J. Phys. Chem. 1996, 100, 3527-3534
3527
Molecular Structure and Infrared Spectra of Adenine. Experimental Matrix Isolation and Density Functional Theory Study of Adenine 15N Isotopomers Maciej J. Nowak,† Leszek Lapinski,† Jo´ zef S. Kwiatkowski,‡ and Jerzy Leszczyn´ ski*,§ Institute of Physics, Polish Academy of Sciences, 02-668 Warszawa, Poland, Institute of Physics, Nicholas Copernicus UniVersity, 87-100 Torun´ , Poland, and Department of Chemistry, Jackson State UniVersity, Jackson, Mississippi 39217 ReceiVed: October 10, 1995X
The infrared spectra of matrix-isolated adenine and its 15N isotopomers with 15N at the N(9) or N(7) positions have been studied. The experimentally observed IR spectra were compared with the spectra predicted at the DFT(B3-LYP)/6-31G(d,p) level. This method was also used to calculate molecular parameters (rotational constants, dipole moments) of both N(9)H and N(7)H tautomers of adenine. The agreement between experimental and theoretical spectral positions, intensities, and isotopic shifts of the IR bands is good. That allows for reliable assignment of the IR spectra and for conclusion that the amino-N(9)H tautomer of adenine strongly dominates in low-temperature matrices.
Introduction
SCHEME 1
Adenine in polar solutions exists as a mixture of two amino tautomers N(9)H and N(7)H.1 In water solution, the N(9)H tautomer predominates over the N(7)H form by a factor of 4.2 The IR spectra of adenine isolated in low-temperature argon matrices3,4 might suggest, at first glance, that both the aminoN(9)H and amino-N(7)H tautomers of the compound coexist in this environment in equal amounts. Especially suggestive is the high-frequency (3600-3300 cm-1) region of the IR spectrum, in which each of the three bands (due to antisymmetric NH2 stretching, symmetric NH2 stretching, and NH stretching vibrations) is split in two.3,4 We have shown, however, that this splitting is caused by the matrix effect3 and that, most probably, only one tautomeric form is present in the matrix. The IR spectra of adenine isolated in neon and nitrogen matrices3 supported this conclusion. Considering the slight difference in energy between the N(9)H and N(7)H forms in polar media, we postulated that the N(7)H tautomer, which has much higher dipole moment and hence can be more strongly stabilized in polar media, should have a less favorable relative energy in an inert environment. The statement of the unique presence of the N(9)H tautomer in low-temperature matrices was further confirmed by experimental and theoretical analysis of the lower frequency region of the IR spectrum of adenine, with special attention paid to the band due to out-of-plane wagging NH vibration (γNH).5 Although many arguments supporting the conclusion of the unique presence of the N(9)H tautomer of adenine in lowtemperature matrices have been accumulated, the ultimate solution to the adenine tautomerism problem needed additional support. Infrared spectra of adenine 15N isotopomers should yield important information concerning tautomerism of the compound. To differentiate between the two amino tautomers N(9)H and N(7)H (Scheme 1), we studied, in the present work, the infrared spectra of the isotopomer of adenine with 15N in the N(9) position (Ade 15N(9)) and of the isotopomer of adenine with 15N in the N(7) position (Ade 15N(7)). The most straightforward information might be derived from the spectral †
Polish Academy of Sciences. N. Copernicus University. § Jackson State University. X Abstract published in AdVance ACS Abstracts, January 15, 1996. ‡
0022-3654/96/20100-3527$12.00/0
position of the band due to the NH stretching vibration (νNH) in the spectra of isotopomers. This band was observed at 3498, 3489 cm-1 (argon matrix) in the spectrum of “all 14N” isotopomer of adenine (Ade 14N). According to theoretical predictions for adenine adopting the amino N(9)H tautomeric form, this band should be observed at exactly the same position in the spectrum of the Ade 15N(7) isotopomer and at a frequency 8.6 cm-1 lower in the spectrum of the Ade 15N(9) isotopomer. For adenine adopting the N(7)H tautomeric form, it should be just opposite: the band due to νNH vibration would be observed at the same frequency (as for the Ade 14N isotopomer) in the spectrum of Ade 15N(9) and at a frequency 8.6 cm-1 lower in the spectrum of Ade 15N(7). The infrared spectra of matrix-isolated adenine have been interpreted in several works. In all cases, the assignments of bands observed in the experimental spectrum were based on comparison with the spectra predicted theoretically within the Hartree-Fock method using the 3-21G, 4-21G, or 6-31G(d,p) basis sets.6-8 None of the assignments presented so far can be treated as reliable, mainly because of the limitations of the applied theoretical methods. In this work we have carried out theoretical simulations of the harmonic frequencies and IR intensities of the bands in the spectrum of adenine N(9)H and N(7)H tautomers. The calculations were performed using the density functional theory with the mixed Becke3-LYP functionals using the 6-31G(d,p) basis set. The spectra of three experimentally studied isotopomers (Ade 14N, Ade 15N(9), and Ade 15N(7)) were predicted theoretically for both the adenine tautomers in question. Comparison of the experimentally measured and theoretically predicted “isotopic shifts” of the IR bands proved to be a good criterion of the assignment of the observed IR bands to the calculated normal modes. © 1996 American Chemical Society
3528 J. Phys. Chem., Vol. 100, No. 9, 1996
Nowak et al.
