Journal of Molecular Structure (Theochem) 545 (2001) 7±15

www.elsevier.nl/locate/theochem

AM1 and PM3 study of tautomerism of xanthine in the gas and

aqueous phases
È . Civcir*
P.U
Ankara University, Faculty of Health Education, Ankara, Turkey
Received 6 October 2000; accepted 1 November 2000

Abstract
Heats of formation, entropies, Gibbs free energies, relative tautomerisation energies, tautomeric equilibrium constants,
relative proton af®nities, dipole moments, and ionisation potentials for the fourteen possible tautomers of xanthine 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 two diketo tautomers X1,3,7 and X1,3,9 are the predominant species at room temperature in the gas
and aqueous phase. But, the ®rst more stable tautomer is the dioxo-7H tautomer, X1,3,7. Comparison with available experi-
mental data provides support for quality of results derived from theoretical computations. The entropy effect on the Gibbs free
energy of the xanthine is very small and there is little signi®cance for the tautomeric equilibria of the base. The enthalpic term is
dominant also in the determination of the equilibrium constant. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: AM1, PM3 semiempirical calculations; Dipole moment; Ionisation potential; Tautomerism; Tautomeric equilibrium constant;
Xanthine

1. Introduction oxidised by the molybdenum- and iron-containing

The oxopurines are of biological importance,
because they are metabolic intermediate products of
purine metabolism formed by degradation of nucleic
acids. Alkylated xanthines are bronchodilators.
Xanthine (3,7-dihydro-1H-purine-2,6-dione) is a
purine base which is not common in ribonucleic
acid or deoxyribonucleic acid, but deamination of
guanine results in xanthine base, and this base is
still able to pair with cytosine by two hydrogens. In
man, the end product of purine metabolism is uric acid
and xanthine oxidase. Actually, hypoxanthine, which
formed by enzymatic degradation of nucleic acid, is
* Tel.: 190-312-3808172; fax: 190-312-3575323.
E-mail address: civcir@health.ankara.edu.tr (P.U. Civcir).
0166-1280/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0166- 128 0( 00) 00821-6
enzyme xanthine oxidase via xanthine to uric acid.
This enzyme found in the liver, is capable of oxidising
xanthine to uric acid [1]. Defects in purine metabo-
lism result in an increase in the uric acid level and in
the deposition of sodium hydrogen urate monohydrate
crystals in joints. This disease known as gout and is
clinically treated by allopurinol, which has also been
used in conjunction with anticancer drugs combined
with 6-mercaptopurine in treatment of leukaemia [2±
4]. Enzymes attack purines at preferred positions, and
therefore the tautomerisation of the purine molecules
may play an important role in the replication process
and the equilibrium is strongly sensitive to the inter-
action of these molecules with their environment [5].
In an equilibrium, xanthine base exists in different
tautomeric forms, the protropic tautomerism can
8 È . Civcir / Journal of Molecular Structure (Theochem) 545 (2001) 7±15
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Fig. 1. Representation of tautomers of xanthine considered in the present work.

