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Infrared spectra of 6-thioguanine tautomers. An experimental and theoretical approach
Spectrochimica Acta Part A 58 (2002) 1793– 1808 www.elsevier.com/locate/saa
Infrared spectra of 6-thioguanine tautomers. An experimental and theoretical approach Okuma E. Kasende * Faculte des Sciences, Uni6ersite de Kinshasa, B.P. 190, Kinshasa XI, Congo Received 27 August 2001; accepted 2 October 2001
Abstract Both amino-thiol N9H and amino-thiol N7H tautomeric forms of 6-thioguanine have been identified in approximately equal abundance in infrared studies of these molecules isolated in the hydrophobic environment of an argon matrix at 12 K. The relative concentrations of the amino-thiol N9H and amino-thiol N7H ([SH, N9H]/[SH, N7H]=KN9H − N7H = 1.0090.02) are estimated from the observed relative infrared absorbances. From these relative concentrations, the difference in the Gibbs free energy of these two tautomers (DG500 N9H − N7H = − 0.0129 0.005 kJ mol − 1) have been estimated. The infrared and Raman spectra of 6-thioguanine in solid state are also discussed in terms of hydrogen bonding and stacking interactions in the crystal which are not considered in the calculation. In an effort to interpret the experimental results, ab initio calculation of the infrared spectrum has been made for the amino-thione N7H tautomer at 3-21G level. Comparison with experimental spectra is of some help in the assignment of the infrared and Raman spectra for 6-thioguanine in the solid state. © 2002 Elsevier Science B.V. All rights reserved. Keywords: 6-Thioguanine; Tautomerism; Infrared spectrum; Raman spectrum; Matrix-isolation; Ab initio calculation
1. Introduction Thioguanine (TG) is a known metabolic inhibitor [1–3] with antitumor and antineoplastic activity used in cancer research [4 – 9]. One of its interesting features is that, similar to guanine [10 –13], a number of different tautomeric forms are possible. They are shown in Fig. 1. For the N9-substituted thioguanine, a residue of thioguanosine, only tautomers 2, 4, 6, 8, 10 and 12 are possible. * Tel.: +243-990-5757. E-mail address: [email protected] Kasende).
(O.E.
According to X-ray data [14] only the aminothione tautomer with the proton at N7 (scheme 2 in Fig. 1) is present in the crystal, in contrast to guanine for which the N9H oxo tautomer is detected [15]. However, the amino-thione N7H tautomer is not the most stable form predicted by recent ab initio calculations [16,17]. These calculations predict that the amino-thiol with the proton at N9 is the most stable tautomer [16,17]. The Leszczynski’s calculation at MP(full)/DZP level predicts the amino-thione N7H tautomer to be less stable by 1.8 kJ mol − 1 than the most stable amino-thiol form (scheme 3 in Fig. 1)[16]. Such a discrepancy between the theoretical prediction of
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O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
the relative stability and X-ray data is the effect of hydrogen bonding and stacking interactions in the crystal which are not considered in the calculation. The amino-thione N7H tautomer (scheme 2), identified in the crystal by the X-ray experiment, has the lowest calculated dipole moment (1.78 D) [16] of any of the thioguanine tautomers shown in Fig. 1. The largest dipole moment (7.42 D) [16]) is predicted for the amino-thione N9H tautomer (scheme 2 in Fig. 1). The presence of the least polar tautomer in the crystal instead of one of the more polar forms is indeed very surprising. It must be a result of packing in the crystal to fulfill the best hydrogen bonding pattern between the molecules in the crystal, and clearly overpowers the electrostatic dipole– dipole forces usually thought to dictate the predominant tautomer expected in a polar medium. In view of these differences between predictions
from the calculation and the structures observed from the X-ray studies, it is particularly interesting to establish experimentally which tautomer(s) of TG is/are present in the absence of hydrogen bonding and stacking interactions. Because of the difficulty of studies of TG in the gas phase, related to its high melting point and possible thermal decomposition, and because of very low solubility of TG in any inert solvent, the only practical way to study isolating TG is in an inert, rigid, argon matrix. The environment in the inert Ar matrix is non-perturbing and close to that in the vapor state; that is, the conditions of the TG molecules are equivalent to those for which calculations have been performed. The main goal of this study is to establish experimentally which tautomers are present in an inert argon matrix and to verify existing ab initio results.
Fig. 1. Structure of the tautomeric forms of 6-thioguanine.
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
We used matrix isolation infrared spectroscopy as the experimental method to determine which tautomeric forms occur for the isolated molecules. This technique has proven to be the most useful procedure for studies of the tautomeric equilibria for a number of pyrimidine and purine bases [18]. The infrared spectrum of TG isolated in an argon matrix is compared with infrared spectra calculated at the HF/6-31 G (d, p) level for aminothione and amino-thiol tautomers shown in schemes 1–4 in Fig. 1. Such a comparison is very helpful in establishing which tautomers are present in the matrix sample. Verification and additional assignments are achieved by comparing the infrared spectra of matrix-isolated TG with infrared spectra for several related model compounds.
2. Methods
2.1. Experimental The experimental procedure and conditions for preparing the matrix-isolated samples and studying the infrared spectra were the same as those described by Person et al. [19]. The infrared spectra were recorded with a Nicolet Model 740 FTIR spectrometer at 1 cm − 1 resolution. The matrix sample was obtained by passing argon gas over the subliming sample of TG (from Sigma) and condensing the resulting gaseous mixture onto a cold (about 12 K) CsI window mounted in a closed cycle He cryostat (Displex model CSA 202E). The infrared spectrum of a polycrystalline sample was recorded for a solid sample dispersed in KBr with the same Nicolet FTIR instrument at 1 cm − 1 resolution. The Raman spectrum was taken for a polycrystalline powder sample of TG placed in a small cavity (about 0.5 mm radius) in an aluminum block and recorded on a Brucker model 66 FTIR spectrometer equipped with the Raman station (FRA 106) using the near infrared line (1.064 mm) from a Cw Nd-YAG laser (CVI Laser Co.) as the exciting line, operated at 4 cm − 1 resolution.
1795
2.2. Calculation Ab initio calculations of the vibrational spectra were carried out using the GAUSSIAN 86 program [20] with a split valence 6-31 G (d, p) basis set. The geometries of the tautomers were optimized by the gradient procedure [21], without requiring the restriction of planarity for the molecules. The frequencies calculated for all the non-planar structures were real, whereas those for planar structures have one imaginary frequency, showing that the structure corresponds to a saddle point in the potential energy. In the nonplanar structures the amino group nitrogen atom lies above the purine ring plane (2° or 0.05 A, ). Relatively large deviations (30° or 0.4 A, ) from the molecular plane were calculated for the hydrogen atoms of the amino group.
3. Results and discussion Fig. 2 shows a comparison of the experimental spectrum of matrix-isolated TG with the calculated spectra of four tautomers of TG. The reason for starting the discussion with such a comparison is to find out how much the spectra of these tautomers differ from each other and what chances we may have to distinguish one tautomer from the other. At this point we shall focus our attention only on the stronger bands, which should be well identified in the experimental spectrum. As seen in Fig. 2 the most dramatic differences between the calculated spectra for different tautomers occur in the following regions: 3560– 3510; 3430–3400; 1550–1400; 1360–1290 cm − 1. Examining both the experimental and calculated spectra one can see that the experimental spectrum is most closely related to the calculated spectra of the two amino-thiol tautomers, suggesting that these two species are predominant in the matrix sample. In Fig. 3 we present a more detailed comparison of the experimental spectrum of the isolated molecule (spectra c) with the calculated spectra of the two amino-thiol tautomers (spectrum a and b) and with the equally weighted sum of the latter (spectrum d).
1796
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
Fig. 2. Comparison of the experimental spectrum of matrix isolated TG with the calculated spectra of amino-thiol N9H (SH, N9H), amino-thiol N7H (SH, N7H), amino-thione N9H (CS, N9H) and amino-thione N7H (CS, N7H) tautomers of 6-thioguanine using a 3-21G basis set.
