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Conformational structures and vibrational spectra of isolated pyrimidine nucleosides: Fourier transform infrared matrix isolation study of 2-deoxyuridine
Spectrochimica Acta Part A 59 (2003) 1959 /1973 www.elsevier.com/locate/saa
Conformational structures and vibrational spectra of isolated pyrimidine nucleosides: Fourier transform infrared matrix isolation study of 2-deoxyuridine A.Yu. Ivanov a,*, S.A. Krasnokutski b, G. Sheina a, Yu.P. Blagoi a a
Institute for Low Temperature Physics and Engineering, Ukrainian Academy of Sciences, Lenin Avenue 47, 310103 Kharkov, Ukraine b Max-Planck-Institut fur stromungsforschung, Busenstr 10, D-37073 Go¨ttingen, Germany Received 14 August 2002; received in revised form 15 November 2002; accepted 21 November 2002
Abstract The conformational structures of 2-deoxyuridine (dU) were investigated using Fourier transform infrared (FTIR) matrix isolation spectroscopy. For the first time the FTIR spectra of dU in Ar matrices were obtained in the range 4000 /200 cm 1. The stabilities of conformers were estimated by the methods HF/3-21G (p), HF/6-31G (d,p) and MP2/ 6-31G (d,p). Ab initio calculations of the infrared spectra were performed by the methods HF/3-21G (p) and HF/6-31G (d,p). The actual occupancy of conformational isomers in matrix samples was determined. It was shown that anti conformers of dU are dominant. The ribose rings of the main anti -conformers dU _a0, dU _a1 are in the C2?-endo conformation, but the ribose rings of minor anti -conformers dU_a2, dU_a3 have the C3?-endo conformation, stabilized by intramolecular hydrogen bonds O3?H O5? and O5?H O3?, accordingly. Syn -conformers of dU are stabilized by the intramolecular hydrogen bond O5?H O2 and the dominant conformation of the ribose ring is C2?-endo . # 2002 Elsevier Science B.V. All rights reserved. Keywords: FTIR spectroscopy; Matrix isolation; Nucleosides; Intramolecular H-bond; MP2
1. Introduction There is an intense interest to studies of conformational isomerism of DNA structural components such as nucleoside by various experimental and theoretical methods [1 /9]. Up to the present, the main experimental methods of the investigation of nucleosides are presented by
* Corresponding author. Fax: /380-57-233-5593. E-mail address: [email protected] (A.Yu. Ivanov).
crystallography and NMR spectroscopy. It is well known that the results of these methods depend strongly on intermolecular interactions. For example, in the crystals only one form of several possible conformers is stabilized and its choice can be affected by the nature of the solvent from which the crystallization was made [1]. Owing to interactions with the solvent, NMR spectroscopy gives no way to obtain the direct data about intramolecular hydrogen bonding of nucleosides in aqueous solutions. [4,5]. In view of these problems, up to now the conformational
1386-1425/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S1386-1425(02)00416-X
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A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
isomerism of the isolated nucleosides and the structure of their intramolecular hydrogen bonds remain unexplored questions. Hitherto, X-ray data [1] and semi-empirical calculations [3] have pointed to the possibility of the formation of intramolecular hydrogen bonds O5?H O2 in the syn -conformations of pyrimidine nucleosides (Fig. 1). Also, the formation of hydrogen bonds C6H O5? in the anti -conformers of pyrimidine nucleosides was based on the crystallographic data [1] and DFT calculations [7,8]. The first experimental data for pyrimidine nucleosides (uridine and thymidine) isolated in the low temperature Ar matrices were obtained using FTIR matrix isolation spectroscopy [10,11]. The high resolution, sensitivity and absence of the strong intermolecular interactions in the inert low temperature matrices makes the spectroscopy of matrix isolation a very useful method to study of intramolecular hydrogen bonding [12]. This allows us to show that syn -conformations of isolated uridine and thymidine are stabilized by the intramolecular H-bond O5?H O2 [10,11]. Recent DFT calculations [7 /9] were also restricted by consideration of this type intramolecular H-bond only. In the present research, for the first time FTIR spectra of 2-deoxyuridine in Ar matrices were obtained using an enhanced experimental setup. FTIR spectra of auxiliary compounds (thymidine, 1methyl-thymine, 1-methyl-uracil) were also obtained. New spectral data suggest the existence of more types of intramolecular H-bonds for the isolated pyrimidine nucleosides than considered before [7 /11]. The new experimental data were
Fig. 1. The molecular structure and atom numbering of 2deoxyuridine in the syn -conformation.
supplemented by ab initio calculations of energy of different conformers and their vibrational spectra.
2. Experimental methods The basic features of the experimental setup have been described previously [11,13/15]. For the present paper, the FTIR spectra of 2?-deoxyuridine and auxiliary compounds were obtained in the ranges: 4000 /1300 cm 1 (KBr beamsplitter (b.)), 2500 /500 cm 1 (CaF2 b.), and 500 /180 cm 1 (Mylar b.), with apodized resolution 0.4 cm 1 (the full spectra of the auxiliary compounds will be published in the future). As was pointed out, nucleosides are very thermally labile molecules and for their evaporation the special Knudsen cell with reduced molecular beam losses was used [11]. The general scheme of evaporation from this cell is presented in Fig. 2. This evaporation cell is characterized by a working vapor pressure around 10 5 Torr and Knudsen’s number of over 100. Efficiency of the new cell is enlarged by a factor of more than 500 /1000 over one of a Knudsen cell used in our previous experiments with more stable compounds [13 /15]. As is shown in Fig. 3 the geometry of the Knudsen cell and its
Fig. 2. The general scheme of matrix isolation setup based on liquid He cryostat: (1) rotating vacuum seal; (2) cryogen block with cold mirrors and QCM; (3) rotating nitrogen shield; (4) flange with indium seal; (5) Knudsen cell; (6) electrical heater of Knudsen cell; (7) Ar gas duct.
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
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K for Ar, and 5 K for Ne. To improve of optical characteristics of Ar matrices, before the deposition of matrix samples, a thin layer of pure Ar was deposited on the mirrors over the temperature range of 35/20 K [15]. As before [14], UVirradiation was used to shift the conformational equilibrium of dU in the matrix samples.
Fig. 3. The relationship of the efficiency of Knudsen cell (I ) as function of distances L between cell and cold mirror. L is in the unit R , R */ radius of outlet nozzle of Knudsen cell.
disposition to the low temperature mirror are very important for effective works. The data in Fig. 3 were obtained by the statistical Monte Carlo method, which is very advantageous for the simulation of complicated vacuum systems [16]. In comparison with previous work [11] the distance L (Fig. 3) was decreased from 4.5 to 2.5 R . The low temperature differential quartz crystal microbalance (QCM) was used for the measurements of absolute intensity of molecular beams and matrix-to-sample ratio (M/S) [13]. Owing to QCM, we have a capability to work not only with an Ar flux passing through the Knudsen cell (Fig. 2) but with an outside flux of cold Ar gas also. In the present experiments the typical intensity of molecular beams of nucleosides was about 60 ng/(s cm2) without any thermodestruction. The test experiments with the evaporation cell from previous work [13 /15] showed that thermodestruction can be easy detected by intense spectral bands due to H2O, CO2, and the narrow characteristic bands of the appropriate bases. Commercial (Sigma) substances (2?-deoxyuridine, thymidine) were used without additional purification. The model substances (1-methyl-uracil, 1-methyl-thymine) were synthesized in the Kharkov State University (Kharkov, Ukraine). Before the creation of matrix samples all substances were annealed to remove impurities just as sorbed H2O, CO2 and N2. The annealing was controlled with the help of QCM. The inert gases (Ne, Ar) were more than 99.99% pure. The matrices were deposited at the temperatures of the mirrors: namely, 11
3. Computational methods The quantum-chemical calculations of the relative energies and vibrational spectra of 2-deoxyuridine conformers have been performed by the program PC GAMESS version 6.0 [17] of the GAMESS (US) QC package [18]. The first phase of full geometry optimization of six dU conformers (Fig. 4) was performed at a restricted Hartree /Fock (HF) level with 3-21G(p) basis sets. Furthermore, the stable structures were optimized by the methods HF/6-31G(d,p) and MP2/6-31G(d,p) sequentially. For all structures vibrational spectra were obtained by the methods HF/3-21G(p), HF/6-31G(d,p). The choice of natural internal coordinates for the dU base was based on the data for uracil [19]. Other natural internal coordinates were constructed using recommendations from literature [20 /22]. The method implemented in GAMESS was used to determine the contribution of the internal coordinates into the normal modes and the calculation of the potential energy distribution (PED). Because of this, the PED for deformation vibrations of rings and some groups of ribose was presented in a simplified form, as the sum of the contributions of separate modes. For the estimations of the rotational /vibrational contribution to the Gibbs free energy G (approximation: harmonic oscillator/rigid rotor) at the HF/3-21G(p) level the standard capabilities of the GAMESS program were used. The total number of basis functions used in the calculations for dU was 204 (321G(p)), 300 (6-31G(d,p)). All calculations were performed on PC with processors Celeron-500, PIII-800, and Duron-700.
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Fig. 4. The conformational structures of 2-deoxyuridine which are stable at the different level of calculation (HF/3-21G(p), HF/631G(d,p) and MP2/6-31G(d,p)). Intramolecular H-bonds are represented with dashed lines.
4. Results and discussion 4.1. The spectral region of stretching vibrations n (OH), n (NH) The region of the stretching (n) vibration n (OH), n (NH) is very important for the identification of nucleoside conformers in matrix samples and for the determination of their occupancy. In this region, only the N3H group of dU plays no part in the intramolecular interactions of dU
conformers. The n(N3H) spectral band can be easily defined by comparison with the auxiliary compounds. As shown in Fig. 5, the frequency of the n(N3H) stretching vibration has no significant changes in the substances studied in Ar matrices: 2-deoxyuridine, 1-methyluracil, 1-methyl-thymine. But for others, four absorption bands in this region (Fig. 5) can belong to two OH groups of 2?-deoxyribose-O3?H, O5?H only (Fig. 1). This spectral pattern is valid for Ar and Ne matrices at 5 K also (Fig. 6). The magnification of the
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
Fig. 5. The FTIR spectra of 2-deoxyuridine (1), 1-methyl-uracil (2), 1-methyl-thymine (3) isolated in Ar matrices (T/12 K, M/ S /700) in the O /H, N /H stretching region (3690 /3390 cm 1).