 È . Civcir / Journal of Molecular Structure (Theochem) 545 (2001) 7±15
P.U
occur in both rings (pyrimidine and imidazole) and
thus two type of equilibria can be observed:
keto Y enol and N(7)H Y N(9)H. Knowledge of the
geometric and electronic structure as well as the rela-
tive stability of tautomeric forms provides a basis for
understanding biological activity of oxopurine bases.
In addition, knowing how these tautomerisation ener-
gies change in different environments can give an
insight into the in¯uence of solvent effects on mole-
cular stability. Several experiments and theoretical
studies on the tautomerism of xanthine are reported
in the literature [6±11]. The highest level calculations
[9] published to date (MP2/6-31G(d) including zero
point energy correction) suggest a similar stability for
the N7(H) dioxo and N9(H) dioxo species in aqueous
solution. This conclusion supported by results of UV
and NMR measurements show that the xanthine is
found mainly in the N7(H) dioxo tautomeric form in
aqueous solution [6±8]. On the other hand, X-ray
experiments show that the sodium salt of xanthine is
found mainly in the N9(H) dioxo tautomeric form in
the solid state [12]. Calculations by the CNDO
method and ab initio method predict that the N7(H)
dioxo tautomeric forms energetically favoured over
N9(H) tautomeric form in the gas phase [9±11]. To
our knowledge, there have not been reported experi-
mental data on the tautomeric preference of xanthine
in the gas phase. The ®rst aim is to provide a set of
consistent theoretical data over large variety of DNA
bases, which can hardly be obtained by experimental
studies.
As part of our continued interest in the tautomerism
of nucleic acid bases and related compounds [13±16],
we have carried out a theoretical study using the AM1
and PM3 methods for the fourteen possible tautomers
of xanthine both in the gas and aqueous phase.
Previous work on xanthine has also described the
structures and the relative stabilities for different
tautomers of the base. Most of these do not consider
the effects of the entropy on the equilibrium. Analysis
of the entropy effect allows for better understanding of
the tautomerisation process. If several tautomers exist
in comparable concentrations, the entropy contribu-
tions are important parts of relative Gibbs free ener-
gies. Because the exact equilibrium concentration
depends on the Gibbs free energies of each tautomers.
Both entropy and enthalpy should be included for a
proper comparison of the calculated and experimental
9
tautomeric stability of the bases. Experimental infor-
mation 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 tautomerisation DGa,b(T ) can be estimated at the
de®ned temperature T.
The present work is directly related to previous
studies of Sponer and Leszczynski [9] who used the
ab initio LCAO-MO method to investigate tautomer-
ism of xanthine both in the gas phase and aqueous
phase, but they have considered only eight tautomeric
forms of xanthine. The second aim of this work is,
therefore, to investigate the relative stability of this
molecule as presented in Fig. 1, using semiempirical
AM1 and PM3 methods, but also considering the
effects of entropy on the equilibrium both in the gas
and aqueous phases. We also extend the previous
theoretical works [9±11] to include tautomeric equi-
librium constants calculated from the Gibbs free ener-
gies of the xanthine. Also, there are no data available
on the prediction of the tautomeric equilibrium
constants for this molecule by means of quantum
chemical calculations in the literature.
The present paper reports the heats of formation,
entropy, Gibbs free energy, relative stability, tauto-
meric equilibrium constants, relative proton af®nities,
dipole moments and ionisation potentials for xanthine
at 298.15 K. After having predicted the relative stabi-
lity of tautomers, we have found the tautomeric equi-
librium constants with respect to the more stable
tautomer both in the gas phase and in aqueous solu-
tion.
2. Method of calculation
Theoretical calculations were carried out at the
restricted Hartree±Fock level (RHF) using AM1
[17] and PM3 [18] semiempirical SCF-MO methods
with the Mopac 7.0 program [19], implemented on an
Pentium II 300 MHz computer. In aqueous phase
calculations, the COSMO (conductor-like screening
model) solvation model [20] is used to construct a
solvent accessible surface area based on Van der
Waals radii. The relative permitivity of water was
taken to be e ˆ 78:4 and the model incorporated up
to 30 surface segments per atom, then we set the
10 È . Civcir / Journal of Molecular Structure (Theochem) 545 (2001) 7±15
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Table 1
The AM1 calculated thermodynamic properties for the tautomers of xanthine in the gas phase …e ˆ 1†

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

X3,9,10 16.26 90.97 210.86 23.50 9.09 8.72

X1,7,10 3.68 87.77 222.49 11.87 9.09 0.87
X1,9,10 2.57 87.79 223.60 10.76 8.96 5.56
X3,7,10 9.10 89.01 217.25 17.11 9.19 3.02
X9,10,11 11.67 88.27 214.65 19.71 9.35 5.42
X1,10,11 24.18 88.54 22.22 32.14 9.24 4.91
X3,10,11 20.39 87.94 25.83 28.53 9.19 3.36
X7,10,11 19.86 92.36 27.68 26.68 9.43 5.88
X1,3,7 28.32 87.35 234.36 0.00 9.34 3.97
X1,3,9 24.11 87.98 230.34 4.02 9.27 6.64
X1,9,11 7.79 88.28 218.53 15.83 8.91 5.03
X1,7,11 19.73 88.48 26.65 27.71 8.91 9.41
X3,9,11 9.77 88.35 216.57 17.79 9.25 8.05
X3,7,11 13.76 90.60 213.25 21.11 9.27 8.11
a
From DGf ˆ DHf2TDS.

