Journal of Molecular Structure (Theochem) 532 (2000) 157±169

www.elsevier.nl/locate/theochem

A theoretical study of tautomerism of cytosine, thymine, uracil

and their 1-methyl analogues in the gas and aqueous phases using
AM1 and PM3
È . Civcir*
P.U
Faculty of Health Education, Ankara University, Ankara, Turkey
Received 4 March 2000; accepted 4 April 2000

Abstract
Heats of formation, entropies, Gibbs free energies, relative tautomerisation energies, relative proton af®nities, tautomeric
equilibrium constants, dipole moments, and ionisation potentials for the tautomers of cytosine, uracil, thymine and their 1-
methyl analogues have been studied using semiempirical AM1 and PM3 quantum-chemical calculations at the SCF level in the
gas and aqueous phases, with full geometry optimisation. The COSMO solvation model was employed for aqueous solution
calculations. The calculations show that the oxo-amino or dioxo tautomers are the most stable both in the gas and aqueous
phase. The results are in good agreement with the available experimental and theoretical data. The entropy effect on the Gibbs
free energy of the pyrimidine bases is very small and there is little signi®cance for the tautomeric equilibria of pyrimidine bases.
The enthalpic term is dominant also in the determination of the equilibrium constant. q 2000 Elsevier Science B.V. All rights
reserved.
Keywords: Pyrimidine bases; Tautomerism; Tautomeric equilibrium constant; Dipole moment; Ionisation potential; AM1; PM3 semiempirical
calculation

1. Introduction mispairing in DNA and thus, mutagenesis is corre-

Studies of tautomerism phenomena have been valu-
able in many areas of chemistry as demonstrated by
several reviews of experimental and theoretical
studies in chemistry and biochemistry. [1±5]. Tauto-
meric equilibria of pyrimidine bases are of continuing
interest both from the theoretical [6±20] and experi-
mental [21±24] points of view, partly due to sugges-
tions that the presence of unusual tautomers may have
important biological properties, such as mutagenesis.
One hypothesis suggests that the frequency of
* Tel.: +90-312-3808172; fax: +90-312-3575323.
È . Civcir).
E-mail address: pervinunal@hotmail.com (P.U
0166-1280/00/$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0166-128 0(00)00556-X
lated with equilibrium constants for the keto-enol or
amino±imino tautomerisation [25,26]. Purine and
pyrimidine bases exist in different tautomeric forms
in an equilibrium and the equilibrium is strongly
sensitive to the interaction of these molecules with
their environment [8,13]. Therefore, knowledge of
the energetics of tautomerism in simple model for
molecules such as cytosine or other pyrimidine
bases, which show an imino±amino equilibrium, can
provide useful information on the intrinsic stability of
various tautomers of molecules. In addition, knowing
how these tautomerisation energies change in differ-
ent environments can give an insight into the in¯uence
of solvent effects on molecular stability.
158 È . Civcir / Journal of Molecular Structure (Theochem) 532 (2000) 157±169
P.U
Fig. 1. Tautomers of cytosine (1±7), uracil (8±13) and thymine (14±19) considered in the present work.
Previous work on pyrimidine bases have described
the structures and relative stability of different tauto-
mers of these bases [7±13]. Most of these do not
consider the effects of entropy on the equilibrium.
The temperature-dependent components of the
enthalpy and entropy contribution to the Gibbs free
tautomerisation energies of DNA bases and their
1-methyl derivatives are known to be important.
Analysis of the entropy effect thus allows for better
understanding of the tautomerisation process. If
several tautomers exist in comparable concentrations,
the entropy contributions are important parts of rela-
tive Gibbs free energies. Because the exact equili-
brium concentration depends on the Gibbs free
energies of each tautomers. Consequently, both
entropy and enthalpy should be included for a proper
comparison of the calculated and experimental tauto-
meric stability of the bases. Experimental information
about the relative stability of two tautomeric forms of
a molecule …a Y b† is obtained from the measurement
of the tautomeric equilibrium constant Ka;b …T†: As a
consequence, the Gibbs free energy of the tautomer-
isation DGa;b …T† can be estimated at the de®ned
