. RESULTS AND DISCUSSIONS A. Steady-state IR absorption spectra

Figure 3 displays the IR absorption spectra of the N3 antisymmetric stretching mode of N3-Ala, N3-Pro, and N3− in H2O. The peak wavenumbers of N3-Ala and N3-Pro are 2116.3 cm−1 and 2115.8 cm−1, respectively, and that of N3− is 2046.5 cm−1. To assign the observed bands, we performed the optimizations of molecular structures and normal mode analyses for N3Ala, N3-Pro, and N3− in gas phase using density functional theory (DFT) with Gaussian 09 at B3LYP/6-311++G(3df,2pd) level.27 The optimized structures of N3-Ala, N3-Pro, and N3− are shown in Fig. 4. We found that these molecules have vibrational modes in 2050-2250 cm−1 region which approximately correspond to the N3 anti-symmetric stretching mode. We summarize DFT results of normal mode analyses and Mulliken charges on nitrogen atoms in the N3 group in Table I. As shown in Table I, in N3-Ala and N3-Pro, the charges are not equally distributed on the covalently bonded and terminal nitrogen atoms due to the breaking of the structural symmetry. Therefore, the potential energy surfaces of the N3 antisymmetric stretching mode are different between the covalently bonded N3 group and free N3− (see force constants in Table I). This causes the frequency difference of the N3 antisymmetric stretching modes. For the line shape of IR absorption spectra, the IR band of N3− can be fitted by a single Lorentz function. On the other hand, the IR bands of N3-Ala and N3-Pro can be fitted by a sum of Gaussian and/or Lorentzian functions (three Gaussian functions for N3-Ala, a single Lorentzian function plus a single Gaussian function for N3-Pro). Comparisons between the experimental and fitted results are shown in Fig. 3. The parameters of fits are given in Table II. For N3-Ala, although there is a small band at 2047.8 cm−1 which has not been assigned yet, there is no signal related to the vibrational coupling between this mode and the N3 anti-symmetric stretching mode in 2D-IR spectra of N3-Ala (see Fig. 7(a)). Therefore, we do not discuss this vibrational mode any more.

FIG. 3. IR absorption spectra of the N3 anti-symmetric stretching mode of (a) N3-Ala, (b) N3-Pro, and (c) N3− in H2O. Red line indicates the experimental data. Blue lines represent the fitted result of the IR band, and green lines are each component of the involved transitions.

The multiple components observed in the IR spectrum may arise from either a Fermi resonance or multiple conformers existed in solution. Such an asymmetric band shape of the N3 anti-symmetric stretching mode of the azide group was also observed in the previous studies.28–32 For example, Bazewicz et al. studied the vibrational spectra of 4-azido--phenylalanine and 4-azidomethyl--phenylalanine in water, and reported that these solutes showed asymmetric band shape of IR spectra.28 They interpreted that this complexity of the IR spectrum is likely due to an accidental Fermi resonance. Nydegger et al. examined the vibrational dynamics of 3-azidopyridine in CH2Cl2 with 2D-IR spectroscopy.32 They found that the 2D-IR spectra of 3-azidopyridine had cross-peaks at early population time which resulted from the vibrational coupling between the N3 anti-symmetric stretching and a combination band or overtone. However, in this study, we did not observe the noticeable amplitudes in the off-diagonal region of the 2D-IR spectra of N3-Ala and N3Pro at T = 0.2 ps (see Figs. 7(a) and 7(b)). Therefore, the multiple components in the IR band of N3-Ala and N3-Pro are not likely to result from the Fermi resonance.33 Similar


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FIG. 4. Molecular structures of (a) N3Ala, (b) N3-Pro, and (c) N3− in the gas phase optimized by DFT calculations at B3LYP/6-311++G(3df,2pd) level. Red and green lines indicate the rotational mode around the CN bond axis and the CC bond axis, respectively (see Sec. III C).

