Vibrational Spectra and Ab Initio DFT Calculations of 4

J. Phys. Chem. B 2000, 104, 4253-4265

4253

Vibrational Spectra and Ab Initio DFT Calculations of 4-Methylimidazole and Its Different Protonation Forms: Infrared and Raman Markers of the Protonation State of a Histidine Side Chain Koji Hasegawa,*,† Taka-aki Ono,† and Takumi Noguchi*,‡ Laboratory for Photo-Biology, RIKEN Photodynamics Research Center, Aoba, Sendai, Miyagi 980-0868, Japan, and Photosynthesis Research Laboratory, RIKEN, Wako, Saitama 351-0198, Japan ReceiVed: January 11, 2000

The imidazole group of a histidine side chain has four different protonation forms, i.e., the two neutral tautomers (N1- and N3-protonated forms), imidazolium cation, and imidazolate anion. Owing to the presence of these convertible protonation forms, histidine plays important roles in proton-transfer reactions in various enzymes. Vibrational spectroscopy is one of the most powerful methods to study the protonation state of histidine in proteins. For systematic investigation of IR and Raman markers of the protonation state of histidine, we have performed ab initio normal-mode calculations using the density function theory (DFT) method for all of the four protonation forms of 4-methylimidazole (a simple model compound of a histidine side chain) and their N-deuterated analogues. FTIR and Raman spectra of all of these compounds were measured, and the observed bands were assigned according to the calculated frequencies and intensities. Differences in the optimized geometries and changes in the vibrational couplings explained the differences in band frequencies and N-deuteration shifts among the protonation forms. These analyses provided theoretical bases for the IR and Raman markers of the protonation state, including known markers, such as the C4C5 stretching and the C5N1 stretching bands, as well as some new potential markers.

Introduction The imidazole group of a histidine side chain possesses two nitrogen atoms, which can be protonated or deprotonated. As a result, it takes four different protonation forms: the two neutral tautomers in which either of the two nitrogen atoms (N1 or N3) is protonated, the imidazolium cation that is fully protonated, and the imidazolate anion that is deprotonated at both the nitrogen sites (Figure 1). In aqueous solution, the neutral N1- and N3-protonated forms are in tautomeric equilibrium.1,2 Because pKa of the imidazolium form is around 6.0, histidine can undergo protonation and deprotonation reactions at physiological pH. Therefore, it functions as a competent protontransfer mediator in various proteins.3-6 Histidine also serves as both a hydrogen-bond donor and an acceptor, and this hydrogen-bonding property is of importance in proton-transfer reactions5 and in organizing the active centers of enzymes. Furthermore, the imidazole group can coordinate the metal ion through the deprotonated nitrogen atom(s), and thus histidine residues are often found in the ligands of metalloenzymes.7,8 Although pKa of neutral histidine (to imidazolate form) is as high as 14, metal binding decreases the pKa value,7,9 and hence, the histidine ligand can directly participate in proton-transfer reactions at the metal center. Owing to such a versatile nature, histidine is often a key amino acid in enzymatic reactions, and therefore, to elucidate the reaction mechanisms, it is essential to know the structures of histidine side chains in the proteins. In recent years, the structures of extensive numbers of proteins have been clarified by X-ray crystallography. However, because of the weakness of X-ray crystallography in detecting hydrogen * Corresponding authors. † Laboratory for Photo-Biology. ‡ Photosynthesis Research Laboratory.

Figure 1. Optimized geometries and atom numbering of the four protonation forms of 4-MeIm: (a) N1-protonated form (N1H-MeIm); (b) N3-protonated form (N3H-MeIm); (c) imidazolium cation (MeImH2+); and (d) imidazolate anion (MeIm-).

atoms, the protonation structures and hydrogen-bonding interactions of amino acid side chains were not well resolved in many cases. For obtaining such information, vibrational spectroscopy, which directly detects chemical bonds and molecular interactions, is one of the most powerful methods. As for histidine, its protonation state, metal binding, and hydrogen bonding interaction have been investigated using both Raman2,10-30 and

10.1021/jp000157d CCC: $19.00 © 2000 American Chemical Society Published on Web 04/01/2000

4254 J. Phys. Chem. B, Vol. 104, No. 17, 2000 FTIR31-36 spectroscopy. Resonance Raman measurements with UV excitation around 210 nm14,17-19 have been effective in detecting histidine signals in proteins.20-24,30 Also, the recent development of FTIR difference spectroscopy made it possible to detect the signals of individual amino acid residues at the active sites of proteins,37 and using this method, the structures and interactions of histidine residues have been studied.31-36 To obtain the structural information of histidine from the spectra, the IR and Raman markers and the empirical rules have been developed. Ashikawa and Itoh2 studied the Raman spectra of neutral histidine in aqueous solution and found that the splitting of the bands at 1568/1585, 1282/1260, and 983/1004 cm-1, which were assigned to the C4C5 stretching, the ring breathing, and the CH in-plain bending modes, respectively, is due to the N1- and N3-protonated tautomers. Takeuchi and coworkers24,26 also investigated the Raman markers of the neutral tautomers with a bound metal ion and reported the upshifts of the C4C5 stretching bands and the intensity enhancement of several bands in the 1500-1200 cm-1 region, upon metal binding. For the N-deuterated imidazolium form (HisD2+), an intense Raman band at about 1408 cm-1 has been observed,12,16,20,23,29 which is a Raman marker of the imidazolium form. For the metal-bridging imidazolate, Hashimoto et al.22,24 observed strong Raman bands at ∼1560 and ∼1285 cm-1 as its markers. As for the IR markers, Noguchi et al.36 recently showed that the frequency and the N-deuteration shift of the C-N stretching band, around 1100 cm-1, are useful to identify the protonation state. Also, a series of subbands above 2500 cm-1,11,38-40 which has been ascribed to Fermi resonance of the overtones and combinations of the ring modes with the hydrogen-bonding NH stretching vibration,11,39,40 has been used as a marker of the presence of an NH group(s) (i.e., exclude the imidazolate form) and its hydrogen bonding interaction.34-36 The normal-mode analysis of a histidine side chain has been performed using 4-methylimidazole (4-MeIm) and 4-ethylimidazole (4-EtIm) as model compounds.2,41-43 Majoube et al.42 reported the complete sets of normal modes of 4-MeIm and 5-MeIm (identical to the N3-protonated form of 4-MeIm) and their N-deuterated analogues calculated at the ab initio 6-31G level using the Hartree-Fock method. More recently, Gallouj et al.43 calculated the normal modes of 4-EtIm and its Ndeuterated analogue using the density function theory (DFT) method and revised some assignments by Majoube et al.42 Hashimoto et al.41 performed a vibrational analysis of the imidazolate form of 4-MeIm using the empirical force field obtained by least-squares refinements. However, ab initio calculations of the imidazolium and imidazolate forms of 4-MeIm or 4-EtIm have not yet been reported, to our knowledge. The significance of the protonation structures of histidine side chains in proteins and the present situation of having quite insufficient theoretical bases for the analytical methods of histidine signals in vibrational spectra prompted us to systematically study the vibrations of histidine and its different protonation forms. We have performed ab initio DFT calculations for all of the four protonation forms of 4-MeIm, and their N-deuterated analogues, and assigned the experimentally observed FTIR and Raman bands of these compounds. These measurements and theoretical analyses have established the IR and Raman markers of the protonation state of histidine and provided theoretical bases for the rules to determine the protonation structure. Methodology Experimental. Raman spectra were measured with a JASCO TRS-600/S monochromator equipped with an 1800 groove/mm

