Isonitrile as an Ultrasensitive Infrared Reporter of Hydrogen-Bonding

Article pubs.acs.org/JPCB

Isonitrile as an Ultrasensitive Infrared Reporter of Hydrogen-Bonding Structure and Dynamics Michał Maj,†,‡,§,∥ Changwoo Ahn,‡,∥ Bartosz Błasiak,†,‡ Kyungwon Kwak,‡ Hogyu Han,*,‡ and Minhaeng Cho*,†,‡ †

Center for Molecular Spectroscopy and Dynamics, Institute for Basic Science (IBS) and ‡Department of Chemistry, Korea University, Seoul 02841, Korea S Supporting Information *

ABSTRACT: Infrared (IR) probes based on terminally blocked β-isocyanoalanine (AlaNC) and p-isocyanophenylalanine (PheNC) amino acids were synthesized. These isonitrile (NC)-derivatized compounds were extensively characterized by FTIR and femtosecond IR pump−probe spectroscopies, and a direct comparison was made with popularly used nitrile (CN)- and azide (N3)-derivatized analogs. It is shown that the isonitrile stretch frequency exhibits extremely high sensitivity to hydrogen-bonding interactions. In addition, the IR intensity of the isonitrile group is much higher than that of the nitrile group and almost as intense as that of the azido group. Furthermore, its vibrational lifetime is much longer than that of the nitrile and azido groups. To elucidate the origin of such a high H-bond sensitivity and IR intensity observed for isonitrile, extensive quantum chemical calculations were performed. It is shown that the Coulombic contributions to the vibrational frequency shifts of the isonitrile and nitrile stretch modes have opposite signs but similar magnitudes, whereas the contributions of exchange repulsion and charge delocalization to their frequency shifts are comparable. Therefore, the isonitrile stretch frequency is much more sensitive to H-bonding interactions because the blue-shifting exchange-repulsion effects are additionally enforced by such electrostatic effects. It is also shown that the much higher IR intensity of the isonitrile group compared to that of the nitrile group is due to the configuration reversal of the atomic electronegativity between the NC and CN groups. Owing to these features, we believe that isonitrile is a much better IR reporter of H-bonding structure and dynamics than the widely used nitrile and azide. solvent fluctuations and H-bond making/breaking processes.28 Other desirable properties include large transition dipole moments, relatively narrow bandwidths, and long vibrational lifetimes. It was shown that the nitrile stretch frequency is particularly sensitive to H-bonding interactions,29−40 which manifests as a distinguishable blue-shift due to the exchange-repulsion solute−solvent interactions.41 Unfortunately, the dipole strength of the nitrile stretch mode is very small, especially when the nitrile group is attached to an aliphatic carbon. The consequent low IR intensity makes it practically impossible to apply aliphatic nitrile-derivatized amino acids to the 2DIR studies of protein dynamics. It has been shown that the dipole strength of the nitrile stretch mode can be greatly enhanced when the nitrile group is attached to an aromatic carbon. Therefore, the use of p-cyanophenylalanine, an aromatic nitrile, has led many researchers to successfully carry out 2DIR studies of site-specific protein dynamics.13,42−44 Despite the numerous efforts, it should be noted that its IR intensity is still much lower than that of azide-derivatized molecules. It is also worth

I. INTRODUCTION Two-dimensional infrared (2DIR) spectroscopy1−7 has been extensively used to quantitatively measure the dynamics of conformational changes, 8−12 local environment fluctuations,13−18 and chemical exchange processes8,19,20 in various biomolecules and their model systems. To extract detailed information on site-specific interactions within proteins and DNA, numerous IR probes have been incorporated into their structures, and various stretch vibrations have been used as reporters of site-specific vibrational dynamics.21−27 In many instances, the choice of an appropriate IR probe is absolutely crucial for obtaining meaningful, high-quality nonlinear signals, which can then be related to the ultrafast dynamics of a molecular system. Stretch vibrations of nitrile (CN), azido (N3), thiocyanato (S−CN), and carbonyl (CO) groups are the most commonly used IR reporters of the structure and dynamics of local environment. Nonetheless, to choose an appropriate IR probe for a specific application, one needs to consider all of its structural and vibrational properties, which are often critical for obtaining meaningful spectroscopic data. High sensitivity to the local electric field or hydrogen-bonding interactions is one of the necessary prerequisites to relate the changes in timedependent vibrational spectra to dynamic observables such as © XXXX American Chemical Society

Received: April 29, 2016 Revised: August 16, 2016

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DOI: 10.1021/acs.jpcb.6b04319 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

vibrational solvatochromism theory62,63 to model systems, we could explain the differences in the H-bonding-interactioninduced vibrational frequency shift and IR intensity between isonitrile and nitrile.

mentioning that the nitrile stretch frequency appears far above 2210 cm−1, resulting in a substantial overlap with a broad optical density stretch band, when D2O is used as the solvent. Such spectral congestion greatly reduces the S/N ratio and produces large amounts of heat due to vibrational relaxation of the unnecessarily excited optical density stretch vibration of water, which can subsequently affect the quality of the nonlinear IR spectroscopic data and the possible outcome of the analyses. Another type of IR reporter that has been extensively used in 2DIR measurements is the azido group, which shows asymmetric stretch IR absorption bands.34,45−55 The azido group gained a great deal of popularity due to the exceptionally large dipole strength of its asymmetric stretch mode.34 The possibility of its use for nonlinear spectroscopic study of biomolecules at very low concentrations, including large protein systems, due to the consequent high IR intensity, has led researchers to synthesize a large number of azide-derivatized compounds, making them the most renowned vibrational probes used in 2DIR studies.34,56 Nevertheless, azido probes have also been found to possess a number of drawbacks, among which broad bandwidth and markedly short vibrational lifetime often hinder their potential applications. Also, it is often the case that azido stretch bands are highly contaminated with accidental Fermi resonance bands.34,47,57−60 Because of the many limitations of the currently used IR probes, 2DIR spectroscopy, although being a powerful technique, is still not versatile enough to cover a wide variety of biomolecular systems. Therefore, a new family of highly sensitive and intensive IR probes has long been sought, as advancement in this area is very important for understanding the structure and dynamics of proteins. Previously, we have introduced β-isocyanoalanine (AlaNC) as a promising IR probe of proteins and characterized its vibrational properties with time-resolved IR spectroscopy.61 Here, as a continuation of our work, we present a much more extensive study of IR probes based on terminally blocked isonitrile-derivatized amino acids, which include the previously studied Ac-Ala(NC)-NHMe (AlaNC) as well as the newly synthesized Ac-Phe(NC)NHMe (p-isocyanophenylalanine, PheNC) (Figure 1). We