As seen in this figure the calculated spectra a and b are nearly identical in most spectral regions. The most pronounced differences are observed near 1499, 1260 and 513 cm − 1. Separately neither calculated spectrum a nor b agrees with the experimental spectrum. However, the sum of the spectra a and b resembles the experimental spectrum very closely and this suggests very strongly, or even proves, that these two amino-thiol tautomers of TG strongly predominate in the matrix-isolated sample. The experimental spectrum of isolated molecule shows rather a complex structure in the region of the SH stretching mode (Fig. 4) which could be explained as arising due to Fermi resonance between the fundamental of SH stretching mode and some suitable overtone/combination. However, comparing the calculated spectra of the two amino-thiol tautomers (specta a and b) taken together and the experimental spectrum (spectrum c), suggests that these two amino-thiol tautomers are predominant in the matrix-isolated sample. The analysis of the different regions of the
experimental spectrum and the corresponding regions of the calculated spectra, provides the assignment of the experimental lines to the normal modes of each amino-thiol tautomer. This assignment is given in Table 1, where experimental and calculated frequencies and intensities together with the potential energy distributions are collected. Using experimental values of the integrated absorbencies for the characteristic vibrations of each tautomer (e.g. the NH2 asymmetric and symmetric stretching modes) with the calculated values of the absolute intensities for the corresponding modes, we have estimated the equilibrium constant K(T) K(T)=
[tautomer:SH, N9H] [tautomer:SH, N7H]
and the difference in the Gibbs free energy (DG) of these two tautomers DG = − RT ln K(T)
WN
3558 3490 3433 3049 2599 1631
1593 1582
1546 1454
1390
1366 1353
Q
1
2 3
4 5 6
7
8
9
10
11
12
13
b
Calculation (cm−1)
Amino-thiol N9H
124
6
204
230
27
485
395
3 1 458
129 99
63
AIR (km mol−1)
e
AmH15 s(51+) AmH16 s(49−) N9H s(100+) AmH16 s(51−) AmH16 s(49−) C8H s(99−) SH s(100+) N3C4 s(32+) N1C6 s(11+) C5C4 s(10−) HNH sci(72−) CNH2 s(15−) C6C5 s(17−) N7C8 s(17+) C5C4 s(16+) C2N3 s(11−) N1C2 s(10+) N7C8 s(49+) C2N3 s(10+) CNH2 s(13+) N1C6 s(12+) HNH sci(12−) N9C4 s(10+) CNH2 s(20−) N1C6 s(18+) C5C4 s(13−) N9H be(33+) C8N9 s(21+) C6C5 s(16+) N9C4 s(16−) N1C6 s(12+) C5N7 s(11−) N9H be(11+)
PED
1353
1366
1395
1459
1577
1599
2605 1622
3485 3456
3574
WN
c
(cm−1)
Experiment (Ar)
26
7
17
17
93
14
17
117 121 37
109 127
53
IIR
a
d
1333
1380
1596
1460
1507
1583
1590
3042 2605 1626
3498 3427
3548
WNb (cm−1)
Calculation
52
327
101
134
119
443
371
6 2 382
124 83
57
AIR (km mol−1)
Amino-thiol N7H
N7H be(24−) N7C8 s(17+) C2Am s(15−) C5C4 s(15+) N1C6 s(14−) C5N7 s(18−) C5C4 s(13+) C8H be(13−)
C8N9 s(43+) C8H be(19−) C2N3 s(16+) N1C6 s(13−) N7H be(13+)
AmH15 s(52−) AmH16 s(48+) N7H s(100−) AmH16 s(52−) AmH15 s(48−) C8H s(99−) SH s(100−) C6C5 s(25+) N3C4 s(17−) C2N3 s(13+) HNH sci(75+) C2Am s(12+) C5C4 s(26+) N3C4 s(20−) N1C2 s(13+)
PEDe
1388 1386 1310 1313
1404
1463
1499
1573 1571
1597
2600 1619
3491 3449
3564
WNc (cm−1)
27
29
38
4
13
9
9
125 117 19
68 124
52
IIRd
Experiment (Ar)a
Table 1 Wavenumbers (WN), infrared integrated intensities, AIR, infrared integrated relative intensities, IIR and potential energy distributions (PED) for amino-thiol N9H and amino-thiol N7H tautomers
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808 1797
WN
1299
1190
1171
1073
1036
1028
940
907
893 885
Q
14
15
16
17
18
19
20
21
22
23
b
Calculation (cm−1)
Amino-thiol N9H
Table 1 (Continued)
95
3
11
15
18
27
11
11
59
18
AIR (km mol−1)
e
R5 def1(44+)
C8H owa(103−)
R5 def1(20+) C6S s(16−) C5C4 s(13+) N1C2 s(13+) C2N2 s(12+) CNH2 s(10+) C6SH be(68+)
N1C2 s(22−) NH2 ro(15+) N9C4 s(14+) C6Sh be(12−) C6S be(12−) C8N9 s(61+) N9H be(27−)
NH2 ro(43−) C2N3 s(28−)
N1C6 s(20+) N3C4 s(17−) N1C2 s(13−) NH2 ro(12−) C8H be(26−) C5N7 s(21+) R6 def1(20+)
C8H be(41+) C5N7 s(18+) N7C8 s(10−)
PED
906
840
917
956
1027
1036
1058
1188
1198
1298
WN
c
(cm−1)
Experiment (Ar)
2
1
7
16
92
1
9
3
49
71
IIR
a
d
883
901
920
940
1018
1082
1085
1158
1178
1288
WNb (cm−1)
Calculation
67
13
23
3
6
29
24
13
64
135
AIR (km mol−1)
Amino-thiol N7H
C6S s(18+) C5C4 s(17−) R5 def1(14−) C2N3 s(11−) R5 def1(44+) C6SH be(23+) C6SH be(51−)
N1C2 s(30−) N9C4 s(18+) NH2 ro(11−) C6SH be(10−) C8H owa(104−)
R6 def1(23+) C5N7 s(21+) C8H be(20−) N9C4 s(10−) NH2 ro(33+) C2N3 s(24−) N7C8 s(13−) N7C8 s(46+) N7H be(19+)
C8N9 s(26+) C8H be(25+) N7H be(12+) N9C4 s(10−) NH2 ro(21−) N1C6 s(19−) N3C4 s(12+)
PEDe
903
888
938
1020
1084
1088
1183
1186
1263
WNc (cm−1)
6
2
11
1
4
5
14
9
17
29
IIRd
Experiment (Ar)a
1798 O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
WN
805
797
733
641
617
611
532
505 496 468 406 339
Q
24
25
26
27
28
29
30
31
32
33
34
35
b
Calculation (cm−1)
Amino-thiol N9H
Table 1 (Continued)
4
5
10
164
9
3
1
7
6
9
11
51
AIR (km mol−1)
e
NH15 owa(45+) NH16 owa(43+) C6S s(39+) R6 def2(36−) R5 ode3(45+) R6 ode1(34−) R5 ode1(23−) R6 ode2(10−)
C2Am owa(47+) R5 ode2(39−) R5 ode1(35−) R5 ode2(65−) R5 ode1(49+) C6S owa(14+) R5 def2(32+) C6C5 s(17+) C2N be(15−) R6 def3(10+) R5 ode3(46+) C6S owa(43−) R6 ode2(14−) R6 def2(27+) N9C4 s(16+) R6 def1(10+) R6 def3(49+) C2N be(12+) N9H owa(97−)
C6S s(12+) C2Am owa(26−) R6 odel(22+) R5 ode2(19−) R5 ode1(13−) R6 def1(19−) C5N7 s(16+) R5 def(12−)
PED
–
426
513
510
552
598
638
645
730
815
791
WN
c
(cm−1)
Experiment (Ar)
1
3
2
2
4
11
1
3
28
1
3
2
IIR
a
d
354
371
403
451
507
539
580
616
632
739
795
802
WNb (cm−1)
Calculation
56
331
12
16
7
3
1
1
1
3
14
52
AIR (km mol−1)
Amino-thiol N7H
R5 ode1(79−) C6S owa(31−) R5 ode2(14+) R5 def2(31−) C2Am be(15−) C6C5 s(15−) R6 def3(12−) R5-R6 owa(57−) C6S owa(21+) R6 ode2(14+) R6 def2(26−) N9C4 s(18−) R6 def1(10−) R6 def3(48−) C2Am be(10+) NH16 owa(59−) NH15 owa(32−) C6S s(39−) R6 def2(34+) NH15 owa(48+) NH16 owa(19−) R5-R6 owa(17−) N7H owa(17−) NH15 owa(17−) R6 ode1(16+)
R5 ode2(27+) C2Am owa(22+) R6 ode1(20−) R6 def1(15−) R5 ode(13+)) C5N7 s(13+) R5 def2(12−) R5 ode2(68−) C2Am owa(45+)
PEDe
422
519
548
574
603
730
816
791
WNc (cm−1)
3
1
4
2
6
8
1
4
1
1
4
2
IIRd
Experiment (Ar)a
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808 1799
309 303
291
227
183 158
125
36
37
38
39
40 41
42
b
(cm−1)
1
3 16
4
45
300
39
AIR (km mol−1)
e
R6 ode3(58−) R6 ode2(18−) SH tor(13−) R5 ode3(11−) C6S be(72−) R6 ode1(24−) R5 ode3(24−) C6S owa(18−) C2Am owa(13−) R5 ode2(11−) R6 ode3(52+) R6 ode3(51−) C6S owa(14+)
C2N be(44−) R5 def2(11 NH15 owa(36−) NH16 owa(32+) SH tor(21+) SH tor(63+) NH15 owa(14+) NH16 owa(10−)
PED
WN
c
(cm−1)
Experiment (Ar)
17
2 1
1
2
4
2
IIR
a
d
122
182 153
189
240
308
339
WNb (cm−1)
Calculation
22
3 14
14
6
13
122
AIR (km mol−1)
Amino-thiol N7H
C6S be(75+) R5-R6 owa(32−) R6 ode1(23−) C6S owa(12−) C2Am owa(11−) R5 ode2(51−) C6S owa(15+) R6 ode(53+) R6 ode2(51−) C6S owa(157)
R5 ode1(16+) NH16 owa(13+) N7H owa(74+) R5 ode1(22+) C2Am be(49+) R5 def2(11−) C6S s(10−) R6 ode3(41+) HS tor(27−) R6 ode2(23+) R5-R6 owa(16+) HS tor(62+) R6 ode3(27+)
PEDe
WNc (cm−1)
1
2 1
2
1
2
1
IIRd
Experiment (Ar)a
b
Dilute in argon matrix at 15 K. Wavenumbers scaled by a constant factor of 0.91. c Only the strongest band is given when multicomponent bands are observed. d Integrated relative intensities in unit chosen in such way that the observed intensity sum of the in-plane modes observed in the separated spectra of amino-thiol N9H or amino–thiol N7H tautomer is equal to the calculated intensity sum of these modes. e Abbreviations: s, stretching; be, bending; sci, scissoring; owa, out-of-plane wagging; ro, rocking; def, deformation; ode, out-of-plane deformation; tor, torsion.