temperature T.
The present work is directly related to previous
studies of Katritzky and Karelson [11] who used the
AM1 and PM3 methods to calculate relative stabi-
lities for the tautomers of cytosine, uracil, thymine
and their 1-methyl derivatives based on the heats of
formation of tautomers …dDH f †: The aim of this
work is, therefore, to reinvestigate the relative stabi-
lity of these molecules as presented in Figs. 1 and 2,
using semiempirical AM1 and PM3 methods, but
also considering the effects of entropy on the equi-
librium. We also extend the previous work reported
by Katritzky and Karelson [11] to include tauto-
meric equilibrium constants calculated from the
Gibbs free energies of the pyrimidine bases.
Although some of cytosine, thymine, uracil and
their derivatives were previously studied, there are
no data available on prediction of the tautomeric
equilibrium constants for these molecules by
means of quantum chemical calculations in the
literature.
 È . Civcir / Journal of Molecular Structure (Theochem) 532 (2000) 157±169
P.U
Fig. 2. Tautomers of 1-methylcytosine (20±22), 1-methyluracil
(23±25) and 1-methylthymine (26±28) considered in the present
work.
In the present paper, we report the heats of
formation, entropy, Gibbs free energy, relative
stability, tautomeric equilibrium constants, relative
proton af®nities, dipole moments and ionisation
potentials for the pyrimidine bases at 298.15 K.
After having predicted the relative stability of
tautomers, we have found the tautomeric equili-
brium constants with respect to the more stable
tautomer both in the gas phase and in aqueous
solution. On the basis of these, the predicted equi-
librium constants were compared with the available
experimental data.
2. Method of calculation
Theoretical calculations were carried out at the
restricted Hartree±Fock level (RHF) using AM1
[27] and PM3 [28] semiempirical SCF±MO methods
with the mopac 7.0 program [29], implemented on an
Pentium II 300 MHz computer. In aqueous phase
calculations, the COSMO solvation model is used to
construct a solvent accessible surface area based on
159
Van der Waals radii. The relative permitivity of water
was taken to be 1 ˆ 78:4 and the model incorporated
up to 30 surface segments per atom. Initial geometry
estimates of the all structures were obtained from a
molecular mechanics calculation (CS Chem Of®ce)
[30], and were followed by full optimisation of all
geometrical variables. All structures were optimised
to a gradient norm of ,0.2 in the gas phase and 0.1±2
in the aqueous phase, using eigenvector method (EF).
In order to calculate thermodynamic properties of the
tautomers, the gradient norm is again reduced a value
very close to zero. The Gibbs free energies of the
tautomerisation (DGf) at 298.15 K were predicted by
adding the enthalpic (DHf) and entropic …TDSf †
terms. Katritzky and Karelson [11] also used same
calculations method to obtain the relative stability of
pyrimidine bases. However, they did not make the
thermodynamic calculations.
3. Results and discussion
3.1. Relative stability
The relative stabilities, enthalpies, entropies and
Gibbs free energies, dipole moments and ionisation
potentials for the tautomers of cytosine, thymine,
uracil and their 1-methyl derivatives are given in
Tables 1±4.
The AM1 calculations of Gibbs free energy for
cytosine (Tables 1 and 2) clearly show that both in
the gas phase and in aqueous solution the most stable
form is the 1H-oxo-amino form 2. Experimental
studies indicate that substantial amounts of the
hydoxy-amino form 1 may be present in the case of
isolated molecules [10,31±37]. Two experiments, one
based on the calculated force constant from compar-
isons with IR frequencies obtained in Ar or N2
matrices [10,31±36] and another based on the
moments of inertia calculated from the microwave
spectra [37], give almost the same stability for
forms 1 and 2. Ab initio calculations [8] favour the
1H-oxo-amino form 2. In aqueous solution, the AM1
calculation indicates that the 1H-oxo-amino form
2 is more stable than the 3H-oxo-amino form 3,
by 0.85 kcal/mol, than the hydroxy-amino form 1
by 10.13 kcal/mol, and than the oxo-imino form 7 by
10.03 kcal/mol.
 160
Table 1
The AM1 calculated thermodynamic properties for the tautomers of cytosine, uracil, thymine and their 1-methyl analogues in the gas phase …1 ˆ 1† at 298.15 K