II. EXPERIMENTAL AND COMPUTATIONAL METHODS II.A. Materials. Compounds 1 and 2 were synthesized and characterized (Scheme 1 and the Supporting Information). To Scheme 1. Syntheses of 2a

a

Reagents and conditions: (a) TFEF, HCO2Na, tetrahydrofuran (THF), rt, 99%; (b) POCl3, Et3N, CH2Cl2, −20 oC, 66%; (c) 40% MeNH2, MeOH, rt, (2a, 91%; 2b, 85%; 2c, 89%); (d) MeI, K2CO3, dimethylformamide (DMF), rt, (7, 78%; 11, 99%); (e) TFA, rt, (8, 99%; 12, 99%); (f) Ac2O, Et3N, CH2Cl2, rt, (9, 81%; 13, 83%).

examine the chemical stability of the NC group in isonitrilederivatized compounds, we measured the FTIR spectra of 1a in 10 mM phosphate-buffered saline (pH 7.4) containing 33 mg/ mL bovine serum albumin at room temperature (rt) for 3 days. The IR absorbance of the isonitrile stretch mode for 1a in such solution remains unchanged, which indicates no notable chemical decomposition of the NC group under physiological conditions. Nonetheless, we found that isonitrile is unstable under acidic conditions (complete decomposition within 30 min at 1 N HCl), whereas it is quite stable under basic conditions (no decomposition at all at 40% MeNH2 in MeOH, as indicated in our synthetic Scheme 1). Thus, despite the fact that isonitrile is stable under neutral and basic conditions, caution needs to be exercised to avoid the exposure of this IR reporter to acidic conditions when it is incorporated into amino acids, peptides, and nucleotides. The sample concentrations for

Figure 1. Structures of IR probes 1 and 2.

compared their vibrational properties to those of nitrile- and azide-derivatized compounds by FTIR and femtosecond IR pump−probe spectroscopies. We found that the isonitrile stretch frequency exhibits an extremely high sensitivity to Hbonding interactions. In addition, the isonitrile group shows very high IR intensity and a much longer vibrational lifetime. To elucidate the origin of such a high H-bond sensitivity and IR intensity observed for isonitrile, extensive quantum chemical calculations were performed. Applying the recently developed B


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The Journal of Physical Chemistry B FTIR and IR pump−probe measurements were set to give an optical density of roughly 0.3. II.B. FTIR and IR Pump−Probe Spectroscopies. FTIR spectra were recorded on a Bruker VERTEX 70 spectrometer, with a frequency resolution of 1 cm−1 and at a constant temperature of 22 °C. Experimental details of polarization-controlled IR pump− probe spectroscopy and the femtosecond laser setup used for the measurements have been described in detail before.61,64 The experimental setup consists of a Ti:sapphire oscillator (Tsunami; Spectra-Physics), a regenerative amplifier (Spitfire; Spectra-Physics), and an optical parametric amplifier (OPA-800 C; Spectra-Physics). The mid-IR laser pulses are generated in the OPA using a 2.0 mm thick AgGaS2 crystal and are further split into pump and time-delayed probe pulses by a ZnSe beam splitter. The time delay between the two pulses is controlled using a motorized linear stage, and the time-dependent pump− probe signal is frequency-resolved with a monochromator coupled to a 64-element LN2-cooled MCT array detector. The average power of the pump pulse, measured in front of the sample, was approximately 500 μW. The relative polarization of the probe beam was controlled using a rotating polarizer, and two polarization conditions were considered, namely, parallel (S∥(t)) and perpendicular (S⊥(t)) to the pump beam. Such polarization control allows us to separate the vibrational (population relaxation, P(t), isotropic signal) and rotational (r(t), anisotropic signal) contributions to the overall signal as P(t ) =

ΔωjSolEDS

−

HL HF ΔωjSolEDS = Δωj(10) ,el + Δωj ,ex + Δωj ,del

(1)

(2)

II.C. Quantum Chemical Calculations. Vibrational solvatochromic frequency shifts of the isonitrile and nitrile stretch modes were studied with the vibrational solvatochromism theory based on the variational−perturbational interaction energy decomposition scheme65−68 (SolEDS) developed recently.62 Within the Hartree−Fock approximation,69 the solute−solvent interaction energy can be decomposed into three terms67,68 HF ΔEHF = ΔEel(10) + ΔEexHL + ΔEdel

(4)

(5)

HL HF where Δω(10) j,el , Δωj,ex , and Δωj,del are the Coulombic, exchangerepulsion, and charge-delocalization contributions, respectively. To study the sensitivities of isonitrile and nitrile stretch frequencies to H-bonding interactions, we chose MeNC and MeCN as model molecules, which interact with H2O and CHCl3 via H-bonds. Energy optimizations (with a gradient RMS threshold of 10−6 au) and subsequent harmonic frequency analyses of single MeNC and MeCN molecules as well as MeNC···X and MeCN···X (X = H2O or CHCl3) complexes were performed using the HF/6-311++G** method,70,71 implemented in the Gaussian 09 suite of programs.72 Cubic anharmonic constants were calculated numerically in our inhouse code. In particular, they were obtained from the first derivatives of Hessian matrices when using the B3LYP, HF, and MP2 methods and from the third derivatives of energies when using the CCSD method. The interaction energies between solute and solvent molecules were computed using the variational−perturbational method65−68 implemented by Góra73 in the GAMESS package.74 All calculations of the interaction energy derivatives with respect to the gas-phase normal coordinates were performed numerically. All first derivatives of both interaction energies and Hessian matrix elements were computed in Cartesian coordinates, assuming 0.006 Å Cartesian atomic displacements, and they were subsequently transformed to the normal coordinate space using the eigenvector elements obtained from the harmonic vibrational analyses of the isolated solute molecules. The second derivatives of interaction energies were computed directly in the normal mode space, with a displacement of 0.085 Å. The Cartesian coordinates of the model systems for the SolEDS analysis are given in Section SII of the Supporting Information. Ab initio solvatochromic dipole and quadrupole moments were computed according to the procedure described before.75 The dipole moment derivatives of MeXY and PhXY (XY = NC or CN) at the MP2 and CCSD levels of theory were computed using atomic polar tensors implemented in Gaussian 09 with the 6-311++G**70,71 and aug-cc-pVnZ (n = D, T)76,77 basis sets. The derivatives of electron density with respect to the vibrational normal coordinate were calculated numerically utilizing the 3-point central finite difference method, with a displacement of 0.03 Å. All other numerical derivatives were computed using the 5-point central finite difference method.

