Reactivity of p-nitrostyrene oxide as an alkylating agent. A kinetic approach to biomimetic conditions

Article abstract of DOI:10.1039/c1ob05909b

The alkylating potential of p-nitrostyrene oxide (pNSO) - a compound used as a substrate to study the activity of epoxide hydrolases as well as in polymer production and in the pharmaceutical industry - was investigated kinetically. The molecule 4-(p-nitr

Full text of DOI:10.1039/c1ob05909b

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PAPER
Reactivity of p-nitrostyrene oxide as an alkylating agent. A kinetic approach
to biomimetic conditions
Marina Gonza´lez-Pe´rez, Rafael Go´mez-Bombarelli, M. Teresa Pe´rez-Prior, Jose´ A. Manso,
Isaac F. Ce´spedes-Camacho, Emilio Calle and Julio Casado*
Received 6th June 2011, Accepted 14th July 2011
DOI: 10.1039/c1ob05909b
The alkylating potential of p-nitrostyrene oxide (pNSO)—a compound used as a substrate to study the
activity of epoxide hydrolases as well as in polymer production and in the pharmaceutical
industry—was investigated kinetically. The molecule 4-(p-nitrobenzyl)pyridine (NBP), as a model
nucleophile for DNA bases, was used as an alkylation substrate. In order to gain insight into the effect
of the hydrolysis of pNSO, as well as the hydrolysis of the NBP-pNSO adduct on the pNSO alkylating
efficiency, these two competing reactions were studied in parallel with the main NBP-alkylation
reaction. The following conclusions were drawn: (i) pNSO reacts through an SN2 mechanism, with NBP
to form an adduct, pNSO-NBP (AD). The rate equation for the adduct formation is: r = d[AD]/dt =
k
alk[NBP][pNSO]- kAhyDd [AD] (kalk, and kAhyDd being the alkylation rate constant and the NBP-pNSO
adduct hydrolysis rate constant, respectively); (ii) the alkylating capacity of pNSO, defined as the
fraction of initial alkylating agent that forms the adduct, is similar to that of mutagenic agents as
effective as b-propiolactone. The instability of the pNSO-NBP adduct formed could be invoked to
explain the lower mutagenicity shown by pNSO; (iii) the different stabilities of the a and b-adducts
formed between NBP and styrene oxides show that the alkylating capacity f = kalk[NBP]/(kalk[NBP] +
k
hyd ) (khyd being the pNSO hydrolysis rate constant) as well as the alkylating effectiveness, AL = f /khAyDd
,
are useful tools for correlating the chemical reactivity and mutagenicity of styrene oxides; (iv) a
pNSO-guanosine adduct was detected.
Introduction
Experimental
Substituted styrene oxides are extensively used in the pharma-
ceutical synthesis of antileukemic, antibacterial, antianginal and
antiarrhythmic drugs1–5 since they and their corresponding glycols
afford chiral building blocks to be obtained that are useful in
the synthesis of biologically active molecules. For such purposes,
epoxide hydrolases are used as a cheap and available tool.6,7 p-
Nitrostyrene oxide (pNSO), used in the polymer industry as well
as in the synthesis of compounds such as the antianginal and
The NBP alkylation reaction by pNSO was monitored spectropho-
tometrically at l = 560 nm, where the adduct spectrum shows
a maximum. Because of the insolubility of NBP in water, the
alkylation reactions were performed in a 7 : 3 (vol.) water/dioxane
medium. Aliquots of the alkylation mixture (2.4 ml) were added
over time to a cuvette containing 0.6 ml of 99% triethylamine. The
addition of the base stopped the alkylation reaction and generated
a blue colour due to the deprotonation of the adduct (Scheme 1).
R
antiarrhythmic Nifenalolꢀ,5,8,9 is the main standard substrate used
to investigate the mechanism of action of these enzymes, because
of its slow rate of spontaneous hydrolysis.10–13
Since to our knowledge: i) the alkylating potential of pNSO has
not been investigated in depth considering neither the stability of
pNSO itself nor that of its adduct with the alkylation substrate,
factors which should modulate the efficiency of pNSO as an
alkylating agent; and ii) no clear correlation has been observed to
date between the chemical reactivity and mutagenicity of styrene
oxides,14–16 here we were prompted to address these issues.