Tautomer DHf (kcal mol 21) DS (cal mol 21 K 21) DGf (kcal mol 21) a d DGf (kcal mol 21) b d DHf (kcal mol 21) c IP (eV) IP(exp) (eV) d m (D) m (exp) (D) e

1 4.45 80.34 219.50 1.79 1.70 9.37 3.15

2 2.74 80.59 221.29 0.00 0.00 9.38 8.94 6.32 6±6.50
3 3.30 80.26 220.63 0.66 0.65 8.94 7.24
P.U

4 48.47 82.93 23.74 45.03 45.81 8.14 13.24

5 24.40 81.50 0.10 21.39 16.21 8.90 5.11
6 16.40 80.23 27.52 13.77 12.10 8.90 1.45
7 6.18 79.79 217.61 3.68 1.45 9.40 4.55
8 234.20 79.32 257.85 19.32 14.85 9.85 3.05
9 244.54 78.60 267.97 9.20 9.32 9.55 2.84
10 239.93 78.96 263.47 13.70 13.95 9.52 5.92
11 233.29 79.98 257.14 20.03 20.58 9.70 6.11
12 235.76 40.04 247.70 12.47 12.51 9.56 4.08
13 253.86 78.18 277.17 0.00 0.00 9.97 9.60 4.29 4.16
14 242.01 87.26 268.03 19.69 14.88 9.71 3.09
15 251.99 90.02 278.83 8.89 9.12 9.24 2.51
16 247.64 87.24 273.65 14.07 13.47 9.26 6.01
17 240.95 90.45 267.92 19.80 20.19 9.42 5.82
18 243.34 86.68 269.18 18.54 19.89 9.50 7.28
19 261.12 89.21 287.72 0.00 0.00 9.60 9.14 4.16 4.13
20 8.17 92.10 219.29 0.00 0.00 9.22 8.65 5.94 6.0±70
21 30.12 90.23 3.22 22.51 16.45 8.77 4.42
22 11.79 90.18 215.10 4.19 1.48 9.22 2.30
23 227.61 90.30 254.53 19.90 20.74 9.48 6.01
24 230.32 89.49 257.00 17.43 12.47 9.54 4.03
25 248.03 88.56 274.43 0.00 0.00 9.70 4.65
26 235.23 99.82 264.99 20.14 20.30 9.23 6.27
27 237.86 97.21 266.84 18.29 12.58 9.30 5.19
È . Civcir / Journal of Molecular Structure (Theochem) 532 (2000) 157±169