TABLE 1: Rotational Constants, Dipole Moments, and Energies of the Adenine N(9)H and N(7)H Tautomersa calculation,b adenine N(9)H HF A
2426.53 (2426.75) 1596.17 (1596.20) 962.86 (962.87) 2.47 (2.48)
B C µ energies Ed ZPEe Etotf
-466.030 92 (ref) 317.7 (ref) 0.0
MP2
calculation,b adenine N(7)H experimentc
HF
2369.13
2371.873(4)
2422.52
2388.70
1566.49
1573.3565(8)
1570.23
1536.54
943.11
946.2576(4)
956.73
936.06
2.40
2.75
6.89 (6.74)
6.76
-466.018 69 (+32.1) 318.0 (+0.2) +32.3
-467.318 36 (+34.3) 294.4 (-0.4) +33.9
DFT(B3-LYP)
DFT(B3-LYP)
(2360.11) (1574.07) (944.29)
-467.331 42 (ref) 294.7 (ref) 0.0
a Rotational constants A, B, and C in megahertz; dipole moments in debye units. b Calculations at the HF, MP2(full), or DFT(B3-LYP) optimized geometries with the 6-31G(d,p) basis set. The calculated rotational constants in parentheses are those computed for the planar form (Cs structure) of the base.16 c Experimental rotational constants from ref 18; experimental dipole moment of 9-methyladenine in dioxane from ref 19. d Electronic (HF +MP2(full) and DFT(B3-LYP) energies in atomic units; the corresponding relative energies in kJ/mol. e Zero-point vibrational energies (ZPE) in kJ/mol. f Total (electronic + ZPE) relative energies in kJ/mol.
Experimental Section The samples of adenine isotopomers Ade 15N(9) and Ade were prepared by Dr. M. Bretner and Dr. K. Felczak of the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland. The sample of Ade 14N was purchased from Fluka Chemie AG (Switzerland). The general procedure used for matrix preparation was the same as that described elsewhere.8 Matrices were deposited on a CsI window mounted on the cold finger of continuous flow helium cryostat. The temperature of the cold window was 7-8 K, and the temperature of the oven from which the sample sublimed was about 450 K. Spectral-grade argon and nitrogen matrix gases were supplied by Linde AG (Germany) and VEB Technische Gase Werke Berlin (Germany), respectively. The time of matrix deposition was 1.5 h. The IR spectra were recorded with a Perkin-Elmer 580B grating spectrometer (resolution 1-2 cm-1) and with a Bomem DA3 FTIR spectrometer (resolution 0.5 cm-1). 15N(7)
Calculations The geometries of two adenine tautomers, N(9)H and N(7)H, were optimized using the density functional theory (DFT) with the Becke’s three-parameter exchange functional and the gradient-corrected functional of Lee, Yang, and Parr (DFT(B3-LYP) method).9 Calculations employed the standard 6-31G(d,p) basis set, i.e. the valence double-ζ basis set augmented by one set of six d and one set of p polarization functions on heavy atoms and hydrogens, respectively.10 The geometry optimizations were carried out without any constraint for planarity of the tautomers. The harmonic vibrational wavenumbers and absolute intensities of the corresponding IR bands were calculated at the same DFT(B3-LYP)/6-31G(d,p) level. The GAUSSIAN 92/DFT program11 was used in all DFT calculations. IR spectra of the adenine isotopomers were obtained from the same force field by simply changing the atomic masses. IR spectra have been predicted for the isotopomers of adenine in both N(9)H and N(7)H tautomeric forms. The calculated wavenumbers of all normal modes were scaled down by a factor of 0.98 to correct for systematic shortcomings of the theoretical methodology (mainly to correct for anharmonicity). The results of the present DFT(B3-LYP) studies are compared with our previous results calculated at the Hartree-Fock (HF)/ 6-31G(d,p) level.8 To carry out the normal mode analysis in terms of molecule fixed coordinates, the set of nonredundant internal coordinates
was defined as recommended by Pulay et al.12 These coordinates are the same as those listed previously.8 Transformations of the force constant matrix in Cartesian coordinates to the force constant matrix in internal coordinates allowed ordinary normalcoordinate calculations to be performed as described by Schachtschneider.13 The potential energy distribution was calculated for all normal modes. Results and Discussion Molecular Parameters. Although the geometries of the adenine tautomers are not presented here, we would like to comment on the predicted nonplanarity of the base. All optimized (HF, DFT) planar structures of the tautomers in question show one imaginary vibration, for which the normal mode corresponds to pyramidalization of the amino groups, whereas nonplanar conformers possess exclusively real vibrational wavenumbers. However, the deviations from planarity of the tautomers are small. Similar results have been obtained in other studies of the DNA bases with amino groups (cytosine, guanine, and their thio analogs) by HF/6-31G(d,p),5,8,14 MP2/ 6-31G(d), or MP2/6-31G(d,p)15,16 and DFT studies.17 Although the gas phase data for the bond lengths and bond angles of adenine are unknown, a recent microwave spectroscopic study has provided the rotational constants for adenine.18 Therefore, we are able to make an indirect examination of the reliability of the optimized geometries at the HF/6-31G(d,p) and DFT(B3-LYP)/6-31G(d,p) levels for this base by comparing the predicted rotational constants with the corresponding experimental data. Table 1 collects predicted data, as well as experimental data and the recent MP2/6-31G(d,p) calculated rotational constants for adenine. The HF/6-31G(d,p) level calculations predict the rotational constants larger than the experimental constants, with a rootmean-square (rms) deviation of 43 MHz for the predicted constants. The agreement between the DFT(B3-LYP) and experimental constants is excellent, with an rms deviation of 9 MHz for the predicted constants. Note that recent MP2/6-31G(d,p) calculations for the planar forms of the DNA bases16 predict similar rotational constants (Table 1). As for the dipole moment of adenine, the predicted dipole moments of the N(9)H tautomer by HF, by MP2 at the HF geometry, and by DFT(B3-LYP) approaches are closer to each other and to the experimental dipole moment 9-methyladenine (the methylated analog of the N(9) tautomer of adenine) than to the dipole moment of the N(7)H tautomer.