Hasegawa et al. grating. A 488 nm line from an Ar ion laser (Coherent, Innova 90) was used for excitation, and Raman scattering was collected perpendicular to the laser beam. Rayleigh scattering was removed with a holographic Notch filter (Kaiser Optical Systems, Inc.). The dispersed Raman scattering was detected with a CCD detector (Prinston Instruments, Inc., LN/CCD-1100PBUVAR). 4-MeIm was used as purchased from Aldrich Chemical Co., Inc. For measuring the N1- and N3-protonated (deuterated) forms (N1H(D)- and N3H(D)-MeIm, respectively) in solution, 4-MeIm was dissolved in H(D)2O to the 0.5 M concentration. Note that N1H(D)- and N3H(D)-MeIm are in equilibrium in aqueous solution. For the imidazolium (MeImH(D)2+) and imidazolate (MeIm-) forms, 4-MeIm (0.5 and 0.2 M, respectively) was dissolved in 1 M H(D)Cl and 5 M NaOH(D), respectively. The DCl and NaOD solutions were prepared by diluting 35% DCl/D2O (>99 atom % deuterium, Isotec Inc.) and 40% NaOD/D2O (>99 atom % deuterium, Isotec Inc.), respectively, with D2O (99.9 atom % deuterium, Isotec Inc.). Solution samples were measured using a rotating quartz cell. Polycrystalline 4-MeIm (N1H-MeIm) was measured in a capillary tube. The laser power was 200 mW at the sample point. The baseline correction was performed with the program for the Raman measuring system provided by JASCO Co. FTIR spectra were measured on a Bruker IFS-66/S spectrophotometer using a DTGS detector. For measuring N1H(D)and N3H(D)-MeIm in aqueous solution, 4-MeIm was dissolved in H(D)2O to a 4 M concentration. For MeImH(D)2+ and MeIm-, 4-MeIm (2 and 1 M, respectively) was dissolved in 3 M H(D)Cl and 5 M NaOH(D), respectively. The solution samples were placed between a pair of ZnSe plates with a spacer of ∼7 µm. The spectra of solvents (H(D)2O, H(D)Cl, or NaOH(D)) were measured and subtracted from the sample spectra to eliminate the high background of H(D)2O absorption. At this time, changes in proton (deuteron) concentration and in D/H ratio by dissolving 4-MeIm were taken into consideration. After the solvent subtraction, further baseline correction was performed in the Bruker OPUS program. The 1 M solution of 4-MeIm in 5 M NaOH(D) included a small amount of neutral species other than MeIm-, and hence, the spectra were corrected using the spectra of neutral species in H(D)2O to obtain the pure MeIm- spectra. Polycrystalline 4-MeIm was measured in a KBr disk. All of the Raman and FTIR spectra were measured at room temperature with a spectral resolution of 2 cm-1. Computational Calculation. Ab initio MO calculations were performed by the GAUSSIAN 98 program package.44 The geometry optimizations were carried out by the density function theory (DFT) method using Becke’s three-parameter hybrid functional45 combined with the Lee-Yang-Parr correlation functional (B3LYP)46 with the 6-31G(df,p) basis set. The Cartesian force constants were analytically computed at the fully optimized geometries. The obtained force-constant matrix was transformed from Cartesian to internal coordinates, and the vibrational frequencies and potential energy distributions (PED) were calculated using a modified program of NCTB.47,48 Both internal and symmetry coordinates for N1H- and N3H-MeIm were defined as those described by Majoube et al.42 The internal coordinates for MeImH2+ and MeIm- were given by adding the stretching, in-plane bending, and wagging coordinates of N3H to the coordinates of N1H-MeIm, and by deleting the N1H coordinates from them, respectively. The computed frequencies were scaled with a uniform scaling factor of 0.98 for all of the protonation and deuteration forms. This scaling factor was determined by the least-squares fit to the observed frequencies of the bands below 1700 cm-1, at which most of

4-Methylimidazole and Its Protonation Forms

J. Phys. Chem. B, Vol. 104, No. 17, 2000 4255

TABLE 1: Selected Optimized Geometry Parameters of Four Protonation Forms of 4-MeIm N1H-MeIm N1-C2 C2-N3 N3-C4 C4-C5 C5-N1 C4-C6 C2-H C5-H N1-H N3-H

1.3641 1.3134 1.3833 1.3728 1.3818 1.4959 1.0810 1.0783 1.0066

N1C2N3 C2N3C4 N3C4C5 C4C5N1 N3C4C6

111.83 105.66 109.95 105.65 121.36

MeImH2+

MeIm1.3487 1.3500 1.3728 1.3907 1.3722 1.5009 1.0903 1.0884

1.0072

1.3321 1.3360 1.3916 1.3666 1.3841 1.4901 1.0785 1.0774 1.0116 1.0120

angles (deg) 111.75 107.56 104.26 111.46 123.14

106.96 110.65 105.24 107.31 123.01

117.24 102.23 108.93 109.93 122.15

N3H-MeIm distances (Å) 1.3123 1.3684 1.3841 1.3732 1.3793 1.4925 1.0810 1.0809

the concerned markers are present. PED values (%) were normalized in each vibrational mode so that the sum of the PED becomes 100%. IR and Raman intensities were computed by the GAUSSIAN program. Results and Discussion The optimized geometries of four protonation forms of 4-MeIm, i.e., N1H-MeIm, N3H-MeIm, MeImH2+, and MeIm-, are presented in Figure 1, and the selected geometry parameters are summarized in Table 1. It is noteworthy that the lengths of the N1-C2 and C2-N3 bonds drastically change depending on the protonation state. The differences in these lengths are more than 0.05 Å, whereas changes in the lengths of other bonds are less than 0.025 Å. N1H-MeIm has a short C2-N3 bond (1.3134 Å) with a double-bond character and a long N1-C2 bond (1.3641 Å), whereas this relationship is reversed in N3HMeIm, which has a long C2-N3 (1.3684 Å) and a short N1C2 (1.3123 Å) bond. MeImH2+ and MeIm- exhibit medium N1-C2 and C2-N3 lengths (∼1.33 Å in MeImH2+ and ∼1.35 Å in MeIm-), indicating a partial double-bond character. Both the N3-C4 length and the C5-N1 length (1.37-1.39 Å) are longer than the N1-C2 and C2-N3 bonds in all of the protonation forms. The C4-C5 lengths fall between 1.3666 and 1.3907 Å, which are much shorter than the C4-C6 lengths (1.49-1.50 Å), showing that the double-bond character of C4C5 is conserved in all the forms. Figures 2 and 3 show the high and low-frequency regions, respectively, of FTIR and Raman spectra of polycrystalline 4-MeIm (N1H-MeIm) (panel A), 4-MeIm in H(D)2O (mixture of N1H(D)-MeIm and N3H(D)-MeIm) (panel B), MeImH(D)2+ in H(D)Cl (panel C) and MeIm- in NaOH(D) (panel D). Note that in the given FTIR spectra large backgrounds due to H2O (∼3400, 1640 and < 800 cm-1) or D2O (∼2500, 1200 and < 600 cm-1), absorptions were eliminated by subtraction of the solvent spectra. Such solvent correction was not performed for the Raman spectra, and thus, the steep background above 3000 cm-1 (Figure 2Bb, Cb, Db) or below 2800 cm-1 (Figure 2Bd, Cd, Dd), and a broad feature around 1640 cm-1 (Figure 3Bb, Cb, Db) or 1200 cm-1 (Figure 3Bd, Cd, Dd) are due to the solvents. MeIm- showed basically identical IR and Raman spectra between in NaOH and in NaOD (Figures 2D, 3D), confirming that MeIm- has the same deprotonated structure in both NaOH and NaOD. This observation also indicates that H/D exchange of the water protons hydrogen bonding to MeImhas little effect on the band frequencies.