a
WN
Q
Calculation
Amino-thiol N9H
Table 1 (Continued)
1800 O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
for T=500 K (227 °C) corresponding to the temperature of the gas phase equilibrium at which the sublimation took place. The values obtained are K(T) = 1.00 9 0.02 and DG = − 0.01290.005 kJ mol − 1. The small value of the difference in the Gibbs free energy indicates that both amino-thiol N9H and amino-thiol N7H tautomeric forms of TG have been identified in approximately equal abundance. To evaluate the effects of hydrogen bonding and stacking interactions on the spectrum of the TG molecule, we have also studied infrared and Raman spectra of the crystalline solid and compared with the infrared and Raman spectra calculated for the isolated tautomer (amino-thione N7H, shown in scheme 2 of Fig. 1) identified in the crystal by the X-ray study. These spectral data are collected in Table 2, where the wavenumbers, intensities and descriptions of the normal modes
1801
(potential energy distributions, PED) are summarized. The Raman and infrared spectra of TG have been investigated earlier experimentally [22] but a normal coordinate analysis has not been performed for the in-plane vibrational modes. Except for the CNH2 and CS stretching modes, the assignments proposed by Singh et al. [22] do not agree with ours based on the potential energy distribution. As the complexity of the spectra of nucleic acid bases makes the vibrational assignments rather difficult, assistance was also taken from the vibrational assignment made for 2-thiocytosine because in relation to hydrogen bonding the case of TG resembles that of 2-thiocytosine [23]. From the crystal structure of TG, Bugg and Thewalt have shown that the molecules form a tightly hydrogen bonded network with all eligible
Fig. 3. Comparison of the experimental spectrum of the isolated molecule (spectrum c) with the calculated spectra of the two amino-thiol tautomers (specta a and b) using a 3-21G basis set, and with the equally weighted sum of the latter (spectrum d).
1802
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
Fig. 4. Comparison of the experimental spectrum of the isolated molecule (spectrum c) with the calculated spectra of the two amino-thiol tautomers (spectrum a and b) taken together in the region of the SH stretching mode.
nitrogen and sulfur hydrogen bond donors and acceptors participating in the formation of hydrogen bonds [14]. The bases are hydrogen bonded around screw axes to form approximately planar ribbons running in the b direction. Within these ribbons each base is joined to the two adjacent bases by a total of six hydrogen bonds of N1H···N3, N2H···N9, N2H···S, N7H…S types [14]. Thus the assignments of the following modes of TG more sensitive to hydrogen bonding will be discussed: NH2, N1H, N7H, CNH2 and CS modes.
3.1. NH2 modes For the amino-thione N7H tautomer, the present calculation predicts antisymmetric and symmetric stretching modes of the NH2 group at 3512 and 3401 cm − 1, respectively. However, the presence of intermolecular hydrogen bonding is expected to
lower the magnitude of stretching modes of the NH2 group. In addition, the two NH bonds being not equivalent in the crystal structure of TG, the relation ws = 435.5+0.876was proposed for the NH2 group [24] is not satisfied by ws and was in the present case. Therefore, the first one, being bonded to a sulfur atom, is assigned to the absorption band observed at 3292 cm − 1 in the spectrum shown in Fig. 5, the second one, bonded to a nitrogen atom, is assigned to 3129 cm − 1 infrared frequency. The scissoring mode of the NH2 group gives rise to its characteristic frequency in the region 1600–1700 cm − 1 [23]. Therefore, the band observed at 1666 cm − 1 in the infrared spectrum and at 1663 cm − 1 in the Raman spectrum is assigned undoubtedly to the scissoring mode of the NH2 group. The rocking NH2 mode, usually appearing in the region 900–1150 cm − 1 for nucleic acid bases [25], has been assigned to infrared and
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
1803
Table 2 Wavenumbers (WN), Infrared integrated intensities, AIR, Raman integrated relative intensities, IRA, and potential energy distributions (PED) for amino-thione N7H tautomer Experiment-solid
Calculation b
(cm−1)
AIR (km/mol)
IRA
3512
62
16
2 3 4
3481 3429 3402
148 90 83
12 11 34
5 6
3051 1641
5 563
40 13
7 8
1595 1566
382 794
2 11
9
1532
40
11
10
1481
29
21
11
1427
164
17
12
1360
299
119
13
1347
7
22
14
1323
115
22
15
1252
137
34
16
1172
51
79
17
1163
78
17
18
1107
30
6
19
1086
35
9
Q
WN
1
d
PED
e
WNIR
AmH16 s(51−) AmH15 s(49+) N7H s(100−) N1H s(99−) AmH15 s(50+) AmH16 s(49+) C8H s(99−) C2N3 s(34−) HNH sci(19+) C2Am s(10+)
3292
HNH sci(55−) N1H be(34−) HNH sci(15+) C5C4 s(10+) N3C4 s(23+) C5C4 s(13−) C8N9 s(25+) C8H be(20−) N7H be(42−) N7C8 s(31+) C5C4 s(30+) C5C4 s(30+) N1C2 s(10−) N9C4 s(10−) C2Am s(22+) N1H be(19−) R6 def1(15−) C5N7 s(22+) N3C4 s(18−) C6C5 s(15−) C8N9 s(11+) C8N9 s(28−) C8H be(22−) N9C4 s(13+) C6C5 s(11−) N1C6 s(44−) C5N7 s(14+) C8H be(10−) N1C6 s(17+) N1H be(14−) R6 def1(13+) C8H be(10−) C6S s(10−) NH2 ro(45+) C2N3 s(17−) C2Am s(13−) N9C4 s(12+) N7C8 s(50+) N7H be(31+)
c
(cm−1)
a
AIR (km/mol)
WNRA
3241 3051 3129
700
1635
150
1666 1618
510 1100
1663 1619
1546
81
1546
1483
77
1478
1436
42
1439
3105
1367
1375
299
1372
1259
1260
1231
1228
1143
1135
1201
1202
1105
1032
1033
c
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
1804 Table 2 (Continued)
Experiment-solid
Calculation Q
WN
20
b
(cm−1)
AIR (km/mol)
IRA
999
54
7
21
942
80
12
22 23
934 909
3 121
1 1
24
804
30
4
25
771
45
1
26
722
44
1
27
659
75
2
28
642
3
1
29
618
16
1
30
574
23
5
31
639
75
18
32
527
215
6
33
504
28
10
34 35
491 417
116 6
4 8
36
360
1
2
37
310
26
15
38 39
289 198
92 5
8 1
40
196
8
26
d
PED
e
C8H be(13−) NH2 ro(21−) N1C2 s(21−) N9C4 s(15+) R5 def1(23+) N1C2 s(20+) C6S s(20−) C8H owa(104−) R5 def1(48−) C6S s(14−) R6 def1(24+) C5N7 s(16−) R5 def2(12+) N3C4 s(10−) R5 ode2(80−) C2Am owa(12−) C2Am owa(64+) R5 ode2(35−) N1H owa(84+) C6S owa(18−) R5 ode1(85−) C6S owa(15−) R5 def2(26−) C2Am be(14−) C6C5 s(12−) R5 ode3(44+) C6S owa(42−) R6 ode2(13−) R6 ode1(11+) NH16 owa(23+) N9C4 s(12−) R6 def2(12−) NH16 owa(57−) R6 def3(13−) R6 def3(41−) C2Am s(11+) N7H owa(87−) R6 def2(53+) C6S s(25−) R5 ode1(45+) R6 ode1(29+) R5 ode1(29+) C2Am owa(11+) R6 ode2(10+) C2Am be(42−) R5 def2(11+) NH15 owa(10−) NH15 owa(78−) R6 ode3(68−) R6 ode2(28−) R5 ode3(19−) C6S be(70−)
WNIR
c
(cm−1)
a
AIR (km/mol)
WNRA
972
972
942
921
925 841
921
838
838
777
776
719
715 657 648
621
572
575
563
824 525 570 418 409 369
362
570 234
215
c
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
1805
Table 2 (Continued) Calculation Q
WN
41
42
Experiment-solid b
(cm−1)
AIR (km/mol)
IRA
148
15
3
128
13
0
d
PED
e
WNIR
c
(cm−1)
R6 ode3(34+) R5 ode3(17−) R5 ode1(15+) R6 ode2(11−) C2Am owa(10−) R6 ode1(45−) R6 ode9(41+) R6 ode3(25−) C6S owa(18−)
a
AIR (km/mol)
WNRA
c
135
a Polycrystalline powder sample dispersed in KBr for infrared measurements or pressed into a rotating cell for Raman measurements. b Wavenumbers scaled by a constant factor of 0.91. c Only the strongest band is given when multicomponent bands are observed. d Integrated relative intensities in unit chosen in such way that the observed intensity sum of the in-plane modes observed in the separated spectra of amino-thiol N9H or amino-thiol N7H tautomer is equal to the calculated intensity sum of these modes. e Abbreviations: s, stretching; be, bending; sci, scissoring; owa, out-of-plane wagging; ro, rocking; def, deformation; ode, out-of-plane deformation; tor, torsion.
