Tautomerism of 1-Methyl Derivatives of Uracil, Thymine, and 5-Bromouracil. Is
Tautomerism the Basis for the Mutagenicity of 5-Bromouridine?
Modesto Orozco,*,†Begon ˜a Herna ´ndez,†and F. Javier Luque*,‡
Departament de Bioquı ´mica i Biologı ´a Molecular, Facultat de Quı ´mica, UniVersitat de Barcelona,
Martı ´ i Franque `s 1, Barcelona 08028, Spain, and Departament de Fisicoquı ´mica, Facultat de Farma `cia,
UniVersitat de Barcelona, AVgda Diagonal s/n. Barcelona 08028, Spain
ReceiVed: February 4, 1998; In Final Form: April 10, 1998
The tautomerism of the N-1-methylated derivatives of uracil, thymine, and 5-bromouracil has been studied in
order to analyze its implications in the mutagenicity of 5-bromouridine. The tautomeric preference in the
gas phase was determined by means of state-of-the-art ab initio quantum mechanical calculations. The influence
of solvation in water on the tautomerism was examined by using ab initio self-consistent reaction field and
Monte Carlo free energy perturbation techniques. Finally, the effect of the DNA environment on the relative
stability between tautomers was estimated from Poisson-Boltzmann calculations. The theoretical results
indicate that there are no relevant differences in the intrinsic tautomeric preference of the three pyrimidine
bases. The canonical oxo form is the main, if not the exclusive, form in the gas phase. Indeed, neither
solvation in water nor solvation in the duplex DNA changes sensibly the relative stability between tautomers.
Therefore, our results provide a basis for ruling out the involvement of noncanonical enol tautomers as the
origin of the mutagenic properties of 5-bromouridine.
Nucleic acids are formed with five nucleic acid bases: two
purines (adenine, A; guanine, G) and three pyrimidines (thymine,
T; uracil, U; cytosine, C). The formation of specific purine-
pyrimidine Watson-Crick hydrogen bonds is responsible for
the maintenance of the genetic code (see Figure 1). The
Watson-Crick pairings A-T and G-C are strong and lead to
steps of similar size, which largely stabilizes the double helix.
However, despite the stability of canonical AT and GC steps,
other recognition patterns are possible and can even lead to
stable helical structures. Thus, nucleic acid fragments with
pairings other than Watson-Crick ones, including hydrogen
bonding between purines or between pyrimidines, have been
found.1A much more complex pattern of hydrogen bonds
appear if noncanonical tautomeric forms of the bases are
The ability of nucleic acids to accommodate noncanonical
hydrogen bonds has been related to the occurrence of spontane-
ous mutations in the DNA.1-4Furthermore, it suggests the
possibility of “expanding” the genetic code using nonstandard
bases or forcing the formation of anomalous tautomers.1,2,5,6In
fact, inspection of some nucleic acid structures such as the
transfer-RNA demonstrate that nonstandard bases can be
incorporated without large structural alterations.7,8Numerous
studies have shown the possibility of forming stable DNA
structures with nonstandard bases.1,6,9,10It has been also shown
that at least some of these nonstandard bases can be incorporated
in vivo into nucleic acids during their synthesis by the
polymerases.5,9,11On the other hand, noncanonical tautomeric
or ionized forms were supposed to be very unstable, and their
role in physiological DNA structures was assumed to be
negligible for years. However, an increasing number of results
support the importance of noncanonical tautomers or ionized
bases in the stabilization of certain nucleic acid structures.1c,2,4
Mutagenicity is associated with some of the nonstandard
nucleic acid bases, like 5-bromouracil (Br-U),1-4,12which is a
mimic of thymine. This base binds adenine in the DNA
duplexes without dramatic structural alterations.13In addition,
it binds guanine with great efficiency, and this feature has been
* Correspondence authors.
†Departament de Bioquı ´mica i Biologı ´a Molecular.
‡Departament de Farma `cia.