TABLE I. The parameters of normalized IR absorption spectra of the N3 anti-symmetric stretching mode of N3-Ala, N3-Pro, and N3− in H2O and summary of DFT calculations for N3-Ala, N3-Pro, and N3− in the gas phase. Linear IR absorptiona Solute Ala Pro N 3−

Normal mode analysisb

Mulliken chargec

ν max/cm−1

∆ν/cm−1

ν/cm−1

IR intensity/km mol−1

µ/a mu

k /mDy ne Å−1

q N(1)/e

q N(2)/e

q N(3)/e

2116.3 2115.8 2046.5

31.3 27.2 25.6

2219.21 2220.42 2079.94

621.91 892.99 1274.91

13.62 13.68 14.00

39.52 39.75 35.69

−0.80 −0.78 −0.76

0.89 1.06 0.52

−0.63 −0.72 −0.72

aν

max: wavenumber at band peak, ∆ν : full width at the half maximum. bν : calculated wavenumber of the N anti-symmetric stretching mode, µ : reduced mass, k : force constant. 3 cq N(i): partial charge on i th nitrogen atom in N3 group. N(1); covalently bonded nitrogen atom, N(2); middle

nitrogen atom, N(3); terminal nitrogen atom.

TABLE II. Obtained fitting parameters of normalized IR absorption spectra of the N3 anti-symmetric stretching mode of N3-Ala, N3-Pro, and N−3 in H2O. Solute Ala Pro N3−

A1 0.1 ± 0.1 0.2 ± 0.1 1.0 ± 0.1

ν max,1/cm−1

∆ν 1/cm−1

A2

ν max,2/cm−1

∆ν 2/cm−1

A3

ν max,3/cm−1

∆ν 3/cm−1

2047.8 ± 0.1 2115.8 ± 0.1 2046.5 ± 0.1

20.2 ± 0.7 22.2 ± 0.1 25.6 ± 0.1

0.8 ± 0.1 0.8 ± 0.1 ...

2114.7 ± 0.1 2125.2 ± 0.1 ...

29.7 ± 0.1 47.8 ± 0.1 ...

0.2 ± 0.1 ... ...

2127.2 ± 0.1 ... ...

79.3 ± 0.6 ... ...

A i : Intensity at band peak of i th component, ν max, i : wavenumber at band peak of i th component, ∆ν i : full width at the half maximum of i th component.

to this study, Thielges et al. observed this type of band in the IR spectrum of azide containing myoglobin. They also did not observe cross-peak in the 2D-IR spectra.15 Thus, the asymmetric IR band shapes of N3-Ala and N3-Pro are not due to a Fermi resonance but due to the existence of different environments. Tucker et al. suggested that an asymmetric IR band of 2′-azido-2′-deoxyuridine (N3dU) in THF may be the result from existence of two different conformers of the molecule.34 Lee et al. showed that the IR spectrum of 4-azideproline derivative, Ac-(4S)-Azp-NHMe (SA), in CHCl3 has two peaks which correspond to two different conformers in the N3 antisymmetric stretching region.16,17 An asymmetric band was also observed in the IR spectrum of azidealanine dipeptide in THF, as a result of two distinct conformers of the peptide.35 Therefore, we consider that various kinds of conformers of N3Ala and N3-Pro are present in water, and these conformers yield different environments around the azide group, which cause an inhomogeneous distribution of the vibrational transition frequencies. B. Vibrational energy relaxation

After excitation by the IR pump pulse, we measured the temporal signals at the parallel and perpendicular polarization of the pump and probe pulses. The isotropic and anisotropic pump-probe signals are, respectively, given by the following equation:

∆A//(T) + 2∆A⊥(T) , 3 ∆A//(T) − ∆A⊥(T) , r(T) = ∆A//(T) + 2∆A⊥(T)