The calculated vibrational frequencies for N1H-MeIm, N1D-MeIm, N3H-MeIm, N3D-MeIm, MeImH2+, MeImD2+, and MeIm-, and the assignments of the observed FTIR and Raman bands, are summarized in Tables 2-8. For the band assignments, both the frequencies and the band intensities were taken into consideration. The uniform scaling factor of 0.98 was employed to calculate the frequencies for all of the forms, to compare the calculated frequencies among the different forms. Band Assignments. 1. NH(D) Vibrations. The NH stretching modes give the highest calculated frequencies at 3600-3550 cm-1 for N1H-MeIm, N3H-MeIm, and MeImH2+ (Tables 2, 4, and 6). In fact, the previous IR spectrum of a dilute solution of 4-MeIm in CCl4 showed a free NH band at ∼3500 cm-1.40 Upon hydrogen bonding, the NH stretching band considerably downshifts and is broadened. Crystalline 4-MeIm, in which the NH group is strongly hydrogen bonded, shows a broad IR band centered at ∼2800 cm-1 with numerous subbands (Figure 2Aa).42 These subbands have been attributed to a Fermi resonance of the overtones or combinations of lower frequency fundamentals with the NH stretching vibration.11,39,40 Similar subbands from a histidine side chain have been observed in the FTIR difference spectra of photosynthetic proteins, providing the evidence for its hydrogen-bonding interaction.34-36 A broad feature around 2900 cm-1 in the Raman spectrum of crystalline N1H-MeIm (Figure 2Ab) may be the corresponding NH Raman band, as reported by Majoube et al.42 Upon N-deuteration, the calculated ND stretching frequencies occur at 2650-2600 cm-1 (Tables 3, 5, and 7). Majoube et al.42 reported the ND frequency of polycrystalline N1D-MeIm at 2130-2125 cm-1. The NH in-plane deformation vibrations of N1H-MeIm, N3H-MeIm, and MeImH2+ occur in the two frequency regions, 1500-1420 and 1200-1150 cm-1 (ν11 and ν16 for N1H-MeIm, ν11 and ν16 for N3H-MeIm, and ν10, ν13, ν17 and ν18 for MeImH2+), as a result of the splitting by coupling mainly with the ring stretching vibrations. Because the higher frequency bands are located in the complex band cluster (Figure 3A, Ba,b, Ca,b), it is rather difficult to identify the bands in the observed spectra. By contrast, the lower frequency bands are found isolated from the neighboring bands and thus easy to recognize. The latter NH bands are known to upshift upon hydrogen bonding,42 and hence, the observed frequencies for aqueous solutions and a crystalline form are considerably higher than the calculated ones (Tables 2, 4, and 6). Upon deuteration, N1D- and N3D-MeIm give the ND deformation vibrations at the calculated frequencies of 828 and 851 cm-1, respectively (ν21, Tables 3 and 5). In MeImD2+, the N1D and N3D deformation vibrations contribute to the four modes ν20-23 (978-841 cm-1 by calculation) that are coupled mainly with ring-deformation vibrations. In the observed spectra, weak, broad FTIR bands at 900 cm-1 of N1(3)D-MeIm (Figure 3Bc) and at 983, 919, and 879 cm-1 of MeImD2+ (Figure 3Cc) were temporarily assigned to these ND vibrations. The calculations showed that the NH out-of-plane wagging modes give rise to strong IR bands near 500 cm-1 in N1Hand N3H-MeIm (ν27, Table 2, 4) and near 700 cm-1 in MeImH2+ (ν26,27, Table 6). Van Bael et al.49 showed that the out-of-plane wagging vibration of the N-H group of imidazole upshifts more than 200 cm-1 upon its hydrogen bonding to a water oxygen. The broad features around 930 cm-1 in the IR spectrum of crystalline N1H-MeIm (Figure 3Aa), around 800 cm-1 in N1(3)H-MeIm in aqueous solution (Figure 3Ba), and around 950 cm-1 in MeImH2+ (Figure 3Ca) are the candidates for the NH wagging modes.

4256 J. Phys. Chem. B, Vol. 104, No. 17, 2000

Hasegawa et al.

Figure 2. FTIR and Raman spectra in the higher frequency region of different protonation and N-deuteration forms of 4-MeIm: (A) polycrystalline 4-MeIm (N1H-MeIm) (a, FTIR; b, Raman); (B) 4-MeIm in H2O (mixture of N1H- and N3H-MeIm) (a, FTIR; b, Raman) and in D2O (mixture of N1D- and N3D-MeIm) (c, FTIR; d, Raman); (C) MeImH2+ in HCl (a, FTIR; b, Raman), and MeImD2+ in DCl (c, FTIR; d, Raman); and (D) MeIm- in NaOH (a, FTIR; b, Raman), and in NaOD (c, FTIR; d, Raman). For the FTIR spectra, H(D)2O bands were subtracted from the original spectra. Raman measurements were performed with 488 nm excitation. The FTIR and Raman spectra were measured at room temperature with a 2 cm-1 resolution.

2. C-H Vibrations. The C2H and C5H stretching bands occur between 3170 and 3070 cm-1 with medium-to-strong intensities in both the IR and the Raman spectra (Figure 2A-D). These bands are virtually insensitive to N-deuteration, being consistent with the calculated results (Tables 2-7). The doublet at 3118 and 3112 cm-1 in crystalline N1H-MeIm is probably due to Fermi resonance. The C2H and C5H deformation vibrations couple with the various ring stretching vibrations and widely spread into the modes in the 1600-1000 cm-1 region. Among them, the modes that have major contributions from the CH deformation vibrations are ν15 of N1H(D)-MeIm observed at 1234-1227 (1222) cm-1, ν14 of N3H(D)-MeIm at 1258 (1252) cm-1, ν15 and ν17 of MeImH2+ at 1297-1296 and 1199 cm-1, ν15 and ν16 of MeImD2+ at 1255 and 1239 cm-1, and ν14 of MeIm- at 1217 cm-1.

The CH out-of-plane wagging vibrations show two prominent, relatively broad FTIR bands at 860-827 and 799-764 cm-1 in all of the forms (Figure 3A-D). The corresponding Raman bands have only weak intensities or are not observable, being consistent with the calculated results (Tables 2-8). In H(D)2O solutions, FTIR bands due to the different tautomers are not resolved, probably because of the broad band features (Figure Ba,c). 3. CH3 Vibrations. The two CH3 asymmetric stretching bands occur at 2987-2974 and 2960-2928 cm-1, and the CH3 symmetric stretching band occurs at 2940-2918 cm-1 (Figure 2A-D). These stretching frequencies of the CH3 group are similar to those of toluene.50 In particular, the symmetric vibration, which usually occurs at 2885-2865 cm-1 in aliphatic hydrocarbons, is known to give a band at a higher frequency of 2930-2920 cm-1 when the CH3 group is attached to an



Figure 3. FTIR and Raman spectra in the lower frequency region of different protonation and N-deuteration forms of 4-MeIm: (A) polycrystalline 4-MeIm (N1H-MeIm) (a, FTIR; b, Raman); (B) 4-MeIm in H2O (mixture of N1H- and N3H-MeIm) (a, FTIR; b, Raman) and in D2O (mixture of N1D- and N3D-MeIm) (c, FTIR; d, Raman); (C) MeImH2+ in HCl (a, FTIR; b, Raman), and MeImD2+ in DCl (c, FTIR; d, Raman); and (D) MeIm- in NaOH (a, FTIR; b, Raman), and in NaOD (c, FTIR; d, Raman). For the FTIR spectra, H(D)2O bands were subtracted from the original spectra. Raman measurements were performed with 488 nm excitation. The FTIR and Raman spectra were measured at room temperature with a 2 cm-1 resolution.


Hasegawa et al.

TABLE 2: Observed and Calculated Vibrational Frequencies (cm-1) and Assignments for N1-Protonated 4-MeIm observeda FTIR

Raman

FTIR (crystal)

Raman (crystal)

calculatedb

assignment (PED)c

∼2800s,br

∼2900br 3148sh 3118/3112s 2978m 2942m 2917s 1567m 1508m 1480w 1446m

3599(49,141) 3219(1,84) 3192(5,95) 3058(21,65) 3035(20,91) 2981(31,162) 1579(18,7) 1502(18,7) 1464(6,24) 1449(5,18) 1431(22,10)

N1H st (99) C5H st (99) C2H st (99) CH3 as st (99) CH3 as st (100) CH3 s st (99) C4C5 st (55), C4C6 st (14) C2N3 st (44), C2H def (24) CH3 as def (84) CH3 as def (92) N1H def (33), N1C2 st (32), C5N1 st (12) CH3 s def (86) C2N3 st (30), N3C4 st (22), C5N1 st (10) N3C4 st (34), C4C6 st (16), C5N1 st (12) C5H def (36), C2H def (35), C5N1 st (12) N1C2 st (51), N1H def (33) C5N1 st (44), C5H def (27), N1H def (7) CH3 rock (77) CH3 rock (53), C4C5 st (18), ring def 2 (12) N3C4 st (32), ring def 2 (26), CH3 rock (14) ring def 1 (81) C2H wag (76), ring tor 2 (14) C5H wag (70), ring tor 1 (13), C2H wag (11) ring tor 2 (55), ring tor 1 (24), C4C6 wag (15) C4C6 st (45), ring def 2 (41) ring tor 1 (63), ring tor 2 (18), C4C6 wag (17) N1H wag (67), ring tor 2 (23) C4C6 def (82), CH3 rock (13) C4C6 wag (72), ring tor 1 (13) CH3 twist (86)

ν1 ν2 ν3 ν4 ν5 ν6 ν7 ν8 ν9 ν10 ν11

3165m 3127s 2984sh 2956m 2926s 1575s 1490s 1451s 1451s 1423sh

3130w 2987w 2960w 2930s 1573m 1490m 1451m 1451m 1424m

1580s 1516w 1476s 1444s 1492sh?