Fig. 6. The comparison of FTIR spectra of 2-deoxyuridine in Ar (1) (T /5 K, M/S/700) and Ne matrices (2) (T /5 K, M/ S /700) in the O /H, N /H stretching region (3700 /3390 cm 1).
number of bands observed in the spectra of dU (Figs. 5 and 6) can be explained by intramolecular hydrogen bonds in the different conformational structures. Therefore, the choice of initial conformational structures for optimization (Fig. 4) was
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motivated by the possibility of formation of intramolecular hydrogen bonds. It is well known that the conformational-flexible sugar ring of nucleosides has a nonplanar puckered structure [1]. According to the data of NMR spectroscopy, C2?-endo and C3?-endo are the main equilibrium conformations of the sugar ring in solutions [1]. For contraction of the computational time we have restricted ourselves to these main conformations of the dU sugar. The transition C3?-endo 0/ C2?-endo has no effect on the structure of the Hbonds in the syn -conformers dU_s1, dU_s2 (Fig. 4), but the transition C2?-endo 0/C3?-endo in the anti -conformers furnishes the formation of the Hbonds O3?H O5?, O5?H O3? in the conformers dU_a2, dU_a3 (Fig. 4). Furthermore, only dU_a2, dU_a3 C3?-endo /anti conformations are stable. Hitherto, DFT calculations (6-31G(d) basis set) confirm the formation of the hydrogen bonds C6H O5? in anti -conformers of uridine and cytosine [7,8]. Therefore, the choice of the structure dU_a1 with the orientation of hydroxymethyl group gauche and torsion angle g (/C3? /C4?/ C5?O5?) //508 are close to the structures from literature [7,8]. We also considered the structure dU_a0 with the orientation of hydroxymethyl group gauche ( /g ://658) and the lack of any classical linear H-bonds (Fig. 4). The stabilization of the position of pyrimidine ring in this and the dU_a2, dU_a3 conformation can be determined by electrostatic interaction between atoms: C6H l/O4? and C2O l/H1?. All conformers from Fig. 4 may be considered as local minima, since they were stable on the HF/3-21G(p), HF/631G(d,p) levels of calculation and have no imaginary frequencies in the calculated spectra. The frequencies and intensities of the H-bonded OH vibrations are indicators of these conformational isomers. For the determination of their population in the matrix samples, experimental and calculated spectra are compared in the region of n (OH), n(NH), n(CH) (Table 1). It is well known that ab initio calculations overestimate the frequencies of normal modes, therefore multiplication by a scaling factor is normally required for comparison with experimental frequencies [23 / 25]. As a rule, this scaling factor is a one-off quantity for the vibrations of different molecular
1964
Conformer mode
Experiment (n cm1) I
dU_a0 (n cm 1) Ia dU_a1 (n cm1) Ia dU_a2 (n cm 1) Ia dU_a3 (n cm 1) Ia dU_s1 (n cm1) Ia
dU_s2 (n cm1) Ia
n O5?H n O3?H
3665, 4.1 3641, 2.3
3667, 48 (3673, 77) 3665, 59 (3365, 76) 3674, 63 (3672, 86) 3651, 37 (3655, 63) 3653, 30 (3659, 54) /
n hb_O3?H
3623*, 0.9*
/
/
n hb_O5?H
3609*, 0.3*
/
/
3611, 120 (3637, 107) /
n hb_O5?H
3482, 7.3
/
/
/
n N3H
3428, 10.0
n C5H
3102, 0.9
n C6H
3075, 0.6
3457, 96 (3439, 104) 3074, 4.6 (3044, 1.3) 3100, 9.7 (3071, 3)
3459, 93 (3441, 103) 3078, 4.4 (3054, 6.2) 3057, 57 (3040, 7.8)
3455, 98 (3439, 105) 3455, 98 (3437, 103) 3074, 3.8 (3044, 1) 3074, 5.5 (3045, 1.5) 3105, 8.2 (3078, 4) 3100, 9.7 (3077, 4.4)
/ / 3663, 55 (3659, 66) 3669, 44 (3680, 75) /
/
/ 3652, 30.2 (3660, 54) /
3600, 121 (3639, 85) /
/
/
3578, 344 (3628, 3549, 494 (3593, 149) 330) 3450, 99 (3434, 103) 3451, 101 (3435, 105) 3081, 2.4 (3051, 0.9) 3081, 2.1 (3052, 0.7) 3048, 3.5 (3026, 4.2) 3051, 3.4 (3029, 3.8)
I */ relative integral intensities. Ia */ absolute integral intensities [km/mol]. n hb_OH */ bands of groups involved in the intramolecular H-bonds. * */ bands in Ne matrices (in Ar matrices only one wide band with maximum 3598 cm 1 and I/1.2 was detected).
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
Table 1 The parameters of experimental (from FTIR spectra in Ar matrices) and calculated (3-21G(p), 6-31G(d,p) */ in brackets) spectral bands of 2-deoxyuridine conformers in the 3700 /3000 cm 1 region
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
groups [20,21]. In Table 1 the uniform scaling factor: k /n O5?H (exp.)/nO5?H (calc.) /0.876 calculated for n(O5?H) of conformer dU_a1 at HF/3-21G(p) was used. But with 6-31G(d,p) basis sets, a scaling factor: k /0.875 was used for O5?H, O3?H groups and 0.89 for N3H and C5H, C6H groups (Table 1). From this table we notice that the calculated frequencies of the free O5?H, O3?H and N3H groups are in good agreement with the experimental frequencies for all conformers. These calculations support our previous assignment for thymidine and uridine [10,11], where the band 3482 cm 1 (Fig. 5, Table 1) can be assigned to the H-bonded vibration O5?H O2 in the syn -conformations. In Fig. 4, two syn -conformations: dU_s1 with C3?-endo and dU_s2 with C2?-endo conformation of sugar ring are shown. The geometric parameters of the H-bonds of dU in the different basis sets (Table 2) indicate that dU_s1 has weaker H-bond than dU_s2, as the power of H-bond AH B and the shift of H-bonded vibrations is inversely related to the spacing A B [26]. According to our calculations, the vibration n (hb_O5?H) in conformer dU_s2 has a frequency about 40 cm 1 below than in dU_s1 conformer (Table 1). But we can see in our experimental spectra that the band at 3482 cm 1 has not highfrequency shoulder in Ar matrices (Fig. 5) and this is true for Ne matrices also (Fig. 6). Because of this, the occupancy of conformation dU_s1 can be neglected.
1965
The calculated frequencies and intensities of characteristic bands of conformers dU_a2, dU_a3 and their geometric parameters of H-bonds are in close agreement (Tables 1 and 2). Because of this, it is difficult to distinguish these conformers in the experimental spectra. So, in Ar matrices we can see only the wide band 3598 cm 1 with a highfrequency shoulder (Fig. 5). But in the Ne matrices two bands 3623 cm 1, 3615 cm 1 are well observed (Fig. 6). Based on the data from Table 1, it may be suggested that the band at 3610 cm1 is assigned to n(hb_O3?H) of conformer dU_a2 and 3623 cm 1 to n (hb_O5?H) of conformer dU_a3. 4.2. The spectral region of stretching vibrations n(CH) The anti conformers: dU_a0 and dU_a1 in the n(OH), n (NH) spectral region are indistinguishable practically but appreciable difference between them may occur in the region n(C5H), n (C5H). Relying on comparison with the fragment of spectra of thymidine and 1-methyl-uracil (Fig. 7), the band at 3102 cm 1 was assigned to n(C5H) and 3075 cm 1 to n (C6H) in the matrix spectra of dU (Fig. 7). Contrary to the spectrum of 1-methyluracil, Fermi resonance splitting is absents in the spectra of nucleosides (Fig. 7). The HF calculations do not demonstrate the low-frequency shift of H-bonded n(C6H) (Table 2) and this is con-
Table 2 ˚ and angles in 8) AH B intramolecular H-bonds of 2-deoxyuridine conformers at the The geometric parameters of (distances in A different level of calculations Method conformer
HF/3-21G(p) ˚) d(AH B) (A
dU_a1 /(C6H O5?)8 dU_a2 /(O3?H O5?)8 dU_a3 /(O5?H O3?)8 dU_s1 /(O5?H O2)8 dU_s2 /(O5?H O2)8
2.18 161.1 2.08 131.8 2.05 142.8 1.93 153.8 1.80 163.2
HF/6-31G(d,p) ˚) d(A B) (A 3.21 2.80 2.86 2.81 2.73
˚) d(AH B) (A 2.47 159.8 2.31 126.0 2.34 131.4 2.30 143.9 1.99 161.8
MP2/6-31G(d,p) ˚) d(A B) (A 3.50 2.96 3.05 3.12 2.90
˚) d(AH B) (A 2.34 160.7 2.19 130.3 2.16 139.0 2.12 146.3 1.88 165.4
˚) d(A B) (A 3.38 2.90 2.96 2.98 2.84
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Fig. 7. The FTIR spectra of 2-deoxyuridine (1), thymidine (2), 1-methyl-uracil (3) isolated in Ar matrices (T /12 K, M/S/ 700) in the C5 /H, C6 /H stretching region (3140 /3040 cm1).
sistent with experiment: the shift of n (C6H) in dU_a1 is small (/15 cm 1) with respect to n (C6H) of 1-methyl-uracil (Fig. 7). The calculations on the HF/3-21G(p) level show that the intramolecular hydrogen bond C6H O5? in conformer dU_a1 produces a strong increase in intensity of vibration n (C6H), which is one order of magnitude greater than above the intensity of n (C5H) (Table 1). The comparison of the geometry parameters of H-bonds in dU_a1 at different basis sets indicates that calculations on the HF/321G(p) level produce a very short C6H O5? distance (Table 2) and correspondingly overestimate the power of this H-bond. The distances on the HF/6-31G(d,p) and MP2/6-31G(d,p) level of these calculations are more realistic. So, the MP2 ˚ distance C6H O5? for this conformer (2.34 A from Table 2) correlated with DFT/6-31G(d) data: ˚ for the C2?-endo /anti and 2.37 A ˚ for the 2.28 A C3?-endo /anti conformation of uridine [8]. The increase in intensity of the H-bonded n (C6H) on the HF/6-31G(d,p) level of these calculations is relatively small, but intensity of nC6H is in 1.25 times more than intensity of n(C5H) (Table 1). In experimental spectra we can see the diametrically opposed picture, namely, the intensity of free vibration n(C5H) is greater than intensity of Hbonded vibration n(C6H) (Fig. 7). This effect may
be explained by the overlapping of spectral bands of different conformers. As was calculated (Table 1), the intensity and frequency of n(C6H) in conformer dU_a0 are greater than for n(C5H) (Table 1). It is true for conformers dU_a2, dU_a3 also, which have a similar orientation of the pyrimidine ring. The superposition of the spectra of these conformers can lead to an apparent increase in intensity of n(C5H). It leads us to conclude that the occupancy of conformer dU_a0 cannot be neglected. The frequencies of two CH2 groups and three CH groups of ribose sugar are located in the region 3040/2750 cm 1. Also, Fermi resonance splitting of spectral bands is standard for this type of vibrations of organic molecules [27]. Owing to these factors overlapping spectral bands are seen (Fig. 8). Because of this, we have used the deconvolution of experimental spectra into bands with Lorentzian (or Gaussian) contours. These results and the calculated spectra are presented in Table 3.