N(T) =

(4) (5)

where ∆A//(T) and ∆A⊥(T) are the absorbance change of the parallel and perpendicular polarization conditions, respectively. The normalized frequency-resolved pump-probe spectra of N3-Ala and N3-Pro at 0.4 ps and N3− at 0.5 ps are shown in Fig. 5(a). The spectra can be fitted by two Lorentzian functions for N3-Pro and N3−. On the other hand, four Lorentzian functions are needed for N3-Ala since there is a small band in the lower-frequency region. From these results, the anharmonicity of the N3 anti-symmetric stretching mode of N3− is found to be 27.0 cm−1, which is consistent with the reported value of N3− in D2O.7,13,36 The anharmonicities of N3-Ala and N3-Pro are 29.1 cm−1 and 25.6 cm−1, respectively, which are also in agreement with the reported result of N3dU in water (29 cm−1).34 Temporal profiles of the pump-probe signals at the v = 1-2 transition of N3-Ala (red), N3-Pro (blue), and N3− (green) are shown in Fig. 5(b). The signal shows a sharp spike at around T = 0 ps, which is assigned to a coherent effect between the pump and probe pulses.37 For all the pump-probe signals studied here, we fit the signals from the delay time of 0.2 ps. The temporal profiles of N3-Ala and N3-Pro were fitted to a double exponential function and that of N3− was fitted to a single exponential function to obtain vibrational


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the N3 anti-symmetric stretching mode of azide-derivatized nucleotides, azido-nicotinamide adenine dinucleotide (azideNAD+) and 3-picolyl azide adenine dinucleotide (PAAD+), in water relaxed with time constants of about 1.0 ps.39,40 In the previous study, Morita and Kato performed molecular dynamics (MD) simulation to examine the vibrational relaxation pathways of N3− in H2O theoretically.41 They revealed that the intramolecular vibrational energy redistribution (IVR) to the combination mode between symmetric and bending mode and VER to the vibrational mode of water equally contributes to the relaxation. Moreover, they elucidated that the charge fluctuations caused by the vibrational motion of the solute and the molecular motion of the solvent significantly accelerate the direct VER and IVR process of N3− in water. Skinner and co-workers also performed MD simulation for the same system and suggested that intramolecular relaxation to the symmetric stretch fundamental is a dominant pathway in the relaxation process.42 Although the numbers of the intramolecular vibrational modes of N3-Ala and N3-Pro are larger than that of N3−, the time constant of N3− is smaller than those of N3-Ala and N3-Pro. Covalently bonded azide group has less resonance structure than ionic one (covalently bonded azide group, —N==N+==N− and —N−—N+≡≡N−, ionic azide group, N≡≡N+—N2−, N2−—N+≡≡N, and N−==N+==N−), and it is expected that the amplitude of charge fluctuations in N3-Ala and N3-Pro is smaller than that in N3−. As a result, we consider that the VER rates of N3-Ala and N3-Pro are slower than that of N3−. C. Rotational relaxation

FIG. 5. (a) Time-resolved pump-probe signals of N3-Ala (red) and N3-Pro (blue) at 0.4 ps and N3− (green) at 0.5 ps in H2O. Closed circles indicate the experimental results. Solid lines represent the fitting results to four Lorentzian functions for N3-Ala and two Lorentzian functions for N3-Pro and N3−. (b) Temporal profiles of the pump-probe signals originated from the transient absorption (v = 1-2 transition) of N3-Ala (red) and N3-Pro (blue), and N3− (green) in H2O. Closed circles are the experimental results. Solid black lines represent the fitting results to single (for N3−) and double exponential functions (for N3-Ala and N3-Pro). A baseline for each profile is represented by a dotted line.

relaxation times. The obtained time constants of N3-Ala, N3-Pro, and N3− are listed in Table III. The covalently bonded N3 group to a carbon atom has intramolecular vibrational coupling between the N3 anti-symmetric stretching and the other modes, which causes efficient VER pathways for this mode.38 The slow time constants of N3-Ala and N3-Pro are similar to what has been reported by several groups.34,39,40 Tucker et al. reported that the VER time of the N3 antisymmetric stretching mode of N3dU in water was 1.1 ± 0.1 ps.34 Dutta et al. observed that the vibrational excitation energy of