Figure 1. Representation of the Watson-Crick hydrogen-bonded
pairings between adenine-thymine and guanine-cytosine
J. Phys. Chem. B 1998, 102, 5228-5233
S1089-5647(98)01005-0 CCC: $15.00© 1998 American Chemical Society
Published on Web 06/11/1998
suggested to be the basis for its mutagenicity.4,12The recogni-
tion between Br-U and G was first explained assuming the
formation of the enol form of Br-U (Figure 2). Other authors
suggested that the recognition is mediated by ionization of Br-U
(Figure 2). Finally, a third possibility is the involvement of
“wobble” hydrogen bonds (Figure 2). It is worth noting that
this latter possibility does not seem to depend intrinsically on
the presence of bromine in position 5. Therefore, the mutagenic
properties of Br-U should stem from (a) a larger percentage of
enol tautomer with respect to the parent compound (uracil and
thymine) or (b) an increase in the acidity, which would facilitate
loss of the proton at N3 and formation of the 5-Br-U-‚‚‚G
In this paper we examine the possible involvement of
tautomerism as the molecular basis for the mutagenic properties
of 5-bromouridine. For this purpose, we have studied the
tautomerism of the 1-methyl derivatives of uracil, 5-bromouracil,
and thymine in the gas phase, in aqueous solution, and in the
duplex DNA. Calculations provide a complete picture of the
reactivity of the three bases and give new insights into the
reasons for the mutagenicity of haloderivatives of uracil, and
particularly of the 5-Br-derivative.
The tautomerization energies for the 1-methyl derivatives of
uracil, 5-bromouracil, and thymine in the gas phase were
determined by using high-level ab initio techniques. Three
tautomers (the oxo N3 and the two enol forms O4c and O2c;
Figure 3) were considered. In addition, the enol form O4t
(Figure 3) was also examined in the case of 5-bromouracil. Low
level ab initio calculations showed that other tautomers were
very unstable, and they were not considered in the study.
Geometries were optimized at the HF/6-311+G(d,p) level,14
and the minimum-energy nature of the stationary points was
verified by frequency analysis. Single-point energy calculations
were performed at the SCF and MP215levels using the
6-311+G(d,p) basis set. The MP4(SDTQ), QCISD, and
QCISD(T) estimates were obtained by adding the MP4-MP2,
QCISD-MP2, or QCISD(T)-MP2 energy difference determined
with the 6-31G(d) basis set16to the energy calculated at the
MP2/6-311+G(d,p) level.This technique is expected to
reproduce very closely the MP4, QCISD, or QCISD(T) results
determined with the large 6-311+G(d,p) basis set.17
determine the enthalpies and free energy differences between
tautomers at 298 K and 1 atm, zero-point energy and thermal
and entropic corrections were added to the tautomerization
energies.These terms were computed using the HF/6-
311+G(d,p) frequencies and the standard procedures in Gauss-
Solvent effects on tautomerism were typically introduced
using standard themodynamic cycles (eq 1). Absolute free
energies of solvation (∆Gsol) were determined using self-
consistent reaction field (SCRF) methods, while relative free
energies of solvation (∆∆Gsol) were determined using both
SCRF and Monte Carlo free energy perturbation techniques
The SCRF values were determined using our 6-31G(d)-
optimized version19of the Miertus, Scrocco, and Tomasi (MST)
method.20Standard cavities and van der Waals parameters were
used.19Parameters for bromine, which are not available in the
standard set, were taken from a current parametrization study
(radius ) 1.95 Å, ? ) -0.0767 kcal mol-1Å-2; REF). The
gas-phase HF/6-311+G(d,p) optimized geometries were used
in calculations. However, to examine the influence of geometry
relaxation in water on the results, the free energy of hydration
of O2c and O4c tautomers of uracil was computed using the
full optimized MST/6-31G(d) geometry. The extra contribution
to ∆Gsoldue to geometry relaxation was around 1 kcal/mol in
both cases, and the change in ∆∆Gsolwas only 0.2 kcal/mol. It
is then clear that the solvent-induced relaxation of the solute
geometry does not lead to dramatic effects and that the use of
gas-phase optimized geometries is reasonable.17,21
The complex mutation scheme used in MC-FEP calculations
is shown in Figure 4. To obtain an additional measure of the
consistency in FEP calculations, closure thermodynamic cycles
Figure 2. Proposed molecular basis of the origin of the mutagenic
properties of 5-bromouracil: ionization of the base, formation of enol
tautomers, and change in the hydrogen-bond pattern mediated by a
gas phase+ ∆GB
gas phase+ ∆∆GAfB
Tautomerism of Methyl Derivatives
J. Phys. Chem. B, Vol. 102, No. 26, 1998 5229
were also considered. Free energy changes were computed
using Zwanzig’s theory.22The solute was placed in a cubic
box (around 15 600 Å3) containing 503 TIP4P water mol-
ecules.23Periodic boundary conditions were applied in con-
junction with a residue-based 10 Å cutoff for solute-solvent
and 9 Å for solvent-solvent nonbonded interactions. Simula-
tions were performed in the isothermic-isobaric ensemble
(NPT; 1 atm, 298 K). Solute rotations and translations were
adjusted to obtain around 40% acceptance.