ν12 ν13

1390m 1304m

1388m 1301s

1382m 1303m

1383m 1302vs

1392(1,17) 1326(6,17)

ν14

1265s

1262sh

1265s

1263s

1268(13,8)

ν15

1229s

1227m

1233m

1234s

1225(9,7)

ν16 ν17

1161m,br 1087s

1163m,br 1085w

1191w 1088s

1191m 1087m

1112(6,12) 1075(28,4)

ν18 ν19

1043w 995m

993m

1044w 997s

996s

1046(3,1) 983(9,4)

ν20

975m

975w

979m

977w

954(7,3)

ν21 ν22 ν23

941s? 827s,br 764m,br

935w

928m 827/820s 761s

926m 825w 773/756w

926(0,2) 803(20,0) 724(13,1)

669m

668m

674(0,1)

657w 626s

659vs 629w

650(3,6) 639(2,0)

345m 282s

502(105,3) 330(6,1) 261(9,2) 105(1,0)

ν24 ν25 ν26

664s 619s

ν27 ν28 ν29 ν30

∼800br?

660vs

∼930br? 346w 270w

a Observed vibrational frequencies in H O solution in a polycrystalline form. Approximate relative intensities: vs, very strong; s, strong; m, 2 medium; w, weak; sh, shoulder; br, broad. b Calculated vibrational frequencies were scaled with a uniform scaling factor of 0.98. IR intensities and Raman scattering activities in parentheses are calculated in km mole-1 and Å4/amu, respectively. c Potential energy distributions (%) are given in parentheses. Abbreviations for the internal coordinates: st, stretching; def, deformation; rock, rocking; wag, wagging; tor, torsion; twist, twisting; as, asymmetric; s, symmetric. Definitions of the ring deformation (ring def 1 and ring def 2) and torsion (ring tor 1 and ring tor 2) are followed by those in Majoube et al.42

aromatic ring.51 The calculations showed that the CH3 stretching vibrations are not influenced by N-deuteration (Tables 2-7). Thus, the bands observed commonly in H2O and D2O solutions (or HCl and DCl; NaOH and NaOD) are assigned to the CH3 modes. The calculations also indicated that in MeImH(D)2+ all the three CH3 stretching vibrations have virtually no IR intensities. This was experimentally verified in the FTIR spectrum of MeImD2+ (Figure 2Cc). In the IR spectrum of MeImH2+, however, this region could not be analyzed well because of the presence of several bands possibly due to the NH stretches, overtones or combinations. The bands at 2875-2862 and 27682736 cm-1 observed in all the forms are probably due to the overtones of the CH3 deformation vibrations.50,51 The CH3 asymmetric and symmetric deformation bands are observed at 1480-1444 and 1394-1382 cm-1, respectively (Tables 2-8). The asymmetric bands have relatively strong IR and medium Raman intensities, whereas the symmetric bands are weak to medium in both IR and Raman intensities. One of the two CH3 rocking vibrations is observed at 10491043 cm-1 as a weak IR band, irrespective of the protonation

(deuteration) forms. The other CH3 rocking vibration is coupled with the ring stretching (C4C5 and N3C4 stretches) and deformation vibrations and splits into two bands at 1022-973 cm-1 (Tables 2-7), with the exception of MeIm-, in which this CH3 rocking mostly contributes to a single band at 978974 cm-1 (Table 8). The C-Me (C4C6) stretching bands are observed at 664652 cm-1 with strong Raman and medium IR intensities in all the forms (Figure 3A-D). In the Raman spectra, this C-Me stretching mode gives rise to a sole prominent band in the 900400 cm-1 region. The C-Me in-plane deformation and out-ofplane wagging bands are observed at 347-327 and 282-257 cm-1, respectively, in all of the Raman spectra. 4. Ring Vibrations. The ring CC and CN stretching vibrations occur in the wide range from 1640 to 900 cm-1 (Tables 2-8). They are often coupled with each other and with various deformation vibrations, generating complex mode features. The highest frequency mode among them is the C4C5 stretching vibration that occurs at 1635-1532 cm-1 (Tables 2-8). The high frequencies of the C4C5 vibrations are consistent with the



TABLE 3: Observed and Calculated Vibrational Frequencies (cm-1) and Assignments for N1-deuterated 4-MeIm observeda FTIR Raman

calculated

b

ν1 ν2 ν3 ν4 ν5 ν6 ν7

3155m 3129s 2987m 2955m 2929s

3154m 3131m 2986w 2956m 2931s

1569s

1570s

3219(1,87) 3193(4,97) 3058(21,66) 3035(20,91) 2981(31,162) 2644(36,64) 1574(15,9)

ν8

1485s

1485m

1496(19,6)

ν9 ν10 ν11 ν12

1451s 1451s 1390w 1363m

1452m 1452m 1391m 1364sh

1462(12,24) 1449(4,18) 1392(0,20) 1356(4,4)

ν13

1305m

1303s

1322(8,17)

ν14

1259s

1257m,sh

1264(11,8)

ν15

1223s

1222m

1222(8,8)

ν16

1097m,sh

1094s

1079(18,9)

ν17 ν18

1043w 1017s

1016m

1046(3,1) 1002(17,9)

ν19

978m

ν20

943s

978m

963(8,1) 916(1,2)

ν21

900w

828(4,2)

ν22

832s,br

803(18,0)

ν23

766m,br

724(12,1)

ν24

670(1,1)

ν25

668s?

662s

648(3,7)

ν26

624m

626w

615(6,1)

ν27

390(49,0)

ν28

347w

327(6,1)

ν29

259w

258(12,2)

ν30

105(1,0)

TABLE 4: Observed and Calculated Vibrational Frequencies (cm-1) and Assignments for N3-protonated 4-MeIm observeda

assignment (PED)c C5H st (99) C2H st (99) CH3 as st (99) CH3 as st (100) CH3 s st (99) N1D st (98) C4C5 st (55), C4C6 st (15) N2C3 st (47), C2H def (24) CH3 as def (80) CH3 as def (92) CH3 s def (87) N1C2 st (42), C5N1 st (26), N1D def (15) C2N3 st (28), N3C4 st (25) N3C4 st (30), C4C6 st (16), C5N1 st (15) C5H def (42), C2H def (35) N1C2 st (32), C5N1 st (28), C5H def (16) CH3 rock(77) CH3 rock (30), C4C5 st (20), ring def 2 (15) CH3 rock (39), N3C4 st (30), ring def 1 (12) ring def 1 (63), ring def 2 (15) N1D def (67), ring def 1 (11) C2H wag (76), ring tor 2 (14) C5H wag (70), ring tor 1 (13), C2H wag (11) ring tor 2 (41), ring tor 1 (35), C4C6 wag (19) C4C6 st (45), ring def 2 (42) ring tor 1 (43), ring tor 2 (43) N1D wag (83), ring tor 1 (11) C4C6 def (82), CH3 rock (13) C4C6 wag (73), ring tor 1 (12) CH3 twist (86)

a Observed vibrational frequencies in D O solution. See footnote a 2 to Table 2 for approximate relative intensities. b Calculated vibrational frequencies were scaled with a uniform scaling factor of 0.98. IR intensities and Raman scattering activities in parentheses are calculated in km mole-1 and Å4/amu, respectively. c Potential energy distributions (%) are given in parentheses. Abbreviations for the internal coordinates: st, stretching; def, deformation; rock, rocking; wag, wagging; tor, torsion; twist, twisting; as, asymmetric; s, symmetric. Definitions of the ring deformation (ring def 1 and ring def 2) and torsion (ring tor 1 and ring tor 2) are followed by those in Majoube et al.42

optimized geometries, in which the double-bond character of the C4-C5 bond is conserved in all the protonation forms (Table