Figure 6 shows the anisotropy decay of the N3 antisymmetric stretching of N3-Ala (red), N3-Pro (blue), and N3− (green). After 0.2 ps, the anisotropy decays of N3-Ala, N3-Pro, and N3− can be characterized by a single exponential plus a constant, and the parameters are summarized in Table III. The anisotropy decay often results from the orientational relaxation of the molecules itself. In this case, the rotational dynamics is often interpreted by the Stokes-Einstein-Debye (SED) theory, which is based on the hydrodynamic theory. Rotational relaxation of ions such as N3−, CN−, and SCN− in water was examined by the IR pump-probe measurements.43,44 The reported time constant of the rotational relaxation for CN− in H2O is 0.8 ps,43 and the time constants are 1.3 ps and 2.7 ps for N3− and SCN− in H2O, respectively.44 According to the SED theory, the rotational relaxation time is proportional to the hydrodynamic volume of the rotator. Therefore, the larger the hydrodynamic volume is, the slower the reorientation relaxation is. For example, Sando et al. observed the rotational relaxation of [Fe(CN)5NO]3−, whose volume is larger than that of N3−, in H2O by IR pump-probe spectroscopy. They reported its rotational time constant of 16 ps.45 Therefore, it can be expected that the time scales of the reorientation relaxation of N3-Ala and N3-Pro are much longer than that of N3−. However, the obtained time constants are similar to each other. We consider that these observations can be explained by considering internal rotational modes. In addition to the reorientation of the whole molecules, internal rotation of the azide group around the CN bond axis in N3-Ala and N3-Pro


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TABLE III. Obtained parameters of population relaxation and anisotoropy decay of the N3 anti-symmetric stretching mode of N3-Ala, N3-Pro, and N3− in H2O. Population relaxationa Solute Ala Pro N−3

Anisotropy decayb

A1

T11/ps

A2

T12/ps

∆/cm−1

A∞

A0

TR/ps

0.9 ± 0.1 0.8 ± 0.2 1.0 ± 0.1

0.1 ± 0.1 0.1 ± 0.1 0.6 ± 0.1

0.5 ± 0.1 0.7 ± 0.1 ...

1.1 ± 0.1 1.0 ± 0.1 ...

29.1 25.6 27.0

0 ± 0.1 0.2 ± 0.1 0 ± 0.1

0.3 ± 0.1 0.1 ± 0.1 0.3 ± 0.1

2.6 ± 0.4 4.8 ± 2.6 2.8 ± 0.7

a T , T : time constants in the fitting function; A exp(−T /T ) + A exp(−T /T ). ∆: anharmonicity of the N anti-symmetric 11 12 1 11 2 12 3 stretching mode. b A : constant component in the fitting function; A + A exp(−T /T ). ∞ ∞ 0 R

can also contribute to the anisotropy decay as shown by red line in Fig. 4. Rezus et al. performed the polarization-controlled IR pump-probe measurements for phenol-d in CHCl3, and reported that the rotational relaxation of the OD group around the CO bond axis decays the anisotropy with a time constant of 3.7 ps.46 Recently, Cho and co-workers studied the internal rotational relaxation of the azide group in SA in CHCl3 with 2D-IR spectroscopy. They obtained a time constant of 5.1

± 2 ps, which is related to interconversion between the bound and free azide group.16 Based on the similar anisotropy decays of N3-Ala and N3-Pro to that of N3−, we concluded that the internal rotation of the azide group plays a significant role in the anisotropy decays of N3-Ala and N3-Pro. As mentioned in Ref. 46, the anisotropy decay of the OD group of phenol-d has two components: fast decaying component and long-lasting component which was considered to be related to the slow molecular rotation as a whole. The rotational motion of the OD group around the CO bond axis restricts the reorientation of the OD group to a limited portion of the unit sphere, and the C—O—D angle determines the offset of the anisotropy. As shown in Table III, the decay rate of anisotropy of N3-Ala is faster than that of N3-Pro. Although it is difficult to determine the anisotropy decays of N3-Ala and N3-Pro on a long time scale because of the short lifetimes of the vibrational excited states, only the anisotropy decay of N3-Pro contains nonzero constant component A∞ in the present experimental accuracy. The internal rotational motion around the CN bond axis is restricted because the azide group of N3-Pro connects to the pyrrolidine ring, like the case of phenol-d in CHCl3. As shown in Fig. 6, the values of the anisotropy at long time are different between N3-Ala and N3-Pro. These differences may be explained by different numbers of internal rotational modes which contribute to the reorientation of the azide group. The azide group of N3-Ala binds to the methylene group. Therefore, the rotation around the CC bond axis (shown by green line in Fig. 4) is expected to induce the anisotropy decay and allow the rotation of the azide group of N3-Ala with less angular restriction. However, it is necessary to obtain the accurate parameters of the anisotropy decay to give definitive conclusion for this issue. Recently, researchers theoretically studied the conformational transition of biomolecules in terms of barrier crossing events on the potential energy surface.47–51 In the conformational change of proteins, multiple rotations around the single bonds in the backbone are important. By performing theoretical study for N3-Ala and N3-Pro, we can obtain more detailed information on the orientational dynamics observed in this study. D. Spectral diffusion