Raman bands observed at 972 cm − 1. The wagging and torsion (NH2) modes are respectively associated with infrared bands at 777 and 621 cm − 1. In fact the torsion and wagging (NH2) modes arise due to out-of-plane and in-plane coupling of the two NH out-of-plane bending motions of the NH2 group. In addition, the presence of intermolecular hydrogen bonding is expected to raise the magnitude of the torsion (NH2) mode.
3.2. N1H modes The HF/6-31G assignments attribute N7H and N1H stretching modes to 3481 and 3428 cm − 1, respectively. Despite the presence of intermolecular hydrogen bonding lowering the magnitude of N7H and N1H stretching modes, there is no confusion possible in the assignment of N1H and N7H stretching modes in experimental spectra. X-ray crystal structure [14] shows the presence of intermolecular NH···N and NH···S hydrogen bonds for N7H and N1H groups of 2.97 and 3.33 A, length,
respectively. Considering the Pimentel relation between OH or NH band shifts and hydrogen bond length, the N1H bond being bonded to the nitrogen atom in the crystal structure is expected to be observed in lower frequency than the N7H bond, which is bonded to the sulfur atom. Thus the shoulder observed around 3051 cm − 1, near the superimposed band situated at 3129 cm − 1, in the infrared spectrum (Fig. 5) is attributed to N1H stretching mode. The N1H in-plane bending mode is assigned to the infrared band at 1618 cm − 1 and Raman band at 1619 cm − 1. The band observed at 719 cm − 1 in the infrared spectrum and 715 cm − 1 in the Raman spectrum could be attributed to the N1H out-of-plane bending mode.
3.3. N7H modes The very weak absorption situated around 3241 cm − 1, between the bands 3292 and 3129 cm − 1, in the spectrum of Fig. 5 has been assigned to the N7H
Fig. 5. Comparison of the experimental infrared and Raman spectra of solid TG with the calculated spectra of the amino-thione N7H tautomer using 3-21G basis set, in the region of NH2, N7H, N1H and CH stretching modes (3600 – 2800 cm − 1).
1806
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
Fig. 5.
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
stretching mode. The N7H in-plane bending mode has been attributed to the infrared band at 1436 cm − 1 and Raman band at 1439 cm − 1. The absorption band at 570 cm − 1 has been associated with the N7H out-of-plane bending mode. It is worth noting that NH2, N1H and N7H stretching modes are not observed in the Raman spectrum (Fig. 5). The unique band observed at 3105 cm − 1 in this region in this region of the Raman spectrum is ascribed to the CH stretching mode.
3.4. CNH2 modes According to the crystal structure of TG the CNH2 bond (1.313 A, ) is shorter than the corresponding bond in 2-thiocytosine (1.333 A, ) [26]. The CNH2 stretching mode is, therefore, expected to appear at higher frequency than in 2-thiocytosine. For TG, we assigned to this mode the infrared frequency 1375 cm − 1 corresponding to Raman frequency 1372 cm − 1, in agreement with the assignment proposed by Singh and Yadav [22]. The calculated CNH2 stretching mode has the frequency 1347 cm − 1. In the 2-thiocytosine infrared spectrum this mode has been assigned to the frequency 1369 cm − 1. The CNH2 in-plane bending and out-ofplane bending modes are tentatively assigned in the Raman spectrum to the bands at 362 and 135 cm − 1, respectively.
3.5. CS stretching mode The CS stretching mode is among the most interesting modes as this is involved in the intermolecular hydrogen bonding which plays a very important role in the biological activities of this molecule [1]. In TG crystal structure [14], the sulfur atom accepts hydrogen bonds from N7H and NH2 of two different molecules; the lengths of these hydrogen bonds are 3.30 and 3.33 A, , respectively. By comparison to the case of 2thiocytosine in which the CS stretching mode has been assigned to the infrared band at 1200 cm − 1, in the TG infrared spectrum, this mode is assigned to the band at 1201 cm − 1 corresponding to the Raman band at 1202 cm − 1 in spite the low frequency 1163 cm − 1 predicted by our
1807
calculation. This assignment agrees with the previous assignment [22].
Acknowledgements The author is grateful to Ambassade 6an het Koninkrijk der Nederlanden (Kinshasa, Congo) for its financial support. I also wish to acknowledge Professor W.B. Person and Dr K. Szczepaniak-Person (University of Florida, USA) for experimental assistance and helpful discussions. Finally, I appreciate the hospitality and support provided by Professor D. de Waal at the University of Pretoria, Republic of South Africa, where the final version of this paper was completed.
References [1] M.W. Yu, J. Sedlackk, R.H. Lindsay, Arch. Biochem. Biophys. 111 (1973) 1995. [2] M. Ono, M.J. Kawakami, J. Biochem. 81 (1977) 1247. [3] W.I. Melvin, H.B. Milne, A.A. Slater, H.J. Allen, H.M. Keir, Eur. Biochem. 92 (1978) 373. [4] L.L. Bennet, J.A. Montgomery, in: H. Busch (Ed.), Methods in Cancer Research, vol. III, Academic Press, New York, 1967. [5] P. Calabresi, R.E. Parks, in: L.S. Goodman, A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, Macmillan, New York, 1970. [6] E.B. Astwood, A. Bissel, A.M. Hugles, Endocrinology 67 (1945) 456. [7] D.M. Tidd, A.R.P. Paterson, Cancer Res. 34 (1974) 738. [8] D.A. Clarke, F.S. Phillips, S. Sternberg, C.C. Stock, Ann. N.Y. Aca. Sci. 60 (1954) 235. [9] V.M. Nerstrom, U. Henriksen, P.E. Nielsnen, Buchardt, K. Schiefelow, C. Koch, Bioconjugate Chem. 5 (1994) 357. [10] I.R. Gould, I.H. Hiller, Chem. Phys. Lett. 161 (1989) 185. [11] M. Sabio, S. Topiol, W.C. Lumma Jr., J. Phys. Chem. 94 (1990) 1366. [12] J. Leszczynski, Chem. Phys. Lett. 174 (1990) 347. [13] J.S. Kwiatkowski, J. Leszczynski, J. Mol. Struct. (Theochem) 208 (1990) 350. [14] C.E. Bugg, U. Thewalt, J. Am. Chem. Soc. 92 (1970) 7741. [15] C.E. Bugg, U. Thewalt, R.E. Marsh, Biochem. Biophys. Res. Commun. 33 (1968) 436. [16] J. Leszczynski, Theochem. J. Mol. Struct. 117 (1994) 37. [17] C. Alhambra, F.J. Luque, J. Estebrich, M. Orozco, J. Org. Chem. 60 (1995) 996.