28 255.51 99.36 285.13 0.00 0.00 9.42 4.49 4.10

a
From DGf ˆ DHf 2 TDSf :
b
dDGf ˆ DGf…b† 2 DGf…a† :
c
Ref. [11].
d
Ref. [48] for cytosine, uracil and thymine, Ref. [49] for 1-methylcytosine.
e
Ref. [18] for cytosine, Ref. [47] for uracil, thymine, 1-methylcytosine and 1-methylthymine.
 È . Civcir / Journal of Molecular Structure (Theochem) 532 (2000) 157±169
P.U 161

Table 2
The AM1 calculated thermodynamic properties for tautomers of cytosine, uracil, thymine and their 1-methyl analogues in aqueous phase …1 ˆ
78:4† at 298.15 K

Tautomer DHf (kcal mol 21) DS (cal mol 21 K 21) DGf (kcal mol 21) a d DGf (kcal mol 21) b d DHf (kcal mol 21) c IP (eV) m (D)

1 216.30 80.06 240.47 10.13 7.74 9.42 5.23

2 227.21 79.34 250.87 0.00 0.00 9.63 11.05
3 225.86 81.03 250.02 0.85 2.81 9.26 13.45
4 217.46 80.89 240.58 10.29 17.24 9.41 21.09
5 0.02 81.30 224.22 26.65 20.51 9.30 11.02
6 22.35 80.53 226.35 24.52 19.12 9.21 2.67
7 217.08 79.69 240.84 10.03 8.69 9.44 7.14
8 255.76 79.57 279.48 23.02 17.98 10.12 5.72
9 266.16 78.46 289.55 12.95 11.59 9.78 5.39
10 265.87 78.71 289.34 13.16 9.87 9.52 5.92
11 262.09 79.04 285.66 16.84 16.15 9.7 6.11
12 265.78 78.81 289.27 13.23 11.94 9.92 7.92
13 279.27 77.93 2102.50 0.00 0.00 9.97 4.28
14 261.49 87.65 287.62 22.56 17.10 9.79 5.91
15 272.93 90.45 299.90 10.28 11.08 9.43 4.79
16 270.51 86.83 296.40 13.78 9.69 9.42 10.65
17 265.49 90.60 292.50 17.68 17.12 9.62 14.58
18 271.43 86.29 297.16 13.02 13.05 9.62 12.01
19 284.11 87.45 2110.18 0.00 0.00 9.53 7.12
20 218.54 91.69 245.88 0.00 0.00 9.48 10.62
21 8.88 91.18 218.31 27.57 18.64 9.22 13.06
22 27.54 91.32 234.77 11.11 6.45 9.34 7.19
23 250.59 87.89 276.79 19.45 16.42 9.92 15.22
24 256.61 89.14 283.19 13.05 12.70 9.78 11.64
25 269.55 89.53 296.24 0.00 0.00 9.75 7.62
26 256.31 97.71 285.44 15.87 17.05 9.58 14.62
27 262.64 96.37 291.37 9.94 12.37 9.52 11.73
28 271.32 100.59 2101.31 0.00 0.00 9.65 6.36
a
From DGf ˆ DHf 2 TDS:
b
dDGf ˆ DGf…b† 2 DGf…a† :
c
Ref. [11].

For the uracil and thymine tautomers, the calcula-
tions show that the dioxo forms 13 and 19 appear
as the more stable forms both in the gas phase and
aqueous solution, which is consistent with the
experimental data [38±46]. According to the
AM1 calculated dDGf ; the next most stable tauto-
mers are 9, 15 in the gas phase and in aqueous
solution. Substitution of uracil at the 5-position by
CH3 does not change the order of the stability of
the tautomers.
1-Methylcytosine 20 can occur in the three forms as
20, 21, 22. The oxo-amino tautomer 20 is predicted as
the most stable form by both AM1 and PM3 methods
both in the gas phase and in aqueous solution. The
predominance of the oxo-amino form 20 is in
complete agreement with the experimental IR data
reported by Person et al. [24]. In the gas phase, the
relative stabilities for oxo-amino form 20 and oxo-
imino form 22 are very close, whereas in aqueous
solution their energy values are substantially different
(cf. Tables 1 and 2). These results illustrate the differ-
ential effects of the polar medium on the relative stabi-
lity of tautomers. The AM1 and PM3 calculations for
1-methyluracil and 1-methylthymine tautomers also
show the dominance of the dioxo forms 25 and 28
both in the gas phase and in solution. This result is
consistent with experimental data [24] and the
previous AM1 calculations reported by Katritzky
and Karelson [11]. The stable methyl tautomers (20,
25 and 28) are same as the main tautomers (2, 13 and
162 È . Civcir / Journal of Molecular Structure (Theochem) 532 (2000) 157±169
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Table 3
The PM3 calculated thermodynamic properties for tautomers of cytosine, uracil, thymine and their 1-methyl analogues in the gas phase …1 ˆ 1†
at 298.15 K