4.3. The spectral region of stretching vibrations n(C /O), n (C5 /C6) The experimental and calculated spectral data for this region are presented in Table 3. In this
Fig. 8. The FTIR spectrum of 2-deoxyuridine (1) isolated in Ar matrix (T /12 K, M/S/700) in the C /H stretching region (3040 /2750 cm 1).
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
1967
Table 3 The experimental (from Ar matrices) and calculated (6-31G(d,p)) frequencies and intensities of vibrations of the 2-deoxyuridine conformers in the 3000 /200 cm 1 region Mode no.
Experiment
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
30 31 32 33 34 35 36 37 38
39 40 41 42 43 44
)
dU_a1
dU_s2
1
n (cm
I
1
Assignment PED (%) for dU_a1 )
a
n (cm
2997 2946 2924 2893 2864 2838 2801 1762 1734 1710 1640 1520 1467 1456 1452 1434 1422
0.9 7 0.8 2.7 0.5 0.3 0.3 5.1 64.0 47.0 6.8 0.3 0.5 11.1 3.6 0.6 0.1
2976 2943 2895 2871 2864 2852 2815
6 8 14 65 34 87 47
2977 2906 2892 2886 2876 2850 2829
3 19 51 29 55 91 35
1714 1735 1641
955 710 206
1700 1744 1644
1072 746 172
1408 1389 1376 1357 1322 1308 1297
0.4 4.8 3.3 2.6 0.9 2.2 1.3
1485 1465 1456 1439 1410 1408 1405 1391 1384 1354 1333 1307 1306
4 148 51 3 8 8 17 149 28 25 13 31 49
1473 1468 1452 1434 1409 1406 1405 1388 1381 1372 1342 1330 1304
10 58 115 44 65 9 16 160 35 46 26 3 13
1284 1272 1264 1259 1247 1239 1227 1216 1204 1190 1179 1147 1125 1115 1103 1092 1079 1064 1057 1037 1001 989 964 956
6.4 6.1 1.5 3.4 2.1 0.9 1.2 5.0 3.0 2.7 1.5 0.4 4.2 3.9 7.8 2.4 5.2
1268
280
1286
7
1249
53
1264
168
1216 1214 1192 1186 1179
44 59 34 135 43
1223 1216 1207 1179 1174
77 60 41 40 51
1150 1121 1104
316 8 9
1152 1131 1099
206 66 71
1074 1061
8 25
1070 1068
27 18
n C3?O(11), n N1C2(10) n C1?O4(16), n C5?O(12)
1032 997 992 960
0.4 64 28 0.5
1002 1000 991 962
33 1.7 80 0.5
gC6H(90) bC2?H2(24), n C3?O(16) n C4?O4?(16) b6ring(20), n C4C5(18)
7.3 4.8 1.5 1.2 0.2 1.5
)
a
I
n (cm 6 7 8 9 10 11 12
1
I
n C1?H(95) n C2?H2as(95) n C2?H2sym(94) n C3?H(84) n C5?H2as(88) n C4?H(80) n C5?H2sym(94) n C2O(76) n C4O(70) n C5C6(57), bC6H(15) bC5?H2(41) bC1?H(27), bC2?H2(14) bC2?H2(50) bC5?H2(52) bC6H(28), bN3H(18) bC3?H(33) bN3H(16), n N1C2(10) n C2N3(22), n N1C2(13) bC4?H(30), bC1?H(26) bC1?H(40) bC4?H(30), bC3?H(18) bC3?H(36), bC4?H(30) bC1?H(23), bC2?H2(25) bC6H(20), n N1C6(19)
bC2?H2(58) bC5H(16), n N3C4(13) bC2?H2(20) bO5?H(22), n N1C1?(20) bO3?H(17), bO5?H(16) bO5?H(24), bO3?H(12) n C1?O4?(27) n C3?O(16), n C5?O(9) n C5?O(33)
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
1968 Table 3 (Continued ) Mode no.
Experiment n (cm 1)
45 46 47 48 49 50
51 52 53 54 55 56 57 58 59
60 61 62 63
64 65 66 67 68 69 70
71 72 73 74 75 76 77 78
dU_a1 n (cm 1)
I
dU_s2 Ia
n (cm1)
Assignment PED (%) for dU_a1 Ia
950 945
0.8
938
23
944
31
bC2?H2(22), n C1?C2?(10)
916 878 868 859 840
0.8 1.2 0,2 0.1 0.4
922 881 875
15 16 13
921 879 869
10 10 27
n C2?C3?(16), n C5?O(8) n C4?O4?(14) n C3?C2?(23), bC2?H2(10)
822
4
830
8
807 805 776 766 760 752 734 714 659 644 605 598 578 549 544 525 516 467 430 418 407 402 392 377 351 326
3.1 1 1.4
816
79
815
81
4.2 0.8 0.2 0.3 6.5 0.3 0.6 2.2 0.2 1.5 0.4 1.6 0.8 0.4 1.1 0.7 1.2 1.0 0.9 1.0 0.4 0.6
780 752 740 721 657 636 634 595
90 11 14 3 71 20 5 18
786 747 738 717 665 651 630 585
98 33 3 0.2 70 4 36 184
545
18
565
32
b6ring(42),bC2O(16)
516 458
18 12
553 519
6 36
b6ring(25), bC4O(16) bN1C1?(26), bC3?O3?(18)
422 401
11 28
457 414
25 11
t6ring(41), bC3?O3?(20) bC4O(42)
277 257 246 240 232 224 217
1.0 1.7 0.5 1.0 0.6 0.7 1.0
367 344 323 292 272
20 14 19 198 49
395 367 344 321 299
29 13 20 0.4 123
t6ring(16), bC1?N1(15) bC3?O3?(39), tO3?H(13) bC4?C5?(34), tO5?H(11) tO3?H(88) tO5?H(72)
252
15
272
4
bC4?C5?(14), n N1C1?(12)
237
14
232
5
bC1?N1(19), bC4?C5?(12) [n (O5?H O2)(14)]*
201 186 177 158 126 93 63 48
1 3 4 7 2 0.4 1.3 0.1
bC1?N1(27) t6ring(55) t6ring(98) tC4?C5?(55) [n (O5?H O2)(34)]* t5ring(57), tC4?C5?(14) n (C6H O5?)(72) [n (O5?H O2)(24)]* tC1?N1(34), gC1?N1(28) tC1?N1(84)
187 180 176 141 110 65 57 42
3 6 1 4 0.6 0.7 0.3 0.4
b5ring(17), n N1C1?(11) gC5H(57), gC4O(44)
gC2O(85) n C4C5(27), n N1C2(13) b5ring(23), n C1?C2?(13) gC6H(32), gC5H(23) gN3H(93) b5ring(14) bC4O(13), bC2O(10) b5ring(19), bC2O(10)
I */ relative integral intensity from the experimental FTIR spectra. Ia */ absolute integral intensity in km/mol. * */ PED for Hbond of conformer dU_s2.
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
region individual scaling factors were used: namely, 0.77 for n (C /O) vibrations and 0.89 for n (C5/C6). Fermi resonance is a marked property of the n(C /O) region in the matrix spectra of nucleic acid bases [13,24]. For example, in the spectrum of 1-methyl-uracil, four bands are shown in the n(C /O) region (Fig. 9). Also, resonance splitting is marked for the n(C5 /C6) bands of 1methyl-uracil: 1650 cm 1, 1636 cm 1 (Fig. 9). The surprising thing is that Fermi resonance is absent practically in the n(C /O), n(C5 /C6) region of the matrix spectra of dU (Fig. 9). It is evident (Fig. 9) that the ribose ring has no strong influence on the frequencies of the spectral bands of the base: n(C / O), n(C5 /C6). As for 1-methyl-uracil [24], the HF method calculated frequencies of n(C2O) and n(C4O) in the order reverse to experiment (Table 3). It was shown that the MP2 frequencies coincide with the experiment and that the intensities of n (C4O) and n (C2O) are in good agreement with experiment for both methods [24]. Because of this, the assignment of n(C2O) to 1734 cm 1 and n(C4O) to 1710 cm 1 was made on a basis of comparison between calculated and experimental intensities of these vibrations (Table 3). It was shown that in comparison with planar ring molecules (uracil), nonplanar molecules (5,6-
Fig. 9. The FTIR spectra of 2-deoxyuridine (1), 1-methyl-uracil (2) isolated in Ar matrices (T /12 K, M/S/700) in the C2/O, C4/O, C5/C6 stretching region (1820 /1625 cm1).
1969
dyhydrouracil) have broad spectral bands in the matrix spectra owing to disordered matrix sites [13]. It may be concluded that this effect is stronger for nonplanar flexible molecules, such as nucleosides. In this region the spectral bands of dU are noticeably wider in comparison with 1-methyluracil (Fig. 9) and this is also true for the bands in the deformation spectral region. 4.4. The spectral region of deformation vibrations (1500 /200 cm 1) In the range of frequencies below 1500 cm 1 the bands of deformation vibrations, stretching vibrations of ribose and pyrimidine rings and n(C3?/O), n(C5?/O) are located. The experimental and calculated spectral data in the region below 1500 cm 1 are presented in Table 3. In this region, the connection of the scaling factor with the type of vibration is not well defined and the uniform scaling factor 0.9 was used which is close to some previous works for nucleic acid bases [23,24]. In the range of 1500 /1000 cm1, 25 spectral bands are located (Fig. 10, Table 3). Most of these bands belong to ribose sugar and attachment groups (Table 3). As in the n(C /O) region the spectral bands are broad in comparison with nucleic acid bases and overlapping is evident (Fig. 10) and the decomposition spectra on the Lorentzian and Gaussian contours was used. In the range of 1000/500 cm 1 the bands of pyrimidine base have the greatest intensities and are well marked in the matrix spectra (Fig. 10); the ribose ring has little influence on the spectral bands of the base. Their frequencies coincide closely with analogous bands of 1-methyl-uracil. The vibrational band g (C2O) around 760 cm1 has a marked splitting (Fig. 10), perhaps connected with conformer dU_s2 since the C2O group take part in the intramolecular H-bond formation. In this spectral range our 6-31G(d,p) calculations (Table 3) and analogous 6-31G(d,p) calculations for uracil [19] are in good agreement. In the 500/200 cm 1 range, calculated and experimental data are markedly worse than in the other region. In this region, the number of observed spectral bands is significantly greater than the calculated bands (Fig. 10, Table 3)
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are presented in Table 4. For comparison with real experimental data the relative Gibbs free energy DG was estimated by the standard method [28]: T
DGAB (T)DE DZPE
g DCdT TDS(T) 0
Fig. 10. The FTIR spectra of 2-deoxyuridine isolated in Ar matrices (T /12 K, M/S/700) in the 1540 /200 cm 1 region.