Scheme 1 Method for monitoring NBP alkylation by pNSO.
Departamento de Qu´ımica f´ısica, Universidad de Salamanca, E-37008,
Salamanca, Spain. E-mail: jucali@usal.es; Fax: +34 923 294574; Tel: +34
923 294486
Acetate, phosphate and borate buffers were used to maintain
pH constant in the 4–9 range.
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Table 1 Theoretical energy barriers for the reaction of pyridine with
For each kinetic run of the pNSO hydrolysis reaction, 20
ml of the stock solution (epoxide (2.4–4.8)¥10-3 M in dioxane)
was added to a cuvette containing 3.0 ml of thermostatted
reaction mixture (water+HClO4+dioxane). The rate of pNSO
hydrolysis was determined spectrophotometrically by monitoring
absorbance at l = 310 nm. The reaction was followed until no
change in absorbance was observed.
A Shimadzu LC20A system coupled with a Shimadzu SPD-
M20A diode array detector was used to follow the kinetics of
pNSO hydrolysis. Chromatographic separation was achieved on a
Mediterranean Sea C18 column (25 mm ¥ 1 mm, 5 mm). Mobile
phase A was acetate buffer (pH = 4.75 and 0.1 M) and mobile
phase B was acetonitrile. 35% B was maintained for 5 min, after
which a gradient up to 90% B was run over 10 min and later held
for 20 min. The flow rate was set at 1 ml min-1 and 200 ml of the
hydrolysis mixture was injected.
Computational calculations were carried out using Gaussian
03W. Energy barriers were obtained by optimization of the
geometries of the reactants and transition states in the DFT-
B3LYP/6-31++G(d,p) basis set and the structures were charac-
terized by harmonic analysis. The IEFPCM model Self Consistent
Reaction Field (SCRF) with the default parameters was used in
the solvation calculation.
Absorbance measurements were performed with a Shimadzu
UV-2401 PC spectrophotometer with a thermoelectric six-cell
holder temperature control system. Temperature was kept con-
stant ( 0.05 ◦C) with a Lauda Ecoline RE120 thermostat. For
pH measurements, a Metrohm 827 pH-meter was used.
Negative and positive-mode electrospray ionization mass spec-
tra were recorded on a Waters ZQ4000 spectrometer by direct
injection.
several epoxides
Epoxide
Addition
DE (kJ mol-1)
Styrene oxide
p-Nitrostyrene oxide
Propylene oxide
Butylene oxide
b
a
b
a
b
a
b
91.4
93.5
86.9
97.7
89.9
104.7
105.1
as the nucleophile, the substituent of the styrene oxide and the
medium.17–19
It is known that a high b- to a-adduct concentrations ratio
results in the reaction of pNSO with nucleophiles20–23 (this ratio
increases to 91% when pyridine is used as a nucleophile.)14
The energy barriers between the reactants and the transition
states for the reaction between pyridine and several epoxides were
calculated and are reported in Table 1. In the reaction with NBP
(a substituted pyridine), no evidence of a-adduct formation was
found according to analyses of the mass spectra and fragmentation
patterns (Fig. 1a). The absorption spectra, as well as the kinetic
profiles for adduct formation, are in agreement with these results
(Fig. 1b).
All kinetic runs were performed in triplicate.
In order to study the formation of the pNSO-guanosine adduct,
guanosine (9.6 ¥ 10-4 M) was treated with pNSO (1.1 mM) in 1 : 4
(vol.) dioxane/acetate buffer (0.02 M and pH 3.99) by incubation
at 45 ◦C for 5 days. The reaction mixture was separated by HPLC.
A gradient consisting of 20 mM ammonium formate, pH 4.6, (A)
and acetonitrile (B) with a flow rate 0.2 ml min-1 was used. Initial
elution was 100% A, followed by an increase in the proportion of
B to 50%, which was maintained for 5 min and increased to 100%
in 1 min. Peak detection was carried out by UV-absorption with a
diode array detector and by negative electrospray ionization mass
spectrometry. An HPLC Waters Alliance 2795 equipped with a
C-18 column was used (Atlantis 100 ¥ 2.1 mm, 3.5 mm).