Tautomer DHf (kcal mol 21) DS (cal mol 21 K 21) DGf (kcal mol 21) a d DGf (kcal mol 21) b IP (eV) IP(exp) (eV) c m (D) m (exp) (D) d

1 213.92 80.60 237.95 2.30 9.27 2.66

2 216.20 80.66 240.25 0.00 9.28 8.94 6.68 6.0±6.50
3 211.44 81.09 235.62 4.63 9.10 6.26
4 18.34 84.07 26.73 33.52 8.09 12.94
5 3.64 81.68 220.71 19.54 8.8 4.46
6 23.88 82.73 228.55 11.70 8.71 1.83
7 210.73 81.77 235.11 5.14 9.06 4.28
8 256.71 80.11 280.60 10.72 9.95 3.44
9 261.02 79.71 284.79 6.53 9.45 2.46
10 257.03 80.07 280.90 10.42 9.33 5.88
11 245.78 82.78 270.46 20.86 9.72 7.41
12 255.36 80.1 279.24 12.08 9.50 6.79
13 267.63 79.47 291.32 0.00 9.71 9.60 3.99 4.16
14 266.87 87.80 293.05 9.63 9.67 3.66
15 269.82 88.48 296.20 6.48 9.22 2.16
16 265.71 87.96 291.74 10.92 9.13 5.95
17 254.91 91.60 282.22 20.46 9.52 7.19
18 264.44 87.67 290.58 12.10 9.31 6.97
19 276.34 88.33 2102.68 0.00 9.44 9.14 3.97 4.13
20 214.27 92.91 241.97 0.00 9.13 8.65 5.58 6.0±70
21 8.60 89.89 218.20 23.77 8.83 6.70
22 211.28 91.06 238.43 3.54 9.03 4.35
23 247.41 89.40 274.06 21.04 9.56 7.95
24 255.77 91.96 283.19 11.91 9.35 6.81
25 268.01 90.87 295.10 0.00 9.52 4.37
26 256.49 98.20 285.77 20.28 9.38 7.71
27 264.81 99.73 294.54 11.51 9.16 6.96
28 276.81 98.08 2106.05 0.00 9.36 4.20 4.10
a
From DGf ˆ DHf 2 TDSf :
b
dDGf ˆ DGf…b† 2 DGf…a† :
c
Ref. [48] for cytosine, uracil and thymine, Ref. [49] for 1-methylcytosine.
d
Ref. [18] for cytosine, Ref. [47] for uracil, thymine, 1-methylcytosine and 1-methylthymine.

19), in other words, substitution of pyrimidine bases at
N-1 position by CH3 does not change the order of the
stability of the tautomers.
The PM3 method gives the same order of stability
for the pyrimidine bases, and their 1-methyl analo-
gues. AM1 numerical data for relative stabilities are
close to those of previous theoretical values reported
by Katritzky and Karelson [11]. Our calculated rela-
tive stability values …dDGf † are very similar their
calculated relative stability values …dDH f † numeri-
cally (cf. Tables 1 and 2). As we can see from Tables
1±4, the calculated entropy values are generally small
(smaller than 100 cal/mol). This results indicate that
the entropy effect on the Gibbs free energy is very
small and the entropy term and TDS value can be
neglected for the tautomeric equilibria of pyrimidine
bases. Thus, the enthalpic term is dominant in the
determination of the equilibrium constant. However,
when the concentration ratio of two considered tauto-
mers is very close, it is necessary to consider entropy
effects. This is particularly important when accurate
calculations are carried out to estimate relative stabi-
lities of the tautomers.
3.2. Dipole moments and ionisation potentials
The calculated dipole moments and the ®rst ionisa-
tion potentials of studied pyrimidine bases are also
 È . Civcir / Journal of Molecular Structure (Theochem) 532 (2000) 157±169
P.U 163

Table 4
The PM3 calculated thermodynamic properties for tautomers of cytosine, uracil, thymine and their 1-methyl analogues in aqueous phase …1 ˆ
78:4† at 298.15 K

Tautomer DHf (kcal mol 21) DS (cal mol 21 K 21) DGf (kcal mol 21) a d DGf (kcal mol 21) b IP (eV) m (D)