parameters NPPA to 1082 and NSPA to 30. Initial
geometry estimates of the all structures were obtained
from a molecular mechanics calculation (CS Chem
Of®ce) [21], and were followed by full optimisation
of all geometrical variables. All structures were opti-
mised to a gradient norm of ,0.2 in the gas phase and
0.1±2 in the aqueous phase, using the eigenvector
method (EF) at PRECISE level. In order to calculate
thermodynamic properties (DHf, DS) of the tautomers,
the gradient norm is again reduced a value very close
to zero. Entropy term is obtained from FORCE calcu-
lations for all the possible tautomers and the Gibbs
free energies of the tautomerisation (DGf) at 298.15 K
were predicted by adding the enthalpic (DHf) and
entropic (TDS) terms.
3. Results and discussion
3.1. Relative stability
Xanthine (X) is capable of existing in the fourteen
tautomeric forms which are given in Fig. 1. To name a
given tautomer we have used the following notations:
Xi, j,k where i, j and k stand for the number of the
nitrogen or oxygen atoms to which the hydrogens are
attached. The AM1 and PM3 calculated relative stabi-
lity energies, enthalpies, entropies and Gibbs free
energies, dipole moments and ionisation potentials
for the tautomers of xanthine are given in Tables 1±
4. From Table 1, it can be inferred that xanthine can be
found in the gas phase as a mixture of two predomi-
nant species: the two diketo tautomers X1,3,7 and
X1,3,9. According to the present results, the tautomer
X1,3,7, i.e. a diketo form containing a hydrogen
attached to N7 of imidazolic ring, is 4.02 kcal mol 21
more stable than X1,3,9. The third most stable tauto-
mer is the X1,9,10, which is 10.76 kcal mol 21 above
the X1,3,7. According to Mezey et al. [22,23] a rela-
tive energy of 10 kcal mol 21 is a reasonable limit for
the existence of the stable species and hence the two
diketo tautomers is predicted to be detectable in the
gas phase, the other tautomers would possess no
signi®cant concentrations at room temperature.
Tautomerism between six-membered ring nitrogens,
N1 and N3, clearly favours the N1±H form, the only
exception is presented by the dihydroxy tautomers
X1,10,11 and X3,10,11. The relative free energy
difference between tautomers X1,9,10 and X3,9,10
amounts to around 13 kcal mol 21. This is probably
due to increased repulsion between the hydrogen
atoms bound N3 and N9 in this latter form. The
presented gas phase result is also in reasonable agree-
ment with previous semiempirical results determined
at the CNDO method [11] and also ab initio results
determined at the HF/6-31G level [9,10]. However,
there is no experimental data for the tautomeric
preference of xanthine in the gas phase, preventing
 È . Civcir / Journal of Molecular Structure (Theochem) 545 (2001) 7±15
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Table 2
The AM1 calculated thermodynamic properties for the tautomers of xanthine in aqueous phase …e ˆ 78:4†

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

X3,9,10 221.25 88.83 247.73 17.31 9.37 19.64

X1,7,10 226.75 87.83 252.94 12.10 9.34 1.32
X1,9,10 227.75 87.95 253.97 11.07 9.26 9.58
X3,7,10 222.99 88.72 249.44 15.60 9.44 6.05
X9,10,11 215.50 88.81 241.98 23.06 9.48 8.34
X1,10,11 27.14 89.03 233.68 31.36 9.43 9.89
X3,10,11 28.10 88.46 234.66 30.38 9.46 6.24
X7,10,11 213.94 88.86 240.43 24.61 9.59 9.70
X1,3,7 238.93 87.56 265.04 0.00 9.39 6.36
X1,3,9 238.33 87.57 264.44 0.60 9.32 11.48
X1,9,11 224.15 87.92 250.36 14.68 9.24 9.14
X1,7,11 222.80 88.18 249.09 15.95 9.30 17.51
X3,9,11 225.04 87.70 251.19 13.85 9.37 13.63
X3,7,11 223.58 88.11 249.85 15.19 9.47 14.64
a
From DGf ˆ DHf2TDS.

