Uracil and Thymine Reactivity in the Gas Phase: The SN2
Reaction and Implications for Electron Delocalization in
Anna Zhachkina and Jeehiun K. Lee*
Department of Chemistry and Chemical Biology, Rutgers, The State UniVersity of New Jersey,
New Brunswick, New Jersey 08901
Received August 11, 2009; E-mail: email@example.com
Abstract: The gas-phase substitution reactions of methyl chloride and 1,3-dimethyluracil (at the N1-CH3)
are examined computationally and experimentally. It is found that, although hydrochloric acid and
3-methyluracil are similar in acidity, the leaving group abilities of chloride and N1-deprotonated 3-methyluracil
are not: chloride is a slightly better leaving group. The reason for this difference is most likely related to the
electron delocalization in the N1-deprotonated 3-methyluracil anion, which we explore further herein. The
leaving group ability of the N1-deprotonated 3-methyluracil anion relative to the N1-deprotonated
3-methylthymine anion is also examined in the context of an enzymatic reaction that cleaves uracil but not
thymine from DNA.
Uracil and thymine are pyrimidine nucleobases that differ in
structure by only a methyl group at C5 (1a and 3a). Uracil
naturally occurs in RNA, while thymine is its DNA counterpart.
Although uracil and thymine are very similar in structure,
the presence of uracil in DNA is problematic.1-4Uracil can
arise in DNA from cytosine deamination, which is mutagenic;
uracil can also be misincorporated into DNA, leading to
cytotoxic uracil·adenine base pairs.1,2,4-6Uracil is removed
from the genome by the enzyme uracil DNA glycosylase
The mechanism of UDG has been shown to involve N1-
deprotonated uracil as the leaving group (LG).5,9,14-30Depro-
tonated uracil as a leaving group seems somewhat surprising
and begs the question: How good of a leaving group is
deprotonated uracil? The N1-H pKa in water is 9.8, which
would indicate a fair or mediocre leaving group ability. In the
enzyme, uracil has a depressed pKaof 6.4.19,20
Examining properties in the gas phase is useful for elucidating
inherent reactivity in the absence of solvent.3,17,31-40In previous
studies, we calculated and measured the gas-phase acidity of
uracil and found it to be as acidic as hydrochloric acid, indicating
that in the gas phase, deprotonated uracil might be, relatively
speaking, a good LG.3Furthermore, because enzyme environ-
ments are sometimes quite nonpolar, reactivity in the gas phase,
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Published on Web 11/24/2009
10.1021/ja906814d 2009 American Chemical Society
18376 9 J. AM. CHEM. SOC. 2009, 131, 18376–18385
a kind of “ultimate” nonpolar medium, can yield insight into
biological reactivity.3,17,35-43Uracil is particularly intriguing,
we found, because the acidities at the N1 and N3 sites are very
different in the gas phase, but coalesce in aqueous solution.3,17
Having established that uracil (1a) is quite acidic in the gas
phase, we now examine the leaving group ability of the
conjugate base, N1-deprotonated uracil (2a), in a substitution
reaction. Because uracil is as acidic as HCl, is deprotonated
uracil as good of a leaving group as chloride in the gas phase?
We are also interested in comparing the leaving group abilities
of deprotonated uracil versus deprotonated thymine. The oc-
currence of uracil in the human genome is typically one uracil
per >107normal DNA base pairs. The ability of the UDG
enzyme to find and excise the few uracils present while leaving
the structurally similar thymine untouched is of interest.44A
recent study indicates that the uracil versus thymine discrimina-
tion could be due in part to base-pair dynamics.4We wanted to
probe the possibility that another contribution to the favorable
excision of uracil over thymine could be due to the relative
leaving group abilities of the corresponding conjugate bases
(deprotonated at N1). If N1-deprotonated thymine (4a) is a
poorer leaving group than N1-deprotonated uracil (2a), this
could presumably contribute to the favorable excision of uracil
over thymine.3,17,37,40Leaving group ability has been implicated
SN2 reactions in the gas phase have been studied for more
than two decades. They follow the “classic” gas-phase double-
well potential energy surface, where an initial ion-molecule
complex is formed that can either dissociate back to reactants
or react to products.34,46- 60Previously measured second-order
reaction rate constants and efficiencies for SN2 reactions of
methyl chloride with different anionic nucleophiles are shown
in Table 1 (where reaction efficiency is defined as the ratio of
the observed rate constant to the estimated collision rate constant
calculated by parametrized trajectory calculations).34,61,62
Model Systems for Gas-Phase Study. The excision of uracil
from DNA involves nucleophilic attack of the C1′ of ribose
(Figure 1). Kinetic isotope effects point to a “dissociative SN2”
reaction mechanism (DN*AN).5,16,25Our interest is in testing
the leaving group ability of deprotonated uracil in a substitution
reaction. The simplest model would be to examine reactivity at
the N1-CH3group of 1-methyluracil (1c). However, experi-
ments with 1-methyluracil are limited by the acidity of the
N3-H, which has been measured to be 348 ( 3 kcal mol-1in
the gas phase.3,17Therefore, any nucleophile with a proton
affinity (PA) of 348 or greater will likely deprotonate the N3-H.