FTIR

Raman

calculatedb

assignment (PED)c N3H st (99) C2H st (91) C5H st (91) CH3 as st (91) CH3 as st (99) CH3 s st (91) C4C5 st (44), C4C6 st (14), N3H def (10) N1C2 st (32), CH3 as def (19), C2H def (18) CH3 as def (53), N1C2 st (18), C2H def (6) CH3 as def (92) CH3 s def (28), N3H def (22), C2N3 st (15) CH3 s def (52), N3C4 st (10), C4C5 st (7) N1C2 st (22), N3C4 st (16), C5N1 st (15) C5H def (48), C2H def (27), C2N3 st (6) C5N1 st (25), C2H def (15), C4C6 st (14) C2N3 st (43), N3H def (36), C2H def (5) C5N1 st (57), C5H def (15), C2H def (8) CH3 rock(80) ring def 2 (29), C4C5 st (17), CH3 rock (16) CH3 rock (55), N3C4 st (17), ring def 1 (11) ring def 1 (66), ring def 2 (14) C5H wag (85), ring tor 1 (10) C2H wag (84), ring tor 2 (14) ring tor 2 (73), ring tor 1 (22) C4C6 st (43), ring def 2 (38), C4C5 st (6) ring tor 1 (66), C4C6 wag (21), ring tor 2 (6) N3H wag (65), ring tor 2 (20), ring tor 1 (8) C4C6 def (82), CH3 rock(11) C4C6 wag (74), ring tor 1 (8), CH3 rock (7) CH3 twist (89)

ν1 ν2 ν3 ν4 ν5 ν6 ν7

3127s 3127s 2984sh 2956m 2926s 1594s

3130w 3130w 2987w 2960w 2930s 1593m

3588(39,100) 3193(1,129) 3187(12,55) 3067(11,62) 3010(26,99) 2966(44,194) 1594(13,13)

ν8

1490s

1490m

1494(16,15)

ν9

1451s

1451m

1461(16,29)

ν10 ν11

1451s 1423sh

1451m 1424m

1453(7,19) 1403(16,21)

ν12

1390m

1388m

1393(5,11)

ν13

1344w

1340m

1354(4,18)

ν14

1258sh

1258s

1257(1,9)

ν15

1229s

1231sh

1235(12,5)

ν16

1153m,br

1151m,br

1091(11,6)

ν17

1104s

1101m

1126(12,7)

ν18 ν19

1043w 1013w

1011m

1044(2,1) 1008(10,9)

ν20

975m

975w

965(1,2)

ν21

941s

935w

910(13,3)

ν22

827s,br

832(17,2)

ν23

764m,br

787(14,1)

ν24

668(3,1)

ν25

664s

ν26

619s

637(16,0)

ν27

∼800br?

515(79,2)

660vs

647(1,7)

ν28

334w

317(3.1)

ν29

270w

245(1,2)

ν30 a

125(0,1)

Observed vibrational frequencies in H2O solution. See footnote a to Table 2 for approximate relative intensities. b Calculated vibrational frequencies were scaled with a uniform scaling factor of 0.98. IR intensities and Raman scattering activities in parentheses are calculated in km mole-1 and Å4/amu, respectively. c Potential energy distributions (%) are given in parentheses. Abbreviations for the internal coordinates: st, stretching; def, deformation; rock, rocking; wag, wagging; tor, torsion; twist, twisting; as, asymmetric; s, symmetric. Definitions of the ring deformation (ring def 1 and ring def 2) and torsion (ring tor 1 and ring tor 2) are followed by those in Majoube et al.42

4260 J. Phys. Chem. B, Vol. 104, No. 17, 2000 TABLE 5: Observed and Calculated Vibrational Frequencies (cm-1) and Assignments for N3-deuterated 4-MeIm observeda FTIR

Raman

calculatedb

assignment (PED)c C2H st (92), C5H st (7) C5H st (92), C2H st (7) CH3 as st (92) CH3 as st (100) CH3 s st (91) N3D st (98) C4C5 st (51), C4C6 st (16), C5H def (11) N1C2 st (35), CH3 as def (20), C2H def (19) CH3 as def (55), N1C2 st (17), C2H def (7) CH3 as def (93) CH3 s def (88) N3C4 st (41), ring def 2 (10), N1C2 st (10) C2N3 st (34), N1C2 st (14), C5N1 st (13) C5H def (53), C2H def (22), C5N1 st (10) C2H def (25), C2N3 st (20), C5N1 st (17) C5N1 st (59), C5H def (17), ring def 2 (9) CH3 rock(81) CH3 rock (34), ring def 2 (24), C4C5 st (18) CH3 rock (36), N3C4 st (16), ring def 1 (13) ring def 1 (58), ring def 2 (15), N3C4 st (10) N3D def (66), ring def 1 (15), C2N3 st (7) C5H wag (86), ring tor 1 (11) C2H wag (84), ring tor 2 (14) ring tor 2 (56), ring tor 1 (36) C4C6 st (40), ring def 2 (38), N3C4 st (7) ring tor 1 (47), ring tor 2 (28), C4C6 wag (19) N3D wag (85), ring tor 2 (10) C4C6 def (82), CH3 rock (11) C4C6 wag (76), ring tor 1 (8), CH3 rock (8) CH3 twist (90), C4C6 wag (9)

ν1

3129s

3131m

3194(1,131)

ν2

3129s

3131m

3187(12,55)

ν3 ν4 ν5 ν6 ν7

2987m 2955m 2929s

2986w 2956m 2931s

1575m,sh

1575m,sh

3067(11,62) 3010(26,99) 2966(44,194) 2636(29,45) 1579(6,15)

ν8

1485s

1485m

1491(16,14)

ν9

1451s

1452m

1460(16,32)

ν10 ν11 ν12

1451s 1390w 1369m

1452m 1391m 1370m

1453(7,19) 1396(2,22) 1364(2,1)

ν13

1323w

1322s

1322(9,25)

1252m

1254(1,10)

ν14 ν15

1198w

1198w

1220(13,2)

ν16

1104s

1101m,sh

1125(13,7)

ν17 ν18

1043w 1006sh

1006m

1044(2,1) 1000(9,9)

ν19

978m

978m

962(4,2)

ν20

943s

914(15,4)

ν21

900w

851(7,4)

ν22

832s,br

831(13,2)

ν23

766m,br

786(12,1)

ν24

668s

665(12,1)

ν25

655sh

655m,sh

639(1,6)

ν26

624m?

626w

616(0,1)

ν27

399(46,0)

ν28

337w

312(3,1)

ν29

259w

244(0,2)

ν30

124(0,0)

a Observed vibrational frequencies in D O solution. See footnote a 2 to Table 2 for approximate relative intensities. b Calculated vibrational frequencies were scaled with a uniform scaling factor of 0.98. IR intensities and Raman scattering activities in parentheses are calculated in km mole-1 and Å4/amu, respectively. c Potential energy distributions (%) are given in parentheses. Abbreviations for the internal coordinates: st, stretching; def, deformation; rock, rocking; wag, wagging; tor, torsion; twist, twisting; as, asymmetric; s, symmetric. Definitions of the ring deformation (ring def 1 and ring def 2) and torsion (ring tor 1 and ring tor 2) are followed by those in Majoube et al.42