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[18] K. Szczepaniak, M. Szczesniak, W. Szajda, W.B. Person, Can. J. Chem. 69 (1991) 1705. [19] W.B. Person, J.E. Delbene, W. Szajda, K. Szczepaniak, M. Szczeniak, J. Phys. Chem. 95 (1991) 2700. [20] M.J. Frish, J.S. Binkley, H.B. Schlegel, K. Raghavachari, C.F. Melius, L. Martin, J.J.P. Stewart, F.W. Bobrowicz, C.M. Rohfing, L.R. Kahn, D.J. Defrees, R. Seeger, H.B. Whitesides, D.J. Fox, E.M. Fluder, J.A. Pople, CarnegieMellon Quantum Chemistry, Publishing unit, Pittsburgh, PA, 1989.
[21] H.B. Schlegel, J. Compt. Chem. 3 (1982) 314. [22] K. Singh, R.A. Yadav, J.S. Yadav, Spectrochimica Acta 47A (1991) 819. [23] R.A. Yadav, P.N.S. Yadav, J.S. Yadav, Spectrochimica Acta 44A (1988) 1201. [24] L. Bellamy, R.L. Williams, Spectrochimica Acta 9A (1957) 341. [25] J.M. Delabar, M. Majoube, Spectrochimica Acta 34A (1978) 129. [26] S. Furberg, L.H. Jensen, Acta Crystallogr. B26 (1970) 1260.
Infrared spectra of 6-thioguanine tautomers. An experimental and theoretical approach Okuma E. Kasende * Faculte des Sciences, Uni6ersite de Kinshasa, B.P. 190, Kinshasa XI, Congo Received 27 August 2001; accepted 2 October 2001
Abstract Both amino-thiol N9H and amino-thiol N7H tautomeric forms of 6-thioguanine have been identified in approximately equal abundance in infrared studies of these molecules isolated in the hydrophobic environment of an argon matrix at 12 K. The relative concentrations of the amino-thiol N9H and amino-thiol N7H ([SH, N9H]/[SH, N7H]=KN9H − N7H = 1.0090.02) are estimated from the observed relative infrared absorbances. From these relative concentrations, the difference in the Gibbs free energy of these two tautomers (DG500 N9H − N7H = − 0.0129 0.005 kJ mol − 1) have been estimated. The infrared and Raman spectra of 6-thioguanine in solid state are also discussed in terms of hydrogen bonding and stacking interactions in the crystal which are not considered in the calculation. In an effort to interpret the experimental results, ab initio calculation of the infrared spectrum has been made for the amino-thione N7H tautomer at 3-21G level. Comparison with experimental spectra is of some help in the assignment of the infrared and Raman spectra for 6-thioguanine in the solid state. © 2002 Elsevier Science B.V. All rights reserved. Keywords: 6-Thioguanine; Tautomerism; Infrared spectrum; Raman spectrum; Matrix-isolation; Ab initio calculation
1. Introduction Thioguanine (TG) is a known metabolic inhibitor [1–3] with antitumor and antineoplastic activity used in cancer research [4 – 9]. One of its interesting features is that, similar to guanine [10 –13], a number of different tautomeric forms are possible. They are shown in Fig. 1. For the N9-substituted thioguanine, a residue of thioguanosine, only tautomers 2, 4, 6, 8, 10 and 12 are possible. * Tel.: +243-990-5757. E-mail address: [email protected] Kasende).
(O.E.
According to X-ray data [14] only the aminothione tautomer with the proton at N7 (scheme 2 in Fig. 1) is present in the crystal, in contrast to guanine for which the N9H oxo tautomer is detected [15]. However, the amino-thione N7H tautomer is not the most stable form predicted by recent ab initio calculations [16,17]. These calculations predict that the amino-thiol with the proton at N9 is the most stable tautomer [16,17]. The Leszczynski’s calculation at MP(full)/DZP level predicts the amino-thione N7H tautomer to be less stable by 1.8 kJ mol − 1 than the most stable amino-thiol form (scheme 3 in Fig. 1)[16]. Such a discrepancy between the theoretical prediction of
1386-1425/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 1 4 2 5 ( 0 1 ) 0 0 6 2 8 - X
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O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
the relative stability and X-ray data is the effect of hydrogen bonding and stacking interactions in the crystal which are not considered in the calculation. The amino-thione N7H tautomer (scheme 2), identified in the crystal by the X-ray experiment, has the lowest calculated dipole moment (1.78 D) [16] of any of the thioguanine tautomers shown in Fig. 1. The largest dipole moment (7.42 D) [16]) is predicted for the amino-thione N9H tautomer (scheme 2 in Fig. 1). The presence of the least polar tautomer in the crystal instead of one of the more polar forms is indeed very surprising. It must be a result of packing in the crystal to fulfill the best hydrogen bonding pattern between the molecules in the crystal, and clearly overpowers the electrostatic dipole– dipole forces usually thought to dictate the predominant tautomer expected in a polar medium. In view of these differences between predictions
from the calculation and the structures observed from the X-ray studies, it is particularly interesting to establish experimentally which tautomer(s) of TG is/are present in the absence of hydrogen bonding and stacking interactions. Because of the difficulty of studies of TG in the gas phase, related to its high melting point and possible thermal decomposition, and because of very low solubility of TG in any inert solvent, the only practical way to study isolating TG is in an inert, rigid, argon matrix. The environment in the inert Ar matrix is non-perturbing and close to that in the vapor state; that is, the conditions of the TG molecules are equivalent to those for which calculations have been performed. The main goal of this study is to establish experimentally which tautomers are present in an inert argon matrix and to verify existing ab initio results.
Fig. 1. Structure of the tautomeric forms of 6-thioguanine.
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
We used matrix isolation infrared spectroscopy as the experimental method to determine which tautomeric forms occur for the isolated molecules. This technique has proven to be the most useful procedure for studies of the tautomeric equilibria for a number of pyrimidine and purine bases [18]. The infrared spectrum of TG isolated in an argon matrix is compared with infrared spectra calculated at the HF/6-31 G (d, p) level for aminothione and amino-thiol tautomers shown in schemes 1–4 in Fig. 1. Such a comparison is very helpful in establishing which tautomers are present in the matrix sample. Verification and additional assignments are achieved by comparing the infrared spectra of matrix-isolated TG with infrared spectra for several related model compounds.
2. Methods
2.1. Experimental The experimental procedure and conditions for preparing the matrix-isolated samples and studying the infrared spectra were the same as those described by Person et al. [19]. The infrared spectra were recorded with a Nicolet Model 740 FTIR spectrometer at 1 cm − 1 resolution. The matrix sample was obtained by passing argon gas over the subliming sample of TG (from Sigma) and condensing the resulting gaseous mixture onto a cold (about 12 K) CsI window mounted in a closed cycle He cryostat (Displex model CSA 202E). The infrared spectrum of a polycrystalline sample was recorded for a solid sample dispersed in KBr with the same Nicolet FTIR instrument at 1 cm − 1 resolution. The Raman spectrum was taken for a polycrystalline powder sample of TG placed in a small cavity (about 0.5 mm radius) in an aluminum block and recorded on a Brucker model 66 FTIR spectrometer equipped with the Raman station (FRA 106) using the near infrared line (1.064 mm) from a Cw Nd-YAG laser (CVI Laser Co.) as the exciting line, operated at 4 cm − 1 resolution.
1795
2.2. Calculation Ab initio calculations of the vibrational spectra were carried out using the GAUSSIAN 86 program [20] with a split valence 6-31 G (d, p) basis set. The geometries of the tautomers were optimized by the gradient procedure [21], without requiring the restriction of planarity for the molecules. The frequencies calculated for all the non-planar structures were real, whereas those for planar structures have one imaginary frequency, showing that the structure corresponds to a saddle point in the potential energy. In the nonplanar structures the amino group nitrogen atom lies above the purine ring plane (2° or 0.05 A, ). Relatively large deviations (30° or 0.4 A, ) from the molecular plane were calculated for the hydrogen atoms of the amino group.