FIG. 6. Time-resolved anisotropy decays r (t) of N3-Ala (red), N3-Pro (blue), and N3− (green) in H2O. Closed circles represent the experimental results. Solid black lines correspond to the fitting results to a single exponential plus a constant. A baseline for each profile is represented by a dotted line.

We measured a series of 2D-IR spectra for N3-Ala, N3Pro, and N3− in H2O at different population times T from 0 ps to 2 ps. Figure 7 shows the 2D-IR spectra measured at T = 0.1 ps and 1 ps. The positive (red) and negative (blue)


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FIG. 7. 2D-IR spectra of (a) N3-Ala, (b) N3-Pro, and (c) N3− in H2O at T = 0.1 ps (left) and 1 ps (right). Red and blue colors represent the positive and negative signals, respectively. Red line in the 2D-IR spectrum indicates the diagonal direction (ω 1 = ω 3) of the 2DIR spectrum. Green lines indicate the center lines of the 2D-IR signals and the CLS is determined from the slope of this line.

bands correspond to the vibrational transitions of the N3anti-symmetric stretching mode between v = 0-1 and v = 1-2, respectively. At early population times, a 2D-IR spectrum is elongated along the diagonal because of the correlation of the two transition frequencies, ω1 and ω3. As the population time increases, the correlation of the vibrational frequency fluctuation is lost due to the change of the local environments around the vibrational probe (spectral diffusion). The shape of the 2D-IR spectrum becomes more circular by reflecting the loss of correlation. Therefore, the temporal evolution of the line shape of the 2D-IR spectra gives information on frequencyfrequency time correlation functions (FFTCFs) of the probe molecules. We quantified the change in the 2D-IR spectral shapes with the center line slope (CLS) method developed by Fayer and coworkers to extract FFTCF from the 2D-IR spectra.52,53 In this analysis, we take the slices of the 2D-IR spectrum at certain ω1 frequencies, and we obtain the center line by connecting

the maximum positions of each slice in the ω3 axis. Under the appropriate approximations, it was theoretically shown that the slope of this line as a function of T is proportional to FFTCF. According to the procedure described by Kwak et al., we determined the center line of each 2D-IR spectrum (shown by green line in Fig. 7), and the obtained CLSs for N3-Ala (red), N3-Pro (blue), and N3− (green) are plotted against the population time T in Fig. 8. As shown in Fig. 8, the initial values of CLS are not equal to one, reflecting that the correlation at T = 0 ps is reduced by the motional narrowing. In addition to the component, the obtained CLS contains the decaying component which is not motionally narrowed. A motionally narrowed component can be expressed by a delta function in FFTCF. Therefore, to represent these behaviors of FFTCF, we assumed that the form of the correlation function C(T) can be expressed as follows: C(T) = δ(T)/T2∗ + ∆21 exp (−T/τC ) + ∆22,

(6)


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FIG. 8. Plots of CLS (solid line with close circles) of N3-Ala (red), N3-Pro (blue), and N3− (green) obtained from the center line of the 2D-IR spectrum at each population time. Solid lines represent the simulated CLS with optimized parameters of C(T ) listed in Table IV.