1 240.37 82.97 265.11 5.12 8.92 5.63

2 246.36 80.06 270.23 0.00 8.98 11.76
3 243.33 81.16 267.52 2.71 8.93 13.89
4 235.09 82.33 259.64 10.59 9.39 20.58
5 220.76 81.27 244.99 25.24 8.97 11.76
6 222.14 81.29 256.47 13.66 9.06 2.58
7 236.31 79.98 260.15 10.08 9.09 7.26
8 278.84 80.00 2102.69 13.96 9.95 3.44
9 286.74 79.15 2110.34 6.31 9.45 2.46
10 282.55 79.53 2106.57 10.08 9.58 11.14
11 281.29 80.40 2105.26 11.39 9.65 15.63
12 282.79 79.56 2106.51 10.14 9.52 7.29
13 293.36 78.12 2116.65 0.00 9.71 3.99
14 286.09 87.76 2112.26 13.78 9.78 5.25
15 291.70 88.98 2118.23 7.81 9.39 7.05
16 289.54 88.67 2115.98 10.06 9.34 11.16
17 287.60 90.00 2114.43 11.61 9.42 14.97
18 293.88 86.90 2119.79 6.25 9.42 12.06
19 2100.04 87.19 2126.04 0.00 9.28 4.44
20 240.05 92.35 267.58 0.00 8.95 11.42
21 219.07 90.04 245.92 21.66 9.08 13.90
22 229.71 91.52 257.00 10.58 9.07 7.21
23 278.01 88.30 2104.34 19.71 9.69 15.30
24 283.81 89.34 2110.45 13.60 9.59 11.65
25 297.41 89.34 2124.05 0.00 9.27 7.13
26 284.57 97.75 2113.71 13.08 9.46 14.74
27 290.89 97.47 2119.95 6.84 9.39 11.74
28 297.38 98.63 2126.79 0.00 9.27 7.13
a
From DGf ˆ DHf 2 TDS:
b
dDGf ˆ DGf…b† 2 DGf…a† :

listed in Tables 1±4. For the most stable tautomer of
cytosine, the calculated dipole moments with the
AM1 and PM3 methods are 6.32 and 6.68 D, respec-
tively (cf. Tables 1 and 3). The results are in good
agreement with experimental data (6±6.5 D) [18]
and with an ab initio prediction (7.2 D) [8]. The calcu-
lated dipole moments for the most stable tautomers of
uracil and thymine (Tables 1 and 3) are in good agree-
ment with the experimental data obtained [47] in the
gas phase. The calculated dipole moment of 1-
methylthymine is in line with experimental data
[47]. For 1-methylcytosine and 1-methyluracil the
calculated dipole moments are 5.94 and 4.65 D,
respectively. Although there are no experimental
dipole moment data available for these compounds,
a theoretical dipole moment for 1-methylcytosine is
5.86 D [18]. This value is in agreement with our
calculated value of 5.94 D.
As we can see from Tables 1±4, the calculated
dipole moments are changed on moving from the
gas phase …1 ˆ 1† to solution …1 ˆ 78:4† and the dipole
moments are sensitive to the polarity of the medium.
The calculated dipole moments are substantially
higher in a medium of high relative permitivity,
mainly due to major charge redistribution in the mole-
cule, and also by changes in the distances between the
charge separations. The magnitude of the in¯uence of
the solvent reaction ®eld on electronic structure is
different in different tautomers. This may also explain
the great variation of the calculated dipole moments
of the tautomers. The calculated dipole moments are
rather sensitive to the tautomeric form of the molecule
164 È . Civcir / Journal of Molecular Structure (Theochem) 532 (2000) 157±169
P.U

Table 5
The AM1 and PM3 predicted equilibrium constants with respect to the most stable tautomers of cytosine, uracil, thymine and their 1-methyl
analogues in the gas phase …1 ˆ 1† and in aqueous solution …1 ˆ 78:4† at 298.15 K

Tautomeric equilibria pKT (1 ˆ 1) a pKT (ab initio) b pKT (1 ˆ 78.4) a pKT (exp) pKT (ab initio) b

AM1 PM3 AM1 PM3

2±1 1.31 1.68 2.7±3.4 7.62 3.76 10.40

2±3 0.48 3.39 0.30 0.62 1.99 2.9 c 0.80
2±4 33.01 24.58 ± 7.54 7.76
2±5 15.68 14.33 16.70 19.53 18.50
2±6 10.09 8.57 13.39 17.98 10.08
2±7 2.70 3.76 5.22 7.35 7.39 4.0±5.0
13±8 14.16 7.86 17.70 16.87 10.23
13±9 6.74 4.78 12.70 9.49 4.63 5.0 d
13±10 10.04 7.64 14.30 9.65 7.39
13±11 14.68 15.29 19.15 12.34 8.35
13±12 9.14 8.86 21.00 9.70 7.43 3.6 d 13.52
19±14 14.43 7.06 18.52 16.54 10.10
19±15 6.52 4.75 12.40 7.53 5.73
19±16 10.31 9.02 24.00 10.10 7.38
19±17 14.52 15.00 18.69 12.96 8.51
19±18 13.59 8.87 15.22 9.55 4.58
20±21 16.50 17.43 20.21 15.88
20±22 3.07 2.60 8.14 7.76
25±23 14.59 15.42 14.26 14.45
25±24 12.78 8.73 9.57 9.97 3.3±4.0 e
28±26 14.76 14.87 11.63 9.59
28±27 13.41 8.44 7.29 5.01
a
pKT ˆ 2log KT :
b
Ref. [8].
c
Refs. [53,54].
d
Ref. [56].
e
Ref. [60].