Water was deionized with a MilliQ-Gradient (Millipore).
Guanosine (98%), (R)-pNSO (99%) and NBP were from Sigma–
Aldrich (98%). Dioxane was from Panreac.
Fig. 1 a) Positive-mode mass spectra obtained for the alkylation mixture
after 10 days in NBP excess. T = 37.5 ◦C; pH = 6.78; [NBP]o = 1.56 ¥ 10-2 M;
[pNSO]o = 4.93 ¥ 10-5 M. b) Kinetic profiles obtained in 7 : 3 water/dioxane
(vol) media with different acidities, T = 37.5 ◦C; l = 560 nm; pH = 4.43;
[NBP]o = 1.29 ¥ 10-2 M ( ); pH = 6.14; [NBP]o = 1.56 ¥ 10-2 M ( ).
᭹
᭺
Since the alkylation reaction of NBP by pNSO occurs in
concurrence with the hydrolysis reactions of pNSO and of the
b-NBP-pNSO adduct (AD) (Scheme 2), in the kinetic treatment
of the NBP alkylation reaction it is necessary to take into account
these side reactions.
Numerical treatment of the data was performed using the 7.1.44
Data Fit (Oakdale Engineering) software.
The reaction conditions are specified in the captions of the
Figures, Schemes and Tables.
Since pH remains constant over time, the adduct formation rate
can be expressed as
d AD
[
]
(1)
r =
= kalk NBP pNSO − k AD AD
[ ][ ] [ ]
hyd
dt
Results
AD
hyd
where kalk is the NBP alkylation rate constant and k is the
hydrolysis rate constant of the adduct.
b-adduct formation
NBP was in large excess, such that its concentration was
assumed to remain constant and a pseudo-first order rate constant
was defined as k¢alk = kalk[NBP].
The nucleophilic ring-opening reaction of substituted styrene
oxides can proceed at two sites: a-CH and b-CH2 (Scheme 1).
The ratio of products formed depends on different factors, such
Eqn (2) gives the concentration of pNSO.
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Table 2 Rate constants and f values for the NBP alkylation reaction by
pNSO as a function of temperature
T/◦C
102kalk (M-1 min-1)a
f
30.0
32.5
35.0
37.5
40.0
42.5
0.84 0.05
1.03 0.06
1.28 0.06
1.49 0.09
1.8 0.1
0.92
0.87
0.85
0.84
0.84
0.83
2.1 0.1
a pH = 7.0. The relative error associated with f values is 10%.
DA
Dt
= kalkel NBP n pNSO m
] [
(5)
vo = lim
[
]
o
o
t→o
Scheme 2 Reactions involved in the alkylation of NBP by pNSO.
k ′ +k
t
)
(
alk
hyd
pNSO = pNSO e−
(2)
Molar absorption coefficient of the NBP-pNSO adduct
[
]
[
]
o
Determination of the molar absorption coefficient of the adduct
(e) is of interest because it allows the NBP alkylation rate constant
(kalk) to be determined simply by measuring measurement of the
initial rate (eqn (5)).
[pNSO]o being the initial concentration of pNSO and khyd the
observed hydrolysis rate constant.
The absorbance of the adduct at time t, AAD (eqn (3)), was
obtained by substitution of eqn (2) into eqn (1), subsequent
integration and applying the Lambert–Beer law.
At t = • and pH £ 4.5, eqn (3) can be written as eqn (6):
khyd
[pNSO]o
1
1
AD
=
+
(6)
′
⎡
⎢
⎢
⎣
⎤
⎥
⎥
⎦
−k
t
kalk pNSO el
[
]
AAD
=
e
−e− k ′
+k
t
)
(
hyd
alk
hyd
o
A∞
kalkel [NBP] el
(3)
′
k
alk + khyd −k AD
(
)
hyd
Experiments performed at pH = 4.43 afforded e = (3.7 0.2) ¥
104 M-1 cm-1(Fig. 2b).
e being the adduct molar absorption coefficient, and l the cuvette
light path.
Eqn (3) can be written in terms of three parameters: a, b, and c,
whose values can be obtained by non-linear optimization.