3. Results and discussion Fig. 2 shows a comparison of the experimental spectrum of matrix-isolated TG with the calculated spectra of four tautomers of TG. The reason for starting the discussion with such a comparison is to find out how much the spectra of these tautomers differ from each other and what chances we may have to distinguish one tautomer from the other. At this point we shall focus our attention only on the stronger bands, which should be well identified in the experimental spectrum. As seen in Fig. 2 the most dramatic differences between the calculated spectra for different tautomers occur in the following regions: 3560– 3510; 3430–3400; 1550–1400; 1360–1290 cm − 1. Examining both the experimental and calculated spectra one can see that the experimental spectrum is most closely related to the calculated spectra of the two amino-thiol tautomers, suggesting that these two species are predominant in the matrix sample. In Fig. 3 we present a more detailed comparison of the experimental spectrum of the isolated molecule (spectra c) with the calculated spectra of the two amino-thiol tautomers (spectrum a and b) and with the equally weighted sum of the latter (spectrum d).
1796
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
Fig. 2. Comparison of the experimental spectrum of matrix isolated TG with the calculated spectra of amino-thiol N9H (SH, N9H), amino-thiol N7H (SH, N7H), amino-thione N9H (CS, N9H) and amino-thione N7H (CS, N7H) tautomers of 6-thioguanine using a 3-21G basis set.
As seen in this figure the calculated spectra a and b are nearly identical in most spectral regions. The most pronounced differences are observed near 1499, 1260 and 513 cm − 1. Separately neither calculated spectrum a nor b agrees with the experimental spectrum. However, the sum of the spectra a and b resembles the experimental spectrum very closely and this suggests very strongly, or even proves, that these two amino-thiol tautomers of TG strongly predominate in the matrix-isolated sample. The experimental spectrum of isolated molecule shows rather a complex structure in the region of the SH stretching mode (Fig. 4) which could be explained as arising due to Fermi resonance between the fundamental of SH stretching mode and some suitable overtone/combination. However, comparing the calculated spectra of the two amino-thiol tautomers (specta a and b) taken together and the experimental spectrum (spectrum c), suggests that these two amino-thiol tautomers are predominant in the matrix-isolated sample. The analysis of the different regions of the
experimental spectrum and the corresponding regions of the calculated spectra, provides the assignment of the experimental lines to the normal modes of each amino-thiol tautomer. This assignment is given in Table 1, where experimental and calculated frequencies and intensities together with the potential energy distributions are collected. Using experimental values of the integrated absorbencies for the characteristic vibrations of each tautomer (e.g. the NH2 asymmetric and symmetric stretching modes) with the calculated values of the absolute intensities for the corresponding modes, we have estimated the equilibrium constant K(T) K(T)=
[tautomer:SH, N9H] [tautomer:SH, N7H]
and the difference in the Gibbs free energy (DG) of these two tautomers DG = − RT ln K(T)
WN
3558 3490 3433 3049 2599 1631
1593 1582
1546 1454
1390
1366 1353
Q
1
2 3
4 5 6
7
8
9
10
11
12
13
b
Calculation (cm−1)
Amino-thiol N9H
124
6
204
230
27
485
395
3 1 458
129 99
63
AIR (km mol−1)
e
AmH15 s(51+) AmH16 s(49−) N9H s(100+) AmH16 s(51−) AmH16 s(49−) C8H s(99−) SH s(100+) N3C4 s(32+) N1C6 s(11+) C5C4 s(10−) HNH sci(72−) CNH2 s(15−) C6C5 s(17−) N7C8 s(17+) C5C4 s(16+) C2N3 s(11−) N1C2 s(10+) N7C8 s(49+) C2N3 s(10+) CNH2 s(13+) N1C6 s(12+) HNH sci(12−) N9C4 s(10+) CNH2 s(20−) N1C6 s(18+) C5C4 s(13−) N9H be(33+) C8N9 s(21+) C6C5 s(16+) N9C4 s(16−) N1C6 s(12+) C5N7 s(11−) N9H be(11+)
PED
1353
1366
1395
1459
1577
1599
2605 1622
3485 3456
3574
WN
c
(cm−1)
Experiment (Ar)
26
7
17
17
93
14
17
117 121 37
109 127
53
IIR
a
d
1333
1380
1596
1460
1507
1583
1590
3042 2605 1626
3498 3427
3548
WNb (cm−1)
Calculation
52
327
101
134
119
443
371
6 2 382
124 83
57
AIR (km mol−1)
Amino-thiol N7H
N7H be(24−) N7C8 s(17+) C2Am s(15−) C5C4 s(15+) N1C6 s(14−) C5N7 s(18−) C5C4 s(13+) C8H be(13−)
C8N9 s(43+) C8H be(19−) C2N3 s(16+) N1C6 s(13−) N7H be(13+)
AmH15 s(52−) AmH16 s(48+) N7H s(100−) AmH16 s(52−) AmH15 s(48−) C8H s(99−) SH s(100−) C6C5 s(25+) N3C4 s(17−) C2N3 s(13+) HNH sci(75+) C2Am s(12+) C5C4 s(26+) N3C4 s(20−) N1C2 s(13+)
PEDe
1388 1386 1310 1313
1404
1463
1499
1573 1571
1597
2600 1619
3491 3449
3564
WNc (cm−1)
27
29
38
4
13
9
9
125 117 19
68 124
52
IIRd
Experiment (Ar)a
Table 1 Wavenumbers (WN), infrared integrated intensities, AIR, infrared integrated relative intensities, IIR and potential energy distributions (PED) for amino-thiol N9H and amino-thiol N7H tautomers
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808 1797
WN
1299
1190
1171
1073
1036
1028
940
907
893 885
Q
14
15
16
17
18
19
20
21
22
23
b
Calculation (cm−1)
Amino-thiol N9H
Table 1 (Continued)
95
3
11
15
18
27
11
11
59
18
AIR (km mol−1)
e
R5 def1(44+)
C8H owa(103−)
R5 def1(20+) C6S s(16−) C5C4 s(13+) N1C2 s(13+) C2N2 s(12+) CNH2 s(10+) C6SH be(68+)
N1C2 s(22−) NH2 ro(15+) N9C4 s(14+) C6Sh be(12−) C6S be(12−) C8N9 s(61+) N9H be(27−)
NH2 ro(43−) C2N3 s(28−)
N1C6 s(20+) N3C4 s(17−) N1C2 s(13−) NH2 ro(12−) C8H be(26−) C5N7 s(21+) R6 def1(20+)
C8H be(41+) C5N7 s(18+) N7C8 s(10−)
PED
906
840
917
956
1027
1036
1058
1188
1198
1298
WN
c
(cm−1)
Experiment (Ar)
2
1
7
16
92
1
9
3
49
71
IIR
a
d
883
901
920
940
1018
1082
1085
1158
1178
1288
WNb (cm−1)
Calculation
67
13
23
3
6
29
24
13
64
135
AIR (km mol−1)
Amino-thiol N7H
C6S s(18+) C5C4 s(17−) R5 def1(14−) C2N3 s(11−) R5 def1(44+) C6SH be(23+) C6SH be(51−)
N1C2 s(30−) N9C4 s(18+) NH2 ro(11−) C6SH be(10−) C8H owa(104−)
R6 def1(23+) C5N7 s(21+) C8H be(20−) N9C4 s(10−) NH2 ro(33+) C2N3 s(24−) N7C8 s(13−) N7C8 s(46+) N7H be(19+)