attributed to intense bands of low-lying conformers. The influence of the matrix cage also complicates the assignment of vibrations in this region. Practically, the vibrations of intramolecular H-bonds are beyond the range of our spectral measurements (for example see frequency of dU_s2 vibration of H-bond n(O5?H O2) in Table 3). The calculations overestimate the intensity of the torsion vibration of the groups O5?H, O3?H. (Table 3). The matrix influence on the barrier height of frozen rotation must also be taken into account for large fragments of a molecule; this can lead to increase in frequencies of deformation vibrations and uniform scaling factor of 0.9 cannot be used. 4.5. The populations of dU conformational isomers in the low temperature matrices The calculated and experimental relative energies the anti -, syn -conformers of 2-deoxyuridine
Relative electronic energy DE was estimated at the MP2/6-31G(d,p) level, and the relative zero-point vibrational energy DZPE and temperature dependent contributions of rotation and vibration were estimated at the HF/6-31G(d,p) level for temperatures of 298 and 420 K (Table 4). According to the relative electronic energy calculations, the syn conformer dU_s2 with the intramolecular hydrogen bond O5?H O2 and conformation of the sugar ring-C2?-endo has the minimal energy at all levels of optimization (Table 4). dU_a1 has minimal electronic energy among anti -conformers (Table 4). The influence of the vibrational /rotational contribution to the free energy reduces the difference of conformers’ energies significantly, and this is especially noticeable with the growth of evaporation temperature (Table 4). But the calculations of the relative free energy DG did not demonstrate principal modifications of the conformational distribution (Table 4). The association of DG and experimental spectral data can be expressed by the standard equation: DGAB (T)RTlnKAB RTln(hA =hB ) where KA,B is the equilibrium constant of conformers A, B and hA, hB are the populations of these conformers. To define the populations of conformers in experimental spectra it is necessary to know the molar extinction coefficients of characteristic spectral bands or their ratios. There are several ways to determine these quantities; in the first place, the ratios of molar extinction coefficients of characteristic bands can be determined through the redistribution intensities of characteristic bands under the influence of changing evaporation temperature or through the UV irradiation of matrix samples. If related experiments are difficult, the calculations of the intensities of characteristic spectral bands may be used. Ab initio calculations
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Table 4 Calculated and experimental relative energies (kcal/mol) of 2-deoxyuridine anti -, syn -conformers Conformer method
d_a0
d_a1
d_a2
d_a3
d_s1
d_s2
HF/3-21G(p) HF/6-31G(d,p) MP2/6-31G(d,p) DG (298 K)b MP2/6-31G(d,p) DG (420 K)b MP2/6-31G(d,p) DG (420 K) experiment
5.0 2.7 5.6 3.4 2.8 /
2.0 1.3 2.8 1.7 1.4 0
2.4 1.4 3.6 2.4 2.1 1.8
4.4 3.1 4.8 3.8 3.4 2.2
3.1 1.5 2.7 1.9 1.6 /
0 (/826.510784)a 0 (/831.066722) 0 (/833.487562) 0 0 1.1
a b
Absolute energies in a.u. are indicate in brackets. Vibrational /rotational contribution was estimated at HF/6-31G(d,p) level.
usually overestimate the absolute infrared intensity of vibrational bands [23 /25], however the relationship of experimental and calculated intensities coincides much better. So, the equilibrium constant of isomers a and b has been adopted [29,30]: P P Ia(eksp:) Ib(calcl:) KAB P (1) P Ia(calcl:) Ib(eksp:) Unlike some studies [29,30] we have applied Eq. (1) to OH, NH and CH stretching vibrations only. With our data the best agreement between the calculated and experimental results is observed for the relationship between the intensities of stretching vibrations. This is true for stretching vibrations of dU also (Table 5), but data for H-bonded stretching vibrations were not available. Because of this, we have used UV irradiation of matrix samples for the shift of conformation equilibrium and determination of band ratios. The influence of UV irradiation on the spectra of the matrix samples is presented in Fig. 11. As some minor UV-induced degradation was detected in our spectra, it was presumed that the UV-induced
degradation is equal for all dU isomers. With this approximation, the growth of the band intensities of H-bonded n(OH) modes demonstrated a good correlation between experimental and calculated data (Table 5). As was discussed above, the characteristic bands of dU_a0 and dU_a1 were not clearly defined in the matrix spectra. Also, our calculations predict very small occupancies of the dU_a0 conformer (Table 4). Therefore for the sake of simplicity, the conformer dU_a0 was ignored and only four conformers: dU_s2, dU_a1, dU_a2, dU_a3 were considered. For the determination of KAB the different combinations of the experimental and calculated intensities from Table 1 and experimental ratios from Table 5 were used. The occupancies of conformers dU_a2, dU_a3 were estimated on the basis of their spectra in Ne matrices (Table 1). The net results of these experimental estimations are presented in Table 4. The experimental and calculated data coincide well for the dU_a2 and dU_a3 conformers (Table 4), but the discrepancy between experimental and calculated occupancies of the conformers’ dU_a1
Table 5 The experimental and calculated ratio of intensities of the stretching vibrations for the 2-deoxyuridine conformers Method ratio I (n AB)/I (n CD)
Experiment Ar matrices
Calculation HF/3-21G(p)
Calculation HF/6-31G(d,p)
I(N3H)a/[I (C2O)/I (C4O)]a I(C2O)a/I (C4O)a I (N3H)a/[I (C5H)/I (C6H)]* I (hb_O5?H)/[I (O5?H)a] in dU_a3 I (hb_O3?H)/[I (O3?H)a] in dU_a2 I (hb_O5?H)/[I (O5?H)a] in dU_s2
0.09 1.4 6.8 2 2 7
0.07 1.1 6.7 2.3 3.5 8.4
0.06 1.3 7.4 1.9 1.1 4.3
a
Vibrations of conformer dU_a1.
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For example, some authors [31] used averaging of energies calculated at several levels of theory for the enhancement of reliability, but obviously this increases the magnitude of the numerical calculations. It is possible that on the way to intensive calculations the usage of correlation consistent (cc) basis sets is more reasonable. As was noted by Jensen [32], the main advantage of cc-basis sets ‘is the ability to generate a sequence basis sets, which converges toward the basis sets limit’. Because of this, it is interest to test the convergence of DE and DG in the row of basis sets: aug-ccpVDZ, aug-cc-pVTZ, aug-cc-pVQZ to the experimental data.
Fig. 11. Effect of UV-irradiation on the FTIR spectrum of 2deoxyuridine isolated in Ar matrix (T /12 K, M/S/700) in the O /H, N /H stretching region (3690 /3400 cm 1). (1) Spectrum before UV-irradiation; (2) difference spectrum after UV-irradiation (t/40 min).
and dU_s2 is obvious (Table 4). Possible reason for this discrepancy are that the conformers’ occupancies in matrices may differ widely from their analogous in the gas phase. This effect is observed at low barriers between conformational isomers [12]. According to experimental data interconversion in an Ar matrix at 30 K is possible for barriers B/2.5 /3 kcal/mol [12]. We tested the effect of interconversion by using deposition of the samples in matrices with different temperatures and annealing of matrix samples. No differncies were see in our spectra in Ar matrices at 5 and 12 K (Figs. 5 and 6). Annealing of matrix samples at 30 K has no influence on the conformational equilibrium. It follows that the barriers heights of dU conformers /2.5 /3 kcal/mol and their conformational equilibrium in gas phase at evaporation temperature must be close to equilibrium in Ar matrices at 12 K. The conformer dU_a0 may be a exception, since clear spectral identification of the transition dU_a0 0/dU_a1 is difficult in these experiments. In the second place, ab initio calculations of electronic energies or free energies are not sufficiently reliable for the large unrigged molecules, even with large basis sets and using the method of electronic correlation at the MP2 level.
5. Conclusions For the first time FTIR spectra of 2-deoxyuridine were obtained in Ar and Ne matrices. For the first time the conformational equilibrium and structures of intramolecular H-bonds of 2-deoxyuridine in a isolated state were investigated by FTIR matrix isolation spectroscopy and ab initio calculations. It was found that anti conformers of dU are dominant in the isolated state. Their occupancy in the matrix samples was estimated at 81%. The ribose rings of the main anti -conformers dU _a1, dU _a0 are in the C2?endo conformation, but the ribose rings of minor anti -conformers dU_a2, dU_a3 have the C3?-endo conformation, stabilized by intramolecular hydrogen bonds O3?H O5? and O5?H O3? accordingly. Intramolecular hydrogen bonds O3?H O5?, O5?H O3? may be treated as a spectroscopic indicator of the transition C2?-endo 0/C3?-endo between conformations of ribose ring in the anti conformers of dU. Syn -conformers of dU are stabilized by the intramolecular hydrogen bond O5?H O2 and the dominant conformation of ribose ring is C2?endo . Their occupancy in the matrix samples was estimated as 19%.
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Acknowledgements This investigation was supported by the Ukrainian Academy of Sciences and in part by the INTAS-International Association under grant no. INTAS 00-00911.
References [1] W. Saenger, Principles of Nucleic Acid Structure, Springer /Verlag, New York, 1984. [2] G.A. Jeffrey, W. Saenger, Hydrogen Bonding in Biological Structures, Springer /Verlag, Berlin, 1991. [3] S.N. Rao, Nucleosides Nucleotides 14 (6) (1995) 1179. [4] F. Seela, H. Debelak, H. Reuter, G. Kastner, I.A. Mikhailopulo, Tetrahedron 55 (1999) 1295. [5] J.J. Barchi, Jr, L.-S. Jeong, M.A. Siddiqui, V.E. Marquez, J. Biochem. Biophys. Methods 34 (1997) 11. [6] J.M. Gavira, M. Campos, G. Diaz, A. Hernanz, R. Navarro, Vibrat. Spectrosc. 15 (1997) 1. [7] N. Leulliot, M. Ghomi, G. Scalmani, G. Berthier, J. Phys. Chem. A 103 (1999) 8716. [8] N. Leulliot, M. Ghomi, H. Jobic, O. Bouloussa, V. Baumruk, C. Coulombeau, J. Phys. Chem. B 103 (1999) 10934. [9] O.V. Shishkin, A. Pelmenschikov, D.M. Hovorun, J. Leszczynski, J. Mol. Struct. 526 (2000) 329. [10] S.A. Krasnokutski, A.Yu. Ivanov, V. Izvekov, G.G. Sheina, Yu.P. Blagoi, XXIV European Congress on Molecular Spectroscopy, Prague, Czech Repablic, 1998, p. 470. [11] S.A. Krasnokutski, A.Yu. Ivanov, V. Izvekov, G.G. Sheina, Yu.P. Blagoi, J. Mol. Struct. 482 /483 (1998) 249. [12] A.J. Barnes, J.Mol. Struct. 113 (1984) 161. [13] A.Yu. Ivanov, A.M. Plokhotnichenko, E.D. Radchenko, G.G. Sheina, Yu.P. Blagoi, J. Mol. Struct. 372 (1995) 91. [14] A.Yu. Ivanov, A.M. Plokhotnichenko, V. Izvekov, G.G. Sheina, Yu.P. Blagoi, J. Mol. Struct. 408 /409 (1997) 459.