Adenine
15N
Isotopomers
Figure 1. High-frequency region of the FTIR absorption spectra of adenine isotopomers (A) Ade 14N, (B) Ade 15N(7), and (C) Ade 15N(9) isolated in argon matrices.
Figure 2. High-frequency region of the IR absorption spectra of adenine isotopomers (A) Ade 14N, (B) Ade 15N(7), and (C) Ade 15N(9) isolated in nitrogen matrices.
J. Phys. Chem., Vol. 100, No. 9, 1996 3529 The agreement of these values with the experimental data for the N(9)H tautomer suggests the predominance of that form over the N(7)H form in the vapor phase. This is also in agreement with the predictions, based on a comparison of the total relative energies of the N(9)H and N(7)H tautomers (Table 1), of the relative stabilities of both tautomeric forms. The total energies Etot (electronic + zero-point vibrational energies) predict the N(9)H tautomer to be about 32-34 kJ mol-1 lower in energy than the N(7)H tautomer at 0 K. Note that temperature-dependent thermodynamic corrections do not change this prediction for room temperature.20 The other quantum-mechanical estimates give qualitatively similar values for the relative total energies of the N(9)H and N(7)H tautomers of adenine.21 Tautomerism and IR Spectra of Adenine. The experimental IR spectra of adenine isolated in low-temperature Ne, Ar, and N2 matrices have been reported in our previous work.6 The high-frequency regions of the spectra of Ade 14N, Ade 15N(9), and Ade 15N(7) isolated in Ar and N2 matrices are presented in Figures 1 and 2, respectively. In the lower frequency region (1900-200 cm-1), the spectra of the three isotopomers in question are very similar in their shapes (Figure 3). This similarity is very favorable as far as the spectral assignment is concerned. The more commonly applied deuteration of studied compounds is not so convenient in this respect. In the case of deuterated species, the spectral pattern is often significantly disturbed because of considerable changes in the normal mode forms (described by PEDs (potential energy distributions)). The spectra of deuterated and nondeuterated compounds need almost completely separate assignments. However, for Ade 14N and Ade 15N the spectral positions of the IR bands differ from isotopomer to isotopomer only by several wavenumbers. The shifts of the bands in the spectra of Ade 15N(7) and Ade 15N(9) with respect to the band positions in the spectrum of Ade 14N are given in Table 2. The theoretically predicted (DFT and HF) isotopic shifts are also presented in this table. The normal mode frequencies and PEDs calculated at the HF/6-31G(d,p) level were published previously.8 Comparison of the experimentally observed and theoretically calculated isotopic shifts of the IR bands provides a valuable basis for the proposed assignments. Table 3 presents the assignments based on the calculated frequencies and intensities and their similarity (Figures 4 and 5) to the experimental observations, as well as on the agreement between the theoretical and experimental isotopic shifts.
Figure 3. Comparison of the IR spectra in the fingerprint spectral range of adenine isotopomers (A) Ade 14N, (B) Ade 15N(7), and (C) Ade 15N(9) isolated in argon matrices.
3530 J. Phys. Chem., Vol. 100, No. 9, 1996
Nowak et al.
TABLE 2: Experimentally Observed (Ar Matrix) and Theoretically Predicted Isotopic Shifts of the IR Bands of Adeninea experiment (Ar matrix) Ade14N ν˜ (cm-1)
Ade15N(9) (cm-1)
∆ν˜
theoryb HF/6-31G(d,p)
theory DFT(B3-LYP)/6-31G(d,p)
Ade15N(7) (cm-1)
∆ν˜
mode no.
Ade14N ν˜ (cm-1)
Ade15N(9) (cm-1)
∆ν˜
Ade15N(7) (cm-1)
∆ν˜
mode no.
Ade14N ν˜ (cm-1)
Ade15N(9) ∆ν˜ (cm-1)
Ade15N(7) ∆ν˜ (cm-1)
3565 3557 3555 3552 3506 3503 3502 3498 3494 3489 3448 3441 3438 3057 3041 1910 1693 1659 1651 1645 1639 1633 1626 1618 1612 1599
0.0 0.0 0.0 0.0 8.2 8.2 8.6 8.6 8.5 8.2 0.0 0.0 0.0 1.5 2.0 0.0 1.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.0 1.5 2.0 0.0 0.2
Q1
3676
0.0
0.0
Q1
3601
0.0
0.0
Q2
3589
8.7
0.0
Q2
3530
8.7
0.0
Q3
3589
0.0
0.0
Q3
3471
0.0
0.0
Q4 Q5
3195 3115
0.0 0.0
0.0 0.0
Q4 Q5
3082 3026
0.0 0.0
0.0 0.0
c
c
Q6
1641
0.2
0.1
Q7
1635
0.1
0.5
3.2 1.9 c
1.7 1.4 c
Q7
1617
1.8
1.0
Q6
1639
2.1
1.1
1482 1474 1421 1419 1389 1358 1345 1334 1328
1.7 1.2 2.2 2.2 9.5 ? 3.3 2.0 0.5
d 0.8 3.6 3.6 0.7 0.0 0.7 6.9 7.8
Q8 Q9 Q10
1584 1502 1487
0.5 1.7 0.8
0.1 9.1 0.4
Q8 Q9 Q10
1602 1549 1485
0.4 1.2 0.7
0.2 12.3 0.4
Q11 Q12
1416 1400
2.2 11.1
3.0 0.5
Q11 Q12
1419 1404
4.2 11.4
2.6 0.3
Q13
1350
3.9
1.3
Q14
1342
0.2
4.0
1290 1246 1240 1229 1133 1127 1078 1061 1032 1017 1005 958 927 887 869 848 802 717 698 687 678
0.2 1.1 1.7 1.1 1.5 4.6 1.2 6.5 6.3 4.0 0.4 0.0 7.5 2.9 0.0 0.4 0.0 1.0 0.0 0.0 0.1
1.2 0.7 5.0 2.6 0.0 1.0 0.0 0.8 0.0 0.1 0.4 0.0 12.5 1.7 ? 0.0 0.8 2.5 0.0 0.0 0.0
Q15
1317
0.3
4.6
Q13 Q14 Q15
1346 1330 1269
1.1 1.1 0.7