C8N9 s(26+) C8H be(25+) N7H be(12+) N9C4 s(10−) NH2 ro(21−) N1C6 s(19−) N3C4 s(12+)
PEDe
903
888
938
1020
1084
1088
1183
1186
1263
WNc (cm−1)
6
2
11
1
4
5
14
9
17
29
IIRd
Experiment (Ar)a
1798 O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
WN
805
797
733
641
617
611
532
505 496 468 406 339
Q
24
25
26
27
28
29
30
31
32
33
34
35
b
Calculation (cm−1)
Amino-thiol N9H
Table 1 (Continued)
4
5
10
164
9
3
1
7
6
9
11
51
AIR (km mol−1)
e
NH15 owa(45+) NH16 owa(43+) C6S s(39+) R6 def2(36−) R5 ode3(45+) R6 ode1(34−) R5 ode1(23−) R6 ode2(10−)
C2Am owa(47+) R5 ode2(39−) R5 ode1(35−) R5 ode2(65−) R5 ode1(49+) C6S owa(14+) R5 def2(32+) C6C5 s(17+) C2N be(15−) R6 def3(10+) R5 ode3(46+) C6S owa(43−) R6 ode2(14−) R6 def2(27+) N9C4 s(16+) R6 def1(10+) R6 def3(49+) C2N be(12+) N9H owa(97−)
C6S s(12+) C2Am owa(26−) R6 odel(22+) R5 ode2(19−) R5 ode1(13−) R6 def1(19−) C5N7 s(16+) R5 def(12−)
PED
–
426
513
510
552
598
638
645
730
815
791
WN
c
(cm−1)
Experiment (Ar)
1
3
2
2
4
11
1
3
28
1
3
2
IIR
a
d
354
371
403
451
507
539
580
616
632
739
795
802
WNb (cm−1)
Calculation
56
331
12
16
7
3
1
1
1
3
14
52
AIR (km mol−1)
Amino-thiol N7H
R5 ode1(79−) C6S owa(31−) R5 ode2(14+) R5 def2(31−) C2Am be(15−) C6C5 s(15−) R6 def3(12−) R5-R6 owa(57−) C6S owa(21+) R6 ode2(14+) R6 def2(26−) N9C4 s(18−) R6 def1(10−) R6 def3(48−) C2Am be(10+) NH16 owa(59−) NH15 owa(32−) C6S s(39−) R6 def2(34+) NH15 owa(48+) NH16 owa(19−) R5-R6 owa(17−) N7H owa(17−) NH15 owa(17−) R6 ode1(16+)
R5 ode2(27+) C2Am owa(22+) R6 ode1(20−) R6 def1(15−) R5 ode(13+)) C5N7 s(13+) R5 def2(12−) R5 ode2(68−) C2Am owa(45+)
PEDe
422
519
548
574
603
730
816
791
WNc (cm−1)
3
1
4
2
6
8
1
4
1
1
4
2
IIRd
Experiment (Ar)a
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808 1799
309 303
291
227
183 158
125
36
37
38
39
40 41
42
b
(cm−1)
1
3 16
4
45
300
39
AIR (km mol−1)
e
R6 ode3(58−) R6 ode2(18−) SH tor(13−) R5 ode3(11−) C6S be(72−) R6 ode1(24−) R5 ode3(24−) C6S owa(18−) C2Am owa(13−) R5 ode2(11−) R6 ode3(52+) R6 ode3(51−) C6S owa(14+)
C2N be(44−) R5 def2(11 NH15 owa(36−) NH16 owa(32+) SH tor(21+) SH tor(63+) NH15 owa(14+) NH16 owa(10−)
PED
WN
c
(cm−1)
Experiment (Ar)
17
2 1
1
2
4
2
IIR
a
d
122
182 153
189
240
308
339
WNb (cm−1)
Calculation
22
3 14
14
6
13
122
AIR (km mol−1)
Amino-thiol N7H
C6S be(75+) R5-R6 owa(32−) R6 ode1(23−) C6S owa(12−) C2Am owa(11−) R5 ode2(51−) C6S owa(15+) R6 ode(53+) R6 ode2(51−) C6S owa(157)
R5 ode1(16+) NH16 owa(13+) N7H owa(74+) R5 ode1(22+) C2Am be(49+) R5 def2(11−) C6S s(10−) R6 ode3(41+) HS tor(27−) R6 ode2(23+) R5-R6 owa(16+) HS tor(62+) R6 ode3(27+)
PEDe
WNc (cm−1)
1
2 1
2
1
2
1
IIRd
Experiment (Ar)a
b
Dilute in argon matrix at 15 K. Wavenumbers scaled by a constant factor of 0.91. c Only the strongest band is given when multicomponent bands are observed. d Integrated relative intensities in unit chosen in such way that the observed intensity sum of the in-plane modes observed in the separated spectra of amino-thiol N9H or amino–thiol N7H tautomer is equal to the calculated intensity sum of these modes. e Abbreviations: s, stretching; be, bending; sci, scissoring; owa, out-of-plane wagging; ro, rocking; def, deformation; ode, out-of-plane deformation; tor, torsion.
a
WN
Q
Calculation
Amino-thiol N9H
Table 1 (Continued)
1800 O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
for T=500 K (227 °C) corresponding to the temperature of the gas phase equilibrium at which the sublimation took place. The values obtained are K(T) = 1.00 9 0.02 and DG = − 0.01290.005 kJ mol − 1. The small value of the difference in the Gibbs free energy indicates that both amino-thiol N9H and amino-thiol N7H tautomeric forms of TG have been identified in approximately equal abundance. To evaluate the effects of hydrogen bonding and stacking interactions on the spectrum of the TG molecule, we have also studied infrared and Raman spectra of the crystalline solid and compared with the infrared and Raman spectra calculated for the isolated tautomer (amino-thione N7H, shown in scheme 2 of Fig. 1) identified in the crystal by the X-ray study. These spectral data are collected in Table 2, where the wavenumbers, intensities and descriptions of the normal modes
1801
(potential energy distributions, PED) are summarized. The Raman and infrared spectra of TG have been investigated earlier experimentally [22] but a normal coordinate analysis has not been performed for the in-plane vibrational modes. Except for the CNH2 and CS stretching modes, the assignments proposed by Singh et al. [22] do not agree with ours based on the potential energy distribution. As the complexity of the spectra of nucleic acid bases makes the vibrational assignments rather difficult, assistance was also taken from the vibrational assignment made for 2-thiocytosine because in relation to hydrogen bonding the case of TG resembles that of 2-thiocytosine [23]. From the crystal structure of TG, Bugg and Thewalt have shown that the molecules form a tightly hydrogen bonded network with all eligible
Fig. 3. Comparison of the experimental spectrum of the isolated molecule (spectrum c) with the calculated spectra of the two amino-thiol tautomers (specta a and b) using a 3-21G basis set, and with the equally weighted sum of the latter (spectrum d).
1802
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
Fig. 4. Comparison of the experimental spectrum of the isolated molecule (spectrum c) with the calculated spectra of the two amino-thiol tautomers (spectrum a and b) taken together in the region of the SH stretching mode.
nitrogen and sulfur hydrogen bond donors and acceptors participating in the formation of hydrogen bonds [14]. The bases are hydrogen bonded around screw axes to form approximately planar ribbons running in the b direction. Within these ribbons each base is joined to the two adjacent bases by a total of six hydrogen bonds of N1H···N3, N2H···N9, N2H···S, N7H…S types [14]. Thus the assignments of the following modes of TG more sensitive to hydrogen bonding will be discussed: NH2, N1H, N7H, CNH2 and CS modes.