Mutations were performed using 21 double-wide sampling
windows, which allowed us to determie the hysteresis in the
calculations. Each window consisted of 2 × 106configurations
for equilibration and 3 × 106configurations for averaging. The
average part of each window was divided into five blocks to
estimate the standard errors in the free energy differences. Gas-
phase geometries were used in all cases, and they were not
explicitly sampled during the MC runs. Atomic charges were
determined by fitting quantum mechanical ESP charges.24The
van der Waals parameters for most atom types were taken from
the OPLS force field.25The parameters for the bromine atom
(not available in the OPLS force field) were determined by
fitting HF/6-31G(d) and classical energies for different con-
figurations of the CH3Br:OH2system.26
Poisson-Boltzmann calculations27were used to obtain a
qualitative measure of the work necessary to generate a
thymidine, uridine, or 5-bromouridine in a piece of standard
DNA compared with the work necessary to generate the same
molecule in water. The three bases were considered in their
oxo (N3) and enol (O4c) forms. The oxo forms were paired
with adenine, while the enol forms were paired with guanine.
In all the cases the same structure (a decamer of standard
B-DNA28with sequence d(A4XA5), with X ) A or G) was used.
The same set of charges and van der Waals parameters used in
MC calculations was considered here to make calculations more
comparable. The dielectric response of DNA and water was
defined by dielectric constants of 2 and 80 relative to that of
the gas phase. This implies that a dielectric response of 2 is
also assumed for the nucleic acid bases. The solution of
Poisson-Boltzmann equations was performed using the lineal
approach,27assuming an ionic strength in the solvent of 1 M
for DNA simulations. To reduce problems due to the limited
size of the grid, we used a three-step focusing process, leading
to a final grid density of around 34 points × Å3. The same
grid is used for the aqueous and DNA calculations to reduce
numerical errors in calculations.
Gas-phase calculations were performed with Gaussian-94.18
SCRF calculations were carried out with a locally modified
version of MonsterGauss29and HONDO-830programs. ESP
charges were determined using MOPETE/MOPFIT.31MC-FEP
calculations were done using BOSS-3.4.32Finally, Poisson-
Boltzmann calculations were carried out using DelPhi.27All
calculations were performed on the SP2 computer of the Centre
de Supercomputacio ´ de Catalunya (CESCA), as well as on
workstations in our laboratory.
Results and Discussion
The HF/6-311+G(d,p) optimized geometries of the most
stable tautomers of 1-methyluracil (U), 1-methylthymine (T),
and 1-methy-l,5-bromouracil (Br-U) are shown in Figure 5. The
structural parameters for U and T are found to be almost
identical, and they are also very similar to those of Br-U. This
suggests that the presence of the methyl and bromine substituents
in position 5 does not change sensibly the electronic distribution
in the pyrimidine ring, which allows us to expect small
differences in the chemical properties of the three bases.