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[15] A.Yu. Ivanov, G. Sheina, Yu.P. Blagoi, Spectrochim. Acta A 55 (1999) 219. [16] G.L. Saksagansky, Molecular Beams in the Composite Vacuum Structures (in Russian), Atomizdat, Moscow, 1980. [17] A.A. Granovsky, http://classic.chem.msu.su/gran/gamess/ index.html [18] M.W. Schmidt, K.K. Baldridge, J.A. Boatz, S.T. Elbert, M.S. Gordon, J.J. Jensen, S. Koseki, N. Matsunaga, K.A. Nguyen, S. Su, T.L. Windus, M. Dupuis, J.A. Montgomery, J. Comput. Chem. 14 (11) (1993) 1347. [19] A. Les, L. Adamowicz, M.J. Nowak, L. Lapinski, Spectrochim. Acta A 48 (10) (1992) 1385. [20] P. Pulay, G. Fogarasi, F. Pang, J.E. Boggs, J. Am. Chem. Soc. 101 (1979) 2550. [21] G. Fogarasi, X. Zhou, P.W. Taylor, P. Pulay, J. Am. Chem. Soc. 114 (1992) 8191. [22] P. Pulay, G. Fogarasi, X. Zhou, P.W. Taylor, Vibrat. Spectrosc. 1 (1990) 159. [23] M.J. Nowak, L. Les, L. Adamowicz, Trends Phys. Chem. 4 (1994) 137. [24] M.J. Nowak, L. Lapinski, D.C. Bienko, D. Michalska, Spectrochim. Acta A 53 (1997) 855. [25] Y. Yamaguchi, M. Frisch, J. Gaw, I.I.I.H.F. Schaefer, J.S. Binkley, J. Chem. Phys. 84 (4) (1986) 2262. [26] N.D. Sokolov (Ed.), Hydrogen bond, Nauka, Moscow, 1981 (in Russian). [27] M.P. Lisitsa, A.M. Yaremko, Fermi Resonanse (in Russian), Naukova Dumka, Kiev, 1984. [28] J.S. Kwiatkowski, J. Leszczynsky, Chem. Phys. Lett. 204 (1993) 430. [29] M.J. Nowak, L. Lapinski, J. Fulara, Spectrochim. Acta A 45 (2) (1989) 229. [30] H. Vranken, J. Smets, G. Maes, L. Lapinski, M.J. Nowak, L. Adamovicz, Spectrochim. Acta A 50 (5) (1994) 875. [31] S.E. Barrows, F.J. Dulles, C.J. Cramer, A.D. French, D.G. Truhlar, Carb. Res. 276 (1995) 219. [32] F. Jensen, Introduction to Computational Chemistry, Wiley, New York, 1999.
Conformational structures and vibrational spectra of isolated pyrimidine nucleosides: Fourier transform infrared matrix isolation study of 2-deoxyuridine A.Yu. Ivanov a,*, S.A. Krasnokutski b, G. Sheina a, Yu.P. Blagoi a a
Institute for Low Temperature Physics and Engineering, Ukrainian Academy of Sciences, Lenin Avenue 47, 310103 Kharkov, Ukraine b Max-Planck-Institut fur stromungsforschung, Busenstr 10, D-37073 Go¨ttingen, Germany Received 14 August 2002; received in revised form 15 November 2002; accepted 21 November 2002
Abstract The conformational structures of 2-deoxyuridine (dU) were investigated using Fourier transform infrared (FTIR) matrix isolation spectroscopy. For the first time the FTIR spectra of dU in Ar matrices were obtained in the range 4000 /200 cm 1. The stabilities of conformers were estimated by the methods HF/3-21G (p), HF/6-31G (d,p) and MP2/ 6-31G (d,p). Ab initio calculations of the infrared spectra were performed by the methods HF/3-21G (p) and HF/6-31G (d,p). The actual occupancy of conformational isomers in matrix samples was determined. It was shown that anti conformers of dU are dominant. The ribose rings of the main anti -conformers dU _a0, dU _a1 are in the C2?-endo conformation, but the ribose rings of minor anti -conformers dU_a2, dU_a3 have the C3?-endo conformation, stabilized by intramolecular hydrogen bonds O3?H O5? and O5?H O3?, accordingly. Syn -conformers of dU are stabilized by the intramolecular hydrogen bond O5?H O2 and the dominant conformation of the ribose ring is C2?-endo . # 2002 Elsevier Science B.V. All rights reserved. Keywords: FTIR spectroscopy; Matrix isolation; Nucleosides; Intramolecular H-bond; MP2
1. Introduction There is an intense interest to studies of conformational isomerism of DNA structural components such as nucleoside by various experimental and theoretical methods [1 /9]. Up to the present, the main experimental methods of the investigation of nucleosides are presented by
* Corresponding author. Fax: /380-57-233-5593. E-mail address: [email protected] (A.Yu. Ivanov).
crystallography and NMR spectroscopy. It is well known that the results of these methods depend strongly on intermolecular interactions. For example, in the crystals only one form of several possible conformers is stabilized and its choice can be affected by the nature of the solvent from which the crystallization was made [1]. Owing to interactions with the solvent, NMR spectroscopy gives no way to obtain the direct data about intramolecular hydrogen bonding of nucleosides in aqueous solutions. [4,5]. In view of these problems, up to now the conformational
1386-1425/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S1386-1425(02)00416-X
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isomerism of the isolated nucleosides and the structure of their intramolecular hydrogen bonds remain unexplored questions. Hitherto, X-ray data [1] and semi-empirical calculations [3] have pointed to the possibility of the formation of intramolecular hydrogen bonds O5?H O2 in the syn -conformations of pyrimidine nucleosides (Fig. 1). Also, the formation of hydrogen bonds C6H O5? in the anti -conformers of pyrimidine nucleosides was based on the crystallographic data [1] and DFT calculations [7,8]. The first experimental data for pyrimidine nucleosides (uridine and thymidine) isolated in the low temperature Ar matrices were obtained using FTIR matrix isolation spectroscopy [10,11]. The high resolution, sensitivity and absence of the strong intermolecular interactions in the inert low temperature matrices makes the spectroscopy of matrix isolation a very useful method to study of intramolecular hydrogen bonding [12]. This allows us to show that syn -conformations of isolated uridine and thymidine are stabilized by the intramolecular H-bond O5?H O2 [10,11]. Recent DFT calculations [7 /9] were also restricted by consideration of this type intramolecular H-bond only. In the present research, for the first time FTIR spectra of 2-deoxyuridine in Ar matrices were obtained using an enhanced experimental setup. FTIR spectra of auxiliary compounds (thymidine, 1methyl-thymine, 1-methyl-uracil) were also obtained. New spectral data suggest the existence of more types of intramolecular H-bonds for the isolated pyrimidine nucleosides than considered before [7 /11]. The new experimental data were
Fig. 1. The molecular structure and atom numbering of 2deoxyuridine in the syn -conformation.
supplemented by ab initio calculations of energy of different conformers and their vibrational spectra.
2. Experimental methods The basic features of the experimental setup have been described previously [11,13/15]. For the present paper, the FTIR spectra of 2?-deoxyuridine and auxiliary compounds were obtained in the ranges: 4000 /1300 cm 1 (KBr beamsplitter (b.)), 2500 /500 cm 1 (CaF2 b.), and 500 /180 cm 1 (Mylar b.), with apodized resolution 0.4 cm 1 (the full spectra of the auxiliary compounds will be published in the future). As was pointed out, nucleosides are very thermally labile molecules and for their evaporation the special Knudsen cell with reduced molecular beam losses was used [11]. The general scheme of evaporation from this cell is presented in Fig. 2. This evaporation cell is characterized by a working vapor pressure around 10 5 Torr and Knudsen’s number of over 100. Efficiency of the new cell is enlarged by a factor of more than 500 /1000 over one of a Knudsen cell used in our previous experiments with more stable compounds [13 /15]. As is shown in Fig. 3 the geometry of the Knudsen cell and its
Fig. 2. The general scheme of matrix isolation setup based on liquid He cryostat: (1) rotating vacuum seal; (2) cryogen block with cold mirrors and QCM; (3) rotating nitrogen shield; (4) flange with indium seal; (5) Knudsen cell; (6) electrical heater of Knudsen cell; (7) Ar gas duct.
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K for Ar, and 5 K for Ne. To improve of optical characteristics of Ar matrices, before the deposition of matrix samples, a thin layer of pure Ar was deposited on the mirrors over the temperature range of 35/20 K [15]. As before [14], UVirradiation was used to shift the conformational equilibrium of dU in the matrix samples.
Fig. 3. The relationship of the efficiency of Knudsen cell (I ) as function of distances L between cell and cold mirror. L is in the unit R , R */ radius of outlet nozzle of Knudsen cell.
disposition to the low temperature mirror are very important for effective works. The data in Fig. 3 were obtained by the statistical Monte Carlo method, which is very advantageous for the simulation of complicated vacuum systems [16]. In comparison with previous work [11] the distance L (Fig. 3) was decreased from 4.5 to 2.5 R . The low temperature differential quartz crystal microbalance (QCM) was used for the measurements of absolute intensity of molecular beams and matrix-to-sample ratio (M/S) [13]. Owing to QCM, we have a capability to work not only with an Ar flux passing through the Knudsen cell (Fig. 2) but with an outside flux of cold Ar gas also. In the present experiments the typical intensity of molecular beams of nucleosides was about 60 ng/(s cm2) without any thermodestruction. The test experiments with the evaporation cell from previous work [13 /15] showed that thermodestruction can be easy detected by intense spectral bands due to H2O, CO2, and the narrow characteristic bands of the appropriate bases. Commercial (Sigma) substances (2?-deoxyuridine, thymidine) were used without additional purification. The model substances (1-methyl-uracil, 1-methyl-thymine) were synthesized in the Kharkov State University (Kharkov, Ukraine). Before the creation of matrix samples all substances were annealed to remove impurities just as sorbed H2O, CO2 and N2. The annealing was controlled with the help of QCM. The inert gases (Ne, Ar) were more than 99.99% pure. The matrices were deposited at the temperatures of the mirrors: namely, 11
3. Computational methods The quantum-chemical calculations of the relative energies and vibrational spectra of 2-deoxyuridine conformers have been performed by the program PC GAMESS version 6.0 [17] of the GAMESS (US) QC package [18]. The first phase of full geometry optimization of six dU conformers (Fig. 4) was performed at a restricted Hartree /Fock (HF) level with 3-21G(p) basis sets. Furthermore, the stable structures were optimized by the methods HF/6-31G(d,p) and MP2/6-31G(d,p) sequentially. For all structures vibrational spectra were obtained by the methods HF/3-21G(p), HF/6-31G(d,p). The choice of natural internal coordinates for the dU base was based on the data for uracil [19]. Other natural internal coordinates were constructed using recommendations from literature [20 /22]. The method implemented in GAMESS was used to determine the contribution of the internal coordinates into the normal modes and the calculation of the potential energy distribution (PED). Because of this, the PED for deformation vibrations of rings and some groups of ribose was presented in a simplified form, as the sum of the contributions of separate modes. For the estimations of the rotational /vibrational contribution to the Gibbs free energy G (approximation: harmonic oscillator/rigid rotor) at the HF/3-21G(p) level the standard capabilities of the GAMESS program were used. The total number of basis functions used in the calculations for dU was 204 (321G(p)), 300 (6-31G(d,p)). All calculations were performed on PC with processors Celeron-500, PIII-800, and Duron-700.