S (t ) − S⊥(t ) S (t ) + 2S⊥(t )

⎫ gijj ⎛ ∂ΔEHF ⎞ ⎪ ⎜ ⎟ ⎬ ⎜ ⎟ Miωi2 ⎝ ∂Q i ⎠ ⎪ Q 0⎭

where Mi and ωi are the solute’s gas-phase reduced mass and harmonic frequency associated with the ith normal coordinate Qi, whereas gijj is the cubic anharmonic constant defined as gijj = (∂3E/∂Qi∂Q2j )Q0, with E denoting the total energy of the isolated solute molecule. All of the derivatives are computed at the gas-phase geometry of the solute (Q0). Because of the additivity of the interaction energy components in eq 3, it is useful to recast the frequency shift in eq 4 as

and r (t ) =

∑ i

S (t ) + 2S⊥(t ) 3

⎧ ⎛ ⎞ 1 ⎪⎜ ∂ 2ΔEHF ⎟ ⎨⎜ ≅ 2Mjωj ⎪⎝ ∂Q j2 ⎟⎠ Q0 ⎩

(3)

where ΔE(10) is equal to the Coulombic interaction energy el HL is the between the unperturbed charge densities, ΔEex associated exchange-repulsion energy due to the Pauli exclusion principle, and the third term, ΔEHF del , collectively takes into account all solute−solvent interaction-induced charge-delocalization phenomena (such as polarization of charge density distribution, charge transfer process, as well as other effects not included in ΔEel(10) and ΔEHL ex ). The resulting vibrational frequency shift of the jth normal mode can be then expressed as follows62 C


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III. RESULTS AND DISCUSSION III.A. H-Bond Sensitivity and IR Intensity of the Studied IR Probes. The ability of an IR probe to quantitatively report on the local electric field or the magnitude of the interaction energy between solute and hydrogen-bonded solvent molecules is crucial for its successful application to studying the local structure at a site-specific position in proteins. Numerous IR spectroscopic studies have been reported for nitrile-derivatized molecules, and their high sensitivity to H-bonding interactions has been frequently shown and discussed.33−35,37,38,42,78,79 Nonetheless, the origin of vibrational frequency shifts upon H-bonding, as well as their magnitudes, turned out to be quite complicated in nature.41,80 Indeed, the number of publications in which solvatochromism of various IR probes is directly compared in a quantitative manner is rather scarce. Thus, for the sake of direct comparison, isonitrile (NC)-, nitrile (CN)-, and azide (N3)-derivatized alanine and phenylalanine were studied by FTIR to elucidate the effects of solvent polarity and its Hbonding ability on their vibrational frequencies and absorption lineshapes. The solvent-dependent FTIR spectra are presented in Figures 2 and 3. Figure 3. (a−c) Solvent-dependent FTIR spectra of derivatized phenylalanines 2. Color codes for the solvents are indicated in (c). For abbreviations of the solvents, see Figure 2. Contrary to the case of 1, FTIR spectra of 2 in D2O could not be obtained because of solubility problems. Similar to AlaNC, PheNC also shows band splitting in Hbonding solvents, but the lineshapes and relative peak areas are somewhat different. Interestingly, PheN3 appears to be insensitive to either the electric field or H-bonding interactions, contrary to the case of AlaN3.

shifts are observed in our study, it is not certain why the vibrational frequencies of PheN3 are not quite sensitive to the solvent electric field and are blue-shifted only in the presence of strongly H-bonding solvents (Figure 3c). The vibrational frequencies of AlaN3, on the other hand, show quite a remarkable sensitivity to the local electric field as well as Hbonding interactions (Figure 2c). Nevertheless, the vibrational frequencies of AlaN3, as well as those of PheN3, do not follow any well-defined trend. It is not clear why relatively weak Hbond donors, like 3-phenyl-1-propanol (PP), give a larger blueshift than methanol, whereas in the case of PheN3, those weakly H-bonding solvents have nearly no effect on the vibrational frequency, as compared to that of all non-H-bonding solvents. Such a strong dependence of the solvatochromic response on the molecular structure of azide-derivatized compounds may limit their successful application as IR probes; therefore, we believe that further investigations are needed to fully understand their solvatochromism. Considering the vibrational solvatochromic behaviors of nitrile and isonitrile probes, we shall discuss their spectral properties in H-bonding and non-H-bonding solvents separately. In the case of aprotic solvents, it has been shown by Levinson et al. that the vibrational frequency of the nitrile stretch mode in aromatic nitriles correlates well with solvent polarity, as expressed by the Onsager factor, which is related to the solvent dielectric constant.36 However, the method used by Levinson et al. is based on the interaction between the solute dipole moment and the continuum reaction field that cannot encompass the specific electrostatic effects like the non-

Figure 2. (a−c) Solvent-dependent FTIR spectra of derivatized alanines 1. Color codes for the solvents are indicated in (c). HMPA, hexamethylphosphoramide; PP, 3-phenyl-1-propanol; DFE, 2,2,diluforoethanol; TFE, 2,2,2-trifluoroethanol. FTIR spectra of AlaCN in CHCl3 could not be obtained because of a solubility problem. Significant splitting of the vibrational bands of AlaNC is observed in H-bonding solvents such as MeOH, PP, DFE, and TFE. On the other hand, the peaks are not as well-resolved in the case of AlaCN. Although AlaN3 was found to be quite sensitive to the electric field, the vibrational frequencies in the above-mentioned H-bonding solvents are rather similar.

Aliphatic and aromatic azide-derivatized compounds have been thoroughly investigated by Wolfshorndl et al.,56 and it was suggested that the azido group, with asymmetric stretch vibrations, may act as a good IR reporter of H-bonding interactions. In particular, it was found that the vibrational frequency of the azido stretch mode correlates linearly with the H-bonding ability of solvents.56 Although similar frequency D