The tautomerization energies, enthalpies, and free energies
for U, T, and Br-U are given in Table 1. The results show
clearly that the oxo form is the most stable tautomer in the gas
phase in all cases. The difference in stability with regard to
the other tautomers is so large as to guarantee that the oxo
tautomer N3 is the only important tautomer in the gas phase,
as previously suggested both experimentally33and theoretically34
for similar systems. It is also clear that entropic effects play a
minor role in the oxo-enol tautomerism. The results also reveal
the importance of electron correlation effects for a quantitative
description of the oxo-enol tautomerism.17,21,34Thus, electron
correlation stabilizes by 2-3 kcal/mol the enol forms for the
three molecules. This finding mimics the trends found in other
systems (see, for example, ref 17a). However, comparison of
Figure 3. Representation of the oxo and enol tautomers considered in the study.
Figure 4. Representation of the mutations performed in MC-FEP
5230 J. Phys. Chem. B, Vol. 102, No. 26, 1998
Orozco et al.
MP2, MP3 (data not shown), MP4, QCISD, and QCISD(T)
results demonstrate that the energy values are reasonably well
converged even at the MP2 level, in disagreement with the
situation found for other tautomeric processes, where large levels
of correlation are necessary to obtain converged results (see,
for example, ref 17c).
The tautomerization free energies in the gas phase are similar
for the three molecules and are also similar to the results for
related molecules.34In all cases the tautomer O4c is the most
stable enol form, whereas the species O2c is disfavored by 6-7
kcal/mol. It is worth noting that theoretical estimates of
tautomerization enthalpy are clearly smaller than those suggested
by calorimetric measures,33cwhich is probably due to the very
large range of error of such experimental estimates.33c,34bThe
substituents at position 5 of U leads to changes in the free energy
of tautomerization of around 1 kcal/mol or even less. In
particular, attachment of bromine and methyl reduces the
stability of tautomer O4c by 0.6 and 1.3 kcal/mol and increases
that of O2c by 1.2 and 0.3 kcal/mol. Therefore, the intrinsic
tautomeric preferences of U and T are not greatly affected upon
inclusion of bromine at position 5. Indeed, the changes in
relative stability due to the substituent does not change at all
the preference of the oxo form in the gas phase.
The solvent effect on the tautomerism of U, T, and Br-U
was analyzed by means of the use of standard thermodynamic
cycles (see Methods) from SCRF-MST and MC-FEP calcula-
tions. The MC-FEP estimates of the relative hydration free
energies were determined from the mutations shown in Figure
4. The results in Table 2 demonstrate the statistical goodness
of the simulations, as noted in the hysteresis and standard errors
Figure 5. Optimized structures of the different species considered in this study. Bond lengths are in angstroms, and angles are in degrees.
TABLE 1: Themodynamic Quantities (kcal/mol) for Selected Tautomers of 1-Methyl Derivatives of Uracil, 5-Bromouracil, and
Thymine in the Gas Phasea
aCalculations were performed at the ab initio level using a 6-311+G(d,p) basis set and the HF/6-311+G(d,p) optimized geometry (see Methods).
All the values given relative to the N3 tautomer.
Tautomerism of Methyl Derivatives
J. Phys. Chem. B, Vol. 102, No. 26, 1998 5231
in the free energy values (less than 0.2 kcal/mol in all cases).
An additional test of the quality of the simulations is provided
by closure thermodynamic cycles (Figure 4). Thus, the cycle
Br-U(O2c) f U(O2c) f U(N3) f Br-U(N3) f Br-U(O2c) is
closed with an error of 0.3 kcal/mol, and the cycle U(N3) f
Br-U(N3) f Br-U(O4c) f U(O4c) f U(N3) is closed with an
error of 0.4 kcal/mol. Finally, the most challenging cycle
U(O4c) f Br-U(O4c) f Br-U(N3) f Br-U(O2c) f U(O2c)
f U(N3) f U(O 4c) is closed with a negligible error of 0.1
The values of ∆∆Ghydare given in Table 3. There is general
agreement between SCRF-MST and MC-FEP results, which is
remarkable considering the methodological differences between
the two techniques. Thus, the rms and the average deviation
between MST and MC-FEP estimates of ∆∆Ghydare only 1.1
and 1.0 kcal/mol, respectively. Enol forms are better hydrated
than the corresponding oxo tautomers, even though the differ-
ences are not very large. The tautomer O2c is more stabilized
by water than O4c by 1-2 kcal/mol in all cases. The presence
of substituents (methyl, bromine) at position 5 does not affect
greatly the relative hydration of the different tautomers, the
largest changes being around 1 kcal/mol.