3.1. NH2 modes For the amino-thione N7H tautomer, the present calculation predicts antisymmetric and symmetric stretching modes of the NH2 group at 3512 and 3401 cm − 1, respectively. However, the presence of intermolecular hydrogen bonding is expected to
lower the magnitude of stretching modes of the NH2 group. In addition, the two NH bonds being not equivalent in the crystal structure of TG, the relation ws = 435.5+0.876was proposed for the NH2 group [24] is not satisfied by ws and was in the present case. Therefore, the first one, being bonded to a sulfur atom, is assigned to the absorption band observed at 3292 cm − 1 in the spectrum shown in Fig. 5, the second one, bonded to a nitrogen atom, is assigned to 3129 cm − 1 infrared frequency. The scissoring mode of the NH2 group gives rise to its characteristic frequency in the region 1600–1700 cm − 1 [23]. Therefore, the band observed at 1666 cm − 1 in the infrared spectrum and at 1663 cm − 1 in the Raman spectrum is assigned undoubtedly to the scissoring mode of the NH2 group. The rocking NH2 mode, usually appearing in the region 900–1150 cm − 1 for nucleic acid bases [25], has been assigned to infrared and
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
1803
Table 2 Wavenumbers (WN), Infrared integrated intensities, AIR, Raman integrated relative intensities, IRA, and potential energy distributions (PED) for amino-thione N7H tautomer Experiment-solid
Calculation b
(cm−1)
AIR (km/mol)
IRA
3512
62
16
2 3 4
3481 3429 3402
148 90 83
12 11 34
5 6
3051 1641
5 563
40 13
7 8
1595 1566
382 794
2 11
9
1532
40
11
10
1481
29
21
11
1427
164
17
12
1360
299
119
13
1347
7
22
14
1323
115
22
15
1252
137
34
16
1172
51
79
17
1163
78
17
18
1107
30
6
19
1086
35
9
Q
WN
1
d
PED
e
WNIR
AmH16 s(51−) AmH15 s(49+) N7H s(100−) N1H s(99−) AmH15 s(50+) AmH16 s(49+) C8H s(99−) C2N3 s(34−) HNH sci(19+) C2Am s(10+)
3292
HNH sci(55−) N1H be(34−) HNH sci(15+) C5C4 s(10+) N3C4 s(23+) C5C4 s(13−) C8N9 s(25+) C8H be(20−) N7H be(42−) N7C8 s(31+) C5C4 s(30+) C5C4 s(30+) N1C2 s(10−) N9C4 s(10−) C2Am s(22+) N1H be(19−) R6 def1(15−) C5N7 s(22+) N3C4 s(18−) C6C5 s(15−) C8N9 s(11+) C8N9 s(28−) C8H be(22−) N9C4 s(13+) C6C5 s(11−) N1C6 s(44−) C5N7 s(14+) C8H be(10−) N1C6 s(17+) N1H be(14−) R6 def1(13+) C8H be(10−) C6S s(10−) NH2 ro(45+) C2N3 s(17−) C2Am s(13−) N9C4 s(12+) N7C8 s(50+) N7H be(31+)
c
(cm−1)
a
AIR (km/mol)
WNRA
3241 3051 3129
700
1635
150
1666 1618
510 1100
1663 1619
1546
81
1546
1483
77
1478
1436
42
1439
3105
1367
1375
299
1372
1259
1260
1231
1228
1143
1135
1201
1202
1105
1032
1033
c
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
1804 Table 2 (Continued)
Experiment-solid
Calculation Q
WN
20
b
(cm−1)
AIR (km/mol)
IRA
999
54
7
21
942
80
12
22 23
934 909
3 121
1 1
24
804
30
4
25
771
45
1
26
722
44
1
27
659
75
2
28
642
3
1
29
618
16
1
30
574
23
5
31
639
75
18
32
527
215
6
33
504
28
10
34 35
491 417
116 6
4 8
36
360
1
2
37
310
26
15
38 39
289 198
92 5
8 1
40
196
8
26
d
PED
e
C8H be(13−) NH2 ro(21−) N1C2 s(21−) N9C4 s(15+) R5 def1(23+) N1C2 s(20+) C6S s(20−) C8H owa(104−) R5 def1(48−) C6S s(14−) R6 def1(24+) C5N7 s(16−) R5 def2(12+) N3C4 s(10−) R5 ode2(80−) C2Am owa(12−) C2Am owa(64+) R5 ode2(35−) N1H owa(84+) C6S owa(18−) R5 ode1(85−) C6S owa(15−) R5 def2(26−) C2Am be(14−) C6C5 s(12−) R5 ode3(44+) C6S owa(42−) R6 ode2(13−) R6 ode1(11+) NH16 owa(23+) N9C4 s(12−) R6 def2(12−) NH16 owa(57−) R6 def3(13−) R6 def3(41−) C2Am s(11+) N7H owa(87−) R6 def2(53+) C6S s(25−) R5 ode1(45+) R6 ode1(29+) R5 ode1(29+) C2Am owa(11+) R6 ode2(10+) C2Am be(42−) R5 def2(11+) NH15 owa(10−) NH15 owa(78−) R6 ode3(68−) R6 ode2(28−) R5 ode3(19−) C6S be(70−)
WNIR
c
(cm−1)
a
AIR (km/mol)
WNRA
972
972
942
921
925 841
921
838
838
777
776
719
715 657 648
621
572
575
563
824 525 570 418 409 369
362
570 234
215
c
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
1805
Table 2 (Continued) Calculation Q
WN
41
42
Experiment-solid b
(cm−1)
AIR (km/mol)
IRA
148
15
3
128
13
0
d
PED
e
WNIR
c
(cm−1)
R6 ode3(34+) R5 ode3(17−) R5 ode1(15+) R6 ode2(11−) C2Am owa(10−) R6 ode1(45−) R6 ode9(41+) R6 ode3(25−) C6S owa(18−)
a
AIR (km/mol)
WNRA
c
135
a Polycrystalline powder sample dispersed in KBr for infrared measurements or pressed into a rotating cell for Raman measurements. b Wavenumbers scaled by a constant factor of 0.91. c Only the strongest band is given when multicomponent bands are observed. d Integrated relative intensities in unit chosen in such way that the observed intensity sum of the in-plane modes observed in the separated spectra of amino-thiol N9H or amino-thiol N7H tautomer is equal to the calculated intensity sum of these modes. e Abbreviations: s, stretching; be, bending; sci, scissoring; owa, out-of-plane wagging; ro, rocking; def, deformation; ode, out-of-plane deformation; tor, torsion.
Raman bands observed at 972 cm − 1. The wagging and torsion (NH2) modes are respectively associated with infrared bands at 777 and 621 cm − 1. In fact the torsion and wagging (NH2) modes arise due to out-of-plane and in-plane coupling of the two NH out-of-plane bending motions of the NH2 group. In addition, the presence of intermolecular hydrogen bonding is expected to raise the magnitude of the torsion (NH2) mode.
3.2. N1H modes The HF/6-31G assignments attribute N7H and N1H stretching modes to 3481 and 3428 cm − 1, respectively. Despite the presence of intermolecular hydrogen bonding lowering the magnitude of N7H and N1H stretching modes, there is no confusion possible in the assignment of N1H and N7H stretching modes in experimental spectra. X-ray crystal structure [14] shows the presence of intermolecular NH···N and NH···S hydrogen bonds for N7H and N1H groups of 2.97 and 3.33 A, length,
respectively. Considering the Pimentel relation between OH or NH band shifts and hydrogen bond length, the N1H bond being bonded to the nitrogen atom in the crystal structure is expected to be observed in lower frequency than the N7H bond, which is bonded to the sulfur atom. Thus the shoulder observed around 3051 cm − 1, near the superimposed band situated at 3129 cm − 1, in the infrared spectrum (Fig. 5) is attributed to N1H stretching mode. The N1H in-plane bending mode is assigned to the infrared band at 1618 cm − 1 and Raman band at 1619 cm − 1. The band observed at 719 cm − 1 in the infrared spectrum and 715 cm − 1 in the Raman spectrum could be attributed to the N1H out-of-plane bending mode.
3.3. N7H modes The very weak absorption situated around 3241 cm − 1, between the bands 3292 and 3129 cm − 1, in the spectrum of Fig. 5 has been assigned to the N7H
Fig. 5. Comparison of the experimental infrared and Raman spectra of solid TG with the calculated spectra of the amino-thione N7H tautomer using 3-21G basis set, in the region of NH2, N7H, N1H and CH stretching modes (3600 – 2800 cm − 1).
1806
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
Fig. 5.
O.E. Kasende / Spectrochimica Acta Part A 58 (2002) 1793–1808
stretching mode. The N7H in-plane bending mode has been attributed to the infrared band at 1436 cm − 1 and Raman band at 1439 cm − 1. The absorption band at 570 cm − 1 has been associated with the N7H out-of-plane bending mode. It is worth noting that NH2, N1H and N7H stretching modes are not observed in the Raman spectrum (Fig. 5). The unique band observed at 3105 cm − 1 in this region in this region of the Raman spectrum is ascribed to the CH stretching mode.
3.4. CNH2 modes According to the crystal structure of TG the CNH2 bond (1.313 A, ) is shorter than the corresponding bond in 2-thiocytosine (1.333 A, ) [26]. The CNH2 stretching mode is, therefore, expected to appear at higher frequency than in 2-thiocytosine. For TG, we assigned to this mode the infrared frequency 1375 cm − 1 corresponding to Raman frequency 1372 cm − 1, in agreement with the assignment proposed by Singh and Yadav [22]. The calculated CNH2 stretching mode has the frequency 1347 cm − 1. In the 2-thiocytosine infrared spectrum this mode has been assigned to the frequency 1369 cm − 1. The CNH2 in-plane bending and out-ofplane bending modes are tentatively assigned in the Raman spectrum to the bands at 362 and 135 cm − 1, respectively.
3.5. CS stretching mode The CS stretching mode is among the most interesting modes as this is involved in the intermolecular hydrogen bonding which plays a very important role in the biological activities of this molecule [1]. In TG crystal structure [14], the sulfur atom accepts hydrogen bonds from N7H and NH2 of two different molecules; the lengths of these hydrogen bonds are 3.30 and 3.33 A, , respectively. By comparison to the case of 2thiocytosine in which the CS stretching mode has been assigned to the infrared band at 1200 cm − 1, in the TG infrared spectrum, this mode is assigned to the band at 1201 cm − 1 corresponding to the Raman band at 1202 cm − 1 in spite the low frequency 1163 cm − 1 predicted by our
1807
calculation. This assignment agrees with the previous assignment [22].
Acknowledgements The author is grateful to Ambassade 6an het Koninkrijk der Nederlanden (Kinshasa, Congo) for its financial support. I also wish to acknowledge Professor W.B. Person and Dr K. Szczepaniak-Person (University of Florida, USA) for experimental assistance and helpful discussions. Finally, I appreciate the hospitality and support provided by Professor D. de Waal at the University of Pretoria, Republic of South Africa, where the final version of this paper was completed.
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