Reaction of the tamoxifen cation and the bis-(4-methoxyphenyl)methyl cation in aqueous solutions containing 2′-deoxyguanosine

Article abstract of DOI:10.1139/v02-011

The competition between 2′-deoxyguanosine (dG) and water has been quantitatively evaluated for the allylic carbocation derived from tamoxifen and for the stabilized diarylmethyl cation (bis-(4-methoxyphenyl)methyl). Both systems were examined by the competition kinetics method, in which the products were quantitatively analyzed after the S_N1 solvolysis of the corresponding acetate esters in aqueous solutions containing the nucleoside. The principal product of the reaction of both cations with dG is the adduct at the NH₂ group, a characteristic of delocalized carbocations. The tamoxifen cation was also examined by laser flash photolysis, with absolute rate constants for the reaction with dG and water being obtained and converted into rate constant ratios. The principal result of this study is that there is a three orders of magnitude difference in the reactivity of these cations towards the neutral form of dG and its conjugate base. Under acidic conditions where the reaction occurs with neutral dG, the guanine-water selectivity is low. Even at relatively high concentrations of dG, the majority of the product is alcohol derived from the water reaction. At pH 10 to 11, in contrast, dG is present as the anion and this is highly competitive. Yields of adduct as high as 90% can be attained. A consequence of the large difference in reactivities is that at neutral pH the majority of the reaction of the cation with dG is actually occurring via the small amount of conjugate base present. A further feature of the results is that the NH₂ adduct is the predominant stable product from the anion. To explain the high rate constant for the reaction forming this product, a mechanism is proposed whereby one of the protons of the NH₂ group is transferred to N1 as the N2-cation bond is forming.

Full text of DOI:10.1139/v02-011

269
Reaction of the tamoxifen cation and the bis-(4-
methoxyphenyl)methyl cation in aqueous solutions
containing 2′-deoxyguanosine
Robert A. McClelland, Cristina Sanchez, Effiette Sauer, and Sinisa Vukovic
Abstract: The competition between 2′-deoxyguanosine (dG) and water has been quantitatively evaluated for the allylic
carbocation derived from tamoxifen and for the stabilized diarylmethyl cation (bis-(4-methoxyphenyl)methyl). Both sys-
tems were examined by the competition kinetics method, in which the products were quantitatively analyzed after the
SN1 solvolysis of the corresponding acetate esters in aqueous solutions containing the nucleoside. The principal product
of the reaction of both cations with dG is the adduct at the NH2 group, a characteristic of delocalized carbocations.
The tamoxifen cation was also examined by laser flash photolysis, with absolute rate constants for the reaction with
dG and water being obtained and converted into rate constant ratios. The principal result of this study is that there is a
three orders of magnitude difference in the reactivity of these cations towards the neutral form of dG and its conjugate
base. Under acidic conditions where the reaction occurs with neutral dG, the guanine–water selectivity is low. Even at
relatively high concentrations of dG, the majority of the product is alcohol derived from the water reaction. At pH 10
to 11, in contrast, dG is present as the anion and this is highly competitive. Yields of adduct as high as 90% can be
attained. A consequence of the large difference in reactivities is that at neutral pH the majority of the reaction of the
cation with dG is actually occurring via the small amount of conjugate base present. A further feature of the results is
that the NH2 adduct is the predominant stable product from the anion. To explain the high rate constant for the reac-
tion forming this product, a mechanism is proposed whereby one of the protons of the NH2 group is transferred to N1
as the N2–cation bond is forming.
Key words: guanine, DNA adduct, carbocation, tamoxifen.
Résumé : On a évalué de façon quantitative la compétition entre la 2′-désoxyguanosine (dG) et l’eau pour le carboca-
tion allylique dérivé du tamoxifène et pour le cation diarylméthyle stabilisé, bis-(4-méthoxyphényl)méthyle. On a exa-
miné les deux systèmes par la méthode des cinétiques en compétition dans laquelle les produits sont analysés de façon
quantitative après une solvolyse SN1 des esters acétiques correspondants en solutions aqueuses contenant le nucléoside.
Le produit principal de chacune des réactions des deux cations avec la dG est un adduit au niveau du groupe NH2, une
caractéristique des carbocations délocalisés. On a aussi étudié le cation du tamoxifène par photolyses éclairs au laser à
partir desquelles on a obtenu des constantes absolues de vitesse pour la réaction avec la dG et l’eau, celles-ci étant
transformées en rapports de constantes de vitesses. Le principal résultat de cette étude est qu’il y a trois ordres de
grandeur de différence dans la réactivité de ces cations vis-à-vis de la forme neutre de la dG et de sa forme conjuguée.
Dans les conditions acides où la réaction se produit avec la dG neutre, la sélectivité guanine–eau est faible. Même à
des concentrations élevées de dG, la plus grande partie du produit est l’alcool qui provient de la réaction avec l’eau.
Par ailleurs, à des pH de 10 à 11, la dG est présente sous la forme d’anion qui est fortement compétitif. On peut alors
obtenir des rendements d’adduit pouvant atteindre 90%. Une conséquence de la grande différence dans les réactivités
est que, au pH neutre, la plus grande partie de la réaction du cation avec la dG se produit par le biais de la faible
quantité de la base conjuguée qui est présente. Une caractéristique additionnelle des résultats est que l’adduit avec le
NH2 est le produit stable prédominant à partir de l’anion. Pour expliquer la constante de vitesse élevée pour la réaction
conduisant à la formation de ce produit, on propose un mécanisme dans lequel un des protons du groupe NH2 est
transféré vers N1 alors que la liaison N2–cation se forme.
Mots clés : guanine, adduit avec l’ADN, carbocation, tamoxifène.
[Traduit par la Rédaction] McClelland et al. 280
Received 30 July 2001. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 22 March 2002.
R.A. McClelland,1 C. Sanchez, E. Sauer, and S. Vukovic. Department of Chemistry, University of Toronto, Toronto,
ON M5S 3H6, Canada.
1Corresponding author (e-mail: rmcclell@alchemy.chem.utoronto.ca).
Can. J. Chem. 80: 269–280 (2002)
DOI: 10.1139/V02-011
© 2002 NRC Canada

270
Can. J. Chem. Vol. 80, 2002
Scheme 1. Ar = C6H4-4-OCH2CH2N(CH3)2.