Hasegawa et al. 1). The frequency, band intensity, and N-deuteration shift are significantly affected by the protonation state (see below). The second high-frequency band in the ring vibrations of the neutral forms is the C2N3 stretch in N1H(D)-MeIm and the N1C2 stretch in N3H(D)-MeIm (ν8, Tables 2-5). The frequencies of these vibrations are higher than other CN stretching vibrations because of their double-bond character in each form (Table 1). In H(D)2O solution, the strong FTIR and medium Raman bands at 1490 (1485) cm-1 are probably assigned to these vibrations, N1H(D)- and N3H(D)-MeIm, overlapping each other (Figure 3B). In MeImH(D)2+, in which both the N1C2 and the C2N3 bonds have similar lengths of ∼1.33 Å (Table 1), their stretching vibrations couple with each other and give rise to a band at 1533 (1518) cm-1 (ν9, Table 6, 7), the frequency of which is even higher than those of the neutral forms. This mode has only weak intensity both in FTIR and in Raman spectra (Figure 3C). In MeIm-, which has longer N1C2 and C2N3 lengths of ∼1.35 Å (Table 1), the corresponding N1C2 and C2N3 vibration (ν9, Table 8) occurs at much lower frequency and is coupled with the CH3 symmetric deformation and the CH deformation. The very strong FTIR and medium Raman bands at 1439 cm-1 are assigned to this mode (Figure 3D). The band cluster in the 1370-1200 cm-1 region comprises three to four modes of the mixed CN stretching and CH deformation vibrations (ν13-15 in N1H- and N3H-MeIm; ν12-15 in N1D- and N3D-MeIm; ν15-17 in MeImH2+; and ν14-16 in MeImD2+; ν11-14 in MeIm-; Tables 2-8). The way of mixing is rather complex, and the mode structures are not well conserved among different forms. The band at 1106-1087 cm-1, which is rather isolated from the neighboring bands, has the main contribution from the C5N1 stretching vibration (Tables 2-8). This band has strong IR and medium-to-strong Raman intensities (Figure 3A-D). The band frequency and the N-deuteration shift are sensitive to the protonation state (see below). The two bands at 1022-995 and 989-973 cm-1 include considerable contribution of the C4C5 and N3C4 stretching vibrations, respectively, as well as the CH3 rocking and the ring deformation vibrations (Tables 2-8). The higher-frequency band is highly sensitive to the protonation state and N-deuteration (see below), whereas the lower-frequency band seems insensitive. The nearly pure ring-deformation mode occurs at 943-923 cm-1 as a relatively strong IR and a weak Raman band in all the forms (Figure 3A-D), with the exception of MeImD2+. In MeImD2+, the ring deformation is significantly coupled with the ND deformation vibration and spreads into several bands at 990-870 cm-1 (ν20-23, Table 7). The two-ring torsion modes occur at 669-619 cm-1 in all the forms (Tables 2-8). As shown by the calculation, these torsion modes are Raman inactive. Hence, the C-Me stretching mode, which gives a strong Raman band at a close position (see above), can be easily distinguished from the ring torsion bands. Interestingly, either of the two torsion modes has strong IR intensity. In most of the forms, this stronger IR band is the lower frequency mode at 629-619 cm-1, which occurs below the C-Me stretching band. In N3D-MeIm and MeIm-, the higher frequency band at 668 cm-1 is strong (Figure 3B,D) and occurs above the C-Me band. IR and Raman Markers of the Protonation State. 1. C4C5 Stretching Band. The C4C5 stretching band near 1600 cm-1 has been known as a marker of the protonation state, especially of neutral tautomers, of histidine and 4-MeIm.2,24,26-28,42 Table 9 summarizes the observed and calculated vibrational frequen-



TABLE 6: Observed and Calculated Vibrational Frequencies (cm-1) and Assignments for the Imidazolium Form of 4-MeIm (4-MeImH2+) observeda FTIR ν1 ν2 ν3 ν4 ν5 ν6 ν7 ν8 ν9 ν10 ν11 ν12 ν13 ν14 ν15 ν16 ν17 ν18 ν19 ν20 ν21 ν22 ν23 ν24 ν25 ν26 ν27 ν28 ν29 ν30 ν31 ν32 ν33

3156s 3156s

1633vs 1533w 1491sh 1466m 1466m 1447sh 1394m 1297w 1267m 1199m,br 1182m,br 1088s 1049w 1007w 973m 925m 836s,br 794sh ∼950br? ∼950br? 659m

Raman

3160m 3160m 2933s 1634m 1534w 1487s 1440m 1393m 1296w 1267s 1199s,br 1183s,br 1088m 1005m 973m 923w

657s

628s 337w 267w

calculatedb

assignment (PED)c

3562(96,104) 3553(252,5) 3249(14,80) 3243(49,45) 3097(0,57) 3058(1,80) 2997(1,164) 1636(66,7) 1537(11,1) 1474(8,38) 1461(17,17) 1447(15,15) 1412(15,15) 1401(4,13) 1279(2,3) 1255(6,8) 1178(7,10) 1150(24,9) 1081(21,2) 1047(4,0) 999(3,5) 964(4,2) 919(3,3) 844(0,0) 799(39,0) 721(186,0) 677(0,1) 642(0,6) 633(0,1) 617(36,1) 324(1,1) 257(7,2) 110(0,0)

N1H st (87), N3H st (12) N3H st (87), N1H st (12) C5H st (93), C2H st (6) C2H st (93), C5H st (6) CH3 as st (95) CH3 as st (100) CH3 s st (95) C4C5 st (47), N3H def (12), C4C6 st (11) N1C2 st (36), C2N3 st (29), C2H def (24) N1H def (33), C5N1 st (15), N1C2 st (14) CH3 as def (62), CH3 rock (9), N3C4 st (7) CH3 as def (91) N3H def (28), CH3 as def (18), C4C5 st (13) CH3 s def (87) C5H def (39), C5N1 st (12), N3C4 st (9) N3C4 st (24), C4C6 st (19), C5N1 st (18) C2H def (39), N1C2 st (27), N1H def (25) N3H def (32), C2N3 st (23), N1C2 st (11) C5N1 st (54), C5H def (19), N1H def (8) CH3 rock (75), CH3 as def (9) CH3 rock (42), ring def 2 (20), C4C5 st (16) CH3 rock (27), ring def 1 (27), N3C4 st (23) ring def 1 (61), ring def 2 (15), N3C4 st (11) C2H wag (50), C5H wag (16), ring tor 2 (15) C5H wag(64), C2H wag(13), ring tor 1 (10) N3H wag(30), N1H wag(30), C2H wag (30) N3H wag(50), N1H wag(46) C4C6 st (45), ring def 2 (39), N3C4 st (6) ring tor 1 (65), C4C6 wag (24) ring tor 2 (71), N1H wag(16) C4C6 def (83), CH3 rock (10) C4C6 wag (66), ring tor 1 (16), CH3 rock (8) CH3 twist (90)

a Observed vibrational frequencies in HCl solution. See footnote a to Table 2 for approximate relative intensities. b Calculated vibrational frequencies were scaled with a uniform scaling factor of 0.98. IR intensities and Raman scattering activities in parentheses are calculated in km mole-1 and Å4/amu, respectively. c Potential energy distributions (%) are given in parentheses. Abbreviations for the internal coordinates: st, stretching; def, deformation; rock, rocking; wag, wagging; tor, torsion; twist, twisting; as, asymmetric; s, symmetric. Definitions of the ring deformation (ring def 1 and ring def 2) and torsion (ring tor 1 and ring tor 2) are followed by those in Majoube et al.42

cies, N-deuteration shifts, and assignments with PED of this mode for all of the four protonation forms. In the neutral forms, the observed IR and Raman frequencies are higher in N3HMeIm than in N1H-MeIm, by about 20 cm-1. Upon protonation (MeImH2+), the band shifts to the higher frequency by about 60 cm-1 from the frequency of the neutral N1H form. On the contrary, upon deprotonation (MeIm-), the band shifts to the lower frequency by about 40 cm-1 from the N1H-MeIm frequency. The large increase and decrease in the frequencies upon protonation and deprotonation, respectively, are consistent with the differences in the optimized distances of the C4-C5 bond (Table 1). The C4-C5 distance is 0.0062 Å shorter and 0.0179 Å longer in MeImH2+ and MeIm-, respectively, than in the neutral N1H form. However, the C4C5 frequency of N3H-MeIm, which is about 20 cm-1 higher than that of N1HMeIm, cannot be explained simply by the C4-C5 distance because that of the N3H form is slightly longer (0.0004 Å) than that of the N1H form. This frequency gap may be derived from the difference in the coupling with other vibrations. One possibility is that the larger coupling with the NH deformation in N3H-MeIm pushes its C4C5 frequency upward. In fact, upon decoupling of the NH vibration by N-deuteration, the frequencies of the N1H and N3H forms fall in the close values (Table 9). The calculations reproduced the frequency differences among the protonation forms well (Table 9). The observed N-deuteration shift of the C4C5 stretching frequency is in the order: MeIm- (0 cm-1) > N1H-MeIm