8.0 0.3 1.0
Q16 Q17
1250 1228
1.6 1.0
5.5 2.3
Q16 Q17
1228 1213
0.7 0.5
3.2 3.2
Q18
1129
5.6
1.2
Q18
1120
5.7
1.0
Q19
1065
6.6
0.8
Q19
1050
7.2
0.5
Q20 Q21 Q22 Q23
1000 953 925 882
0.5 0.0 7.2 3.0
0.3 0.0 12.4 1.7
Q21 Q20 Q22 Q24
996 1009 922 882
0.8 0.0 7.1 3.1
0.4 0.0 12.3 1.8
Q24 Q25 Q26
831 793 713
0.4 0.0 1.4
0.0 0.9 3.1
Q23 Q25 Q26
904 809 702
0.5 0.2 1.3
0.3 0.5 3.0
655 610 591 583 566
0.9 1.9 0.0 0.0 0.0
6.8 1.2 0.0 0.0 0.0
Q27 Q28 Q29
672 658 607
0.9 1.1 2.6
0.0 7.0 1.7
Q27 Q28 Q29
694 652 601
1.1 1.2 2.7
0.0 7.3 1.6
513 503
2.8 2.6e
0.0 0.6e
276 242 214
0.0 0.0 0.0
1.0 0.0 0.0
Q30 Q31 Q32 Q33 Q34 Q35 Q36 Q37 Q38
568 528 521 514 504 298 269 244 205
0.1 0.9 0.4 3.7 2.2 1.7 0.3 0.0 0.2
0.0 0.4 1.8 0.3 0.7 0.9 1.2 0.0 0.0
Q30 Q32 Q31 Q34 Q33 Q35 Q36
557 515 517 500 505 298 271
0.2 0.4 0.1 3.0 2.9 1.9 0.3
0.0 1.3 1.2 0.3 0.5 0.9 1.3
Q39
162
0.8
1.2
Q37 Q38 Q39
215 168 93
0.1 0.9 0.0
0.0 1.2 0.0
}
}
a Wavenumbers, ν ˜ , correspond to adenine with all nitrogen atoms being 14N isotopes (Ade14N). Isotopic shift, ∆ν˜ , is the difference between the wavenumber of a band in the spectrum of Ade14N and the wavenumber of the corresponding band in the spectrum of adenine isotopomer: Ade15N(9) or Ade15N(7). b The calculations of IR spectra of adenine at the HF/6-31G(d,p) level were previously published.8 Mode numbering is the same as it was in Table 3 in ref 8. Theoretical wavenumbers and spectral shifts calculated at the DFT(Becke3LYP)/6-31G(d,p) level were scaled by a single factor of 0.98. Theoretical wavenumbers and spectral shifts calculated at the HF/6-31G(d,p) level were scaled by a single factor of 0.90. c The spectral isotopic shift was not measured; see text. d In the spectrum of Ade15N(7) this band overlaps with the intense band at 1473 cm-1.e The shift taken from the nitrogen matrix.
Adenine
15N
Isotopomers
J. Phys. Chem., Vol. 100, No. 9, 1996 3531
TABLE 3: Experimental Frequencies (ν˜ ), Relative Integral Absorbancesa (I), and Assignment to the Normal Modes of the Absorption Bands of Isolated Adenine in an Ar Matrixb experiment (Ar matrix)
}
ν˜ (cm-1) 3565 3557 3555 3552 3506 3503 3502 3498 3494 3489 3448 3441 3438 3057 3041 1909 1693 1659 1651 1645 1639 1633 1626 sh 1618 sh 1612 1599
}}
1482 1474 1421 sh 1419 1389 1358 1345 1334 1328 1290 1246 1240 1229 1133 1127 1078 1061 1032 1017 1005 958 927 887 869 848 802 717 698 687 678 655 610 591 583 566
}
}
theory DFT(B3-LYP)/6-31G(d,p) ν˜ (cm-1)
Ath (km/mol)
Q1
3676
53
ν NH2 antisym (100)
135
Q2
3589
80
ν N9H (100)
110
Q3
3540
90
ν NH2 sym (100)
6 3 2 11 9 6 33
Q4 Q5
3195 3115
1 30
ν C8H (99) ν C2H (100)
447
Q6
1641
597
β NH2 scis (28), ν C6N10 (21), ν C5C6 (19)
219 49
Q7
1617
108
ν N3C4 (27), ν C5C6 (12)
Q8 Q9 Q10
1584 1502 1487
7 15 57
β NH2 scis (48), ν C4C5 (12) ν N7C8 (48), β C8H (12) ν C6N1 (24), β C2H (22), ν C6N10 (13), β NH2 scis (12), ν C2N3 (12)
Q11 Q12
1416 1400
18 11
ν C4C5 (27), ν C4N9 (21) β N9H (27), β C2H (27), ν C8N9 (14), β R1 (12)
Q13
1350
38
ν N9C8 (20), β C8H (13), β N9H (10), ν C6N1 (10)
Q14 Q15
1342 1317
30 67
ν N1C2 (31), ν C5N7 (19), β C2H (11) ν C2N3 (43), ν C5N7 (13), ν N1C2 (10)
Q16 Q17
1250 1228
30 11
β C8H (36), ν N7C8 (16), β N9H (10) β NH2 rock (26), ν C5N7 (22)
Q18
1129
20
ν C4N9 (20), β r4 (10), ν C6N10 (10)
Q19
1065
17
ν C8N9 (55), β N9H (32)
Q20 Q21 Q22 Q23
1000 953 925 882
5 2 15 13
β NH2 rock (47), ν C6N1 (28), ν N1C2 (10) γ C2H (108) β r4 (42), β r5 (32), ν C4C5 (11) β R1 (49), β R3 (15)
Q24 Q25 Q26
831 793 713
4 14 3
γ C8H (90) τ R1 (46), τ r4 (20), γ C6N10 (19), γ C8H (15) ν N3C4 (20), β r4 (14), ν C5N7 (11), ν C4N9 (10)
Q27 Q28 Q29
672 658 607
3 6 1
γ C6N10 (47), τ r5 (20), τ R3 (13), τ Rr (13) τ r4 (52), τ r5 (36) β r5 (29), ν C5C6 (21), β R2 (17)
Q30 Q31 Q32 Q33 Q34 Q35 Q36 Q37 Q38 Q39
568 528 521 514 504 298 269 244 205 162
64 13 8 61 5 0.5 22 177 90 2
τ R2 (27), γ N9H (24), τ R1 (23), τ r5 (12) twist NH2 (62) β R3 (56), β R2 (14) γ N9H (59), τ R2 (10) twist NH2 (28), β R2 (21), β C6N10 (14) τ R3 (42), τ r5 (18), τ Rr (12), τ R2 (12) β C6N10 (51), β R2 (12) inv NH2 (74), τ Rr (13) τ Rr (54), inv NH2 (24), τ R3 (16) τ R2 (50), τ R1 (17), τ R3 (16), γ C6N10 (15)
I (rel)
mode no.
84
11 71 49 45 2 21 7 40 68 9 28 13 7 6 1 13 27 4 9 3 13 8 2 6 9 5 2 3 2
}
6 5
PED
99 46
513 503
92 4
276 242 214
12 66 75
a For better comparison of experiment with theory the integrated absorbances of absorption bands are normalized in such a way that the sum of integrated absorbances of all normal modes observed experimentally was equalized to the sum of absolute intensities obtained in calculations. sh ) shoulder. b The theoretically calculated wavenumbers (ν˜ ), absolute intensities (Ath), and potential energy distribution (PED) of the absorption bands of the amino-N(9)H tautomer of adenine.
3532 J. Phys. Chem., Vol. 100, No. 9, 1996