any comparison with the calculated values could be
not made.
The effect of the polar environment was estimated
by the COSMO solvation model. These calculations
did not change the gas phase stability order of the
tautomers. The most important effect of COSMO
solvation model is a signi®cant lowering of the energy
gap between the two most stable diketo tautomers.
However, the Gibbs free energy differences between
X1,3,7 and X1,3,9 decreased from 4.02 kcal mol 21 in
the gas phase to 0.60 kcal mol 21 in aqueous phase (cf.
Tables 1 and 2). The stability of the two diketo tauto-
mers becomes more similar upon solvation and the
changes are moderate. Only the diketo tautomers
X1,3,7 and X1,3,9 can also exist in appreciable
amounts at room temperature while the other tauto-
mers can not exist in aqueous solution. The diketo
forms (X1,3,7 and X1,3,9) would have a clear

Table 3
The PM3 calculated thermodynamic properties for the tautomers of xanthine in the gas phase …e ˆ 1†

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

X3,9,10 222.05 93.07 249.80 26.78 9.51 9.87

X1,7,10 238.52 89.55 265.22 11.36 9.08 1.59
X1,9,10 239.27 89.61 265.99 10.59 9.02 5.31
X3,7,10 230.02 91.46 257.29 19.10 9.34 4.73
X9,10,11 234.54 90.20 261.43 15.15 9.36 4.96
X1,10,11 216.40 90.62 243.42 33.16 9.28 5.47
X3,10,11 223.72 89.73 250.47 26.11 9.30 3.60
X7,10,11 226.70 91.09 253.86 22.72 9.30 5.82
X1,3,7 249.97 89.25 276.58 0.00 9.22 4.09
X1,3,9 243.04 92.87 270.73 5.85 9.28 6.68
X1,9,11 232.00 90.21 258.90 17.68 8.74 4.64
X1,7,11 223.01 89.94 249.83 26.75 8.96 8.14
X3,9,11 232.11 91.43 259.37 17.21 9.21 8.07
X3,7,11 231.28 91.43 258.54 18.04 9.12 8.20
a
From DGf ˆ DHf2TDS.
12 È . Civcir / Journal of Molecular Structure (Theochem) 545 (2001) 7±15
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Table 4
The PM3 calculated thermodynamic properties for the tautomers of xanthine in aqueous phase …e ˆ 78:4†

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

X3,9,10 268.11 91.00 295.24 14.57 9.26 20.97

X1,7,10 272.95 88.95 299.47 10.34 9.32 3.06
X1,9,10 270.6 89.12 297.17 12.64 9.25 9.62
X3,7,10 270.91 90.05 297.76 12.05 9.31 9.47
X9,10,11 263.02 89.60 289.73 20.08 9.50 7.86
X1,10,11 252.21 90.02 279.05 30.76 9.52 11.70
X3,10,11 256.03 89.57 282.74 27.07 9.51 6.75
X7,10,11 264.62 89.23 291.22 18.59 9.56 10.60
X1,3,7 283.67 87.68 2109.81 0.00 9.20 6.73
X1,3,9 280.52 87.99 2106.75 3.06 9.15 12.25
X1,9,11 267.40 88.85 293.89 15.92 9.17 8.96
X1,7,11 269.10 89.62 295.82 13.99 9.25 19.46
X3,9,11 270.84 88.79 297.31 12.50 9.24 14.38
X3,7,11 273.09 88.71 299.54 10.27 9.31 15.16
a
From DGf ˆ DHf2TDS.