Introduction
OX
A common property of chemical carcinogens is the forma-
tion of DNA adducts (1). These adducts arise from chemical
reactions in which DNA reacts with an electrophile, often an
electrophile resulting from metabolism of the parent com-
pound. Tamoxifen 1 (Scheme 1), an antiestrogen used in the
treatment and control of breast cancer, is a hepatocarcinogen
in the rat (2–4) and causes a small number of endometrial
cancers in women (5–7). DNA adducts have been observed
in both the animal models (8, 9) and in women treated with
the drug (10–14). These are explained by the pathway of
Scheme 1 (15–22), whereby an allylic carbocation 3 forms
in an SN1 ionization of a sulfate ester 2b. The latter is de-
rived from metabolic α-hydroxylation of the parent drug to
α-hydroxytamoxifen 2a followed by sulfate transfer. The
principle DNA adducts 4 are isomers (see below) where the
the α-carbon of the tamoxifen is attached to the NH2 group
of guanine residues (20–22). This position of attachment to
guanine is characteristic of delocalized carbocations (1).
We have described experiments where the cation 3, the
“tamoxifen carbocation,” was observed with laser flash
photolysis (LFP) (23). The LFP approach has now been em-
ployed to study a number of carbocations (24). These are
ground-state cations, identical to ones formed in SN1
solvolyses. The significant advantage of the LFP method is
that it is capable of providing direct information on reactiv-
ity, both towards the solvent (i.e., water) and to added
nucleophiles. A nucleophile of interest because of the rela-
tion to carcinogenicity discussed above is guanine or a gua-
nine derivative such as 2′-deoxyguanosine (dG). Our
previous study (23) showed that the tamoxifen cation is
quenched by dG in an aqueous solution. However, the selec-
tivity over the reaction with solvent is poor, so that even at
the highest concentrations of dG, the majority of the cation
reacts with solvent. This behavior is typical of carbocations
in water (25), but it contrasts dramatically with arylnitren-
ium ions, the DNA-binding electrophiles derived from carci-
nogenic amines. Both LFP studies (25, 26) and product
analyses (27, 28) show that guanine derivatives are excellent
nucleophiles for arylnitrenium ions, even in water.
Ph
Ar
CH2CH3
Ph
Ph
Ar
CHCH3
Ph
1
2a; X = H
2b; X = SO3
-
2c; X = COCH3
DNA
+
Ph
Ar
CHCH3
Ph
Ar
CHCH3
Ph
Ph
4
3
λmax at 460 nm was identified as the tamoxifen cation on the
basis of several criteria employed to assign carbocation in-
termediates (24). A key observation was the quenching by
azide ion, in particular, the identity of the azide:water rate
constant ratio with one obtained by competition kinetics
(product analysis) for the ground-state solvolysis of the sul-
fate in aqueous solutions containing azide ion.
The sulfate ester has a high reactivity towards solvolysis
in the ground state. This limits LFP experiments to solutions
with considerable organic component (i.e., 40% acetonitrile)
where the lower ionizing power slows the SN1 reaction.
Since acetate esters have been successfully employed as pre-
cursors of carbocations in LFP experiments (24), we investi-
gated the appropriate tamoxifen derivative 2c. This also
produces the tamoxifen cation upon irradiation in aqueous
solutions. This was established by comparing LFP results
with the acetate and the sulfate in 40% acetonitrile. The two
gave the same transient with the same decay kinetics.
The acetate is much more stable towards ground-state
solvolysis, with a half-life in 100% water of 2 h. We there-
fore started by carrying out a detailed kinetic study of the
decay of the tamoxifen cation in 100% aqueous solutions.
Since the buffers accelerated the decay, experiments were
performed with serial buffer dilutions at constant buffer ratio
(and constant pH), and the rate constant obtained as the in-
tercept of the plot of k vs. buffer concentration. These rate
constants, along with ones obtained in solutions of
perchloric acid and sodium hydroxide, are plotted as log ko
vs. pH in Fig. 1. Also plotted in this figure are rate constants
obtained in a similar manner for the other cation of this
study, the bis-(4-methoxyphenyl)methyl cation.
The profile for the latter cation is straightforward. There is
a pH-independent region from acidic pH to a pH of about 11
that corresponds to the reaction of the cation with solvent wa-
ter molecules. The rate constant is 1.0 × 105 s–1. (Table 1 pro-
vides a summary of all rate and equilibrium constants
measured in this work, along with their errors provided as one
standard deviation.) At pH 11, there is a break to a reaction
that is first order in hydroxide ion. This obviously represents
the cation–hydroxide combination; the second-order rate con-
stant is 3.8 × 106 M–1 s–1. One noteworthy feature is the rela-
We have now performed a detailed study of the reaction of
the tamoxifen cation with dG, employing LFP to measure ab-
solute rate constants and product analyses (competition kinet-
ics) for relative rate constants. Competition kinetic experiments
have also been carried out with a diarylmethyl cation, the bis-
(4-methoxyphenyl)methyl cation, which has a similar lifetime
in water to the tamoxifen cation (29). These studies show that
there is very pronounced effect of pH on the dG reaction. The
neutral form of dG is quite unreactive as previously found
(25). Its conjugate base, however, is far more reactive, so much
so that at pH 7 the principal species that is reacting with the
cations is the anion. The difference between the two forms of
guanine has been recognized (30), but we are not aware of a
detailed kinetic study demonstrating the magnitude of the ef-
fect, especially for short-lived carbocations.
Results and kinetic analysis
Rate pH-profiles in 100% aqueous solutions
In our original paper, the sulfate ester 2b was employed as
the precursor in the LFP experiments (23). A transient with
tively little difference (a factor of 13) between kw and kOH,
typical of short-lived cations in aqueous solutions (31–33).
© 2002 NRC Canada

McClelland et al.
271
the allylic cation. Parameter a (2.5 × 104 s–1), is equal to
kw2+, the rate constant for water addition to the dication. In
terms of the pH profile, this reaction dominates from acidic
pH to a pH just before 9; the downward break between pH 7
and 8 is a reflection of the changing acid–base equilibrium.
Parameter c (kO2H+ KwKa(T) = 2.2 × 10–17) explains the in-
crease in base. This must be due to a reaction of the mono-
cation T+ with hydroxide ion. With Ka(T) known, the rate
constant kO+H can be calculated; the value is 1.6 × 105 M–1 s–1.
Parameter b (8.4 × 10–5) accounts for the pH-independent
region between pH 9 and pH 11. There are two kinetically
equivalent processes that could be responsible, the reaction
of hydroxide ion with the dication T2+ and the reaction of
water with the monocation T+. The former can be ruled out,
Fig. 1. pH dependence of the logarithm of the rate constants for
the decay of the tamoxifen cation (squares) and the bis-(4-meth-
oxyphenyl)methyl cation (circles) in aqueous solutions. Tempera-
ture is 20°C and ko has units of s–1. The data for the tamoxifen
cation have been obtained at a constant ionic strength of 0.10 M
maintained with NaClO4. The data for the bis-(4-methoxy-
phenyl)methyl cation were obtained in solutions with ionic
strength less than 0.01 M. The cations were generated by LFP of
acetate precursors with irradiation at 248 nm and detection at
460 nm (tamoxifen cation) and 500 nm (bis-(4-methoxyphenyl)-
methyl cation). Rate constants in the region pH 3–11 were ob-
tained in buffer solutions, and are based upon extrapolation to
zero buffer concentration. The points are experimental. The line
for the tamoxifen cation has been drawn on the basis of eq. [2]
and the parameters given in the text.
since it requires a value of k of 8.4 × 109 M–1 s1, several
2+
OH
orders of magnitude greater than expected. As noted above,
hydroxide would not react with a cation of the aqueous life-
time of T2+ at anywhere near this rate. Our conclusion,
therefore, is that the pH-independent region in weakly basic
solution represents water reacting with the monocation T+.