Proton transfers are enthalpically generally barrierless (∆Hq)
0) and will, if exothermic, compete with the SN2 reaction.33
Using nucleophiles with PAs less than 348 kcal mol-1would
be too limiting; for substitution reactions with CH3Cl, anions
with a PA at or below 348 kcal mol-1yield very low efficiencies
(Table 1). Therefore, to allow the use of more basic nucleophiles,
we chose to examine the 1,3-dimethyl substrates (1d, 3d)
wherein the methyl group at N3 acts as a sort of “protecting
Acidity Studies. With the chosen model systems, our leaving
groups are no longer the deprotonated uracil and deprotonated
thymine, but rather the 3-methyl derivatives (Figure 2).
As a starting point toward ascertaining whether N1-depro-
tonated-3-methyluracil is a better leaving group than N1-
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Table 1. Second-Order Reaction Rate Constants and Efficiencies
for Reactions of Nucleophiles of Varying Proton Affinity with
PA, kcal mol-1
J. AM. CHEM. SOC. 9 VOL. 131, NO. 51, 2009
Uracil and Thymine Reactivity in the Gas Phase
deprotonated 3-methylthymine, we assessed the acidity of the
N1-H proton in 3-methyluracil (3-MeU, 1b) and compared it
to the acidity of the N1-H proton in 3-methylthymine (3-MeT,
3b). The acidity of 3-MeU has been calculated previously by
us (332.8 kcal mol-1, Figure 3); herein, we calculate the ∆Hacid
of 3-MeT to be 1.4 kcal mol-1less than the ∆Hacidof 3-MeU
(332.8 vs 334.2 kcal mol-1, Figure 3).17On the basis of these
acidities, we would expect deprotonated 3-methyluracil to be a
better leaving group than deprotonated 3-methylthymine.
We have also previously measured the gas-phase ∆Hacidof
3-MeU (1b) to be 333 ( 2 kcal mol-1.17We bracket the acidity
of 3-MeT herein (3b) (Table 2). We find that while the conjugate
base of 3-MeT deprotonates 2-chloropropanoic acid (∆Hacid)
337.0 ( 2.1 kcal mol-1) and acids with lower ∆Hacidvalues, it
cannot deprotonate trifluoro-m-cresol (∆Hacid) 339.3 ( 2.1
kcal mol-1) or reference acids with higher ∆Hacid values.
Consistent with this, 2-chloropropanoate cannot deprotonate
3-MeT, but trifluoro-m-cresolate can. We therefore bracket the
∆Hacidof 3-MeT to be 338 ( 3 kcal mol-1(∆Gacid(3-MeT) )
331 ( 3 kcal mol-1).
We also conducted Cooks kinetic method experiments to
measure the relative acidity of 3-MeT (3b) and 3-MeU
(1b).65-69We accomplished this by dissociating the [(3-
MeU)-·H+·(3-MeT)-] dimer. These experiments indicate that
3-MeU (1b) is 2-3 kcal mol-1more acidic than 3-MeT (3b).
The experiments therefore indicate a difference in acidity
between 3-MeU and 3-MeT that is on the order of 2-5 kcal
mol-1. Although this is a rather large range, what is consistent
is that 3-MeU is more acidic than 3-MeT. This could make the
N1-conjugate base of 3-MeU a potentially better leaving group
than that of 3-MeT.
Another important reason to probe acidities is to establish
the upper limit of proton affinity for the nucleophiles to be
studied experimentally. As we noted earlier, we are using the
N3-methyl substrates because the N3-H of 1-methyluracil has
a ∆Hacidof 348 kcal mol-1, and to avoid competition between
SN2 reaction at the N1-CH3and proton transfer at the N3-H,
we would be limited to using nucleophiles with proton affinities
(PA) under 348 kcal mol-1.3,17Now that we have established
that we will be using the 1,3-dimethyl derivatives 1d and 3d,
we need to assess the acidities of all of the sites of those
substrates (Figure 3). The C6 protons of both of these derivatives
are quite acidic, with values just below that of acetone (the C6
and C5 of 1,3-dMU have also been previously measured by
the Gronert lab and our lab).17,53Therefore, to avoid competition
from deprotonation, nucleophiles with proton affinities below
367 kcal mol-1will be utilized.