Figure 4. Comparison of the 3700-3000 cm-1 region of the experimental FTIR spectrum of adenine (Ade 14N) with the spectrum calculated at the DFT(Becke3LYP)/6-311G(d,p) level. The theoretical spectrum is presented in the stick spectrum form.
(a) Tautomerism. The comparison of the high-frequency regions of the spectra of adenine isotopomers isolated in Ar and N2 matrices (Figures 1 and 2) shows without any ambiguity that the N(9)H tautomer of adenine dominates very strongly. With respect to the spectrum of Ade 14N (Figures 1A and 2A), all the components of the complex structure of the band due to νNH vibration (near 3500 cm-1) retain the same positions in the spectrum of Ade 15N(7) (Figures 1B and 2B) and change their positions by 8.6 cm-1 in the spectrum of Ade 15N(9) (Figures 1C and 2C). This observation corresponds exactly to what would be expected for the N(9)H tautomer (see Table 2). Although for adenine isolated in an argon matrix the shape (resulting from the matrix splitting) of the νNH band is considerably different from that found for adenine in solid nitrogen, the isotope shifts are the same for both matrices. If in the matrix, together with the dominating N(9)H tautomeric form, some experimentally detectable amount of the N(7)H form existed, then the band due to the stretching of the N7H bond should be observed. This band would be observed at ca. 3500 cm-1. No such band was observed, but one should consider the possibility that this band might be hidden under the broad band due to νN9H. If the band due to νN7H was hidden under the low-frequency part of the νN9H band, then in the spectrum of the Ade 15N(7) the band due to νN7H should be red-shifted and should appear on the low-frequency side of the νN9H band. If the νN7H band was hidden under the high-frequency part of the νN9H band, then in the spectrum of the Ade 15N(9) isotopomer this band should be uncovered because of the redshift of the νN9H band. However, no low-intensity band was
Nowak et al. observed on the high-frequency side of the νN9H band in the spectrum of Ade 15N(9), and no bands were observed on the low-frequency side of the νN9H band in the Ade 15N(7) spectrum. These observations indicate that the amount of the N(7)H tautomer in the matrix is lower than the detection limit of the applied measurement system. It is noteworthy that, in the case of the Ade 15N(9) spectrum, the observations mentioned above proves that the 15N isotope enrichment of adenine is certainly not less than the 98% guaranteed by the supplier of the enriched reagent. Because of inherent limitations of the adopted methodology (15N isotope enrichment rate and sensitivity of the measuring system for low-intensity-band detection), the present study may provide only the upper limit of the concentration ratio of the N(7)H and N(9)H tautomers of adenine in a low-temperature matrix environment. It seems reasonable to conclude that, according to the presented results, this ratio must be lower than 0.02. The statement of the unique (within the limits of the applied detection system) presence of the N(9)H tautomer of adenine in low-temperature matrices can be further supported by comparison of experimentally observed and theoretically calculated spectral positions of the band due to γNH vibration. As in our previous paper,5 but this time using the present DFT calculations of the IR spectra of adenine tautomers, we observed that the experimental position of the γNH band (513 cm-1) coincides very well with its theoretically predicted wavenumber (514 cm-1) for the N(9)H tautomer. However, the position of the γNH band predicted for the N(7)H form (403 cm-1) corresponds to the middle of a considerable (more than 200 cm-1 wide) emtpy space in the experimental spectrum. We believe that the ratio of tautomers in the matrix reflects the equilibrium in the gas phase (at the temperature of the microoven) just before the gas mixture condenses onto the cold window in the matrix experiment. This was shown to be the case for several heterocyclic compounds: 4-pyrimidinone,22 4-thiopyrimidine,23 2-thiopyridine,24 2-thiomethyluracil,25 and 2-pyridinone.26 Hence, on the basis of the present matrix isolation experiments, we also can postulate a high predominance of the amino-N(9)H tautomer of adenine in the gas phase. This is in agreement with the Brown et al.18 interpretation of the microwave spectrum of adenine in a continuous-wave seeded supersonic beam, as well as with the results of theoretical predictions of the relative energies of adenine tautomers. The nearly complete, reliable assignment of the observed adenine spectrum compared to the calculated spectrum of aminoN(9)H tautomer (see the next section) supports the above conclusion on the tautomerism of adenine.