where T2∗ is the pure homogeneous dephasing time constant, ∆12 is the amplitude of the frequency fluctuations, τC is the correlation time, and ∆22 is the static inhomogeneous component. Based on the response function formula of the linear and third-order nonlinear optical polarizations,9,52,54 we optimized the parameters of C(T) by the numerical calculations to reproduce the experimental data. Here, to avoid the pulse overlap effects due to coherent interactions among three pulses, we used the value of CLS from T = 0.2 ps for this analysis. The obtained parameters in Eq. (6) are listed in Table IV. Several groups including us experimentally and theoretically examined the frequency fluctuations of ions in water and pure water and revealed that the characteristic time constant of approximately 1 ps reflects fluctuations of hydrogen-bonding network in liquid water.9–13,55–58 Consistent with the results of the threepulse IR photon echo experiment for N3− in D2O,12,13 we also found that FFTCFs decay with a time constant of 1.3 ps for N3− in H2O. Moreover, we showed that FFTCF of N3-Ala and N3-Pro are also characterized by time constants of about 1.5 ps and 1.0 ps, respectively. Based on the previous studies, we consider that the decay component in FFTCF of N3-Ala and N3-Pro reflects hydration dynamics around the solutes. Therefore, according to the result that the time constants of the frequency fluctuations for N3-Ala and N3-Pro in water are not greatly different from that for N3−, it is suggested that hydration dynamics and structures around the non-ionic azide group are similar to that around the ionic one. Similar to our study, the 2D-IR measurements were performed for biomolecules which contain the azide group as a vibrational probe in water, and the similar behaviors are observed in FFTCF.34,39,40 Notable difference in FFTCF between the covalently bonded azide group and ionic one is the static inhomogeneous component ∆2. This term results from the dynamics which

proceed on a longer time scale than our experimental time window of T. As discussed in Sec. III A, for N3-Ala and N3-Pro, multiple transitions are present in the N3 antisymmetric stretching modes. In order to estimate the contribution of the weaker transitions in the 2D-IR spectra, we also simulated the decay of CLS in the presence of two transitions (νN3,low and νN3,high bands in the lower and higher frequency regions, respectively).59 Here, we consider that the time scale of chemical exchange process between the two transitions is slow because the spectral signatures of the cross peaks are not seen clearly in the 2D-IR spectra in our experimental time window. Therefore, we neglect it in the simulations (see the supplementary material for details67). Though we can reproduce of line shape of the linear absorption spectra and temporal profiles of CLS, uncertainty of the parameters in FFTCF of νN3,high band is very large. Note that we performed simulations for different sets of parameters in FFTCF of the νN3,high band, and observed weak dependence of the simulated CLS on parameters in FFTCF of νN3,high band (see Table S1 and Fig. S467). One of the simulated CLS is shown in Fig. 9. The decay of CLS of the whole components is similar to that of the component of νN3,low band. In the IR band of the N3 antisymmetric stretching mode of N3-Ala and N3-Pro, the peak intensity of νN3,low band is about four times larger than that of νN3,high band (see Table II). Moreover, the bandwidth of νN3,high band is broader than that of νN3,low band. As shown in the previous study, the center line data points of the 2D-IR spectra are a weighted average of those for each component under a certain limit.60 Therefore, the obtained 2D-IR spectra and CLS mainly reflect the contribution of νN3,low band and the static component ∆2 results from an inhomogeneity distribution of frequencies in this band.

TABLE IV. Optimized parameters of the FFTCF for N3-Ala, N3-Pro, and N3−. We assumed the form of FFTCF as follows: ⟨∆ω (T )∆ω (0)⟩ = δ(T )/T2∗ + ∆21 exp(−T /τ C ) + ∆22. Solute Ala Pro N3−

Solvent

T2∗/ps

∆1/ps−1

τ C /ps

∆2/ps−1

H 2O H 2O H2O

0.7 1.2 1.0

1.0 1.0 1.0

1.5 1.0 1.3

0.8 0.6 0.1

FIG. 9. Simulated CLS (solid line with close circles) of (a) N3-Ala and (b) N3-Pro at each population time. Red closed circles and blue squares indicate CLS for ν N3,low and ν N3,high bands, respectively, and green triangles represent CLS for whole band.