predominance over the dienol and enol forms in both
phases, most probably because of electronic
stabilisation and thermal corrections. As we can see
from Tables 1±4, the calculated entropy values are
generally small (ca.87±93 cal/mol). These results
indicate that the value of the entropy effect on the
Gibbs free energy is very small and the entropy
term and TDS value can be neglected for the tauto-
meric equilibria of xanthine. Thus, the enthalpic term
is dominant in the determination of the equilibrium
constant.
Experimental evidence clearly indicates that
xanthine exist as the N7(H) diketo species in aqueous
solution [6±8]. Ultraviolet spectra reported by Cava-
lier and co-workers [7], and Ogston [8], suggest that
the diketo tautomers are predominant in aqueous solu-
tion. NMR measurements reported by Lichtenberg
and co-workers [6], which clearly indicates that the
N7(H) diketo tautomer is most stable than N9(H)
tautomer in dimethyl sulphoxide and deuterium
oxide. This result is also in reasonable agreement
with previous ab initio calculation result determined
at the HF/6-31G level in aqueous solution by Sponer
and Leszczynski [9].
The PM3 calculations give the same order of tauto-
mer stability for the xanthine. As previously noted, the
AM1 method performs better than PM3 method in
six-membered nitrogen heterocycles [24].
3.2. Dipole moments and ionisation potentials
The calculated dipole moments and the ®rst ioni-
sation potentials of xanthine tautomers are also
listed in Tables 1±4. The calculated dipole moments
for the most stable tautomers of xanthine, X1,3,7
and X1,3,9 are 3.97 and 6.64 debye, respectively
(cf. Table 1). There is no experimental dipole
moment for xanthine, because of the solubility in
non-polar solution. Dipole moment measurements
for distinguishing between different tautomeric
forms, in particular, between the N7±H and N9±H
tautomer are dif®cult by the physicochemical tech-
niques. However, in order to make possible deter-
mination of the dipole moments of the xanthine
derivatives, it is necessary to introduce a solubilis-
ing substituent (the decylthio-group) at C-8 posi-
tion. In the reasonable assumption that the
introduction of the same group at the same position
in all compounds will not affect the sequence of the
physical properties of the various substance. The
results obtained for derivatives of 8-decylthiox-
anthine [11] are in good agreement with our results
calculated by AM1 and PM3 method. Dipole
moments determined by semiempirical CNDO
method for the tautomers X1,3,7 and X1,3,9 are
4.0 and 7.9 debye, respectively. However, our
calculated dipole moments are quite close to the
 È . Civcir / Journal of Molecular Structure (Theochem) 545 (2001) 7±15
P.U 13