The rate constant for this process (kw+) is 6.3 × 103 s–1, four-
fold smaller than the rate constant kw2+ for the reaction of
water with T2+. This greater reactivity of the dication is not
surprising. There is undoubtedly a significant stabilization in
both cations by the π-interaction with the oxygen of the
dimethylaminoethoxy group. This interaction will be stronger
when that group is neutral as opposed to where the amine is
protonated. In other words, the (CH3)2NCH2CH2O- group must
be a better π donor than the (CH3)2HN+CH2CH2O- group.
Analysis of the rate constants associated with the buffer
revealed that the rate accelerations were due to the basic
component of the buffer, the terms in (B) that appear in
Scheme 2. As with water and hydroxide, reaction in princi-
ple could occur with both T2+ and T+, although of the four
buffers employed, only tris resulted in rate constants for both
the dication and cation that were statistically significant (Ta-
ble 1). The actual reaction occurring with the buffer is not
known. The most likely scenario is a cation–nucleophile
combination. However, a reaction where the buffer base acts
as a general base to assist the addition of water to the cation
is also possible. Arguments have been made that the water
reaction itself is generally base-catalyzed, with a second wa-
ter molecule acting as the base (29).
The profile for the tamoxifen cation can be explained by
recognizing that there is a side-chain amine. This means that
there are two carbocations, a dication T2+ where the amine is
protonated and a monocation T+ where the amine is neutral
(Scheme 2). Both, in principle, can react with water and hy-
droxide, giving rise to eq. [1], where Kw is the autoprotolysis
constant of water.
Decay of the tamoxifen cation in the presence of 2′-
deoxyguanosine
kO+HKwKa(T)
2+
kw2+[H+ ] + k Kw + kw+Ka(T) +
These experiments were carried out in 20% acetonitrile
for solubility reasons. LFP experiments with dG as the trap-
ping nucleophile require irradiation, at 308 nm, with our
equipment where the dG does not absorb. While 2c does
have absorbance at this wavelength, it is weak. Thus, more
concentrated solutions of the precursor were required. These
concentrations could not be achieved in 100% aqueous solu-
tions, especially solutions of higher pH where the side-chain
amine of 2c is not protonated. The use of 20% acetonitrile
also meant that the solubility of dG was increased. Solutions
with a dG concentration of 20 mM were possible, in con-
trast to 100% aqueous where 8 mM is about the maximum
concentration.
OH
[H+ ]
[1]
ko =
[H+ ] + Ka(T)
This takes the form of a four-parameter equation (eq. [2])
where y = ko and x = [H+].
ax + b + c/x
[2]
y =
x + d
The experimental data were fit to eq. [2] to provide the
four parameters. Parameter d (1.34 × 10–8) is the acidity
constant, corresponding to a pKa of 7.87. This means that
the ammonium group in T2+ is one log unit more acidic than
in the parent drug. This reflects the electron withdrawing in-
fluence of the delocalized positive charge associated with
Figure 2 provides the results. The rate constants for the
solvent reaction were obtained as described in the previous
© 2002 NRC Canada

272
Can. J. Chem. Vol. 80, 2002
Table 1. Summary of rate and equilibrium constants for the reactions of tamoxifen dication T2+, tamoxifen monocation T+, and bis-(4-
methoxyphenyl)methyl cation (Ar2CH+).
Parameter
T2+
T+
Ar2CH+
kw (s–1) (water)
kw (s–1) (20% AN)
(2.5 ± 0.1) × 104a
(3.2 ± 0.1) × 104a
(6.3 ± 0.2) × 103a
(8.2 ± 0.3) × 103a
(1.01 ± 0.05) × 105b
(1.3 × 105)c
kOH (M–1 s–1) (water)
d
—
(1.6 ± 0.2) × 105a
(3.8 ± 0.8) × 106b
pKa(T) (water)
pKa(T) (20% AN)
7.87 ± 0.05a
7.26 ± 0.08a
kn (M–1 s–1) (20% AN)
ka (M–1 s–1) (20% AN)
(1.0 ± 0.2) × 105
(6.4 ± 0.8) × 107
~(1.5–3) × 104e
(1.2 ± 0.1) × 107
pKa(G) (20% AN)
pKa(G) (20% AN)
9.27 ± 0.11 (Kinetics)
9.37 ± 0.05 (Spect.)
9.44 ± 0.08 (Fig. 6)
kn:kw (20% AN) (LFP)
kn:kw (20% AN) (Pdts.)
(3.1 ± 0.6)
(2.7 ± 0.3)
d
—
(1.21 ± 0.07)f
ka:kw (20% AN) (LFP)
ka:kw (20% AN) (Pdts.)
(2.0 ± 0.3) × 103
(1.5 ± 0.1) × 103
(7 ± 1) × 102
(5.7 ± 0.6) × 102
(1.52 ± 0.13) × 103f
kAcO (M–1 s–1) (water)
(3.4 ± 0.4) × 105a
(5.5 ± 0.5) × 105a
—
d
s–1) (water)
k
(M–1
—
2 −
d
HPO4
ktris (M–1 s–1) (water)
(8 ± 1) × 104
—
(2 ± 0.5) × 104
(2.1 ± 0.4) × 105
k
(M–1 s–1) (water)
2 −
d
CO 3
aIonic strength = 0.1.
bIonic strength < 0.01.
cFrom ref. 29.
dCannot be measured since not significant in competition with other processes.
eEstimated.
fRatio calculated for DdGa.
Scheme 2.
sence of dG. The observed rate constants were plotted
against the concentration of added dG. These plots were lin-
ear. Their slopes, defined as kdG, are plotted as the circles in
Fig. 2.
+
+2
Ph
kw
kOH+2[OH-]
kB+2[B]
(Z)
Ph
The solvent reaction was analyzed as in the previous sec-
tion. Values of the parameters so obtained were Ka(T) =
5.5 × 10–8 (pKa(T) = 7.27), kw2+ = 3.2 × 104 s–1, and kw+
=
H
8.2 × 103 s–1. The two rate constants are slightly larger than
the ones obtained in the study in 100% aqueous solutions.