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to H-bond strength. Although this trend has been observed for nitrile probes before,84 more focus has been paid to understanding their response to solvent polarity rather than their H-bonding ability.85 Despite the similar behaviors upon H-bonding, isonitriles appear to give strikingly larger frequency shifts in comparison to those of nitriles (Figures 2a and 3a). It is worth noting that even chloroform, which is able to form rather weak H-bonds, gives a substantial 5 cm−1 blue-shift and significant line broadening. More interestingly, in all of the other protic solvents, it is possible to differentiate between strongly H-bonding and weakly or non-H-bonding species, which appear either as an asymmetric or clearly separated spectral feature. These effects are especially strong in alcohols, because they are able to form relatively strong H-bonds, and at the same time are large enough to allow for substantial structural heterogeneity around an IR probe. Although it is apparent that an isonitrile probe is able to provide information on H-bonding interactions with much larger sensitivity as compared to a nitrile probe, there has been no well-established method that allows direct and quantitative comparison of different IR probes and their response to the H-bonding ability of solvent. Throughout the years, considerable efforts have been made to develop and validate simple empirical models that connect vibrational solvatochromic observables to certain solvent molecular descriptors such as polarity, electron-donating/ accepting ability, Brønsted acidity/basicity, and so on.36,37,80,85−94 In the present study, we directly compare the observed vibrational frequency shifts and bandwidths with empirically determined Kamlet−Taft parameter α, which is a parameter directly related to the H-bond donating ability of solvent.85−88,94 Correlation plots for derivatized alanines and phenylalanines are presented in Figure 4. The vibrational frequency shifts of both nitrile and isonitrile probes correlate well with α. However, the magnitudes of their shifts, expressed by the slope of the fitted line, are substantially different, with about twice larger slope obtained for the isonitrile probes. Similar correlation plots were prepared for full width at halfmaximum (FWHM) (Figure S2). The bandwidth of azido probes was found to be almost completely solvent-independent. On the other hand, quite substantial changes in bandwidth were observed for the nitrile and isonitrile probes. Considering aliphatic alanines, higher correlation was found for AlaNC in comparison to AlaCN. Moreover, isonitrile shows much larger spectral shifts and well-separated spectral features, which fit very well to analytical functions, whereas nitrile very often shows highly overlapped peaks and the estimation of their bandwidths may not be that accurate. The situation looks opposite in the cases of aromatic phenylalanines, in which case the FWHM of PheCN shows much better correlation with α than that of PheNC. This indicates that, in this particular case of PheNC, the center frequency may be a more reliable measure of H-bond strength. Here, despite the excellent correlation between nitrile and isonitrile stretch frequencies and Kamlet−Taft parameters of solvents, due to the empirical nature of the Kamlet−Taft parameterization of solvent Hbonding ability, the plots in Figure 4 are still empirical correlations. We therefore further investigated the underlying mechanism behind the solvation-induced frequency shifts theoretically in Section III.B. Now, it should be noted that it is difficult to predict the vibrational frequency shifts of nitriles when they are in non-Hbonding solvents even though H-bonding interaction-induced

vanishing quadrupolar contributions as well as the molecular granularity of the solvent.81 In addition to this, it was previously found by Rey and Hynes that van der Waals forces are very important for the nitrile frequency shifts in the interaction between the CN − anion and water.82 Morales and Thompson made a similar observation in the case of acetonitrile.83 Recently, Błasiak et al. showed, using the firstprinciples vibrational solvatochromism theory, that the solute− solvent interaction-induced frequency shifts cannot be simply represented by first-order electrostatic interactions.41,62 Particularly, the higher-order electrostatic terms, that is, polarization and dispersion, as well as the non-electrostatic exchangerepulsion terms have to be accounted for the complete description of solvatochromic frequency shifts.41,62 The exchange-repulsion and dispersion contributions, which cannot be connected to the electrostatic potential or field distribution around an IR probe at all, are especially important for the vibrational solvatochromism of nitrile probes when their Hbonds with polar solvent molecules like water are involved.41 Similar correlation plots against the Onsager factor are presented in Figure S1. It is interesting to note that the magnitudes of vibrational frequency shifts of PheCN and benzonitrile36 are clearly different, with much larger shifts observed for the latter. This indicates that the introduction of a bulkier substituent into the para position of the aromatic ring may either affect the local solvation structure around the molecule or substantially change the vibrational solvatochromic response of the nitrile probe to the environment. This observation is important if one wants to correlate vibrational frequency shift with the electric field in the active sites of proteins. Not much to our surprise, a similar lack of correlation in aprotic solvents was observed for the isonitrile probes, with center frequencies scattered around 2149 and 2125 cm−1 for AlaNC and PheNC, respectively; note that all frequency shifts reported below are given with respect to these two values. Small, but notable frequency shifts were observed for PheNC only in nitromethane, hexamethylphosphoramide (HMPA), and THF. Note that the former two solvents are highly polar, with dielectric constants of 35.9 and 30.0, respectively, whereas THF is far less polar, with a dielectric constant of 7.6. The values of the Onsager factor are 1.02, 1.24, and 1.29 for THF, nitromethane, and HMPA, respectively. Nevertheless, nitromethane blue-shifts the spectrum of PheNC by 5 cm−1, whereas HMPA red-shifts it by about 3 cm−1, similar to that by THF (see Figure S1b). These results clearly show that the vibrational frequency shifts of (aromatic) isonitriles, like those of (aromatic) nitriles, are not correlated to solvent polarity. This is not only due to the breakdown of the Stark dipole model in the cases of triple-bonded IR probes,41 but also may be due to an additional vibrational frequency shift effect caused by the intramolecular electrostatic interactions of the IR reporter with the neighboring polar peptide groups in our molecules studied here. The contribution of this intramolecular interaction is highly sensitive to molecular conformation,41 which in turn is likely to be solvent-dependent. To provide a detailed description of numerous contributions to the vibrational solvatochromism of the studied IR probes, quantum chemical calculations were performed and are presented in the next subsection. Interestingly, in H-bonding solvents, the vibrational frequency shifts are much more pronounced, showing blueshifting behavior and significant line broadening, in proportion E


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Figure 4. Plot of center frequencies against the Kamlet−Taft parameter α for 1 (a) and 2 (b). High degree of linear correlation was observed for NC (CN)-derivatized compounds, with slightly smaller R2 (0.69) found for PheCN. The azido compounds exhibit very small frequency shifts, which suffer from a large uncertainty due to complicated spectral lineshapes. Fitted constants: AlaNC (R2 = 0.89, slope = 21), AlaCN (R2 = 0.88, slope = 11), AlaN3 (R2 = 0.34, slope = 6), PheNC (R2 = 0.90, slope = 21), PheCN (R2 = 0.69, slope = 9), PheN3 (R2 = 0.60, slope = 5).