The free energy of tautomerization in water was determined
by adding the average value of MST-SCRF and MC-FEP
relative free energies of hydration to the differences in free
energy of tautomerization in the gas phase (computed at the
MP4 level). The results in Table 3 indicate that solvation does
not change sensibly the gas-phase preference between tautomers
and that the oxo form is the only relevant species in water. Our
results agree qualitatively well with the experimental evidence,
which show that the enol forms of uracil derivatives are very
minor in water.35However, the free energy differences found
here are larger (in absolute values) than the values derived from
acidity measurements35b,35cor Hammet constants.35b
The results in Table 3 also indicate that the presence of
substituents at position 5 does not increase the stability of enol
forms in water. On the contrary, the free energy difference
between oxo and enol forms in water is larger for Br-U and T
than for U. This finding does not support early suggestions
derived from basicity measurements, which suggested that enol
tautomers of Br-U were around 1-2 kcal/mol more stable than
those of U,35bbut agree well with recent experiments that show
no evidence of enol species for the brominated derivates of
uridine.4,13In summary, the results point out that the mutagenic
properties of Br-U do not seem to obey to a greater population
of enol forms in aqueous solution.
The influence of the DNA environment on the tautomerism
of U, T, and Br-U was explored using Poisson-Boltzmann
calculations, which allowed us to estimate the difference in
solvation free energy between water and the DNA.36Only the
oxo N3 and O4c enol tautomers were considered, and the DNA
fragments d(A10) and d(A5GA4) were used for the oxo and enol
tautomers, respectively (see Methods).
The mutation of the oxo tautomer N3 of U to the correspond-
ing species of either T or Br-U leads to a free energy change of
1.1 kcal/mol. These results show that the effect of the duplex
DNA environment on the three molecules is very similar and
suggest that a piece of DNA of sequence d(A10) should be
equally stable irrespective of the base (U, T, or 5-Br-U)
hydrogen-bonded to the sixth adenine. The corresponding
mutations for the enol tautomer O4c gives free energy differ-
ences of 1.0 kcal/mol in d(A5GA4) (U f T) and 1.1 kcal/mol
in d(A5GA4) (U f Br-U). This indicates that the shift in the
stability of the enol tautomer on going from water to the duplex
DNA is similar for the three compounds and allows us to
envisage that a fragment of duplex DNA made with the enol
forms of thymidine, uridine, or 5-bromouridine paired with
guanine would have similar stability. Overall, the results in
Table 4 point out that attachment of methyl or bromine at
position 5 of uracil has a small effect on the ratio between oxo
and enol tautomers in the DNA environment.
In summary, the results indicate that the enol tautomers of
uracil, thymine, and 5-bromouracil are intrinsically unstable in
the gas phase and that neither solvation in water nor solvation
in the duplex DNA environment justifies large changes in the
ordering of stability between tautomers. Moreover, the results
point out that the differences in the tautomeric preference of
uracil, thymine, and 5-bromouracil are small.