This is a typical observation for carbocation–water reactions
as acetonitrile is added (29). There is also a difference in the
pKa(T) value. This is likely also a solvent effect. Our previ-
ous study in 40% acetonitrile resulted in an even lower value
of pKa(T) (6.6) (23).
O
Me2
N
+
+
T 2
Ka(T)
+
+
Ph
kw
kOH+[OH-]
kB+[B]
(Z)
An interesting feature of the reaction with dG is that at
about the point where the curve for the solvent reaction
breaks downward, the profile for the dG reaction breaks up-
ward. The reasons for this must be different. The downward
break in the solvent profile occurs because of the conversion
of T2+ to T+. The upward break in the dG profile must rep-
resent the onset of a new reaction, the reaction of the cation
with the conjugate base of dG. This leads to the kinetic
model of Scheme 3, which shows the nucleoside reaction oc-
curring in four ways. Both the neutral form of dG (dG-H)
and its conjugate base (dG–) are nucleophiles, and each, in
principle, reacts with T2+ and T+.
Ph
O
+
Me2
N
T
section, with (where appropriate) serial buffer dilutions pro-
viding the rate constants at zero buffer concentration. The
rate constants for the reaction with dG were obtained using
dilute buffers, with 5 to 6 concentrations of dG ranging
from [dG] ~ 0.02 M to [dG] = 0 at each pH. The rate con-
stants increased in the presence of dG. This acceleration was
modest in the solutions with pH 4–6, with only about a 15%
increase from the solution with no dG to the solution with
the highest concentration. As the pH increased, however, the
effect of dG became much more pronounced, and by pH 9 it
was possible to triple the rate constant over that in the ab-
The kinetic system of Scheme 3 gives rise to eq. [3] for kdG
.
[3]
kdG
(kn2+[H+ ]2 + {ka2+Ka(G) + kn+Ka(T)}[H+ ] + ka+Ka(G)Ka(T))
([H+] + Ka(T))([H+] + Ka(G))
=
© 2002 NRC Canada

McClelland et al.
273
Fig. 2. pH dependence of the logarithm of the rate constants for
the decay of the tamoxifen cation in solvent (squares) and the
quenching by 2′-deoxyguanosine (dG) (circles). Temperature is
20°C, and the solvent is 20:80 (v:v) acetonitrile–water with ionic
strength maintained at 0.10 M with sodium perchlorate. Units of
k are s–1 (solvent reaction) and M–1 s–1 (dG reaction). The cation
was generated by excitation of 2c at 248 nm (solvent data) and
308 nm (dG data). The points are experimental. The lines have
been drawn on the basis of eq. [2] (solvent reaction) and eq. [4]
(dG reaction).
Scheme 3.
kn +[dG-H]
2
+
ka [dG-]
2
T2
Ka(T)
+
kn+[dG-H]
ka+[dG-]
+
T
cesses, neutral dG reacting with T+ (the kn+ process) and an-
ionic dG reacting with T2+ (the ka2+ process). The latter must
be the major contributor. If the former is responsible, b =
kn+Ka(T), which requires kn+ to be 6.2 × 105 M–1 s–1. This is a
sixfold greater than kn2+, the rate constant for the reaction of
neutral dG with the dication. This makes no sense since the
dication is the more reactive species. Based upon results for
other nucleophiles, kn+ will lie in the range 1.5–3 × 104 M–1 s–1,
a factor of three to six times smaller than kn2+. We therefore
conclude that the kn+ process could contribute about 3% to
the term in b. However, 97% of this term must be due to the
process in ka2+. Using this 97% factor, ka2+ is calculated as
6.4 × 107 M–1 s–1. It can be seen that this is consistent with
the greater reactivity of the dication — ka2+ is fivefold larger
than ka+.
Figure 3 shows how the observed rate constants for the re-
action of the tamoxifen cation and dG are broken down into
the contributions from the four reactions. The curves in this
figure were calculated using the rate constants provided in
the previous paragraph (with an estimate for kn+ of 2 × 104).
It can be seen that under acidic conditions the reaction that
occurs involves T2+ and the neutral form of dG, while under
basic conditions, T+ reacts with the anion of dG. The princi-
ple contributor at pH 7 is the reaction of T2+ with the conju-
gate base.
This equation contains two acid acidity constants (Ka(T)
and Ka(G)) referring, respectively, to the ammonium group
of the tamoxifen cation and the N1-H proton of dG. The rate
constants are identified with superscripts “2+” and “+” to
differentiate reactions with T2+ and T+ and with subscripts
“n” and “a” to differentiate reactions with the neutral and
anionic forms of the nucleoside. This equation takes the
form of eq. [4], with five adjustable parameters.
Tamoxifen cation and deoxyguanosine — competition
kinetics
The experiments described in this section were performed
to determine if the pH effect is also observed in the prod-
ucts. The principle adduct of the tamoxifen cation and dG
has been isolated and characterized previously as compound
ax2 + bx + c
[4]
y =
TdG of Scheme
4 (22). This is formed as four
(x + d)(x + e))
diastereomers, arising from the combinations of the (R) and
(S) forms at the α stereocenter and (E) and (Z) forms about
the C=C. The latter arise because the cation undergoes rapid
rotation about this bond before capture by nucleophiles.
HPLC analysis of this mixture then provided retention times
for the four isomers under our HPLC conditions.
Experiments were then performed in which the acetate 2c
was added to solutions of 20% acetonitrile containing 2′-
deoxyguanosine, and the ground-state solvolysis reaction al-
lowed to proceed to completion overnight. The products
were analyzed by HPLC. With appropriate correction for rel-
ative HPLC sensitivities, this provided the ratio of the sum
of the concentrations of the four adducts [TdG] to the sum
of the concentrations of the (E) and (Z) alcohols [TOH].
This ratio was then converted to a competition-kinetic ratio
according to eq. [5], where [dG] was the concentration of
the nucleoside in the solution.
The experimental data were fit to this equation with the
parameter d = Ka(T) fixed at the value of 5.5 × 10–8 obtained
from the analysis of the water reaction. The other four were
allowed to vary to values providing the best fit.
The parameter e (5.3 × 10–9) is the acidity constant for
dG, and corresponds to a pKa value of 9.27. We also mea-
sured this value spectroscopically under the same solvent
conditions, and obtained a value of 9.37. Within the experi-
mental error (Table 1), the value obtained from the kinetic fit
is identical. The parameter a = kn2+ = 1.0 × 105 M–1 s–1 is the
rate constant for the reaction of the neutral form of dG with
the dication T2+. The parameter c (3.4 × 10–10) is equal to
ka+Ka(T)Ka(G) and represents the reaction of the conjugate
base of dG with T+. With values of the acidity constants
known, ka+ is calculated as 1.2 × 107 M–1 s–1. The parameter
b (3.4 × 10–2) represents two kinetically equivalent pro-
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274
Can. J. Chem. Vol. 80, 2002
Fig. 3. Breakdown of the reaction of 2′-deoxyguanosine and the
Scheme 4.
tamoxifen cation into components.