Figure 5. (a) Molar extinction spectra of 1 and 2 in DMF. For the sake of clarity, the spectra of nitrile-derivatized amino acids are additionally shown in the inset. (b) Pseudo-Voigt profile fits to the three components in the spectrum of PheN3 in DMF. Similar Fermi resonance satellite peaks are present in all other solvents too.

was found between the stretch vibrations of nitrile and isonitrile, with the latter having an almost 15 times larger extinction coefficient. It is not to our surprise that aliphatic nitriles have an extremely low intensity, as it is often the case that their vibrational bands are barely found in the FTIR spectra and their 2DIR signal is difficult to measure. Therefore, it is of great advantage to have an IR reporter, like the studied isonitrile, that can be easily incorporated into both aliphatic and aromatic amino acid residues. We also studied how the intensities of the IR reporters change on the basis of whether they are introduced into the aliphatic or aromatic moiety of amino acid side chains. The most substantial change was observed for PheCN, which shows an almost 10-fold increase in intensity as compared to that of AlaCN. Such an intensity enhancement allows for application of PheCN to the 2DIR studies of protein structure and dynamics. Nevertheless, its integrated intensity is still about 3 times smaller than that of PheNC. PheN3 is characterized by the highest integrated intensity; however, its spectrum consists of at least three different absorption bands, as presented in Figure 5b. Such a complicated lineshape is very likely to originate from the presence of accidental Fermi resonances, which have been found to complicate the 2DIR spectra.95 Note that an asymmetric lineshape was also observed for the aliphatic AlaN3. Obviously, the presence of such resonance contributions to the 2DIR spectra may greatly complicate their analyses and subsequent interpretation. Thus, PheNC, due to its simple

frequency blue-shifts are quite notable. This makes it hard in the case of nitrile probes to correlate their frequency shifts with local electrostatics and/or the solvent structure around them because the origin of the frequency shifts is of various natures. On the other hand, the vibrational frequency shifts of isonitriles are most likely to originate purely from specific interactions with H-bonding species because the frequency shifts originating from solvent polarity are mostly negligibly small for isonitrile probes. Therefore, isonitriles once introduced site-specifically into biomolecules could be applied to probe the H-bond strength in their vicinity. This is a huge step toward the accurate and quantitative probing of H-bonding interactions in various molecular systems. Another interesting observation is related to the intensities of the fundamental transitions of the studied compounds. One of the important requirements for successful application of an IR probe is having a high enough intensity so that incorporation of a single IR reporter into a large protein structure produces a measurable absorption signal. The molar extinction spectra of the studied molecules in DMF are presented in Figure 5a, and the numerical values of the extinction coefficients and dipole strengths are additionally summarized in Table 1. Among the derivatized alanines, AlaN3 clearly gives the most intensive integrated band, although the extinction coefficient of AlaNC is slightly larger than that of AlaN3. The most striking difference F


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The Journal of Physical Chemistry B Table 1. Spectral Properties of 1 and 2 in DMF AlaNC

PheNC

ω0 (cm−1) FWHM (cm−1) ε (cm−1 M−1) εarom/εalipha D (10−2 D2) Darom/Daliphb T1 (ps)c

2151.6 10.6 390 1.6 2.21 1.2 5.5 ± 0.2

2125.0 7.6 610 9.6 2.71 6.6 10.5 ± 0.2

τor (ps)c

7.3 ± 0.9

0.4 ± 0.1 (0.12) 72.8 ± 4.4 (0.88)

AlaCN

PheCN

2250.2 11.0 27 2.2 0.15 2.7

AlaN3

PheN3

2227.0 6.6 259

2103.6 26.4 333

2118.9 22.7 756

0.99

4.80

12.9

0.5 ± 0.1 (0.28) 4.8 ± 0.1 (0.72)

0.065 ± 0.004 (0.60) 1.34 ± 0.08 (0.40)

0.22 ± 0.03 (0.68) 1.1 ± 0.4 (0.24) 8.4 ± 3.0 (0.08)

53.9 ± 7.3

3.82 ± 1.98

a Ratio of molar extinction coefficients between aliphatic alanine and aromatic phenylalanine. bRatio of dipole strengths between aliphatic alanine and aromatic phenylalanine. cFrom the multiexponential fitting analyses of the isotropic IR pump−probe data, we obtained the relative amplitudes (in parentheses), in addition to decay constants.

Table 2. Physical Origins of the Vibrational Frequency Shifts of NC/CN Stretch Modes Interacting with Water and Chloroform Molecules via H-Bondsa Δω(10) el ΔωHL ex ΔωHF del ΔωSolEDS full QM

MeNC···H2O (RCH = 2.421 Å)

MeNC···HCCl3 (RCH = 2.575 Å)

MeCN···H2O (RNH = 2.245 Å)

MeCN···HCCl3 (RNH = 2.375 Å)

+9.0 +14.3 −2.8 +20.5 +20.1

+7.8 +11.7 −1.3 +18.2 +18.0

−4.6 +16.0 −4.9 +6.5 +5.9

−5.3 +8 ± 4b ∼0 ± 4b +4.4 +4.1

a

Vibrational frequency shift analysis (with respect to the gas phase) was performed using the SolEDS//HF/6-311++G** method (eq 5) on the basis of constrained cluster calculations (see ref 62 for details). Exact frequency shifts (full QM) computed for the fully energy-optimized structures shown in Figure 6 are also presented here. Frequency shifts are given in cm−1. The lengths of hydrogen bonds are shown in parentheses below the system label. bApproximate estimation due to the numerical instability (see Table S1).

lineshape and relatively large intensity, may be a useful substitute for PheN3. III.B. Molecular Origin of H-Bond Sensitivity and IR Intensity. To elucidate the molecular origin of the extremely high sensitivity of isonitrile stretch frequency to H-bonding interactions, we carried out extensive QM calculations of the interaction energy decomposition, from which individual contributions to vibrational frequency shifts were determined on the basis of the recently developed vibrational solvatochromism theory. For the study, we chose two simple molecules, MeNC and MeCN, that are H-bonded to either a H2O or CHCl3 molecule. The results showing each interaction energy contribution to vibrational frequency shift are given in Table 2, and the molecular structures are depicted in Figure 6. It is clear that the frequency blue-shifts of MeNC and MeCN due to (both proper and improper) H-bonding relative to those in their gas-phase states are significantly larger in the case of isonitriles, with an almost 4-fold increase in the frequency shift as compared to that in nitriles. It is interesting that the origin of these frequency shifts in terms of the exchange-repulsion and charge-delocalization effects is virtually the same. On the other hand, the Coulombic contribution (which is mostly due to the multipolar interactions) changes its sign in the case of isonitriles, causing the electrostatic blue-shift instead of red shift. Note that this change in sign enforces blue-shifts caused by the exchange-repulsion effect, which is in stark contrast to the nitrile frequency shift where Coulombic and exchangerepulsion effects cancel each other. To understand this sign change in the Coulombic contribution to the vibrational frequency shifts of the isonitrile stretch mode, we computed the ab initio vibrational

Figure 6. (a−d) Molecular structures of the model systems used for the SolEDS//HF/6-311++G** analysis.