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Fig. 4. The conformational structures of 2-deoxyuridine which are stable at the different level of calculation (HF/3-21G(p), HF/631G(d,p) and MP2/6-31G(d,p)). Intramolecular H-bonds are represented with dashed lines.
4. Results and discussion 4.1. The spectral region of stretching vibrations n (OH), n (NH) The region of the stretching (n) vibration n (OH), n (NH) is very important for the identification of nucleoside conformers in matrix samples and for the determination of their occupancy. In this region, only the N3H group of dU plays no part in the intramolecular interactions of dU
conformers. The n(N3H) spectral band can be easily defined by comparison with the auxiliary compounds. As shown in Fig. 5, the frequency of the n(N3H) stretching vibration has no significant changes in the substances studied in Ar matrices: 2-deoxyuridine, 1-methyluracil, 1-methyl-thymine. But for others, four absorption bands in this region (Fig. 5) can belong to two OH groups of 2?-deoxyribose-O3?H, O5?H only (Fig. 1). This spectral pattern is valid for Ar and Ne matrices at 5 K also (Fig. 6). The magnification of the
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
Fig. 5. The FTIR spectra of 2-deoxyuridine (1), 1-methyl-uracil (2), 1-methyl-thymine (3) isolated in Ar matrices (T/12 K, M/ S /700) in the O /H, N /H stretching region (3690 /3390 cm 1).
Fig. 6. The comparison of FTIR spectra of 2-deoxyuridine in Ar (1) (T /5 K, M/S/700) and Ne matrices (2) (T /5 K, M/ S /700) in the O /H, N /H stretching region (3700 /3390 cm 1).
number of bands observed in the spectra of dU (Figs. 5 and 6) can be explained by intramolecular hydrogen bonds in the different conformational structures. Therefore, the choice of initial conformational structures for optimization (Fig. 4) was
1963
motivated by the possibility of formation of intramolecular hydrogen bonds. It is well known that the conformational-flexible sugar ring of nucleosides has a nonplanar puckered structure [1]. According to the data of NMR spectroscopy, C2?-endo and C3?-endo are the main equilibrium conformations of the sugar ring in solutions [1]. For contraction of the computational time we have restricted ourselves to these main conformations of the dU sugar. The transition C3?-endo 0/ C2?-endo has no effect on the structure of the Hbonds in the syn -conformers dU_s1, dU_s2 (Fig. 4), but the transition C2?-endo 0/C3?-endo in the anti -conformers furnishes the formation of the Hbonds O3?H O5?, O5?H O3? in the conformers dU_a2, dU_a3 (Fig. 4). Furthermore, only dU_a2, dU_a3 C3?-endo /anti conformations are stable. Hitherto, DFT calculations (6-31G(d) basis set) confirm the formation of the hydrogen bonds C6H O5? in anti -conformers of uridine and cytosine [7,8]. Therefore, the choice of the structure dU_a1 with the orientation of hydroxymethyl group gauche and torsion angle g (/C3? /C4?/ C5?O5?) //508 are close to the structures from literature [7,8]. We also considered the structure dU_a0 with the orientation of hydroxymethyl group gauche ( /g ://658) and the lack of any classical linear H-bonds (Fig. 4). The stabilization of the position of pyrimidine ring in this and the dU_a2, dU_a3 conformation can be determined by electrostatic interaction between atoms: C6H l/O4? and C2O l/H1?. All conformers from Fig. 4 may be considered as local minima, since they were stable on the HF/3-21G(p), HF/631G(d,p) levels of calculation and have no imaginary frequencies in the calculated spectra. The frequencies and intensities of the H-bonded OH vibrations are indicators of these conformational isomers. For the determination of their population in the matrix samples, experimental and calculated spectra are compared in the region of n (OH), n(NH), n(CH) (Table 1). It is well known that ab initio calculations overestimate the frequencies of normal modes, therefore multiplication by a scaling factor is normally required for comparison with experimental frequencies [23 / 25]. As a rule, this scaling factor is a one-off quantity for the vibrations of different molecular
1964
Conformer mode
Experiment (n cm1) I
dU_a0 (n cm 1) Ia dU_a1 (n cm1) Ia dU_a2 (n cm 1) Ia dU_a3 (n cm 1) Ia dU_s1 (n cm1) Ia
dU_s2 (n cm1) Ia
n O5?H n O3?H
3665, 4.1 3641, 2.3
3667, 48 (3673, 77) 3665, 59 (3365, 76) 3674, 63 (3672, 86) 3651, 37 (3655, 63) 3653, 30 (3659, 54) /
n hb_O3?H
3623*, 0.9*
/
/
n hb_O5?H
3609*, 0.3*
/
/
3611, 120 (3637, 107) /
n hb_O5?H
3482, 7.3
/
/
/
n N3H
3428, 10.0
n C5H
3102, 0.9
n C6H
3075, 0.6
3457, 96 (3439, 104) 3074, 4.6 (3044, 1.3) 3100, 9.7 (3071, 3)
3459, 93 (3441, 103) 3078, 4.4 (3054, 6.2) 3057, 57 (3040, 7.8)
3455, 98 (3439, 105) 3455, 98 (3437, 103) 3074, 3.8 (3044, 1) 3074, 5.5 (3045, 1.5) 3105, 8.2 (3078, 4) 3100, 9.7 (3077, 4.4)
/ / 3663, 55 (3659, 66) 3669, 44 (3680, 75) /
/
/ 3652, 30.2 (3660, 54) /
3600, 121 (3639, 85) /
/
/
3578, 344 (3628, 3549, 494 (3593, 149) 330) 3450, 99 (3434, 103) 3451, 101 (3435, 105) 3081, 2.4 (3051, 0.9) 3081, 2.1 (3052, 0.7) 3048, 3.5 (3026, 4.2) 3051, 3.4 (3029, 3.8)
I */ relative integral intensities. Ia */ absolute integral intensities [km/mol]. n hb_OH */ bands of groups involved in the intramolecular H-bonds. * */ bands in Ne matrices (in Ar matrices only one wide band with maximum 3598 cm 1 and I/1.2 was detected).
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
Table 1 The parameters of experimental (from FTIR spectra in Ar matrices) and calculated (3-21G(p), 6-31G(d,p) */ in brackets) spectral bands of 2-deoxyuridine conformers in the 3700 /3000 cm 1 region
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
groups [20,21]. In Table 1 the uniform scaling factor: k /n O5?H (exp.)/nO5?H (calc.) /0.876 calculated for n(O5?H) of conformer dU_a1 at HF/3-21G(p) was used. But with 6-31G(d,p) basis sets, a scaling factor: k /0.875 was used for O5?H, O3?H groups and 0.89 for N3H and C5H, C6H groups (Table 1). From this table we notice that the calculated frequencies of the free O5?H, O3?H and N3H groups are in good agreement with the experimental frequencies for all conformers. These calculations support our previous assignment for thymidine and uridine [10,11], where the band 3482 cm 1 (Fig. 5, Table 1) can be assigned to the H-bonded vibration O5?H O2 in the syn -conformations. In Fig. 4, two syn -conformations: dU_s1 with C3?-endo and dU_s2 with C2?-endo conformation of sugar ring are shown. The geometric parameters of the H-bonds of dU in the different basis sets (Table 2) indicate that dU_s1 has weaker H-bond than dU_s2, as the power of H-bond AH B and the shift of H-bonded vibrations is inversely related to the spacing A B [26]. According to our calculations, the vibration n (hb_O5?H) in conformer dU_s2 has a frequency about 40 cm 1 below than in dU_s1 conformer (Table 1). But we can see in our experimental spectra that the band at 3482 cm 1 has not highfrequency shoulder in Ar matrices (Fig. 5) and this is true for Ne matrices also (Fig. 6). Because of this, the occupancy of conformation dU_s1 can be neglected.
1965
The calculated frequencies and intensities of characteristic bands of conformers dU_a2, dU_a3 and their geometric parameters of H-bonds are in close agreement (Tables 1 and 2). Because of this, it is difficult to distinguish these conformers in the experimental spectra. So, in Ar matrices we can see only the wide band 3598 cm 1 with a highfrequency shoulder (Fig. 5). But in the Ne matrices two bands 3623 cm 1, 3615 cm 1 are well observed (Fig. 6). Based on the data from Table 1, it may be suggested that the band at 3610 cm1 is assigned to n(hb_O3?H) of conformer dU_a2 and 3623 cm 1 to n (hb_O5?H) of conformer dU_a3. 4.2. The spectral region of stretching vibrations n(CH) The anti conformers: dU_a0 and dU_a1 in the n(OH), n (NH) spectral region are indistinguishable practically but appreciable difference between them may occur in the region n(C5H), n (C5H). Relying on comparison with the fragment of spectra of thymidine and 1-methyl-uracil (Fig. 7), the band at 3102 cm 1 was assigned to n(C5H) and 3075 cm 1 to n (C6H) in the matrix spectra of dU (Fig. 7). Contrary to the spectrum of 1-methyluracil, Fermi resonance splitting is absents in the spectra of nucleosides (Fig. 7). The HF calculations do not demonstrate the low-frequency shift of H-bonded n(C6H) (Table 2) and this is con-
Table 2 ˚ and angles in 8) AH B intramolecular H-bonds of 2-deoxyuridine conformers at the The geometric parameters of (distances in A different level of calculations Method conformer
HF/3-21G(p) ˚) d(AH B) (A
dU_a1 /(C6H O5?)8 dU_a2 /(O3?H O5?)8 dU_a3 /(O5?H O3?)8 dU_s1 /(O5?H O2)8 dU_s2 /(O5?H O2)8
2.18 161.1 2.08 131.8 2.05 142.8 1.93 153.8 1.80 163.2
HF/6-31G(d,p) ˚) d(A B) (A 3.21 2.80 2.86 2.81 2.73
˚) d(AH B) (A 2.47 159.8 2.31 126.0 2.34 131.4 2.30 143.9 1.99 161.8
MP2/6-31G(d,p) ˚) d(A B) (A 3.50 2.96 3.05 3.12 2.90
˚) d(AH B) (A 2.34 160.7 2.19 130.3 2.16 139.0 2.12 146.3 1.88 165.4
˚) d(A B) (A 3.38 2.90 2.96 2.98 2.84
1966
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
Fig. 7. The FTIR spectra of 2-deoxyuridine (1), thymidine (2), 1-methyl-uracil (3) isolated in Ar matrices (T /12 K, M/S/ 700) in the C5 /H, C6 /H stretching region (3140 /3040 cm1).