SN2 Reaction Studies: Calculations. a. 1,3-Dimethyluracil
(1,3-dMU). We next calculated the energetics associated with
SN2 reactions of 1,3-dMU. We chose formate and methyl
thiolate as the nucleophiles. Formate was chosen as a probable
slow reaction example, based on the acidity of formic acid
(∆Hacid ) 346.2 ( 1.2 kcal mol-1) and the known methyl
chloride data for nucleophiles in that acidity range (Table 1).
Methyl thiolate (∆Hacid) 357.6 ( 2.0 kcal mol-1) was chosen
as a faster (although still moderate) reaction example.
The reaction of formate with 1,3-dMU first forms the expected
reactant ion-molecule complex, a process that is 23.1 kcal
mol-1exothermic (Figure 4). The ∆Hqbarrier to the SN2 reaction
is 32.3 kcal mol-1from this complex (and 9.2 kcal mol-1from
the separated reactants (Figure 4)). The product ion-molecule
complex is 9.3 kcal mol-1more stable than the separated
reactants, but the separated products are 2.7 kcal mol-1higher
in energy than the separated reactants. Given that both the
transition state (TS) and the separated products are higher in
energy than the separated reactants, this reaction is not likely
to proceed significantly under our gas-phase conditions.
For the reaction of methyl thiolate with 1,3-dMU (figure in
Supporting Information), the transition state is calculated to be
just 4.3 kcal mol-1higher than the energy of the separated
reactants. The energy of the transition structure must be below
the energy of the separated reactants for reaction to be observed,
so we could see reaction depending on how accurate the
calculations are, and how entropically unfavorable the process
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Figure 1. Nucleophilic attack at C1′ to excise uracil.5
Figure 2. SN2 reactions studied.
Figure 3. Calculated N1-H acidities of 3-methyluracil17(3-MeU, 1b) and
3-methylthymine (3-MeT, 3b) and all of the sites of 1,3-dimethyluracil (1d)
and 1,3-dimethylthymine (3d) (B3LYP/6-31+G*, ∆H298K, kcal mol-1).
Experimental values where known are in parentheses.17,63
18378J. AM. CHEM. SOC. 9 VOL. 131, NO. 51, 2009
Zhachkina and Lee
b. Methyl Chloride. We also calculated the energetics for
the reaction of formate (Figure 5) and methyl thiolate (figure
in Supporting Information) with methyl chloride, to provide a
benchmark for comparison. Interestingly, the methyl chloride
reactions are consistently more exothermic and have lower
barriers than the uracil reactions. With formate, the ∆Hqis 1.9
kcal mol-1below the separated reactants (Figure 5). For methyl
thiolate, the ∆Hqis 7.5 kcal mol-1below the separated reactants.
Our calculations therefore indicate that, although 3-MeU and
HCl have similar acidities, the 1,3-dMU SN2 reactions are
expected to be slower than those of CH3Cl.
c. 1,3-Dimethylthymine (1,3-dMT). Our calculations indicate
that in keeping with the fact that thymine is less acidic than
uracil, the SN2 reactions wherein deprotonated thymine is a
leaving group do have slightly higher barriers than the corre-
sponding reactions with deprotonated uracil. Using 1,3-dMT
as the model system, the energy surfaces for reaction with
formate and methyl thiolate were calculated (figures in Sup-
porting Information). For the formate reaction, the barrier is
1.2 kcal mol-1higher with 1,3-dMT than with 1,3-dMU (relative
to separated reactants) and is also more endothermic. For the
methyl thiolate reaction, the barrier for 1,3-dMT is 1 kcal mol-1
higher than that with 1,3-dMU. We also find computationally
that for a wide range of nucleophiles (formate, acetate, n-
pentylthiolate, methyl thiolate, and anilide), the exothermicities
of the 1,3-dMU versus the 1,3-dMT reactions are always roughly
1.4 kcal mol-1apart (with 1,3-dMU being more exothermic;
data in Supporting Information).
Table 2. Summary of Results of Acidity Bracketing of 3-Methylthymine (3-MeT, 3b)
343.8 ( 2.1
340.80 ( 0.60
339.3 ( 2.1
337.0 ( 2.1
335.8 ( 2.1
333.5 ( 2.9
331.0 ( 2.2
328.3 ( 2.9
336.7 ( 2.0
332.4 ( 2.0
330.4 ( 2.0
328.1 ( 2.0
326.5 ( 2.8
323.8 ( 2.0
322.0 ( 2.0
aAcidities are in kcal mol-1.64 bA “+” indicates the occurrence, and a “-” indicates the absence of proton transfer.