Figure 5. Comparison of the 1700-200 cm-1 region of the experimental IR spectrum of adenine (Ade 14N) with the spectrum calculated at the DFT(Becke3LYP)/6-31G(d,p) level. The theoretical spectrum is presented in the stick spectrum form.
Adenine
15N
Isotopomers
(b) Vibrational Assignment. In the high-frequency (36003400 cm-1) range (see Figure 4), the present assignment is the same as that proposed earlier.6,8 The experimentally observed isotopic shifts, which are 0.0 cm-1 for bands due to antisymmetric and symmetric stretching vibrations of the amino group and 8.6 cm-1 for the band due to the N(9)H stretching vibration (in the spectrum of Ade 15N(9)), are in very good agreement with the theoretical prediction (see Table 2). Hence, the assignment in this region (given in Table 3) seems unquestionable. In the lower frequency range, the most intense band in both the experimental and theoretical spectra is placed at 1640-1630 cm-1 (Figures 3 and 5). In the spectra of adenine in lowtemperature matrices, this band is split into several components. We believe that the splitting of this band is caused by the Fermi resonance. This assumption is supported by our observation that the pattern of the splitting varies in the spectra of the three isotopomers in question (Figure 3). This is why it was impossible for us to evaluate isotopic shifts for this band. Nevertheless, the agreement between the observed and calculated frequencies and intensities is so good that misassignment seems very unlikely in this case. For almost all of the bands at lower frequencies, the proposed assignment is supported by the comparison of experimentally measured and theoretically predicted isotopic shifts. For the bands due to the normal modes: Q7, Q11-Q13, Q16-Q20, Q22-Q26, Q28, Q29, Q33, and Q36, the nearly perfect agreement of the observed and calculated isotopic spectral shifts suggests very strongly that the assignment is correct. Moreover, in all cases, the agreement concerns simultaneous shifts in the spectra of both Ade 15N(9) and Ade 15N(7). The absence of a spectral shift upon isotopic substitution can also be informative. If a band like that at 958 cm-1 (Ar) is in exactly the same position in the spectra of all three isotopomers, but all other bands in that spectral range are subject to isotopic shifts, then its assignment to the calculated normal mode Q21, which is the only one in that spectral region characterized by a 0.0 cm-1 isotopic shift, seems very reliable. The assignment of a few other bands deserves some comment. The band at 1482 cm-1 (Ar) has been assigned to the mode Q9. According to our calculations, its spectral shift in the spectrum of Ade 15N(9) should be 1.7 cm-1, which is in agreement with the experimental observation. For the spectrum of Ade 15N(7), a considerable shift (9.1 cm-1) was theoretically predicted. In the recorded spectrum, however, the band in question simply vanishes. We believe that this band is shifted, as theoretically predicted, and due to this shift is hidden under the intense band at 1474 cm-1. The shifts of the bands at 1328 and 1290 cm-1 (Table 2) in the spectrum of Ade 15N(7) differ to some extent from the theoretical prediction. For the first band the predicted shift is too small, while for the second band it is too big. One suggestion for the correction of this disagreement comes from the HF/6-31G(d,p) calculation, which predicts a larger contribution by the νC5N7 vibration to the normal mode at higher frequency (and hence its larger shift in the spectrum of Ade 15N(7)) and a smaller contribution by this vibration to the normal mode at lower frequency (and hence its lower shift in the spectrum of Ade 15N(7)). Special attention should be given to the low-frequency (600200 cm-1) region of the adenine IR spectrum. Among the bands present in this region, the assignment of those at 566, 513, and 276 cm-1 (Ar) can be treated with confidence. The band at 503 cm-1 (Ar) is an absorption of low intensity, in the vicinity of the much stronger band (at 513 cm-1) that is due to the γN9H vibration. It is, then, difficult to determine precisely its isotopic shifts. This task is facilitated by consideration of the spectra of adenine isotopomers isolated in N2 matrices. In the case of
J. Phys. Chem., Vol. 100, No. 9, 1996 3533 these matrices, the band due to the γN9H vibration is shifted considerably toward higher frequencies, and the isotopic shifts of the band assigned to the normal mode Q34 can be determined. These turned out to be in agreement with our theoretical predictions. The two doublets of bands at 591, 583 cm-1 and at 242, 214 cm-1 correspond, most probably , to the out-of-plane vibrations of the amino group. All of these bands are considerably shifted toward higher frequencies and appear as broad bands in the spectrum of adenine isolated in a N2 matrix. This feature is characteristic for the bands due to out-of-plane vibration of the amino group. The band at 591, 583 cm-1 is split into two components due to the matrix effect. Annealing of the Ar matrix leads to an increase in intensity of one of the components and to a decrease of the other. In the spectrum of adenine isolated in a Ne matrix, only one band at 583 cm-1 appears6 instead of the pair of 591, 583 cm-1 observed in the Ar matrix. The origin