The whole of the results strongly argues against the hypothesis
that the existence of enol tautomers of 5-bromouracil is the
origin of its mutagenic properties.2This finding confirms recent
results from NMR analysis, which were not able to indicate
TABLE 2: Differences in Free Energy of Hydration
(kcal/mol) between Tautomers of 1-Methyl Derivatives of
Uracil, 5-Bromouracil, and Thyminea
U(N3) f U(O4c)
U(N3) f U(O2c)
Br-U(N3) f Br-U(O4c)
Br-U(N3) f Br-U(O2c)
Br-U(N3) f Br-U(O4t)
U(N3) f Br-U(N3)
U(O4c) f Br-U(O4c)
U(O2c) f Br-U(O2c)
U(N3) f T(N3)
U(O4c) f T(O4c)
aMutations were performed between tautomers of the same com-
pound, as well as between different compounds in the same tautomeric
form. SE: statistical error in the free energy difference. HST: hysteresis
in the free energy difference.
TABLE 3: Absolute (∆Ghyd) and Relative (∆∆Ghyd) Free
Energy of Hydration and Tautomerization Free Energies
(∆Gt) in Water (kcal/mol) of Tautomers of 1-Methyl
Derivatives of Uracil, 5-Bromouracil, and Thyminea
aSCRF calculations were performed at the MST/6-31G(d) level. The
∆∆Ghydvalues determined from MC-FEP results in Table 2 are given
in italics. The ∆Gtvalues were computed by adding the MP4 results
in the gas phase to the average solvent effect determined from MC-
FEP and MST/6-31G(d) calculations.
TABLE 4: Free Energy Changes (kcal/mol) for Replace-
ment of the Oxo and Enol Tautomers of 1-Methyluracil by
the Corresponding Forms of 1-Methylthymine and
1-Methyl-5-bromouracil in Two DNA Sequences
U(N3) f T(N3)
U(N3) f Br-U(N3)
U(O4c) f T(O4c)
U(O4c) f Br-U(O4c)
5232 J. Phys. Chem. B, Vol. 102, No. 26, 1998
Orozco et al.
the presence of the enol tautomer for the 5-bromoracil bound
to guanine.4On the contrary, our results indirectly favor the
alternative hypothesis, which implies that the formation of
complexes between guanidine and 5-bromouridine occurs after
ionization of this latter compound.3,4At this point, it is worth
noting that the experimental pKa(in water) of 5-bromouracil is
suggested to be around 8,13b,35bi.e., around 1.5 units below the
pKa of uracil.1,13a,35a
The larger acidity of 5-bromoracil
compared with that of the unsubstituted compound is also
observed in the gas phase, since calculations (data not shown)
at the MP4/6-311+G(d,p) level indicate that the ionization of
5-bromouracil is 7 kcal/mol easier than that of uracil, and
preliminary PB calculations suggest that the same situation
might occur in the DNA.37The preceding discussion provides
a basis to suggest that the mutagenic properties of 5-bromo-
uridine stems from its ability to lose a proton at N3 rather than
from its tendency to form enol tautomers.
Acknowledgment. We are indebted to Dr. Ramo ´n Eritja for
many helpful discussions. We thank Professor J. Tomasi for a
copy of his MonsterGauss code, which was modified by us to
perform SCRF-MST calculations, and for providing a copy of
the HONDO-8 program modified by the Pisa group to include
the MST model. We also thank Professor W. L. Jorgensen for
a copy of BOSS 3.4. This work has been supported by the
Spanish DGICYT (PB96-1005) and by the Centre de Super-
comptacio ´ de Catalunya (CESCA, Mol. Recog. Project-97).
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(36) Poisson Boltzman calculations give only the electrostatic contribu-
tion to the solvation free energy. PB is probably less precise than MC-FEP
or MST calculations owing to the use of a rigid geometry and a rigid and
oversimplified expression of the solute charge distribution. It is not simple
to compare PB with MC-FEP or MST calculations. But as a reference, the
electrostatic contribution to the free energy of solvation computed from
PB calculations for the N3 forms considered here ranges from -22 to -24
kcal/mol (? ) 1 for solute) and from -12 to -13 kcal/mol (? ) 2 for the
solute). The equivalent MST estimates range between -15 and -17 kcal/
(37) Orozco, M.; Hernandez, B.; Luque, F. J. To be published.
Tautomerism of Methyl Derivatives
J. Phys. Chem. B, Vol. 102, No. 26, 1998 5233