may be used with experimental data to decide which
tautomer is most stable.
Calculated orbital energies as well as experimental
ionisation potentials for pyrimidine bases and their
1-methyl derivatives are listed in Tables 1 and 3. The
ionisation potentials were obtained from the energies of
the highest occupied molecular orbitals using
Koopmans' theorem. The ®rst ionisation potentials
for the most stable tautomers of cytosine, uracil
and thymine are close to experimental values [48].
The experimental ionisation potentials for 1-
methylcytosine is 8.65 eV [49]. Unfortunately, no
experimental data are available for the ionisation
potentials of 1-methyluracil and 1-methylthymine.
Experimental ionisation potentials for pyrimidine
bases are closer to our calculated PM3 values, than to
the AM1 values. As previously noted, the ionisation
potentials are systematically overestimated by AM1 as
well as MNDO [9,50] by ca. 0.5 eV for p-type orbitals
[50]. Results obtained by the PM3 method are not too
different from those obtained by AM1 and MNDO [9].
However, the PM3 method yields somewhat lower
values for ionisation from pyridine-like lone pairs
and, thus, in better agreement with experimental
values than either AM1 or MNDO. The AM1 results
are also close to the ab initio calculations [51,52].
3.3. Tautomeric equilibrium constants
The AM1 and PM3 calculated tautomeric equili-
brium constants with respect to the most stable
tautomers of uracil, thymine, cytosine and their 1-
methyl analogues both in the gas and in aqueous solu-
tion are listed in Table 5. The tautomeric equilibrium
 È . Civcir / Journal of Molecular Structure (Theochem) 532 (2000) 157±169
P.U
between tautomers a and b is described as
KT
aOb
Equilibrium constants for each species were calcu-
lated by using the following equation
KT ˆ e2…dDGf =RT† …
where KT is the tautomeric equilibrium constant
between the tautomers, the gas constant R is 1:987 £
1023 kcal=mol; and the temperature T is 298.15 K.
The quantity dDGf is the difference between the
Gibbs free energies of the given tautomer with respect
to the most stable one, dDGf ˆ DG…b† 2 DG…a† : DG…a†
and DG…b† are Gibbs free energies of each tautomer (a
or b) and they are given in Tables 1±4. If the enthalpy
and entropy terms are known we can calculate the
Gibbs free energy of each molecule, dDGf and KT
value. The Gibbs free energy of each tautomer (i) at
a given temperature T can be expressed as
DGf…i† ˆ DHf…i† 2 TDSf…i† …
where i ˆ a; b: The differences in enthalpy DH,
entropy DS and free energy DG between the two
tautomeric species was determined from the tempera-
ture dependence of KT
DG ˆ 2RT ln KT …
The pKT values of the studied molecules were calcu-
lated by means of the following equation
dDG
pKT ˆ …
2:303RT
Calculated values for the equilibrium between the
tautomers 2 and 3 of cytosine show that the 1H-oxo-
amino form 2 is more dominant than the 3H-hydroxy-
amino form 3, both for the gas and aqueous solution
with a pKT value of 0.48 and 0.62, respectively. The
1H-oxo-amino form 2 is also a more dominant tauto-
mer than the hydroxy-amino form 1 and the oxo-
imino form 7. Tautomers 4, 5 and 6 are not present
in detectable amounts. The experimental KT value
reported by Katritzky et al. [53] for 2 Y 3 is ca.
10 2.9 (i.e. pKT ˆ 2:9†; by pKa measurements. Dreyfus
et al. [54] have estimated the equilibrium constant for
K2/3 to be 2:5 £ 1023 in aqueous solution and asso-
ciated DH to be 13.0 kJ/mol. In the gas phase the
order of the stability of cytosine tautomers is 2 . 3 .
165
1 . 7 while tautomers 4, 5 and 6 are not detected.
However, we predict the order of stability in aqueous
solution to be 2 . 3 . 7 . 4 . 1: Furthermore,
comparison of the gas phase equilibrium constants
with experimental values implies that large changes
may occur on going from gas phase to solution.
1† Our results as well as experimental studies [3,5,55]
indicate that the rare lactim tautomers (10, 11, 12) of
uracil have not been observed in either the gas phase
or solution. This has led to the conclusion that the
tautomeric equilibria for uracil is not effected by
phase change. Predicted equilibrium constants in the
gas phase for the equilibria 13 Y 9 and 13 Y 12 are
pKT ˆ 6:74 and pKT ˆ 9:14; respectively while the
equilibrium constants in solution are pK T ˆ 9:49
and 9.70. The experimental equilibrium constants
for the 13 Y 9 and 13 Y 12 in solution [56] are
also 8:7 £ 1026 (i.e. pKT ˆ 5:0† and 2:5 £ 1024 (i.e.
pKT ˆ 3:6†: There is a substantial difference between
our results and the experimental results. Unfortu-
2† nately, the exact pKT value of cytosine is questionable
because uracil does not behave as a Hammett base and
does not follow the Hammett acidity function [57].
The calculations for the gas and solution phase of
the thymine, 19 Y 15 tautomeric equilibrium suggest
that the dioxo form 19 is more dominant than the 3H-
hydroxy-oxo form 15 by pKT value of 6.52 and 7.53,
3†
respectively. The other tautomers of thymine do not
exist in the gas phase and in solution phase. Again,
there is no experimental data for the equilibrium
constants of thymine tautomers, preventing any
4† comparison with the calculated pKT values could be
not made.
Calculations also show that the tautomers of uracil
9 and thymine 15 are more stable than uracil 11 and
thymine 17, respectively, which implies that the
proton at N1 is more acidic than that of N3. The
same results were obtained by Del Bene, who has
shown that H bonding of water to H(N1) is more
stable than that of H(N3) for the most stable tautomers
of uracil [58] and thymine [59].
For the equilibrium of the methyl substituted deri-
vatives 25 Y 24 and 28 Y 27; the AM1 and PM3
calculations show a dominance of dioxo forms 25
and 28 both in the gas phase and in solution. Appar-
ently, there is only one experimental result available
for comparison with our calculated data. Nearly
40 years ago Katritzky and Warring [60] reported
166 È . Civcir / Journal of Molecular Structure (Theochem) 532 (2000) 157±169
P.U