of this relatively strong band is not clear. As is seen in Figure 5, there is no theoretically predicted counterpart for this band. For the compounds with the amino group attached to the ring, the theoretical calculations predict at frequencies of 550-500 cm-1 a low-intensity band due to the twisting vibration of the NH2 group (mode Q31 (DFT) or modes Q31, Q32 (HF)). The experimentally observed band is, however, quite strong. It might be, as well, that the band in question corresponds to a higher than fundamental band, due to the inversional motion of the amino group. For the fundamental bands large transition intensities are expected, and because of considerable anharmonicity, the presence of bands due to higher inversional tones also seems probable. In contrast to the doublet at 591, 583 cm-1, the pair of strong bands at 242 and 214 cm-1 do not seem to originate from matrix splitting. The relative intensities of the two bands are not sensitive to the annealing of the matrix. The present theoretical DFT(B3-LZP)/6-31G(d,p) calculation predicts two strong bands at 244 and 205 cm-1, which coincides well with the experimental spectral pattern. These theoretical bands correspond to the coupled inversion of the amino group and the relative “butterfly” torsions of the two rings of the molecule. Despite the nice coincidence between theory and experiment, the assignment of the bands at 242 and 214 cm-1 (Ar) should be treated with caution, because calculations carried out within a harmonic approximation might not predict properly such modes as those involving the inversional vibration of the amino group, for which considerable anharmonicity is expected. Comparing the results of the DFT calculations with those previously obtained at the HF/6-31G(d,p) level,8 one may conclude that the spectral pattern (normal mode frequencies and IR band intensities) is considerably better predicted using the DFT method. However, the isotopic shifts are predicted using both methods with nearly the same accuracy. Conclusions The main results of the present matrix isolation and DFT studies of adenine and a comparison with the the results of our previous studies may be summarized as follows: (1) Only one amino N(9)H tautomer of adenine is observed for the compound isolated in low-temperature matrices. (2) The DFT(B3-LYP)/6-31G(d,p) calculations predict the IR spectra of adenine well enough to allow for reliable assignment of the bands in the experimental spectra. (3) Studies of IR spectra of the Ade 15N(9) and Ade 15N(7) isotopomers proved to be of crucial importance for determining the tautomeric form present in the matrix and for the positive testing of the IR spectrum assignment.
3534 J. Phys. Chem., Vol. 100, No. 9, 1996 Acknowledgment. We join in congratulating Professor David Shugar (University of Warsaw) on his 80th birthday and gratefully acknowledge his work on nucleic acids. The authors are obliged to Dr. P. Kaczor for his help and for kind allotment of the FTIR instrument. This work was supported at Warsaw by the European Union Grant No. CIPA-CT 93-0108, at Torun´ by the Committee for Scientific Research (KBN) within Grant No. 3 TO9A 026 08, and at Jackson by NIH Grant No. 332090 and a contract (DAAL 03-89-0038) between the Army Research Office and the University of Minnesota for the Army High Performance Computing Research Center under the auspices of the Department of the Army, Army Research Laboratory cooperative agreement no. DAAH04-95-2-0003/contract no. DAAH04-95-C-008, the content of which does not necessarily reflect the position or the policy of the government, and no official endorsement should be inferred. We also acknowledge the Mississippi Center for Supercomputing Research for an allotment of computer time to make some of the calculations presented here. Supporting Information Available: Table of calculated (DFT(B3-LYP)/6-31G(d,p)) harmonic wavenumbers and intensities of the bands for the amino-N(7)H tautomer of adenine (Table 4) (3 pages). Ordering information is given on any current masthead page. References and Notes (1) (a) Chenon, M. T.; Pugmire, R. J.; Grant, D. M.; Panzica, R. P.; Townsend, L. B. J. Am. Chem. Soc. 1975, 97, 4636. (b) Eastman, J. W. Ber. Bunsen-Ges. Phys. Chem. 1969, 73, 407. (c) Dreyfus, M.; Dodin, G.; Bensaude, O.; Dubois, J. E. J. Am. Chem. Soc. 1975, 97, 2369. (2) Shugar, D.; Psoda, A. In Landolt-Børnstein-New Series:Biophysics of Nucleic Acids; Saenger, W., Ed.; Springer: Berlin, 1990; Vol. VII/1d, p 308. (3) Nowak, M. J.; Lapinski, L.; Kwiatkowski, J. S. Chem. Phys. Lett. 1989, 157, 14. (4) (a) Radchenko, E. D.; Plokhotichenko, A. M.; Sheina, G. G.; Blagoi, Yu. Biofizika (Russian) 1984, 29, 553. (b) Stepanian, S. G.; Sheina, G. G.; Radchenko, E. D.; Blagoi, Yu. J. Mol. Struct. 1985, 131, 333. (c) Sheina, G. G.; Radchenko, E. D.; Stepanian, S. G.; Blagoi, Yu. Stud. Biophys. 1986, 114, 123. (5) Nowak, M. J.; Rostkowska, H.; Lapinski, L.; Kwiatkowski, J. S.; Leszczynski, J. J. Phys. Chem. 1994, 98, 2813. (6) Nowak, M. J.; Lapinski, L.; Kwiatkowski, J. S.; Leszczynski, J. Spectrochim. Acta 1991, 47A, 87. (7) Wio´rkiewicz-Kuczera, J.; Karplus, M. J. Am. Chem. Soc. 1990, 112, 5324. (8) Nowak, M. J.; Rostkowska, H.; Lapinski, L.; Kwiatkowski, J. S.; Leszczynski, J. Spectrochim. Acta 1994, 50A, 1081.