(∼-5 cm-1) > N3H-MeIm (∼-20 cm-1) > MeImH2+ (∼30 cm-1) (Table 9). This relationship was again well-reproduced in the calculation (Table 9). The frequency downshifts upon N-deuteration are probably caused by decoupling of the NH deformation vibration(s). The PED value of the NH deformation in each protonation form, i.e., 18, 10, 4, and 0% in MeImH2+, N3H-MeIm, N1H-MeIm, and MeIm-, respectively, is consistent with the extent of downshift (Table 9). In the Raman spectra, the C4C5 bands occur with mediumto-strong intensity in all of the forms (Figure 3A-D). In the FTIR spectra, on the other hand, MeImH2+ and MeImD2+ have very strong bands (Figure 3C), whereas MeIm- has only a weak band (Figure 3D). The calculation also expressed this intensity relationship. The IR intensity of the C4C5 band in MeImH2+ is 3.7 times higher than in N1H-MeIm, whereas no IR intensity is expected in MeIm- (Table 6, 8). Thus, the C4C5 stretching band is an excellent marker of the protonation state by its frequency, N-deuteration shift, and IR intensity. 2. C5N1 Stretching Band. The FTIR band around 1100 cm-1 has been used as a marker of the protonation state of histidine.32,36 This band is especially useful in IR studies because the band has relatively strong IR intensity in all the forms and does not overlap the large amide I (∼1650 cm-1) and II (∼1550 cm-1) bands of peptide backbones. According to the calculation, all of the protonation forms have a similar mode structure: the C5N1 stretching vibration coupled with the C5H deformation (Table 10). As summarized in Table 10, the observed band

4262 J. Phys. Chem. B, Vol. 104, No. 17, 2000 TABLE 7: Observed and Calculated Vibrational Frequencies (cm-1) and Assignments for the N-deuterated Imidazolium Form of 4-MeIm (4-MeImD2+) observeda FTIR

Raman

calculatedb

ν1 ν2 ν3 ν4 ν5 ν6 ν7 ν8

3151s 3151s

1605vs

3155m 3249(15,85) 3155m 3243(52,44) 3097(0,57) 3058(1,79) 2940s 2997(1,164) 2623(28,46) 2611(165,13) 1606s 1612(43,13)

ν9

1518w

1520w 1524(7,1)

ν10 1453s ν11 1453s ν12 1408m

1452w 1455(14,18) 1452w 1447(15,15) 1409vs 1409(1,15)

ν13 1391m

1391m 1398(2,11)

ν14 1366m

1368s

ν15 1255m

1255m 1250(0,9)

ν16 1239m

1233(7,2)

1355(3,13)

ν17 1106s

1106s

ν18 1049m ν19 1022m

1047(4,0) 1018w 1015(8,5)

ν20 983w

989m

1103(11,10)

978(4,5)

ν21

914(0,2)

ν22 919w

903(3,2)

ν23 879sh

841(23,1)

ν24 860m,br

834(23,0)

ν25 796m,br

793(6,0)

ν26 652m ν27 ν28 627s ν29 ν30 ν31 ν32 ν33

652s

332w 264w

635(0,6) 634(0,1) 617(28,1) 531(100,0) 515(0,0) 317(1,1) 252(7,2) 110(0,0)

assignment (PED)c C5H st (91), C2H st (7) C2H st (91), C5H st (7) CH3 as st (95) CH3 as st (100) CH3 s st (95) N1D st (71), N3D st (26) N3D st (71), N1D st (26) C4C5 st (56), C4C6 def (14), C5H def (9) N1C2 st (36), C2N3 st (29), C2H def (24) CH3 as def (78), CH3 rock (9) CH3 as def (91) CH3 s def (30), N1C2 st (19), C5N1 st (11), C2N3 st (10) CH3 s def (60), N1C2 st (11), C5N1 st (9) N3C4 st (31), C2N3 st (18), C4C5 st (11) C5H def (47), C5N1 st (21), C4C6 st (14) C2H def (33), C5H def (12), N3C4 st (10) C5N1 st (34), N1C2 st (20), C2H def (18) CH3 rock (75), CH3 def (9) CH3 rock (44), C4C5 st (16), C5H def (10), ring def 2 (7) ring def 1 (18), CH3 rock(16), N3D def (15), N3C4 st (12) ring def 1 (29), N1D def (21), N3D def (18) N3D def (23), N3C4 st (20), ring def 1 (17) N1D def (40), ring def 1 (21), N3D def (21) C2H wag (49), C5H wag (26), ring tor 2 (15) C5H wag (53), C2H wag (28), ring tor 1 (9) C4C6 st (42), ring def 2 (36) ring tor 1 (60), C4C6 wag (21), N3D wag (10) ring tor 2 (74), N1D wag(14) N1D wag (48), N3D wag (46) N3D wag (48), N1D wag (47) C4C6 def (83), CH3 rock (10) C4C6 wag (66), ring tor 1 (17), CH3 rock (8) CH3 twist (90)

a

Observed vibrational frequencies in DCl solution. See footnote a to Table 2 for approximate relative intensities. b Calculated vibrational frequencies were scaled with a uniform scaling factor of 0.98. IR intensities and Raman scattering activities in parentheses are calculated in km mole-1 and Å4/amu, respectively. c Potential energy distributions (%) are given in parentheses. Abbreviations for the internal coordinates: st, stretching; def, deformation; rock, rocking; wag, wagging; tor, torsion; twist, twisting; as, asymmetric; s, symmetric. Definitions of the ring deformation (ring def 1 and ring def 2) and torsion (ring tor 1 and ring tor 2) are followed by those in Majoube et al.42

frequency is in the order: N3H-MeIm g MeIm- > MeImH2+ g N1H-MeIm, and the N-deuteration shift is in the order: MeImH2+ (∼+20 cm-1) > N1H-MeIm (∼+10 cm-1) > N3H-MeIm ≈ MeIm- (0 cm-1).36 A similar relationship has been observed in DL-histidine in aqueous solution.36 The calculated results reproduced the observed N-deuteration shifts well (Table 10). The order of the band frequency was also

Hasegawa et al. reproduced, although the calculated frequencies of N3H-MeIm and MeIm- were a little too high. The above frequency order seems to be resulted from both the C5-N1 distance and the extent of coupling with the C5H deformation. The optimized C5-N1 distance is in the order: MeImH2+ (1.3841 Å) > N1HMeIm (1.3818 Å) > N3H-MeIm (1.3793 Å) > MeIm- (1.3722 Å) (Table 1). From these distances, the frequency orders between MeImH2+ and N1H-MeIm and between N3H-MeIm and MeIm- should be opposite. However, in N1H-MeIm and MeIm-, the relative contribution of the C5N1 stretch is lower than that found in MeImH2+ and N3H-MeIm, respectively, and instead, the contribution of the C5H deformation vibration is higher (Table 10). The C5H coupling pushes the C5N1 frequency downward (because the original frequency of the C5N1 stretch is lower than that of the C5H deformation), and thus, the high C5H contribution will decrease the frequency more, resulting in the above frequency order. The extent of upshift upon N-deuteration may be explained by the coupling with the NH deformation vibration. Because the NH coupling decreases the C5N1 frequency, its decoupling will increase the frequency. In N3H-MeIm, which showed no N-deuteration shift, there is little coupling with N3H ( MeIm- (3094-3078 cm-1). This frequency order was reproduced by the calculations (Tables 2-8) and inversely correlated to the order of the optimized C-H distances: MeIm- > N3HMeIm > N1H-MeIm > MeImH2+ (Table 1). Because the C-H stretching vibrations are not influenced by N-deuteration, the above relationship may be useful in D2O solution. In neutral MeIm and MeImH2+, the N-H deformation couples with the ring vibrations to various extents, and thus, the FTIR and Raman spectral features in the 1700-900 cm-1 region are significantly affected by N-deuteration (Figure 3B,C). By contrast, the spectra of MeIm- in aqueous solution showed


Hasegawa et al.

TABLE 9: Vibrational Frequencies (cm-1) and N-deuteration Shifts of the C4C5 Stretching Modes of the Four Protonation Forms of 4-MeIm Observed N1H-MeIm N3H-MeIm MeImH2+ MeIm-

in H2O in D2O in H2O in D2O in HCl in DCl in NaOH in NaOD

FTIR

Raman

calculatedc

assignment (PED)d

1575 1569 (-6)a 1594 [+19]b 1575 (-19) 1633 [+58] 1605 (-28) 1535 [-40] 1535 (0)

1573 1570(-3) 1593 [+20] 1575 (-18) 1634 [+61] 1606 (-28) 1532 [-41] 1533 (+1)

1579 1574(-5) 1594 [+15] 1579 (-15) 1636 [+57] 1612 (-24) 1514 [-65] 1514 (0)

C4C5 st (55), C4C6 st (14), C5H def (10), C2C3 st (4), N1H def (4) C4C5 st (44), C4C6 st (14), N3H def (10), C5H def (10) C4C5 st (47), N3H def (12), C4C6 st (11), C2H def (8), N1H def (6) C4C5 st (45), C4C6 st (14), C5H def (13)

a N-deuteration shifts (cm-1) are given in paretheses. b Frequency differences (cm-1) from the N1H form are given in square brackets. c Calculated vibrational frequencies in all the forms were scaled with a uniform scaling factor of 0.98. d See footnote c of Table 2.