solvatochromic multipole moments 75 of NC/CN stretches (Table 3), which are defined as 1 Δω NC/CN = −Δμ NC/CN ·F − ΔΘNC/CN : ∇F + ··· (6) 3 In the above equation, ΔωNC/CN is the frequency shift, F is the electric field, and ΔμNC/CN and ΔΘNC/CN are the vibrational solvatochromic dipole and quadrupole moments, respectively. Generally, the B3LYP and HF values of ΔμCN are in good agreement with those from the experiment. For instance, the B3LYP/6-311++G** ab initio calculation and B3LYP/6-311+ +G(3df,2pd) empirical estimation give absolute magnitudes of 0.29 and 0.32 cm −1 /(MV/cm), respectively, with the experimental range of 0.32−0.39 cm−1/(MV/cm). InterestG


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Table 3. Electric Vibrational Spectroscopic Properties of NC/CN Stretch Modes in Model Aliphatic Compoundsa method

basis set

B3LYP HF MP2 CCSD CCSD CCSD

6-311++G** 6-311++G** 6-311++G** 6-311++G** aug-cc-pVDZ aug-cc-pVTZ

B3LYP

6-311++G(3df,2pd)

MeNC

MeCN

z +0.28 +0.38 +0.014 +0.22 +0.25 +0.24

xx = yy −0.42 −0.38 −0.51 −0.44 −0.45 −0.42

zz +0.85 +0.77 +1.02 +0.87 +0.90 +0.85

z

xx = yy

zz

ab initio z −0.29 −0.38 −0.059 −0.19 −0.18 −0.19 fitting z −0.32b exp.c |z| 0.33−0.39d

|z|

xx = yy −0.54 −0.54 −0.43 −0.47 −0.50 −0.48

zz +1.08 +1.09 +0.86 +0.93 +1.00 +0.96

xx = yy −0.62b

zz +1.24b

Vibrational solvatochromic dipole and traceless quadrupole moments of NC/CN stretches in MeNC and MeCN are given in cm−1/(MV/cm) and 10−8 cm−1/(MV/cm2), respectively. Principal symmetry axes of all of the molecules are collinear with the z axis so that the x and y dipoles as well as the xy, xz, and yz quadrupole components vanish. The z axis is pointing toward the C-atom in the isonitrile or N atom in the nitrile group. The vibrational solvatochromic quadrupole has its origin in the NC or CN mid-bond. bRef 81. Note that the quadrupole moment in ref 81 is defined according to Jackson,100 whereas in refs 41, 62, 101 as well as in the present work, we use Buckingham’s convention.102 To convert values of quadrupole tensor elements from Buckingham’s to Jackson’s convention, multiply them by 2. cRef 103. dAssuming that the local field correction factor is between 1.1 and 1.3. a

nitrile group, we analyzed the dipole moment derivatives with respect to XY (XY = NC or CN) stretch modes (∂μ/∂QXY)Q0 because the IR intensities are proportional to |(∂μ/∂QXY)Q0|2. The dipole moment derivative can be partitioned into nuclear and electronic contributions according to

ingly, MP2 significantly underestimates the absolute magnitude of the CN solvatochromic dipole moment, which is calculated to be less than 0.06 cm−1/(MV/cm). The fact that the HF level provides a correct order of magnitude (0.38 cm−1/(MV/cm)) is surprising, and apparently, a more refined electroncorrelation method than MP2 is necessary in the case of correlated ab initio calculations. The CCSD method gives the result with a correct order of magnitude, and our best ab initio estimations of the Stark tuning rate at the CCSD/aug-cc-pVTZ level are 0.24 and 0.19 cm−1/(MV/cm) for NC and CN in MeNC and MeCN, respectively. Unfortunately, there is no vibrational Stark spectroscopic measurement of ΔμNC for MeNC in the literature. Nevertheless, all of our ab initio and empirical estimates consistently show that ΔμNC and ΔμCN point in opposite directions along the corresponding NC/ CN bond axes and have comparable absolute magnitudes. However, the vibrational quadrupole moment, unlike the dipole moment, is not affected much by flipping their C and N atoms. Therefore, the differences in the sign of Δω(10) j,el between the NC and CN stretch modes are caused primarily by the opposite direction of their solvatochromic dipole moments. Our theoretical approach can be used to validate the purely empirical parameterizations based on the Kamlet−Taft method. Strong correlations of the NC/CN vibrational frequencies with parameter α are likely to be the result of the strongly blueshifting, H-bonding interaction-induced contributions to the (10) vibrational frequency shifts (ΔωHL j,ex and also Δωj,el in the case of NC stretch modes). This supports the interpretation of parameter α that reflects the H-bond donating ability of various solvent molecules interacting with NC/CN groups. Carrying out extensive ab initio frequency shift analyses of larger solute− solvent clusters or even bulk systems could provide a comprehensive theoretical justification of the Kamlet−Taft parameters in the future. Only then could the vibrational solvatochromic empirical correlations be interpreted on the grounds of the intermolecular interaction theory. To understand the reasons behind the significantly higher IR intensities of the isonitrile group compared to those in the

⎛ ∂μ ⎞ ⎜⎜ ⎟⎟ = − ⎝ ∂Q XY ⎠Q

⎛

XY Q 0

0

≡

⎞

∭V ⎜⎜⎝ ∂∂ρQ(r) ⎟⎟⎠

cf δμ XY

+

vib δμ XY

atoms

r dr +

∑ a

⎛ ∂r ⎞ Za⎜⎜ a ⎟⎟ ⎝ ∂Q XY ⎠Q

0

(7)

In the above equation, δμcfXY denotes the charge-flow term due to the electron density redistribution that occurs during the vibrational displacement of atoms,96 whereas δμvib XY is associated with the stretching itself, that is, the structural character of the vibration. Za and ra denote the atomic number and position of the ath atom, respectively. Note that in the case of XY stretches the vibrational motion of the whole molecule is dominated by the change in the XY bond length. Equation 7 is correct for electron density distribution ρ(r), which is localized in a finite volume, V, and vanishes elsewhere. Partitioning according to eq 7 was performed for MeXY and PhXY (XY = NC or CN), and the results are shown in Table 4. We assume here that X and Y atoms are placed along the z axis and the Y atom points toward the positive z axis side. From our analyses, it is clear that the charge-flow term, δμcfXY, contributes the most to the total dipole moment derivative in all of the cf studied model systems. Moreover, the directions of δμXY computed for NC and CN are opposite to each other. In contrast, the structural contributions, δμvib XY, are quite similar in every case. This leads to strengthening of the NC dipole moment derivative and weakening of the CN dipole moment derivative (note that the charge-flow and structural contribution effects act in the same direction in the case of NC, contrary to that in CN). We also performed dipole moment derivative approximate partitioning by the distributed charge method, as discussed elsewhere (Table S2). H