sistent with experiment: the shift of n (C6H) in dU_a1 is small (/15 cm 1) with respect to n (C6H) of 1-methyl-uracil (Fig. 7). The calculations on the HF/3-21G(p) level show that the intramolecular hydrogen bond C6H O5? in conformer dU_a1 produces a strong increase in intensity of vibration n (C6H), which is one order of magnitude greater than above the intensity of n (C5H) (Table 1). The comparison of the geometry parameters of H-bonds in dU_a1 at different basis sets indicates that calculations on the HF/321G(p) level produce a very short C6H O5? distance (Table 2) and correspondingly overestimate the power of this H-bond. The distances on the HF/6-31G(d,p) and MP2/6-31G(d,p) level of these calculations are more realistic. So, the MP2 ˚ distance C6H O5? for this conformer (2.34 A from Table 2) correlated with DFT/6-31G(d) data: ˚ for the C2?-endo /anti and 2.37 A ˚ for the 2.28 A C3?-endo /anti conformation of uridine [8]. The increase in intensity of the H-bonded n (C6H) on the HF/6-31G(d,p) level of these calculations is relatively small, but intensity of nC6H is in 1.25 times more than intensity of n(C5H) (Table 1). In experimental spectra we can see the diametrically opposed picture, namely, the intensity of free vibration n(C5H) is greater than intensity of Hbonded vibration n(C6H) (Fig. 7). This effect may
be explained by the overlapping of spectral bands of different conformers. As was calculated (Table 1), the intensity and frequency of n(C6H) in conformer dU_a0 are greater than for n(C5H) (Table 1). It is true for conformers dU_a2, dU_a3 also, which have a similar orientation of the pyrimidine ring. The superposition of the spectra of these conformers can lead to an apparent increase in intensity of n(C5H). It leads us to conclude that the occupancy of conformer dU_a0 cannot be neglected. The frequencies of two CH2 groups and three CH groups of ribose sugar are located in the region 3040/2750 cm 1. Also, Fermi resonance splitting of spectral bands is standard for this type of vibrations of organic molecules [27]. Owing to these factors overlapping spectral bands are seen (Fig. 8). Because of this, we have used the deconvolution of experimental spectra into bands with Lorentzian (or Gaussian) contours. These results and the calculated spectra are presented in Table 3.
4.3. The spectral region of stretching vibrations n(C /O), n (C5 /C6) The experimental and calculated spectral data for this region are presented in Table 3. In this
Fig. 8. The FTIR spectrum of 2-deoxyuridine (1) isolated in Ar matrix (T /12 K, M/S/700) in the C /H stretching region (3040 /2750 cm 1).
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
1967
Table 3 The experimental (from Ar matrices) and calculated (6-31G(d,p)) frequencies and intensities of vibrations of the 2-deoxyuridine conformers in the 3000 /200 cm 1 region Mode no.
Experiment
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
30 31 32 33 34 35 36 37 38
39 40 41 42 43 44
)
dU_a1
dU_s2
1
n (cm
I
1
Assignment PED (%) for dU_a1 )
a
n (cm
2997 2946 2924 2893 2864 2838 2801 1762 1734 1710 1640 1520 1467 1456 1452 1434 1422
0.9 7 0.8 2.7 0.5 0.3 0.3 5.1 64.0 47.0 6.8 0.3 0.5 11.1 3.6 0.6 0.1
2976 2943 2895 2871 2864 2852 2815
6 8 14 65 34 87 47
2977 2906 2892 2886 2876 2850 2829
3 19 51 29 55 91 35
1714 1735 1641
955 710 206
1700 1744 1644
1072 746 172
1408 1389 1376 1357 1322 1308 1297
0.4 4.8 3.3 2.6 0.9 2.2 1.3
1485 1465 1456 1439 1410 1408 1405 1391 1384 1354 1333 1307 1306
4 148 51 3 8 8 17 149 28 25 13 31 49
1473 1468 1452 1434 1409 1406 1405 1388 1381 1372 1342 1330 1304
10 58 115 44 65 9 16 160 35 46 26 3 13
1284 1272 1264 1259 1247 1239 1227 1216 1204 1190 1179 1147 1125 1115 1103 1092 1079 1064 1057 1037 1001 989 964 956
6.4 6.1 1.5 3.4 2.1 0.9 1.2 5.0 3.0 2.7 1.5 0.4 4.2 3.9 7.8 2.4 5.2
1268
280
1286
7
1249
53
1264
168
1216 1214 1192 1186 1179
44 59 34 135 43
1223 1216 1207 1179 1174
77 60 41 40 51
1150 1121 1104
316 8 9
1152 1131 1099
206 66 71
1074 1061
8 25
1070 1068
27 18
n C3?O(11), n N1C2(10) n C1?O4(16), n C5?O(12)
1032 997 992 960
0.4 64 28 0.5
1002 1000 991 962
33 1.7 80 0.5
gC6H(90) bC2?H2(24), n C3?O(16) n C4?O4?(16) b6ring(20), n C4C5(18)
7.3 4.8 1.5 1.2 0.2 1.5
)
a
I
n (cm 6 7 8 9 10 11 12
1
I
n C1?H(95) n C2?H2as(95) n C2?H2sym(94) n C3?H(84) n C5?H2as(88) n C4?H(80) n C5?H2sym(94) n C2O(76) n C4O(70) n C5C6(57), bC6H(15) bC5?H2(41) bC1?H(27), bC2?H2(14) bC2?H2(50) bC5?H2(52) bC6H(28), bN3H(18) bC3?H(33) bN3H(16), n N1C2(10) n C2N3(22), n N1C2(13) bC4?H(30), bC1?H(26) bC1?H(40) bC4?H(30), bC3?H(18) bC3?H(36), bC4?H(30) bC1?H(23), bC2?H2(25) bC6H(20), n N1C6(19)
bC2?H2(58) bC5H(16), n N3C4(13) bC2?H2(20) bO5?H(22), n N1C1?(20) bO3?H(17), bO5?H(16) bO5?H(24), bO3?H(12) n C1?O4?(27) n C3?O(16), n C5?O(9) n C5?O(33)
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
1968 Table 3 (Continued ) Mode no.
Experiment n (cm 1)
45 46 47 48 49 50
51 52 53 54 55 56 57 58 59
60 61 62 63
64 65 66 67 68 69 70
71 72 73 74 75 76 77 78
dU_a1 n (cm 1)
I
dU_s2 Ia
n (cm1)
Assignment PED (%) for dU_a1 Ia
950 945
0.8
938
23
944
31
bC2?H2(22), n C1?C2?(10)
916 878 868 859 840
0.8 1.2 0,2 0.1 0.4
922 881 875
15 16 13
921 879 869
10 10 27
n C2?C3?(16), n C5?O(8) n C4?O4?(14) n C3?C2?(23), bC2?H2(10)
822
4
830
8
807 805 776 766 760 752 734 714 659 644 605 598 578 549 544 525 516 467 430 418 407 402 392 377 351 326
3.1 1 1.4
816
79
815
81
4.2 0.8 0.2 0.3 6.5 0.3 0.6 2.2 0.2 1.5 0.4 1.6 0.8 0.4 1.1 0.7 1.2 1.0 0.9 1.0 0.4 0.6
780 752 740 721 657 636 634 595
90 11 14 3 71 20 5 18
786 747 738 717 665 651 630 585
98 33 3 0.2 70 4 36 184
545
18
565
32
b6ring(42),bC2O(16)
516 458
18 12
553 519
6 36
b6ring(25), bC4O(16) bN1C1?(26), bC3?O3?(18)
422 401
11 28
457 414
25 11
t6ring(41), bC3?O3?(20) bC4O(42)
277 257 246 240 232 224 217
1.0 1.7 0.5 1.0 0.6 0.7 1.0
367 344 323 292 272
20 14 19 198 49
395 367 344 321 299
29 13 20 0.4 123
t6ring(16), bC1?N1(15) bC3?O3?(39), tO3?H(13) bC4?C5?(34), tO5?H(11) tO3?H(88) tO5?H(72)
252
15
272
4
bC4?C5?(14), n N1C1?(12)
237
14
232
5
bC1?N1(19), bC4?C5?(12) [n (O5?H O2)(14)]*
201 186 177 158 126 93 63 48
1 3 4 7 2 0.4 1.3 0.1
bC1?N1(27) t6ring(55) t6ring(98) tC4?C5?(55) [n (O5?H O2)(34)]* t5ring(57), tC4?C5?(14) n (C6H O5?)(72) [n (O5?H O2)(24)]* tC1?N1(34), gC1?N1(28) tC1?N1(84)
187 180 176 141 110 65 57 42
3 6 1 4 0.6 0.7 0.3 0.4
b5ring(17), n N1C1?(11) gC5H(57), gC4O(44)
gC2O(85) n C4C5(27), n N1C2(13) b5ring(23), n C1?C2?(13) gC6H(32), gC5H(23) gN3H(93) b5ring(14) bC4O(13), bC2O(10) b5ring(19), bC2O(10)
I */ relative integral intensity from the experimental FTIR spectra. Ia */ absolute integral intensity in km/mol. * */ PED for Hbond of conformer dU_s2.
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
region individual scaling factors were used: namely, 0.77 for n (C /O) vibrations and 0.89 for n (C5/C6). Fermi resonance is a marked property of the n(C /O) region in the matrix spectra of nucleic acid bases [13,24]. For example, in the spectrum of 1-methyl-uracil, four bands are shown in the n(C /O) region (Fig. 9). Also, resonance splitting is marked for the n(C5 /C6) bands of 1methyl-uracil: 1650 cm 1, 1636 cm 1 (Fig. 9). The surprising thing is that Fermi resonance is absent practically in the n(C /O), n(C5 /C6) region of the matrix spectra of dU (Fig. 9). It is evident (Fig. 9) that the ribose ring has no strong influence on the frequencies of the spectral bands of the base: n(C / O), n(C5 /C6). As for 1-methyl-uracil [24], the HF method calculated frequencies of n(C2O) and n(C4O) in the order reverse to experiment (Table 3). It was shown that the MP2 frequencies coincide with the experiment and that the intensities of n (C4O) and n (C2O) are in good agreement with experiment for both methods [24]. Because of this, the assignment of n(C2O) to 1734 cm 1 and n(C4O) to 1710 cm 1 was made on a basis of comparison between calculated and experimental intensities of these vibrations (Table 3). It was shown that in comparison with planar ring molecules (uracil), nonplanar molecules (5,6-
Fig. 9. The FTIR spectra of 2-deoxyuridine (1), 1-methyl-uracil (2) isolated in Ar matrices (T /12 K, M/S/700) in the C2/O, C4/O, C5/C6 stretching region (1820 /1625 cm1).