OCOCH3
CHCH3
+
Ph
CHCH3
Ph
Ar
Ar
Ph
Ph
3
2c = TOAc
kdG[dG]
ko
O
N
N
HN
H
N
N
OH
dRibose
Ph
Ar
CHCH3
Ph
Ar
CHCH3
Ph
Ph
TdG
TOH
This means that there are four reactions occurring in
parallel, one forming alcohol and three forming different dG
adducts (Scheme 5). These adducts are designated as DdG1,
DdG2, and DdG3 in order of increasing HPLC retention
times.
Figure 5 shows how the areas of the peaks for the four
products vary with pH for experiments performed with con-
stant concentrations of dG and acetate ester. Under acidic
conditions (pH 3–5) the areas are constant. The alcohol is
the major product, with only small peaks for the three ad-
ducts (note the scale in Fig. 5 is logarithmic). As the pH in-
creases, the alcohol peak decreases significantly, the peaks
for DdG1 and DdG2 grow, and the peak for DdG3 disap-
pears. Under basic conditions there is another constant re-
gion, with the major peak corresponding to DdG1.
A sample of DdG1 was isolated from a scaled-up reaction
at high pH. The NMR spectra in DMSO-d6 clearly estab-
lished that this is the N2-adduct. Features of the NMR lead-
ing to this assignment were the down-field proton
(10.4 ppm) integrating as 1H characteristic of N1H of gua-
nine derivatives, the proton at C8 in its characteristic posi-
tion near 7.8 ppm, and a proton at 7.4 ppm integrating only
as 1H for N2H. The attachment N2H was most clearly seen
by the coupling of this proton and the methine proton of the
diarylmethyl group (6.0 ppm). This coupling was verified by
a 2D COSY experiment.
[5]
kdG/ko = (1/[dG])([TdG]/[TOH])
The results are plotted in Fig. 4, and show the same trend
as observed in the absolute rate constants, with a significant
increase in the selectivity ratio at higher pH. In terms of the
analysis of the previous sections, this ratio is given by
eq. [6] (see below), obtained by dividing eq. [3] by eq. [1].
The line drawn through the experimental points in Fig. 4
is based on a fit to this equation. Because of the number of
parameters involved, the fitting was performed by fixing the
values of Ka(T), Ka(G), and kw+:kw2+ at the values obtained
previously, 5.5 × 10–8, 5 × 10–10, and 0.256, respectively.
This left the three rate constant ratios as variables; values are
provided in Table 1. Also given in this table are values of the
same ratios calculated from the absolute rate constants mea-
sured with LFP. The values of k2n+:kw2+ obtained by the two
methods are not outside of experimental error. The two val-
ues of ka:kw are, however, statistically different, with the
LFP ratio greater than the product ratio in both cases. A pos-
sible explanation is provided in the Discussion.
With a sample of DdG1 available, the HPLC area ratio for
this peak relative to the alcohol was converted to a concen-
tration ratio, and then, with eq. [7], into a ratio of rate con-
stants.
Bis-(4-methoxyphenyl)methyl cation and deoxyguanosine —
Competition kinetics
These experiments were performed following the same
procedure described in the previous section, with the cation
(designated as D+) being obtained by solvolysis of the ace-
tate ester in solutions of 20% acetonitrile containing dG.
The HPLC traces showed the peak for the alcohol product
DOH and three peaks associated with a reaction with dG.
Since this cation does not generate a new stereocenter (or
C=C isomers), each of these peaks must represent a product
derived from a different position of attachment to the dG.
[7]
kdG1/ko = (1/[dG])([DdG1]/[DOH])
The pH dependence of this ratio is shown in Fig. 6. As
with the tamoxifen cation the selectivity is modest in acidic
solutions, but becomes much more significant at higher pH.
Figure 6 can be explained by a kinetic model where the
cation reacts with water in a pH-independent manner to form
(kn2+/kw2+)[H]2+ + (ka2+/kw2+)Ka(G)[H+ ] + (ka+/kw+)(kw+/kw2+)Ka(G)Ka(T)
[6]
kdG/ko =
([H+] + (kw+/kw2+)Ka(T))([H+ ] + Ka(G))
© 2002 NRC Canada

McClelland et al.
275
Fig. 5. pH dependence of the logarithm of the HPLC areas for
the four products observed in the reaction of the bis-(4-methoxy-
phenyl)methyl cation in 20% acetonitrile in the presence of 2′-
deoxyguanosine. The cation was obtained from the solvolysis of
bis-(4-methoxyphenyl)methyl acetate. The experiments were per-
formed with a constant concentration of dG (0.0096 M) and a
constant concentration of the acetate (100 µM).
Fig. 4. pH dependence of the logarithm of the ratio of rate con-
stants kdG:kw for the competition for the tamoxifen cation be-
tween 2′-deoxyguanosine and water. Temperature is 20°C, and
the solvent is 20:80 (v:v) acetonitrile–water with ionic strength
maintained at 0.10 M with sodium perchlorate. The circles are
the experimental data obtained by analysis of the products of the
ground-state solvolysis of the acetate 2c. The solid line was ob-
tained by fitting the data to eq. [6] as described in the text. The
dashed line is the ratio of the absolute rate constants measured
by LFP; this line was calculated as the difference between the
two lines drawn in Fig. 2.
where Ka(G) is the acidity constant of dG, kw is the rate
constant for the reaction with water, and k1n and ka1 are the
rate constants for reactions forming adduct DgD1. As above,
the subscripts “n” and “a” represent reactions of the neutral
and conjugate base forms of dG. The superscript “1” indi-
cates that these are reactions forming DdG1. Equation [8]
contains three variables, two rate constant ratios, and the
acidity constant. The experimental data were fit to this equa-
tion with all three parameters as adjustable parameters. The
results are provided in Table 1. It can be seen that the value
of the acidity constant is within experimental error the same
as values obtained by a spectroscopic analysis and a kinetic
analysis of the tamoxifen system.
Scheme 5.
kion
+
CH
DOAc
MeO
2
D+
kdG1[dG] kdG2[dG]
kdG3[dG]
ko
The other two dG adducts have not been identified. Under
acidic conditions where the neutral form of dG is reacting,
the area of the peak for DdG3 is 60% of that for DdG1.
However, both are minor components of the reaction mix-
ture. With the assumption that the HPLC sensitivity for
DdG3 is the same as that of DdG1, the ratio kn3:kw is only
0.7 M–1. This means that even at the highest concentrations
of dG attainable (~20 mM), the yield of DdG3 is only
around 1%. The peak for this adduct is not observed in the
basic solutions where the anion of dG is reacting. Either this
adduct is not formed by the reaction with the anion, or its
yield is very small in comparison with the other products.