Table 5 the stability of nitrogen atom lone-pair orbitals rela-

The AM1and PM3 predicted tautomeric equilibrium constants with tive to the manifold of p-type orbitals [27]. Calculated
respect to the most stable tautomers of xanthine in the gas phase
…e ˆ 1† and in aqueous solution …e ˆ 78:4† at 298.15 K
dipole moments by PPP method reported by Pullman
and Pullman [11] are 8.8 and 8.7 eV for the most
Tautomeric equilibrium pKT a (e ˆ 1) pKT a (e ˆ 78.4) stable diketo tautomers. Experimental ionisation
potential for xanthine is closer to our calculated
AM1 PM3 AM1 PM3
AM1 and PM3 values, and in better agreement than
X1,3,7±X3,9,10 17.23 19.63 12.69 10.68 those of the CNDO and PPP values. As previously
X1,3,7±X1,7,10 8.70 8.33 8.87 7.58 noted, the ionisation potentials are systematically
X1,3,7±X1,9,10 7.89 7.76 8.12 9.27 overestimated AM1 as well as MNDO by ca. 0.5 eV
X1,3,7±X3,7,10 12.54 14.14 11.44 8.83
X1,3,7±X9,10,11 14.45 11.11 16.91 14.72
for p-type orbitals [28]. Results obtained by the PM3
X1,3,7±X1,10,11 23.56 24.31 22.99 22.55 method are not too different from those obtained by
X1,3,7±X3,10,11 20.92 19.14 22.27 19.85 AM1. However, the PM3 method yields somewhat
X1,3,7±X7,10,11 19.56 16.66 18.04 13.63 lower values for ionisation from pyridine-like lone
X1,3,7±X1,3,9 2.95 4.29 0.44 2.24 pairs and, thus, in better agreement with experimental
X1,3,7±X1,9,11 11.61 12.96 10.76 11.67
X1,3,7±X1,7,11 20.31 19.61 11.69 10.26
values than AM1.
X1,3,7±X3,9,11 13.04 12.62 10.15 9.16
X1,3,7±X3,7,11 15.48 13.23 11.14 7.53 3.3. Tautomeric equilibrium constants
a
pKT ˆ 2log KT.
The AM1 and PM3 calculated tautomeric equili-
brium constants with respect to the most stable tauto-
results determined from ab initio calculations mers of xanthine both in the gas and in aqueous
[9,25], which are 3.97 and 6.87 debye for the most solution are listed in Table 5. The pKT values of the
stable diketo tautomers. studied molecules were calculated by means of the
As we can see from Tables 1±4, the calculated following equation
dipole moments are substantially higher in moving
dDG
from the gas phase …e ˆ 1† to solution …e ˆ 78:4† pKT ˆ
2:303RT
and the dipole moments are sensitive to the polarity
of the medium. The calculated dipole moments are Calculated values for the equilibrium between the
substantially higher in a medium of high relative tautomers X1,3,7 and X1,3,9 of xanthine in the gas
permitivity, mainly due to major charge redistribution phase show that the diketo form N7(H) is more domi-
in the molecule, and also by changes in the distances nant than the diketo form N9(H), with a pKT value of
between the charge separations. The magnitude of the 2.95. The calculation for the aqueous phase of
in¯uence of the solvent reaction ®eld on electronic xanthine, X1,3,7 Y H1,3,9 tautomeric equilibrium
structure is different in different tautomers. This again suggest that the diketo form NH(7) with hydro-
may also explain the great variation of the calculated gen atoms at N1, N3 and N7 is more dominant tauto-
dipole moments of the tautomers. mer than the diketo form with hydrogen atoms at N1,
The ionisation potentials for the tautomers of N3 and N9 with a pKT value of 0.44. Calculations also
xanthine were obtained from the energies of the high- show that two diketo forms of xanthine are most
est occupied molecular orbitals using Koopmans' stable than all other forms in aqueous solution. Gener-
theorem. The ®rst vertical ionisation potentials for ally, 1H tautomers are more stable than the corre-
the most stable tautomers of xanthine are in good sponding 3H tautomers, the only exception is
agreement with the experimental value. The experi- presented by the dihydroxy tautomers in the gas
mental ionisation potential for xanthine is phase, which implies that the proton at N1 is more
9.30 ^ 0.2 eV [26] and the CNDO calculations values acidic than that of N3. There is no experimental
reported by Pullman and Pullman [11] are 10.8 and evidence for the equilibrium constants of the investi-
10.4 eV for the more stable diketo tautomers. As gated compound, preventing any comparison with the
previously noted, CNDO calculations overestimate calculated pKT values could be not made.
14 È . Civcir / Journal of Molecular Structure (Theochem) 545 (2001) 7±15
P.U