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We consider two possible interpretations for the static inhomogeneous component ∆2. First, slower solvation dynamics compared to that of bulk water may exist around the solute. Bakker et al. conducted the anisotropy decay measurements for trimethylamine-N-oxide (TMAO), tertiary butyl alcohol (TBA), and tetramethyl urea (TMU) in water and demonstrated that the reorientational motions of water molecules around the solute become slower than those of bulk waters due to hydrophobic effects of the methyl group in TBA, TMAO, and TMU.61,62 Moreover, they performed the 2D-IR experiments for the same systems and showed that the 2D-IR spectra of water around solute exhibited the long-lived elongation along the diagonal compared to that of bulk water.63,64 On the other hand, recently, Laage et al. simulated reorientational and spectral diffusion dynamics of water surrounding TBA, TMAO, and TMU with classical MD simulations and pointed out that not hydrophobic group but hydrophilic group in these molecules causes slow down of water dynamics.65,66 Both the hydrophobic and hydrophilic groups of N3-Ala and N3-Pro may contribute to the static component of FFTCF. The second possibility is that the structural dynamics of the solute itself causes the correlation of the frequency fluctuation on a slower time scale. As discussed in Sec. III A, the asymmetric band shape of the observed azide stretching modes may arise from distinct inhomogeneous environments of the solute molecules. The structural fluctuation of N3-Ala and N3-Pro in water may take place slower than water dynamics. Such fluctuations can contribute to the static homogeneous component ∆2. In the previous studies, Hochstrasser and coworkers and Cheatum and coworkers revealed that FFTCF of PAAD+ and N3dU in water also contained the static component, which was considered to be related to the structural dynamics of the probe molecule.34,39 IV. CONCLUSION

In this study, we have performed ultrafast nonlinear IR experiments for N3-Ala, N3-Pro, and N3− in water, and examined vibrational dynamics of the N3-anti-symmetric stretching mode of these compounds. From the polarization-controlled IR pump-probe measurements, we obtained the time constants for VER and rotational relaxation of the probe molecules. Based on the similarity of the obtained time constants for VER, it is suggested that the IVR processes including the theoretically suggested one in Ref. 41 are also important for the vibrational relaxation pathway of the azide-derivatized amino acids. Even though the number of energy accepting modes for IVR is expected to be larger for N3-Ala and N3-Pro, we found that the VER rates of N3-Ala and N3-Pro are slower than that of N3−. This suggests that the charge fluctuation in the azide ion plays a significant role in the relaxation process. By comparing the time scales for the anisotropy decays of N3-Ala and N3-Pro with that of N3−, it is expected that internal rotational motions promote the rotational relaxation of the azide group. Moreover, we observed that the structural difference such as structural flexibility around the vibrational probe affects the behavior of the anisotropy decay. We considered that our observations are very useful to understand the structural dynamics of proteins by using an azide label as a vibrational probe.

J. Chem. Phys. 142, 212418 (2015)

From the results of 2D-IR spectra, we found that FFTCF of the solutes can be described by delta function plus an exponential function and constant, which reflect the homogeneous and inhomogeneous components, respectively. From the numerical calculations, it is revealed that the decay time constants of the correlation functions are approximately 1 ps. Similar to the previous studies for azide ions in water, this indicates that the frequency fluctuations of the azide stretching mode of N3Ala and N3-Pro reflect the fluctuations of hydrogen-bonding network. While CLS of N3− decays to zero in our observed time window, those of N3-Ala and N3-Pro have some finite offsets. These slow frequency fluctuations are related to the dynamics of water affected by the solutes or the structural fluctuations of solute itself. Our experimental results suggest that the structural complexity of the probe molecule can affect the vibrational dynamics of the studied modes. Therefore, we must be careful to interpret the experimental results when we incorporate a vibrational tag into a complex system to investigate the microscopic environment around the probe molecules in solutions or biological molecules by ultrafast IR vibrational spectroscopy. ACKNOWLEDGMENTS

Theoretical calculations were performed using Research Center for Computational Science, Okazaki, Japan. This work was partially supported by a Grant-in-Aid for Scientific Research on the Priority Area Molecular Science for Supra Functional Systems (No. 20050019) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. 1I.

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