DdG2 follows the pattern of the major adduct with a sig-
nificant increase relative to alcohol when the dG reacts as
the anion. The assumption of the same HPLC sensitivity re-
sults in ratios kn2:kw = 0.1 M–1 and ka2:kw = 45 M–1. While
the latter shows relatively good selectivity over the alcohol,
DdG1
DdG2
DdG3
DOH
O
N
N
N
HN
H
N
dRibose
Me
CH
O
DdG1
2
the alcohol, while adduct DgD1 is formed from both neutral
dG and its anionic conjugate base. This leads to eq. [8],
(k1n/kw)[H+ ] + (ka1/kw)Ka(G)
[8]
kdG1 / ko =
[H+ ] + Ka(G)
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276
Can. J. Chem. Vol. 80, 2002
Fig. 6. pH dependence of the logarithm of the ratio of rate con-
stants kdG:kw for the competition for the bis-(4-methoxyphenyl)-
methyl cation between 2′-deoxyguanosine and water. Tempera-
ture is 20°C, and the solvent is 20:80 (v:v) acetonitrile:water.
The circles are the experimental data obtained by analysis of the
products of the ground-state solvolysis of the bis-(4-methoxy-
phenyl)methyl acetate. The line was obtained by fitting the data
to eq. [8].
the order of 50 ns in water (34, 35) — the p-methoxybenzyl
cation, a benzylic system that has been studied with guanine
in the past (36–38) — lifetime of 5 ns (39) — and the
diphenylmethyl cation — lifetime of 800 ps (40). The parent
benzyl cation is too short-lived to exist in water (41).
The cations of this study clearly show significant differ-
ences in reactivity towards the neutral form of 2′-deoxy-
guanosine and its conjugate base. This difference is seen in
the guanine:water selectivities obtained by analyzing prod-
ucts of solvolysis reactions. It is also seen in the absolute
rate constants measured with LFP. A greater reactivity of the
conjugate base form of a nucleophile is hardly surprising.
Previous studies of guanine–electrophile interactions have
commented on this in qualitative terms (30, 36–38). We are
not aware of a quantitative study, particularly one where rate
constants and rate constant ratios were obtained for interme-
diate carbocations.
For the cations of this study, the difference between the
rate constants for the neutral form of dG and the conjugate
base is about three orders of magnitude. This results in very
different selectivities over the solvent reaction. Under acidic
conditions where it is the neutral form of dG that reacts, the
cations preferentially react with water even at the highest
concentrations of dG. Arylnitrenium ions, the reactive
electrophiles derived from arylamine carcinogens, provide
an interesting contrast here. These cations react very effi-
ciently with dG, with a high selectivity over the water reac-
tion (25–28). This reaction involves the neutral form of dG,
since the rate constant is unchanged over the pH range 3.5–
8.0 (25). (Studies of arylnitrenium ions with anionic dG are
currently in progress). The following comparison shows how
dramatic the difference is. When the tamoxifen cation (as
the T2+ form) reacts in acidic solutions containing 1 mM
dG, only 0.3% of the products are derived from the
nucleoside, 99.7% being the alcohol obtained from capture
by solvent. The percentage of adducts is even lower for the
bis-(4-methoxyphenyl)methyl cation under the same condi-
tions. The 2-fluorenylnitrenium ion has an aqueous lifetime
that is identical to that of T2+. When this cation reacts in
1 mM dG, 96% of the products are derived from the
nucleoside and only 4% from the solvent (24).
Changing to basic conditions where the dG has been con-
verted to its conjugate base results in much higher
selectivities towards the nucleoside. For example, the bis-(4-
methoxyphenyl)methyl cation reacting at pH 11 in the pres-
ence of 1 mM dG will form 60% dG adduct. This is obvi-
ously an attractive method for the preparation of adducts for
the purpose of structural characterization. The difference be-
tween the two forms of dG is such that by pH 7 to 7.5, the
majority of the reaction that does occur with the nucleoside
is the reaction with the small fraction of anion that is pres-
ent. This is also reflected in an improved selectivity over the
solvent reaction. As one example, the dG:water selectivity
for the bis-(4-methoxyphenyl)methyl cation at pH 7.3 has
increased to 13 from its acidic value of 1.2. This increase
occurs since 91% of the reaction with dG at pH 7.3 is occur-
ring via the conjugate base.
ka2:k1a is only 0.03. Thus the yield of DdG2 is never greater
than 3%.
Finally, we note the evidence that the reaction has pro-
ceeded via SN1 solvolyses. This takes the form of experiments
where the first-order rate constant for the disappearance of
DOAc was measured by HPLC. In 20% acetonitrile with the
pH at 9.5, rate constants of 0.0018 s–1 and 0.0017 s–1 were
measured for solutions with [dG] = 0 and [dG] = 0.0096, re-
spectively. The products in the latter are around 90% ad-
ducts. The observation that the rate constant is unchanged,
within experimental error, shows that the product-
determining step has occurred after the rate-determining
step; i.e., the reaction is SN1. A similar experiment was not
necessary with the tamoxifen system. The observation of
C=C isomers in the products shows that there must have
been a cationic intermediate.
Discussion
An attractive approach to model DNA–electrophile inter-
actions is to investigate the reaction with a monomer such as
2′-deoxyguanosine. In this study electrophiles were two
delocalized carbocations, the allylic cation derived from the
carcinogen tamoxifen, and the bis-(4-methoxyphenyl)methyl
cation. Lifetimes of these electrophiles in aqueous solution
are 10–200 ms. By carbocation standards, these are rela-
tively long-lived (24). Some relevant comparisons are the
benzylic cations derived from ring opening of the diol
epoxides of the carcinogen benzo[a]pyrene — lifetimes of
In the tamoxifen system, a comparison was made between
ratios of rate constants for the dG and water reactions ob-
tained by product analysis and ratios calculated from the ab-
solute rate constants measured with LFP. The two ratios for
© 2002 NRC Canada

McClelland et al.
277
Scheme 6.
Scheme 7.
T
H
N
H
N
O
N
kau[dG-]
N
N
H
H
N
T+
O
N
O
N
N
N
N
N
H
+
ka [dG-]
s
kw
H2 N
H
H
dRibose
N
N
O
O
Unstable
H
ROH
O
N
H
N
H
R
N
N
R+
HN
TH
N
N
H
H
dRibose
O
O
O
N
O
Stable
H
O
H
H
H
-
N
N
O
H
H
H
H
N
N
N
H
the competition involving the neutral form of dG and water
were not outside of experimental error (although this error
was quite large). On the other hand, the ratios for the anion
of dG vs. water were statistically different, with a greater ra-
tio being obtained by the LFP experiments. The difference is
almost a factor of three. There is a very large difference in
the time scales of the two experiments. The LFP measure-
ments occurred on the microsecond time scale. The product
analyses, on the other hand, were carried out after 16–24 h,
the time required for complete solvolysis of the acetate. This
leads to an explanation whereby the reaction of the cation
with dG anion proceeds reversibly to an unstable adduct in
addition to its irreversible reaction forming the stable prod-
uct, the N2 adducts (Scheme 6). The quenching by the dG
anion measured as the second-order rate constant in the LFP
experiment would then correspond to the sum of the rate
constants for the formation of the unstable and stable prod-
ucts (kau + kas). The rate constant in the competition experi-
ment would only refer to the latter (kas). Thus, the ratio for
the latter would be smaller.