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The Journal of Physical Chemistry B Table 4. Partitioning of the Molecular Dipole Moment Derivatives with Respect to the NC/CN Stretch Modes in Model Aliphatic and Aromatic Compoundsa MeNC δμcfz,XY δμvib z,XY

( ) ∂μz

∂Q XY

( ) ∂μz

( ) ∂μz

+0.8599 +0.2017 +1.0616

MP2/6-311++G** +1.2735 −0.1219 +0.0220 +0.1210 +1.2955 −0.0008

−0.0699 +0.0114 −0.0585

+1.2010 +0.1885 +1.3895

CCSD/6-311++G** +1.7827 −0.2442 +0.0182 +0.0996 +1.8010 −0.1447

−0.3524 +0.0089 −0.3435

+1.1701 +0.1944 +1.3644

CCSD/aug-cc-pVDZ +1.7512 −0.2437 +0.0189 +0.1010 +1.7701 −0.1428

−0.3534 +0.0090 −0.3444

+1.1765 +0.1838 +1.3603

CCSD/aug-cc-pVTZ −0.2904 +0.0949 −0.1955

Figure 7. Mechanisms of electron density flow that occurs when (a) NC and (b) CN bonds are elongated. First derivatives of the electron density distribution ((∂ρ/∂QXY)Q0) with respect to the XY stretch mode (XY = NC or CN) were calculated at the CCSD/aug-ccpVDZ level. The positive (negative) sign of QXY corresponds to the elongation (shortening) of the XY bond. The isocontours were plotted for ±0.20 au, with red and blue colors representing the increase and decrease in electronic density, respectively. (∂ρ/∂QXY)Q0 is associated with the charge flow terms (δμcfz,XY) shown in Table 4 (see eq 7).

Q0

δμcfz,XY δμvib z,XY

( ) ∂μz

∂Q XY

PhCN

Q0

δμcfz,XY δμvib z,XY ∂Q XY

MeCN

Q0

δμcfz,XY δμvib z,XY ∂Q XY

PhNC

Q0

a

Charge-flow (δμcfXY) and structural-vibrational (δμvib XY) contributions to the dipole moment first derivative ((∂μ/∂QXY)Q0 for XY = NC or CN) were obtained using the MP2 and CCSD methods and eq 7. Principal symmetry axes of all molecules are collinear with the z axis so that the x and y components vanish. The z axis points toward the C-atom in the isonitrile group or N atom in the nitrile group. The positive (negative) sign of QXY corresponds to XY bond elongation (shortening). All values are in atomic units.

⎛ ∂μ ⎞ ⎟⎟ Δμ XY ∝ ⎜⎜ ⎝ ∂Q XY ⎠Q

0

(8)

III.C. Vibrational Dynamics. Besides the previously mentioned properties, like sensitivity to specific interactions or the IR intensity, it is also important to consider the dynamic properties of the studied compounds. Because most of the IR probes are designed to study proteins and nucleotides, it should be emphasized that time-dependent structural evolution in such systems needs to be fully sampled after times ranging from tens to hundreds of picoseconds or sometimes even longer. Therefore, it is important to find a molecular probe whose vibrational excited state is characterized by a relative long lifetime. Such a probe allows one to capture a substantially long dynamics for the accurate estimation of frequency−frequency correlation functions (FFCF). For a direct comparison between vibrational lifetimes of the studied IR probes, we carried out polarization-controlled IR pump−probe measurements and determined both the vibrational lifetimes and rotational relaxation rates. The fitting results are summarized in Table 1. Here, it should be noted that we were unable to measure the IR pump−probe spectra of AlaCN because of its extremely small transition dipole moment. Moreover, the IR pump− probe spectra of AlaNC and AlaN3 were measured in both D2O and DMF, whereas those of derivatized phenylalanines were measured only in DMF due to their low solubility in water. The IR pump−probe analyses of derivatized alanines is depicted in Figure 8. The vibrational lifetime of AlaNC was determined to be 5.5 ± 0.2 ps and was found to be independent of the solvent used.61 In the case of AlaN3, the situation is slightly more complicated because we observed a biexponentially decaying pattern, with an ultrashort component constituting 60−70% of the total decay, and the other, still considerably fast, component was estimated to be in the range of 1.1−1.3 ps. The almost 5 times longer vibrational lifetime of AlaNC makes it a better probe of local structural fluctuations in proteins. Perhaps the

To get a deeper insight into the origin of the increase in the magnitude of the NC dipole moment derivative, we plotted a 3D map of the electron density flow, (∂ρ/∂QXY)Q0, for an isovalue of ±0.2 au in Figure 7. In Figure S3, we present similar maps for other isovalues. Obviously, as the XY bond length increases (QXY > 0), the electron density on the XY bond decreases, which is indicated by blue color in Figure 7. However, when the NC bond in RNC (R = Me or Ph) elongates, there is a notable electron density flow from the N C bond toward the R−N bond but a relatively much weaker flow to the C-atom lone pair of the NC group. Therefore, the electron density increases asymmetrically around NC, which is indicated by red color in Figure 7. In contrast, when the CN bond in RCN (R = Me or Ph) elongates, the electron density flows from the CN bond in two opposite directions, with roughly similar amounts that counterbalance each other: toward the R−C bond and toward the N-atom lone pair of the CN group. This indicates that the net charge flow is dictated by the position of the more electronegative N atom in cf the XY group, which results in a much smaller δμ XY contribution in the case of nitrile stretching vibrations and, in turn, in quite a small dipole moment derivative. This explains the significant increase in the IR intensity of the isonitrile group relative to that of the nitrile group in both aliphatic and aromatic cases. This phenomenon might also be responsible for the opposite direction of the solvatochromic dipole moments between the NC and CN stretch modes because ΔμXY is approximately proportional to (∂μ/∂QXY)Q063 I


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Figure 8. IR pump−probe data of AlaNC and AlaN3 in DMF at delay time t. Population relaxation (a) and anisotropy (b) decays. For details, see Figures 5 and 6 of ref 61.