1969
dyhydrouracil) have broad spectral bands in the matrix spectra owing to disordered matrix sites [13]. It may be concluded that this effect is stronger for nonplanar flexible molecules, such as nucleosides. In this region the spectral bands of dU are noticeably wider in comparison with 1-methyluracil (Fig. 9) and this is also true for the bands in the deformation spectral region. 4.4. The spectral region of deformation vibrations (1500 /200 cm 1) In the range of frequencies below 1500 cm 1 the bands of deformation vibrations, stretching vibrations of ribose and pyrimidine rings and n(C3?/O), n(C5?/O) are located. The experimental and calculated spectral data in the region below 1500 cm 1 are presented in Table 3. In this region, the connection of the scaling factor with the type of vibration is not well defined and the uniform scaling factor 0.9 was used which is close to some previous works for nucleic acid bases [23,24]. In the range of 1500 /1000 cm1, 25 spectral bands are located (Fig. 10, Table 3). Most of these bands belong to ribose sugar and attachment groups (Table 3). As in the n(C /O) region the spectral bands are broad in comparison with nucleic acid bases and overlapping is evident (Fig. 10) and the decomposition spectra on the Lorentzian and Gaussian contours was used. In the range of 1000/500 cm 1 the bands of pyrimidine base have the greatest intensities and are well marked in the matrix spectra (Fig. 10); the ribose ring has little influence on the spectral bands of the base. Their frequencies coincide closely with analogous bands of 1-methyl-uracil. The vibrational band g (C2O) around 760 cm1 has a marked splitting (Fig. 10), perhaps connected with conformer dU_s2 since the C2O group take part in the intramolecular H-bond formation. In this spectral range our 6-31G(d,p) calculations (Table 3) and analogous 6-31G(d,p) calculations for uracil [19] are in good agreement. In the 500/200 cm 1 range, calculated and experimental data are markedly worse than in the other region. In this region, the number of observed spectral bands is significantly greater than the calculated bands (Fig. 10, Table 3)
1970
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
are presented in Table 4. For comparison with real experimental data the relative Gibbs free energy DG was estimated by the standard method [28]: T
DGAB (T)DE DZPE
g DCdT TDS(T) 0
Fig. 10. The FTIR spectra of 2-deoxyuridine isolated in Ar matrices (T /12 K, M/S/700) in the 1540 /200 cm 1 region.
attributed to intense bands of low-lying conformers. The influence of the matrix cage also complicates the assignment of vibrations in this region. Practically, the vibrations of intramolecular H-bonds are beyond the range of our spectral measurements (for example see frequency of dU_s2 vibration of H-bond n(O5?H O2) in Table 3). The calculations overestimate the intensity of the torsion vibration of the groups O5?H, O3?H. (Table 3). The matrix influence on the barrier height of frozen rotation must also be taken into account for large fragments of a molecule; this can lead to increase in frequencies of deformation vibrations and uniform scaling factor of 0.9 cannot be used. 4.5. The populations of dU conformational isomers in the low temperature matrices The calculated and experimental relative energies the anti -, syn -conformers of 2-deoxyuridine
Relative electronic energy DE was estimated at the MP2/6-31G(d,p) level, and the relative zero-point vibrational energy DZPE and temperature dependent contributions of rotation and vibration were estimated at the HF/6-31G(d,p) level for temperatures of 298 and 420 K (Table 4). According to the relative electronic energy calculations, the syn conformer dU_s2 with the intramolecular hydrogen bond O5?H O2 and conformation of the sugar ring-C2?-endo has the minimal energy at all levels of optimization (Table 4). dU_a1 has minimal electronic energy among anti -conformers (Table 4). The influence of the vibrational /rotational contribution to the free energy reduces the difference of conformers’ energies significantly, and this is especially noticeable with the growth of evaporation temperature (Table 4). But the calculations of the relative free energy DG did not demonstrate principal modifications of the conformational distribution (Table 4). The association of DG and experimental spectral data can be expressed by the standard equation: DGAB (T)RTlnKAB RTln(hA =hB ) where KA,B is the equilibrium constant of conformers A, B and hA, hB are the populations of these conformers. To define the populations of conformers in experimental spectra it is necessary to know the molar extinction coefficients of characteristic spectral bands or their ratios. There are several ways to determine these quantities; in the first place, the ratios of molar extinction coefficients of characteristic bands can be determined through the redistribution intensities of characteristic bands under the influence of changing evaporation temperature or through the UV irradiation of matrix samples. If related experiments are difficult, the calculations of the intensities of characteristic spectral bands may be used. Ab initio calculations
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
1971
Table 4 Calculated and experimental relative energies (kcal/mol) of 2-deoxyuridine anti -, syn -conformers Conformer method
d_a0
d_a1
d_a2
d_a3
d_s1
d_s2
HF/3-21G(p) HF/6-31G(d,p) MP2/6-31G(d,p) DG (298 K)b MP2/6-31G(d,p) DG (420 K)b MP2/6-31G(d,p) DG (420 K) experiment
5.0 2.7 5.6 3.4 2.8 /
2.0 1.3 2.8 1.7 1.4 0
2.4 1.4 3.6 2.4 2.1 1.8
4.4 3.1 4.8 3.8 3.4 2.2
3.1 1.5 2.7 1.9 1.6 /
0 (/826.510784)a 0 (/831.066722) 0 (/833.487562) 0 0 1.1
a b
Absolute energies in a.u. are indicate in brackets. Vibrational /rotational contribution was estimated at HF/6-31G(d,p) level.
usually overestimate the absolute infrared intensity of vibrational bands [23 /25], however the relationship of experimental and calculated intensities coincides much better. So, the equilibrium constant of isomers a and b has been adopted [29,30]: P P Ia(eksp:) Ib(calcl:) KAB P (1) P Ia(calcl:) Ib(eksp:) Unlike some studies [29,30] we have applied Eq. (1) to OH, NH and CH stretching vibrations only. With our data the best agreement between the calculated and experimental results is observed for the relationship between the intensities of stretching vibrations. This is true for stretching vibrations of dU also (Table 5), but data for H-bonded stretching vibrations were not available. Because of this, we have used UV irradiation of matrix samples for the shift of conformation equilibrium and determination of band ratios. The influence of UV irradiation on the spectra of the matrix samples is presented in Fig. 11. As some minor UV-induced degradation was detected in our spectra, it was presumed that the UV-induced
degradation is equal for all dU isomers. With this approximation, the growth of the band intensities of H-bonded n(OH) modes demonstrated a good correlation between experimental and calculated data (Table 5). As was discussed above, the characteristic bands of dU_a0 and dU_a1 were not clearly defined in the matrix spectra. Also, our calculations predict very small occupancies of the dU_a0 conformer (Table 4). Therefore for the sake of simplicity, the conformer dU_a0 was ignored and only four conformers: dU_s2, dU_a1, dU_a2, dU_a3 were considered. For the determination of KAB the different combinations of the experimental and calculated intensities from Table 1 and experimental ratios from Table 5 were used. The occupancies of conformers dU_a2, dU_a3 were estimated on the basis of their spectra in Ne matrices (Table 1). The net results of these experimental estimations are presented in Table 4. The experimental and calculated data coincide well for the dU_a2 and dU_a3 conformers (Table 4), but the discrepancy between experimental and calculated occupancies of the conformers’ dU_a1
Table 5 The experimental and calculated ratio of intensities of the stretching vibrations for the 2-deoxyuridine conformers Method ratio I (n AB)/I (n CD)
Experiment Ar matrices
Calculation HF/3-21G(p)
Calculation HF/6-31G(d,p)
I(N3H)a/[I (C2O)/I (C4O)]a I(C2O)a/I (C4O)a I (N3H)a/[I (C5H)/I (C6H)]* I (hb_O5?H)/[I (O5?H)a] in dU_a3 I (hb_O3?H)/[I (O3?H)a] in dU_a2 I (hb_O5?H)/[I (O5?H)a] in dU_s2
0.09 1.4 6.8 2 2 7
0.07 1.1 6.7 2.3 3.5 8.4
0.06 1.3 7.4 1.9 1.1 4.3
a
Vibrations of conformer dU_a1.
1972
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For example, some authors [31] used averaging of energies calculated at several levels of theory for the enhancement of reliability, but obviously this increases the magnitude of the numerical calculations. It is possible that on the way to intensive calculations the usage of correlation consistent (cc) basis sets is more reasonable. As was noted by Jensen [32], the main advantage of cc-basis sets ‘is the ability to generate a sequence basis sets, which converges toward the basis sets limit’. Because of this, it is interest to test the convergence of DE and DG in the row of basis sets: aug-ccpVDZ, aug-cc-pVTZ, aug-cc-pVQZ to the experimental data.
Fig. 11. Effect of UV-irradiation on the FTIR spectrum of 2deoxyuridine isolated in Ar matrix (T /12 K, M/S/700) in the O /H, N /H stretching region (3690 /3400 cm 1). (1) Spectrum before UV-irradiation; (2) difference spectrum after UV-irradiation (t/40 min).
and dU_s2 is obvious (Table 4). Possible reason for this discrepancy are that the conformers’ occupancies in matrices may differ widely from their analogous in the gas phase. This effect is observed at low barriers between conformational isomers [12]. According to experimental data interconversion in an Ar matrix at 30 K is possible for barriers B/2.5 /3 kcal/mol [12]. We tested the effect of interconversion by using deposition of the samples in matrices with different temperatures and annealing of matrix samples. No differncies were see in our spectra in Ar matrices at 5 and 12 K (Figs. 5 and 6). Annealing of matrix samples at 30 K has no influence on the conformational equilibrium. It follows that the barriers heights of dU conformers /2.5 /3 kcal/mol and their conformational equilibrium in gas phase at evaporation temperature must be close to equilibrium in Ar matrices at 12 K. The conformer dU_a0 may be a exception, since clear spectral identification of the transition dU_a0 0/dU_a1 is difficult in these experiments. In the second place, ab initio calculations of electronic energies or free energies are not sufficiently reliable for the large unrigged molecules, even with large basis sets and using the method of electronic correlation at the MP2 level.
5. Conclusions For the first time FTIR spectra of 2-deoxyuridine were obtained in Ar and Ne matrices. For the first time the conformational equilibrium and structures of intramolecular H-bonds of 2-deoxyuridine in a isolated state were investigated by FTIR matrix isolation spectroscopy and ab initio calculations. It was found that anti conformers of dU are dominant in the isolated state. Their occupancy in the matrix samples was estimated at 81%. The ribose rings of the main anti -conformers dU _a1, dU _a0 are in the C2?endo conformation, but the ribose rings of minor anti -conformers dU_a2, dU_a3 have the C3?-endo conformation, stabilized by intramolecular hydrogen bonds O3?H O5? and O5?H O3? accordingly. Intramolecular hydrogen bonds O3?H O5?, O5?H O3? may be treated as a spectroscopic indicator of the transition C2?-endo 0/C3?-endo between conformations of ribose ring in the anti conformers of dU. Syn -conformers of dU are stabilized by the intramolecular hydrogen bond O5?H O2 and the dominant conformation of ribose ring is C2?endo . Their occupancy in the matrix samples was estimated as 19%.
A.Yu. Ivanov et al. / Spectrochimica Acta Part A 59 (2003) 1959 /1973
Acknowledgements This investigation was supported by the Ukrainian Academy of Sciences and in part by the INTAS-International Association under grant no. INTAS 00-00911.
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