R
H
R+
could well survive the time scale of the LFP experiment, but
it would not be around after several hours.
Despite the fact that the negative charge in the dG anion
is at O6 (and N1), there is still a remarkably large rate con-
stant for the reaction that forms the N2 adduct. This effect is
best seen in the competition kinetic experiments with the
bis-(4-methoxyphenyl)methyl cation where a single carbo-
cation was involved and the N2 adduct:water product ratio
was measured and converted to a rate-constant ratio. The lat-
ter changes from 1.2 (kn1:kw) for neutral dG to 1500 (ka1: kw)
for the anion. The rate constant kw is the same in the two ra-
tios, with a value of 1.3 × 105 s–1 in this solvent (29). Thus,
the rate constant k1n for the formation of the N2 adduct with
neutral dG is 1.6 × 105 M–1 s–1, and k1a for the anion is 2.2 ×
108 M–1 s–1, over three orders of magnitude greater.
Observations of enhanced formation of N2 adducts in the
anion of guanine have been made previously (36–38), al-
though it is not clear if free carbocations were involved in all
cases. The N2 adduct is also the principle product of the re-
action of DNA with the tamoxifen carbocation (20–22), as
well as a number of other electrophiles (1). In DNA one of
the NH2 protons is involved in hydrogen bonding to the
complementary cytosine. This has led to the recent proposal
(42) of a mechanism whereby the proton in the hydrogen
bond transfers to the neighboring cytosine as the nitrogen–
electrophile bond is forming (top reaction of Scheme 7). In
other words, the neighboring cytosine acts as a base to assist
the reaction of the NH2 group by removing one of its pro-
tons as the reaction proceeds.
A similar mechanism would explain the high reactivity of
the NH2 group in the guanine anion. Here the base would be
the adjacent partially negatively charged nitrogen at N1. The
proton at NH2 could be transferred directly to N1 through a
four-center transition state or, perhaps, through the interven-
tion of surrounding water molecules, as shown in the bottom
mechanism in Scheme 7. In any case the reaction would lead
directly to the product, and there with little or no build-up of
positive charge at N2.
This scenario requires that the unstable adduct be “stable”
on the time scale of the LFP experiment, i.e., that it last at
least half a millisecond. On the other hand, it must have
completely reacted before the products were analyzed. A
structure that would satisfy these requirements is the adduct
at O6 shown in Scheme 6. Such an adduct is an obvious
product of the reaction of a carbocation and the anion of
guanine, since it is at O6 that the majority of the negative
charge will reside. O6-(p-methoxybenzyl)guanosine, the an-
alog derived from the p-methoxybenzyl cation, has been
shown to be unstable in aqueous solution (36). Its decompo-
sition was pH independent at high pH, and the reaction was
proposed to involve ionization to the p-methoxybenzyl cat-
ion and the guanosine anion. The half-life for this ionization
was about 0.5 h. Both the tamoxifen cation and the bis-(4-
methoxyphenyl)methyl cation are more stable than p-meth-
oxybenzyl, and thus ionizations that result in these cations
will be considerably faster. This can be seen in the observa-
tion that the acetate esters derived from the two solvolyze at
a reasonable rate in water, while p-methoxybenzyl acetate is
very stable under these conditions. Thus, an O6 adduct
formed with the tamoxifen cation (or the bis-(4-methoxy-
phenyl)methyl cation) is expected to be quite short-lived. It
© 2002 NRC Canada

278
Can. J. Chem. Vol. 80, 2002
of absorbance vs. time to the exponential equation. Rate
constants for the solvent reaction were obtained as the aver-
age of five to eight kinetic runs. For the decay of the tam-
oxifen cation in the presence of 2′-deoxyguanosine, 12–16
kinetic runs were averaged. The pH of the solutions was es-
tablished by the use of perchloric acid (pH < 3), sodium hy-
droxide (pH > 11) with acetate, phosphate, tris, and
carbonate buffers in between. When a buffer was employed,
serial dilutions were performed to provide four to five solu-
tions of the same pH (as verified by recording the pH with a
pH meter). The observed rate constants were linear in the
buffer concentration, and the value of ko, the rate constant in
the absence of the buffer, was obtained as the intercept at
zero buffer concentration. The experiments with dG present
were performed with dilute solutions of the buffers (total
concentration less than 0.01 M) with four to five concentra-
tions of dG ranging up to 20 mM. The rate constants kdG
were obtained by linear regression of the plot of the ob-
served rate constants against the added concentrations of
dG.
The value of the acid dissociation constant of 2′-deoxy-
guanosine was measured in 20% acetonitrile, ionic
strength = 0.1 M, 20°C by preparing a series of buffer solu-
tions to which had been accurately added the same constant
concentration (100 µM) of the nucleoside. Spectra were re-
corded with a HewlettPackard diode array spectrometer, and
titration curves of absorbance vs. pH constructed at several
wavelengths. These were fit to the equation A = (Aacid[H+] +
AbaseKa)/([H+] + Ka), Aacid and Abase are the plateau
absorbances at low pH and high pH, respectively.
HPLC experiments were performed with a Waters HPLC
system comprising a Waters 600E system controller, a Wa-
ters 486 tunable absorbance detector set at 238 nm, a Waters
746 data module, and a Waters U6K injector. The column
employed was a Waters Symmetry C18 column of 5 µm par-
ticle size and dimensions 4.6 mm × 150 mm; the eluting sol-
vent was first passed through a guard column of the same
packing material and 4.6 × 25 mm dimensions. Solvents
were sparged with helium.
Solutions of α-acetoxytamoxifen and bis-(4-methoxy-
phenyl)methyl acetate were prepared as described above, by
adding the concentrated stock solution in acetonitrile to a so-
lution of 20% acetonitrile. The entire set of experiments
with each acetate were performed with the same stock solu-
tion to ensure that the final solutions were all of the same
concentration, around 100 µM. The solutions were allowed
to stand at room temperature for a period of 16–24 h, suffi-
cient time that the signal of the acetate in the HPLC had dis-
appeared.
Experimental section
α-Hydroxytamoxifen and α-acetoxytamoxifen ((E)-1-[4-[2-
(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-buten-3-ol and
its acetate ester) were available from a previous study (43) as
were bis-(4-methoxyphenyl)methanol and bis-(4-methoxy-
phenyl)methyl acetate (44).