TABLE 10: Vibrational Frequencies (cm-1) and N-deuteration Shifts of the C5N1 Stretching Mode of the Four Protonation Forms of 4-MeIm Observed N1H-MeIm N3H-MeIm MeImH2+ MeIm-


FTIR

Raman

calculatedc

assignment (PED)d

1087 1097 (+10)a 1104 [+17]b 1104 (0) 1088 [+1] 1106 (+18) 1101 [+14] 1101 (0)

1085 1094 (+9) 1101 [+16] 1101 (0) 1088 [+3] 1106 (+18) 1100 [+15] 1100 (0)

1075 1079 (+4) 1126 [+51] 1125 (-1) 1081 [+6] 1103 (+22) 1120 [+45] 1120 (0)

C5N1 st (44), C5H def (27), N1H def (7) C5N1 st (57), C5H def (15), C2H def (8) C5N1 st (54), C5H def (19), N1H def (8) C5N1 st (38), C5H def (31), N1C2 st (9)


TABLE 11: Vibrational Frequencies (cm-1) and N-deuteration Shifts of the Mixed Mode of Ring Deformation, CH3 Rocking, and C4C5 Stretching Vibrations of the Four Protonation Forms of 4-MeIm Observed N1H-MeIm N3H-MeIm MeImH2+ MeIm-


IR

Raman

calculatedc

assignment (PED)d

995 1017 (+22)a 1013 [+18]b 1006 (-7) 1007 [+12] 1022 (+15) 1010 [+15] 1009 (-1)

993 1016 (+23) 1011 [+18] 1006 (-5) 1005 [+12] 1018 (+13) 1008 [+15] 1007 (-1)

983 1002 (+19) 1008 [+25] 1000 (-8) 999 [+16] 1015 (+16) 989 [+6] 989 (0)

CH3 rock (53), C4C5 st (18), ring def 2 (12) ring def 2 (29), C4C5 st (17), CH3 rock (16) CH3 rock (42), ring def 2 (20), C4C5 st (16) ring def 2 (36), C4C5 st (19), ring def 1 (12)


virtually no deuteration effect, because of its deprotonated structure and insensitivity to H/D exchange of the protons hydrogen bonded to the imidazole nitrogens. Thus, this spectral insensibility to H/D exchange in the fingerprint region can be a good indication of the MeIm- form. Another potential marker of MeIm- is the relatively intense IR band of the ring torsion, which occurs at a much higher frequency (688 cm-1) than in N1H- and N3H-MeIm and MeImH2+ (627-619 cm-1) in aqueous solution. Conclusion IR and Raman markers of the protonation state of a histidine side chain were systematically studied by spectral measurements and ab initio DFT calculations for all of the protonation forms of 4-MeIm. The C4C5 stretching band near 1600 cm-1 is a good marker to distinguish between all of the protonation forms because both the vibrational frequencies and the N-deuteration shifts are largely separated from each other. The differences in the C4-C5 distance and the coupling with the NH deformation vibration are the main reason for the sensitivity to the protonation state and N-deuteration. The significant difference in IR

intensity between the imidazolium and imidazolate forms, i.e., very strong and weak absorption in the former and the latter, respectively, is also useful to distinguish these structures. The C5N1 stretching band near 1100 cm-1 is also a good marker. Although this mode is less sensitive to the difference in the protonation state than is the C4C5 stretch, combined usage of the band frequency and the N-deuteration shift will be useful to identify the protonation form. The coupling with the CH and NH deformation vibrations as well as the C5-N1 distance is responsible for the frequency change. For practical use for proteins, this band is especially advantageous to FTIR spectroscopy because the band occurs in the region free from the IR absorptions of polypeptide main chains. The band that arises from the mixed mode of the CH3 rocking (around 1000 cm-1), C4C5 stretching and ring-deformation vibrations, is also sensitive to the protonation state, but the large CH3 rocking contribution makes this mode less secure in its practical use for the histidine side chain. Some intense Raman bands in the 1450-1250 cm-1 region, which have been used as Raman markers, i.e., the bands at 1408 cm-1 in MeImD2+, at 1255 cm-1 in MeIm-, and at 1301 and 1258 cm-1 in neutral MeIm,

4-Methylimidazole and Its Protonation Forms are assigned to the ring vibrations, including the various CN stretches and CH deformations. The CH stretching bands at >3000 cm-1 are potential markers, and a practical use is expected in D2O solution, in which solvent absorption does not interfere with this region. The relatively intense IR band at ∼690 cm-1, due to the ring torsion, may distinguish the imidazolate anion from the other forms, which give the ring-torsion bands at ∼620 cm-1. Also, insensitivity to H/D exchange in the 1700900 cm-1 region will indicate the imidazolate form. It is likely that the above IR and Raman markers of the protonation state are influenced by metal binding and hydrogenbonding interactions. In fact, upshifts of the C4C5 bands, and some other ring bands, upon metal binding have been reported.14,18,22,24,26 Also, the effects of hydrogen bonding on a number of modes of imidazole have been found.49 Thus, for definitive identification of the protonation state of histidine residues in proteins, a couple of markers should be used together, and not only the band frequency but also the N-deuteration shift and band intensity should be taken into consideration. Detailed studies of the effects of metal binding and hydrogen bonding on the vibrational spectra of a histidine side chain will be important to establish the IR and Raman markers of these interactions. Such studies are also necessary to make the best use of the markers of the protonation state presented in this study. Theoretical studies of these issues are now under way. Acknowledgment. The authors thank Dr. Hiroshi Yoshida for valuable technical advice about calculations. This research was supported by grants for Photosynthetic Sciences, Biodesign Research Program, and Frontier Research System at the Institute of Physical and Chemical Research (RIKEN), given by the Science and Technology Agency (STA) of Japan. References and Notes (1) Blomberg, F.; Mauer, W.; Ruterjans, H. J. Am. Chem. Soc. 1977, 99, 8149. (2) Ashikawa, I.; Itoh, K. Biopolymers 1979, 18, 1859. (3) Warshel, A.; Russell, S. J. Am. Chem. Soc. 1986, 108, 6569. (4) Tu, C.; Silverman, D. N. Biochemistry 1989, 28, 7913. (5) Frey, P. A.; Whitt, S. A.; Tobin, J. B. Science 1994, 264, 1927. (6) Hays, A. A.; Vassiliev, I. R.; Golbeck, J. H.; Debus, R. J. Biochemistry 1998, 37, 11 352. (7) Sundberg, R. J.; Martin, R. B. Chem. ReV. 1974, 74, 471. (8) Regan, L. Annu. ReV. Biophys. Biomol. Struct. 1993, 22, 257. (9) El Yazal, J.; Pang, Y.-P. J. Phys. Chem. B 1999, 103, 8773. (10) Garfinkel, D.; Edsall, J. T. J. Am. Chem. Soc. 1958, 80, 3807. (11) Bellocq, A. M.; Perchard, C.; Novak, A.; Josien, M. L. J. Chim. Phys. 1965, 62, 1334. (12) Lord, R. C.; Yu, N.-T. J. Mol. Biol. 1970, 51, 203. (13) Yoshida, C. M.; Freedman, T. B.; Loehr, T. M. J. Am. Chem. Soc. 1975, 97, 1028. (14) Salama, S.; Spiro, T. G. J. Am. Chem. Soc. 1978, 100, 1105. (15) Itabashi, M.; Itoh, K. Chem. Lett. 1979, 1331. (16) Tasumi, M.; Harada, I.; Takamatsu, T.; Takahashi, S. J. Raman Spectrosc. 1982, 12, 149. (17) Chinsky, L.; Jolles, B.; Laigle, A.; Turpin, P. Y. J. Raman Spectrosc. 1985, 16, 235.

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Vibrational Spectra and Ab Initio DFT Calculations of 4

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