more serious problem with the azido probe, from the perspective of biomolecular applications, is the fact that the majority of initially observed intensity of the azido stretch mode decays rapidly within 200−300 fs after the excitation. This makes such a large intensity of the azido band in the FTIR spectra less useful, and apparently, the application of azido probes to the 2DIR studies of protein dynamics could be limited. The IR pump−probe spectra of both PheNC and PheCN in DMF are presented in Figure 9. Because of many complications regarding the spectral analysis of PheN3, we shall discuss their results separately in Figure 10. As the intensity of PheCN is much higher than that of AlaCN, we were able to determine its vibrational lifetime. Interestingly, the isotropic IR pump−probe signal of PheCN shows a biexponential decay, with time constants of 0.5 ± 0.1 and 4.8 ± 0.1 ps, whereas that of PheNC decays with a single time constant of 10.5 ± 0.2 ps. The almost 2 times longer vibrational lifetime of PheNC, along with its much higher intensity, looks very promising. It has been shown that the vibrational lifetime of the CN stretch mode can be increased when 13C isotopic labeling is introduced.97 Nonetheless, such an isotopic substitution could be expensive, and it clearly does not solve the problem of a low intensity of the nitrile stretch band. As mentioned above, the FTIR spectra of PheN3 appear to be rather complicated, with a number of additional satellite bands overlapping with the fundamental transition. These were assigned to accidental Fermi resonance bands, as there is no possibility for the azido group to form any structurally distinct conformations in this particular system. The presence of those additional, intrinsically coupled peaks has a strong influence on the lineshape itself as well as its evolution in time. As can be

Figure 9. IR pump−probe data of PheNC and PheCN in DMF at delay time t. (a) Isotropic transient absorption spectra. The lineshapes of 0−1 and 1−2 transitions were found to be nearly identical for PheNC but very different for PheCN. (b) Population relaxation decays. The integrated areas of the transient absorption peaks extracted for both 0−1 and 1−2 transitions were used. (c) Anisotropy decay. The anisotropy signals over the indicated range of probe frequencies for the 0−1 transition were averaged.

seen in Figure 10a, the normalized transient absorption spectra at different time delays reveal complicated features in the excited state absorption band, due to the presence of a number of strongly overlapped signals. Moreover, the vibrational decays were found to be highly frequency-dependent in that region. What complicates the analyses even more is the fact that the time-dependent intensity changes exhibit oscillating features (Figure 10b), which greatly resemble the oscillations observed before for other Fermi resonance coupled systems.98,99 We are J


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was found to be much higher than that of the nitrile group and almost as intensive as that of the azido group. However, contrary to that for the CN stretch, no substantial intensity difference was observed for the NC stretches between aromatic and aliphatic isonitriles. To elucidate the molecular origin of the extremely high sensitivity of the stretch frequency of isonitrile to H-bonding interactions, we carried out extensive QM calculations and quantified the contribution of each individual interaction energy component to H-bonding interaction-induced vibrational frequency shift. We found that only the Coulombic contributions to the vibrational frequency shifts are affected by replacing the nitrile with an isonitrile group. The consequence is a drastic increase in the H-bonding-induced frequency blueshift of the isonitrile stretch mode, which is now strongly enforced by exchange repulsion, contrary to that of the nitrile stretch mode.41 Because of the configuration reversal of the atomic electronegativity between the NC and CN groups, the charge-flow contribution to the dipole moment derivatives changes sign, whereas the purely nuclear contribution is barely affected and relatively weak in strength. This causes a notable increase in the IR intensity of the isonitrile group relative to that of the nitrile group. It is believed that this phenomenon is also responsible for the direction reversal of the vibrational Stark dipole moment, which makes the isonitrile stretch frequency unusually sensitive to H-bonding interactions. Our results support the usual interpretation of Kamlet−Taft parameter α based on the H-bond donating ability of the solvent, but to fully validate the Kamlet−Taft method in terms of the intermolecular interaction theory, more computational analyses should be undertaken in the future. We also carried out polarization-controlled IR pump−probe measurements and determined both the vibrational lifetimes and rotational relaxation rates. The isonitrile probes were found to have significantly longer vibrational lifetimes than those of the azido probes, for which very-rapid-intensity decays were found. Interestingly, the vibrational lifetime of PheNC is about 2 times longer than that of the widely used PheCN. Moreover, both the linear and nonlinear IR spectra of azido probes exhibited many complicated features, which highly limits their potential applicability. In summary, isonitriles possess much better vibrational properties for time-resolved spectroscopic studies as compared to those of nitriles and azides. Although the stretch frequency of isonitrile does not show any noticeable sensitivity to solvent polarity, it must also be noted that the solvatochromism of nitriles and isonitriles cannot be solely explained by their interactions with the local electric field.41 Nevertheless, Hbonding interactions are of tremendous importance from a biomolecular perspective; thus, developing isonitrile as a selective reporter of the H-bonding structure and dynamics is an enormous step forward in the application of 2DIR spectroscopy to proteins and nucleotides.

Figure 10. IR pump−probe data of PheN3 in DMF at delay time t. (a) Normalized isotropic transient absorption spectra. Strong shifts and lineshape changes are observed in the 1−2 transition part of the spectrum. (b) Population relaxation decays. The time-dependent intensities of the transient absorption at three different probe frequencies for the 0−1 transition were used. To show the long time component and oscillations, a smaller part of the plot was zoomed in and shown in the inset.

quite certain that the oscillations are not simple experimental noise but are a real consequence of multiple couplings between the vibrational modes. Although the presence of the oscillations did not affect our determination of vibrational lifetime, it was impossible for us to obtain high-quality anisotropy data. The vibrational lifetime of PheN3 was found to decay triexponentially, with major components of 0.2 and 1.2 ps and a minor component of 8.6 ps (see Table 1 for the relative amplitudes of these components). Similar to that in AlaN3, most of the intensity decays very rapidly in 1 ps so that the nonlinear IR spectroscopic signal after 1 or 2 ps would be very weak, with complicated oscillatory features. This makes those azido probes less useful for the 2DIR studies of protein environment fluctuations.

VI. SUMMARY In the present study, we synthesized IR probes based on terminally blocked isonitrile-derivatized amino acids, AlaNC and PheNC, and compared their vibrational properties with those of nitrile- and azido-derivatized compounds by FTIR and femtosecond IR pump−probe spectroscopies. We found that the stretch frequency of isonitrile exhibits extremely high sensitivity to H-bonding interactions, whereas it showed negligible sensitivity to solvent polarity. Thus, the stretch vibration of isonitrile can be a highly specific reporter of Hbonding interactions. The IR intensity of the isonitrile group

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b04319. Detailed synthetic procedures and characterization for derivatized phenylalanines; figures and tables showing the additional results of FTIR and computational studies (PDF) K


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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (H.H.). *E-mail: [email protected] (M.C.). Present Address §

Department of Chemistry, University of Wisconsin at Madison, Madison, Wisconsin 53706-1396, United States (M.M.). Author Contributions ∥

M.M. and C.A. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by IBS-R023-D1. H.H. is grateful for the financial support from the National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT, and Future Planning (NRF20110020033 and NRF2016R1A2B4013572) and the Ministry of Education (NRF2013R1A1A2010933). SolEDS calculations were performed at the Wroclaw Centre for Networking and Supercomputing (WCSS) of which support is kindly acknowledged.

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