Nowak et al. (9) (a) Hohenberg, P.; Kohn, W. Phys. ReV. 1964, 136, B864. (b) Kohn, W.; Sham, L. J. Phys. ReV. 1965, 140, A1133. (c) Parr, R. G.; Yang, W. Density Functional Theory of Atoms and Molecules; Oxford University Press: New York, 1989. (d) Hedin, L.; Lundquist, B. I. J. Phys. (C) 1971, 4, 2064. (e) Becke, A. D. Phys. ReV. 1988, A38, 3098. (f) Becke, A. D. J. Chem. Phys. 1992, 96, 2155. (g) Becke, A. D. J. Chem. Phys. 1992, 97, 9173. (h) Becke, A. D. J. Chem. Phys. 1993, 98, 5648. (i) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. 1988, 37, B785. (j) Vosko, S. H.; Wilk, L.; Nusair, M. Can. J. Phys. 1980, 58, 1200. (10) Hehre, W. J.; Radom, L.; Schleyer, P. v. R.; Pople, J. A. Ab Initio Molecular Orbital Theory; Wiley: New York, 1986. (11) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Gill, P. M. W.; Johnson, B. G.; Wong, M. W.; Foresman, J. B.; Robb, M. A.; Head-Gordon, M.; Replogle, E. S.; Gomperts, R.; Andres, J. L.; Raghavachari, K.; Binkley, J. S.; Gonzalez, C.; Martin, R. L.; Fox, D. J.; Defrees, D. J.; Baker, J.; Stewart, J. J. P.; Pople, J. A. GAUSSIAN 92/DFT, Revision F.3; Gaussian, Inc.: Pittsburgh, PA, 1993. (12) Pulay, P.; Fogarasi, G.; Pang, F.; Boggs, J. A. J. Am. Chem. Soc. 1979, 101, 2550. (13) Schachtschneider, J. H. Technical Report; Shell Development Co.: Emeryville, CA, 1969. (14) (a) Ha, T.-K.; Gunthard, H. H. J. Mol. Struct. 1993, 300, 619. (b) Gould, I. R.; Vincent, M. A.; Hillier, I. H.; Lapinski, L.; Nowak, M. J. Spectrochim. Acta 1992, 48A, 811. (c) Rostkowska, H.; Nowak, M. J.; Lapinski, L.; Bretner, M.; Kulikowski, T.; Les, A.; Adamowicz, L. Spectrochim. Acta 1993, 49A, 551. (15) (a) Leszczynski, J. Int. J. Quantum Chem. 1992, QBS19, 43. (b) Sponer, J.; Hobza, P. J. Phys. Chem. 1994, 98, 3161. (16) Stewart, E. L.; Foley, C. K.; Allinger, N. L.; Bowen, J. P. J. Am. Chem. Soc. 1994, 116, 7282. (17) (a) Kwiatkowski, J. S.; Leszczynski, J. J. Phys. Chem., submitted. (b) Bakalarski, G.; Grochowski, P.; Kwiatkowski, J. S.; Lesyng, B.; Leszczynski, J. Chem. Phys., in press. (c) Szczepaniak, K.; Person, W. B.; Leszczynski, J.; Kwiatkowski, J. S. In preparation. (18) Brown, R. D.; Godfrey, P. D.; McNaughton, D.; Pierlot, A. P. Chem. Phys. Lett. 1989, 156, 61. (19) (a) Wierzchowski, K. L. in Landolt-Børnstein-New Series. Biophysics of Nucleic Acids; Saenger, W., Ed.; Springer Verlag: Berlin, 1990, Vol. VII/1d, p 295. (b) Mauret, P.; Fayet, J. C. R. Acad. Sci. Paris (C) 1964, 264, 2081. (20) Kwiatkowski, J. S.; Leszczynski, J. Chem. Phys. Lett. 1993, 204, 430. (21) (a) Kwiatkowski, J. S.; Leszczynski, J. J. Mol. Struct. (THEOCHEM) 1990, 208, 35. (b) Sabio, M.; Topiol, S.; Lumma, W. C., Jr. J. Phys. Chem. 1990, 94, 1366. (22) Nowak, M. J.; Szczepaniak, K.; Barski, A.; Shugar, D. J. Mol. Struct. 1980, 62, 47. (23) Nowak, M. J.; Lapinski, L.; Fulara, J.; Les, A.; Adamowicz, L. J. Phys. Chem. 1991, 95, 2404. (24) Nowak, M. J.; Lapinski, L.; Rostkowska, H.; Les, A.; Adamowicz, L. J. Phys. Chem. 1990, 94, 7406. (25) Rostkowska, H.; Barski, A.; Szczepaniak, K.; Szczesniak, M.; Person, W. B. J. Mol. Struct. 1988, 176, 137. (26) Nowak, M. J.; Lapinski, L.; Fulara, J.; Les, A.; Adamowicz, L. J. Phys. Chem. 1992, 96, 1562.
JP9530008