Figure 4. Calculated (B3LYP/6-31+G*) potential energy diagram of the reaction of 1,3-dMU (1d) with formate (∆H, kcal mol-1, 298 K).
J. AM. CHEM. SOC. 9 VOL. 131, NO. 51, 2009
Uracil and Thymine Reactivity in the Gas Phase
d. Methyl Chloride versus Pyrimidine Derivatives Acidity
Calculations Revisited. Experimentally, HCl and uracil have
similar acidities. However, our calculations indicate that,
although the N1-deprotonated uracil derivatives are still fairly
good leaving groups (for example, the reaction of methyl
thiolate, which is not that basic, with 1,3-dMU is exothermic
by 23.4 kcal mol-1and has a barrier of only 4.3 kcal mol-1
above the separated reactants), chloride is still better.
To assess whether this calculated difference in reactivity
might be a computational artifact, we compared the computed
acidities of 3-MeU and HCl. We know by experiment that the
two have comparable acidities.3,17However, we find that the
∆Hacid of HCl calculates to 325.1 kcal mol-1at B3LYP/6-
31+G*, 7.7 kcal mol-1more acidic than the calculated value
for 3-methyluracil, and 8.3 kcal mol-1more acidic than the
known measured ∆Hacid of HCl (333.4 ( 0.1 kcal mol-1).
Essentially, although 3-methyluracil and HCl have the same
experimental acidity, the B3LYP/6-31+G* calculations are not
accurate: HCl appears to be more acidic. To assess whether the
calculated differences in SN2 reaction barriers for the methyl
chloride and pyrimidine derivatives are due to a related
computational artifact or truly reflect the reactivity, we con-
ducted experiments, which are described later in this Article.
e. N1 versus N3 Attack. One possible complication for
examining these reactions experimentally is a potential competi-
tion between N1-CH3 attack (which we wish to see) and
N3-CH3attack (which is not of biological interest; Figure 6).
We computationally examined nucleophilic attack at the
N1-CH3versus N3-CH3for a series of nucleophiles (formate,
acetate, n-pentyl thiolate, n-propyl thiolate, methyl thiolate, and
anilide) and find that the N1-CH3SN2 reaction is consistently
on the order of 12 kcal mol-1more exothermic than the
N3-CH3path (Table 3).
We also calculated the activation enthalpies (∆Hq) for the
reactions with formate and methyl thiolate; for both reactions,
the barrier for attack at the N1-methyl group is 10 kcal mol-1
lower than that at the N3-methyl. These differences in barrier
are significant enough that, experimentally, SN2 reaction at
N1-CH3should be considerably favored over SN2 reaction at
N3-CH3. These results are as expected, given that the N1-H
is more than 10 kcal mol-1acidic than the N3-H.3,17
SN2 Reaction Studies: Experiments. The calculated barriers
for the reaction of formate with 1,3-dMU versus methyl chloride
indicate that the methyl chloride reaction should be faster
(Figures 4 and 5). Furthermore, Table 1 indicates that a
nucleophile with PA of 361.7 (CF3CH2O-) reacts with methyl
chloride with an efficiency of just 11%. We would therefore
expect that the SN2 reactions of nucleophiles whose PAs are
less than the ∆Hacidof C6-H (∆Hacid≈ 370 kcal mol-1) with
1,3-dMU and 1,3-dMT will all be relatively slow.
To establish that we can see SN2 reactivity under our
conditions, we repeated the known reactions of 2,2,2-trifluo-
roethoxide and methyl thiolate with methyl chloride. Our
efficiency values for these SN2 reactions are comparable to those
obtained previously (for 2,2,2-trifluoroethoxide, 13.3% (current
work) vs 11% (literature); for methyl thiolate, 8.0% (current
work) vs 4.7% (literature)).34,49,57
The reactions of a series of nucleophiles with 1,3-dMU and
1,3-dMT were studied (Table 4). The SN2 reaction product was
Figure 5. Calculated (B3LYP/6-31+G*) potential energy diagram of the reaction of methyl chloride with formate (∆H, kcal mol-1, 298 K).
Figure 6. N1 versus N3 attack of 1,3-dimethyluracil.
Table 3. Calculated Enthalpies of SN2 Reactions of 1,3-dMU (1d)
with a Series of Nucleophiles: N1-Methyl versus N3-Methyl
∆Hrxn, kcal mol-1
N1 attack N3 attack
18380J. AM. CHEM. SOC. 9 VOL. 131, NO. 51, 2009
Zhachkina and Lee