Table 6 may arise from hydrogen bonding effects. Solvent

The AM1 and PM3 calculated relative proton af®nities (d PA) effects have been ascribed to two major components
dPA ˆ dDH ˆ DH…B† 2 DH…A† for the tautomers of uracil, thymine,
cytosine and their 1-methyl analogues (in kcal mol 21)
[39], electrostatic solvent±solute interaction and
hydrogen bonding. The hydrogen bonding effects
Tautomers d PA(1 ˆ 1) d PA(1 ˆ 78.4) cannot be estimated in a quantitative manner from
the solvation model, further large-scale calculations.
AM1 PM3 AM1 PM3
A semiquantitave estimation of the solvation differ-
2±1 1.71 2.28 10.91 5.99 ences can be obtained from COSMO solvation model.
2±3 0.56 4.76 0.76 3.03 The electrostatic solvent±solute effects, however, are
2±4 45.73 34.54 9.75 11.27 readily estimated by the reaction ®eld continuum
2±5 21.66 19.84 27.23 25.60
2±6 13.66 12.32 24.86 24.22
model [63] using the dipole moments and molecular
2±7 3.44 5.47 10.13 10.06 polarizabilities. Application of the reaction ®eld
13±8 19.66 10.92 23.51 14.52 continuum model leads to an explanation of the
13±9 9.32 6.61 13.11 6.62 change in order of tautomeric stability on going
13±10 13.93 10.60 13.40 10.81 from gas phase to solution. However, such a treatment
13±11 20.57 21.85 17.18 12.07
13±12 18.10 12.27 13.49 10.57
lacks explicit consideration of base-water hydrogen
19±14 19.11 9.47 22.62 13.95 bonding effects [64], tautomeric equilibrium constants
19±15 9.13 6.52 11.18 8.34 predicted in water are considerably less reliable than
19±16 13.48 10.63 13.60 10.50 those predicted in the gas phase.
19±17 20.17 21.43 18.62 12.44
19±18 17.78 11.90 12.68 6.16
20±21 21.95 22.87 27.42 20.98
20±22 3.62 2.99 11.00 10.34 3.4. Relative proton af®nities
25±23 20.42 20.60 18.96 19.40
25±24 17.71 12.24 12.94 13.60 Since protonation of nucleic acid bases plays an
28±26 20.28 20.32 15.01 12.81 important role in many biochemical (i.e. enzymatic
28±27 17.65 12.00 8.68 6.49
reactions, stabilisation of triplex structures) and muta-
genic process [65], we have calculated the relative
pKT values of 3.3 (or 4.0) for 1-methyluracil and 1.3± proton af®nities for pyrimidine bases in order to put
3.3 for 5-bromouracil in aqueous solution at room in a more reliable basis the qualitative results inferred
temperature. These values are uncertain because from the AM1 and PM3 calculations. Pyrimidine
these compounds also do not behave as Hammett bases may be protonated at different nitrogen or
bases [57,60]. For the 1-methyl cytosine, tautomer oxygen atoms, but experimentally it is not possible
21 is not possibly existent. Mezey et al. [61,62] to discriminate between the various possibilities.
have proposed that any species with a energy larger The protonation energetics depend on the chemical
than 10 kcal/mol above the most stable form would surrounding, and so theoretical investigations repre-
not exist in any appreciable concentration and hence sent a practicable way for obtaining information about
tautomers 20 and 22 can exist in appreciable amounts the energetics of proton attachment to the different
at room temperature. Therefore, only the equilibrium sites of pyrimidine bases and to establish which of
20 Y 22 was considered. Calculations for the gas and these are favoured. The predicted relative proton af®-
solution phase for equilibrium 20 Y 22 show that nities are collected in Table 6 for AM1 and PM3
oxo-amino form 20 more dominants the oxo-imino methods in both phases.
form 22 by a pKT value 3.07 and 8.14, respectively. In the gas phase, we may consider the following
Unfortunately, there is no experimental evidence for equilibrium
20 Y 22 equilibria.
Our predicted pKT values are close to the ab initio B…g† 1 H1 1
…g† O BH…g† …5†
calculation results rather than experimental results.
There is a discrepancy between our predicted values The proton af®nity (PA) of given base B is de®ned as
in solution and that measured experimentally which the negative of the corresponding enthalpy change
 È . Civcir / Journal of Molecular Structure (Theochem) 532 (2000) 157±169
P.U
according to Eq. (6) for the reaction in Eq. (5)
Proton affinity PA…B† ˆ 2DHB
The PA is calculated as
PA…B† ˆ ‰DHf…B† 1 DHf…H1 † 2 DHf…BH1 † Š
In the aqueous phase, equilibrium is written as
B : 1AH1 O BH1 1 A
where B and BH 1 are the neutral and protonated
species of base B; A and AH 1 are the neutral and
protonated species of a reference base, A. The proton
af®nity of a given base B is calculated by means of Eq.
(9) for the reaction in Eq. (8).
Proton affinity :
PA…B† ˆ ‰DHf…B† 1 DHf…AH1 † Š 2 ‰DHf…BH1 † 1 DHf…A† Š
Thus the direction of the proton transfer reactions of
Eqs. (5) and (8) gives the basicity of bases A and B or
the acidities of the conjugated acids. The relative
proton af®nities (d PA), which is the difference
between proton af®nities of molecules A and B, can
be calculated as
dPA ˆ dDH ˆ PA…B† 2 PA…A† or dPA ˆ dDH
ˆ DH…B† 2 DH…A† …
where DH(B) and DH(A) are the heats of formation
values of tautomers A and B taken from Tables 1±
4. Predicted relative proton af®nities show that the
tautomers of cytosine 2 and 5 are more stable than
cytosine 3 and 6, respectively, which implies that
the proton at N1 is more acidic than that of N3. For
this compound, relative proton af®nities are in good
agreement with 15N-NMR experimental result which
show that the N1 is the protonated site in 2-aminopyr-
imidine [66]. Similarly, uracil 9 and 10 are more
stable than 11 and 12, while thymine 15 and 16 are
most stable than 17 and 18. Cytosine gives the PA of
the N1 endocyclic centre, whereas uracil and thymine
produce the N3 PA. The above PA order also
follows a logical trend in the sense that the endo-
cyclic nitrogens will be protonated ®rst, then the
amino group and ®nally the oxygen, which is less
basic centre. However, the ®nal exact theoretical
167
prediction of the proton af®nities of pyrimidine
bases, it is necessary to calculate the PA for each
…6†
tautomer, then it can be given the order of the PA of
pyrimidine bases.
…7†
4. Conclusion
…8† Several conclusions can be made on the basis of the
results of the present theoretical study.
1. The results clearly indicate that pyrimidine bases
and their 1-methyl analogues exist predominantly
in dioxo or amino-oxo tautomeric forms. These
results are in agreement with previous experimen-
tal and theoretical studies.
2. Substitution of uracil at the 5-position by CH3
does not change the order of the stability of the
tautomers.
…9†
3. Substitution of pyrimidine bases at N-1 position
by CH3 does not change the order of the stability
of the tautomers.
4. The results presented in this paper con®rm earlier
observations about the applicability of the AM1
method for the quantitative prediction of relative
stability of heterocyclic tautomers in solution.
5. The comparison of experimental data with the
calculated data by AM1 is also in agreement for
10† gas phase tautomerisation energies of pyrimidine
bases.
6. AM1 also gives a good representation of the
charge distribution in molecules in terms of calcu-
lated dipole moments.
7. The tautomeric equilibria of cytosine are very
sensitive to phase change.
8. The inclusion of the solvent reaction ®eld in quan-
tum-chemical theory is obligatory for accurate
results in solution.
9. Use of the Cosmo solvation model successfully
explains the reordering of the relative tautomeric
stability on passing from the gas phase to the
solution.
10. The entropy effect on the Gibbs free energy of the
pyrimidine bases is very small and there is little
signi®cance for the tautomeric equilibria of
pyrimidine bases. The enthalpic term is dominant
also in the determination of the equilibrium
constant.
168 È . Civcir / Journal of Molecular Structure (Theochem) 532 (2000) 157±169
P.U

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