Table 6 proton af®nities for xanthine in order to put in a

The AM1 and PM3 calculated relative proton af®nities (d PA) with more reliable basis the qualitative results inferred
respect to the most stable tautomer of xanthine (in kcal mol 21)
from the AM1 and PM3 calculations. Xanthine may
Tautomeric equilibrium d PA a …e ˆ 1† d PA a …e ˆ 78:4† be protonated at different nitrogen atoms or oxygen
atom, but experimentally it is not possible to discri-
AM1 PM3 AM1 PM3
minate between the various possibilities. The proto-
X3,9,10 24.58 27.92 17.68 15.56 nation energetics depend on the chemical
X1,7,10 12.00 11.45 12.18 10.72 surrounding, and so theoretical investigations repre-
X1,9,10 10.89 10.70 11.18 13.07 sent a practicable way for obtaining information about
X3,7,10 17.42 19.95 15.94 12.76 the energetics of proton attachment to the different
X9,10,11 19.99 15.43 23.43 20.65
X1,10,11 32.50 33.57 31.79 31.46
sites of xanthine and to establish which of these are
X3,10,11 28.71 26.25 30.83 27.64 favoured. The predicted relative proton af®nities are
X7,10,11 28.18 23.27 24.99 19.05 collected in Table 6 for AM1 and PM3 methods in
X1,3,9 4.21 6.93 0.60 3.15 both phases. The relative proton af®nities (d PA),
X1,9,11 16.11 17.97 14.78 16.27 which is the difference between proton af®nities of
X1,7,11 28.05 26.96 16.13 14.57
X3,9,11 18.09 17.86 13.89 12.83
molecules A and B, can be calculated as
X3,7,11 22.08 18.69 15.35 10.58
dPA ˆ dDH ˆ PA…B† 2 PA…A† or dPA ˆ dDH
a
d PA ˆ d DH ˆ DH(B) 2 DH(A).
ˆ DH…B† 2 DH…A†
Furthermore, comparison of the gas phase equili- where DH(B) and DH(A) are the heats of formation values
brium constants with solution phase values implies of tautomers A and B taken from Tables 1±4. As we
that large changes may occur on going from gas stated before, the two-diketo forms X1,3,7 and X1,3,9
phase to solution. This can be explained by solvent species possess comparable relative energies. This
effects. Solvent effects have been ascribed to two implies that the ®rst protonation of xanthine is imidazole
major components: electrostatic solvent±solute inter- ring, i.e. the N7 (in X1,3,9) and N9 (in X1,3,7). This is in
action and hydrogen bonding. The hydrogen bonding good agreement with 15N, 1H and 13C NMR experimen-
effects can not be estimated in a quantitative manner tal results in DMSO, which show that xanthine is proto-
from the solvation model, further large-scale calcula- nated preferentially at imidazole ring [31,32].
tions. A semiquantitative estimation of the solvation Generally, 1H tautomers are more stable than the corre-
differences can be obtained from COSMO solvation sponding 3H tautomers, the only exception is presented
model. The electrostatic solvent±solute effects, by the dihydroxy tautomers in the gas phase, which
however, are readily estimated by the dielectric conti- implies that the proton at N1 is more acidic than that
nuum model [29]. Application of the COSMO solva- of N3. Thus, the second protonation site of xanthine is
tion model leads to an explanation of the change in the N1 or N3. Predicted relative proton af®nities show
order of tautomeric stability on going from gas phase that C2O is more acidic than the C6O, which shows that
to solution. However, such a treatment lacks explicit third protonation site of xanthine is C2O, then C6O. The
consideration of base±water hydrogen bonding above proton af®nity order also follows a logical trend in
effects, tautomeric equilibrium constants predicted the sense that the endocyclic nitrogens will be proto-
in water are considerably less reliable than those nated ®rst, then the oxygen, which is less basic centre.
predicted in the gas phase.

3.4. Relative proton af®nities 4. Conclusions

Since protonation of nucleic acid bases plays an Several conclusions can be made on the basis of the
important role in many biochemical (i.e. enzymatic results of the present theoretical study.
reactions, stabilisation of triplex structures) and muta-
genic process [30], we have calculated the relative 1. The results clearly indicate that xanthine both in
 È . Civcir / Journal of Molecular Structure (Theochem) 545 (2001) 7±15
P.U
the gas phase and in aqueous phase exists predo-
minantly in the two diketo forms X1,3,7 and
X1,3,9. This result is in agreement with available
experimental and theoretical studies.
2. The energy difference between X1,3,7 and X1,3,9
is predicted to be signi®cantly lowered by the polar
solvent.
3. The results presented in this paper con®rm our
earlier observations about the applicability of the
AM1 method for the quantitative prediction of rela-
tive stability of nucleic acid bases in solution.
4. AM1 also gives a good representation of the charge
distribution in molecules in terms of calculated
dipole moments.
5. The inclusion of the solvent reaction ®eld in quan-
tum-chemical theory is obligatory for accurate
results in solution.
6. The entropy effect on the Gibbs free energy of
xanthine tautomers is very small and there is little
signi®cance for the tautomeric equilibria of the
base. The enthalpic term is dominant also in the
determination of the equilibrium constant.
7. The protonation of xanthine in both phases occurs
at the imidazole nitrogens (N7 or N9), then pyri-
midine nitrogens (N1 or N3).
Acknowledgements
I would like to thank Prof. Knut Faegri and Prof.
Einar Uggered for their helpful comments and discus-
sions.
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