N2-Bis-(4-methoxyphenyl)methyl-2′-deoxyguanosine was
isolated from a scaled-up reaction. 2′-Deoxyguanosine
(286 mg, 1 mmol) was added to a mixture of 20 mL
acetonitrile and 30 mL of water, and 0.9 mL (0.9 mmol) of
sodium hydroxide was added. A solution of bis-(4-methoxy-
phenyl)methyl acetate (200 mg, 0.7 mmol) in 5 mL of
acetonitrile was then slowly added over 8 h, and the mixture
left to stand at room temperature, with periodic monitoring
by HPLC. When, after 3 days, this indicated that there was
no ester remaining, the pH was adjusted to about 5 by the
addition of a small amount of 1 M HCl, and the mixture
concentrated with a rotary evaporator until only water re-
mained. At this point, a precipitate had formed, which was
isolated by filtration. This solid was purified by column
chromatography on silica gel with 100% ethyl acetate as the
eluting solvent. Fractions were analyzed by HPLC and the
fractions containing the adduct were combined and the sol-
vent removed. The solid obtained was purified by dissolving
it in a small amount of methanol and then induction of
precipitaton by the addition of water. The solid showed only
a single peak on HPLC corresponding to the adduct identi-
fied as DdG1. This was characterized as N2-bis-(4-methoxy-
phenyl)methyl-2′-deoxyguanosine by 1H NMR, with coupled
1
protons being identified by gCOSY experiments. H NMR
(500 MHz, DMSO-d6) δ: 2.05–2.10 (m, 1H, H2a′), 2.55–2.6
(m, 1H, H2b′), 3.40–3.45 (m, 1H, H5a′), 3.55–3.60 (m, 1H,
H5b′), 3.70 (s, 6H, OCH3), 3.80 (m, 1H, H4′), 4.30 (m, H3′),
4.83 (t, 1H, C5′-OH), 5.22 (d, 1H, C3′-OH), 6.02 (d, 1H, Cα-
H), 6.08 (t, 1H, H1′), 6.94 (d, 4H, Ar-H), 7.21 (d, 4H, Ar-
H), 7.36 (d, 1H, N2-H), 7.84 (s, 1H, H8), 10.35 (s, 1H, N1-
H). Mass spectra of this material, even with soft ionization
techniques, showed only a very small peak for the molecular
ion (m/z = 492).
LFP experiments involved ca. 20 ns pulses at 248 or
308 nm (60–120 mJ per pulse) from a Lumonics excimer la-
ser. A pulsed xenon lamp provided the monitoring light. Af-
ter passing through a monochromator, the signal from the
photomultiplier tube was digitized and sent to a computer
for analysis. Stock solutions of the acetates (α-
acetoxytamoxifen and bis-(4-methoxyphenyl)methyl acetate)
of concentration 20–50 mM were prepared in acetonitrile.
Immediately before irradiation a small volume was added to
the solution of interest (usually in a 25 mL volumetric
flask). Final concentrations of the acetates were 50 µM (α-
acetoxytamoxifen in water) and 200–300 µM (α-
acetoxytamoxifen in 20% acetonitrile and bis-(4-methoxy-
phenyl)methyl acetate). The solutions were irradiated with
laser light at 248 nm (when studying the reaction with the
solvent) and at 308 nm (α-acetoxytamoxifen in the presence
of 2′-deoxyguanosine). The signals of the transient carbo-
cations were monitored at 460 nm (tamoxifen cation) and
500 nm (bis-(4-methoxyphenyl)methyl cation). These
showed excellent first-order decays. Observed first-order
rate constants were obtained by fitting the experimental data
The products from the α-acetoxytamoxifen were analyzed
at 260 nm. Elution at 1 mL min–1 involved: (i) an initial
0.5 min isocratic run at 25% acetonitrile – 75% acetate buffer
(pH 4.5, 0.05 M); (ii) a linear gradient to 85% acetonitrile –
15% acetate buffer over 13.5 min; (iii) a 3 min isocratic run at
85% acetonitrile – 15% acetate buffer. Peaks were observed at
1 to 2 min (dG), 10.5, 10.6 (overlapping (E)-TdG isomers),
11.8, 11.9 (overlapping (Z)-TdG isomers), 12.8 ((E)-TOH)),
and 13.5 ((Z)-TOH). The areas for these peaks were con-
verted into concentration ratios as follows:
[9]
[TdG]/[TOH] = (1/RF)(Area(TdG)/Area(TOH))
© 2002 NRC Canada

McClelland et al.
279
where [TdG], [TOH], Area(TdG), and Area(TOH) are the
sums of the concentrations and HPLC areas of the four dG
adducts and the two alcohols, respectively, and RF is the rel-
ative HPLC response factor. The latter was obtained as fol-
Acknowledgment
Financial support from the Natural Sciences and Engi-
neering Research Council of Canada (NSERC) is gratefully
acknowledged.
lows. For
a series of solutions of the same total
concentration of products, eq. [10] applies:
References
[10] [TdG] + [TOH] = Area(TdG)/F(TdG)
1. A. Dipple. Carcinogensis, 16, 437 (1995).
2. G.M. Williams, M.J. Iatropoulos, M.V. Djordjevic, and O.P.
Kaltenberg. Carcinogenesis, 14, 315 (1993).
+ Area(TOH)/F(TOH) = [TOAc]o
3. P. Greaves, R. Groonetilleke, G. Nunn, J. Topham, and T.
Orton. Cancer Res. 53, 3919 (1993).
4. G.C. Hard, M.J. Iatropoulos, K. Jordan, L. Radi, O.P.
Kaltenberg, A.R. Imondi, and T. Orton. Cancer Res. 53, 4534
(1993).
5. M.A.-F. Seoud, J. Johnson, and J.C. Weed. Obstet. Gynecol.
82, 165 (1993).
6. B. Fischer, J.P. Costantino, C.K. Redmond, E.R. Fisher, D.L.
Wickerham, and W.M. Cronin. J. Natl. Cancer Inst. 86, 527
(1994).
where F(TdG) and F(TOH) are the HPLC sensitivities of
the adducts and alcohols, respectively, and [TOAc]o is the
constant total concentration of products determined by the
initial concentration of acetate ester. Rearrangement gives
eq. [11]:
[11] Area(TdG) = –(F(TdG)/F(TOH))Area(TOH)
+ F(TdG)[TOAc]o
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where F(TdG):F(TOH) is equal to RF. A plot of the total
area of the adducts was indeed linear in the sum of the area
of the two alcohols. The slope (–2.2) was the negative of RF.
It should be noted that there are two assumptions in this
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= (1/RF)(Area(DdG1)/Area(DOH))
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