Alkylation rate constant
The kalk values obtained by the IRM are in accordance with those
resulting from parameter b (eqn (3)). No variations in kalk values
with pH were observed in the 4.5–9 range. A good fit of the kalk
values at different temperatures (Table 2) to the Arrhenius and
Eyring–Wynne–Jones equations was found (eqn (7)).
a
(4)
AAD
=
−ct −e−bt
⎡ ⎤
e
⎢ ⎥
⎣
⎦
b −c
The observed kinetic profiles (Fig. 1b) show a good fit to eqn (4)
and revealed that adduct hydrolysis in acid media (pH < 4.5) can
be considered negligible (c~0). The reaction orders with respect
to [pNSO] and [NBP] (m and n, respectively; see eqn (5)) were
determined with the Initial Rate Method (IRM).24 Two series of
experiments were carried out varying the concentration of each
reactant. Fig. 2a shows that m = n = 1.
k
D‡So
R
k
D‡ H o
RT
(7)
ln
=
+ ln
−
T
h
The activation parameters were obtained from the slope and
intercept. They are similar to those obtained by Laird and Parker
with p-bromo and p-methylstyrene oxides using benzylamine as a
nucleophile (Table 3).20
Table 3 Activation parameters for the SN2 alkylation and spontaneous
hydrolysis reactions of several compounds
Reaction Compound
D‡H◦ (kJ mol-1) -D‡S◦ (J K-1mol-1)
Alkylation pNSOa
54
2
139
190
157
81 18
99 25
156
95
5
p-Bromostyrene oxideb 42.3
p-Methylstyrene oxideb 54.9
Hydrolysis pNSOa
64
79
59
79
6
8
1
1
Ethylene oxidec,d
Diketenee
BPLe
5
Fig. 2 a) Reaction orders with respect to each reagent (R) (᭡) NBP
3
-[pNSO]o = 3.9 ¥ 10-4 M- and ( ) pNSO -[NBP] = 1.54 ¥ 10-2 M- in
᭹
o
Values taken froma This work. 7 : 3 water/dioxane medium; pH = 7,
[NBP]o = 1.49 ¥ 10-2 M. b Ref. 20. The nucleophile used was benzylamine.
c Ref. 25. d Ref. 26. e Ref. 27.
7 : 3 water/dioxane; T = 37.5 ◦C; pH = 6.97. b) Determination of the
NBP-pNSO adduct molar absorption coefficient in 7 : 3 water/dioxane.
T = 37.5 ◦C; pH = 4.43; [pNSO]o = 4.93 ¥ 10-5 M.
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Table 4 pNSO Hydrolysis catalytic rate constant as a function of the
composition of the medium; T = 37.5 ◦
These values were obtained by direct spectrophotometry (l =
310 nm). Absorbance (A) at time t was the sum of the epoxide and
the glycol contributions (eqn (11)).
C
+
Water/dioxane (vol.)
Dielectric constant (e)a
kH (M-1 min-1)
t
A = l[pNSO]o[epNSG + (epNSO - epNSG)e-k
]
(11)
hyd
10 : 0
9 : 1
8 : 2
74.0
65.5
57.1
1.09 0.06
0.70 0.06
0.53 0.04
In strongly acidic media (pH £2.6; khyd = kH+[H+]), the catalytic
constant kH+ for each medium is given by the slope of the plot
a Values taken from ref. 29.
k
hyd /[H+] (experiments performed for each medium at different
pH-values revealed first order (a = 1) with respect to [H+]; see Fig.
4a).
Hydrolysis of p-nitrostyrene oxide
Since pNSO undergoes acid and base hydrolysis (pH < 4 and pH
> 12, respectively),28 the hydrolysis rate constant is the sum of
the spontaneous hydrolysis rate constant, kohyd = kH2O[H2O], and the
catalytic hydrolysis rate constants (eqn(8)):
-
khyd = kohyd + kH + kOH [OH ]
+
-
(8)
Acid-catalyzed hydrolysis
The hydrolysis reaction in 7 : 3 (v/v) water/dioxane (pH = 3) was
followed by HPLC separation and UV detection. The evolution
of the chromatograms over time and the kinetic profiles are cor-
responding to that of the glycol, appeared in the chromatograms
(Fig. 3). The reaction kinetics was followed by monitoring (l =
254 nm) the decrease in the pNSO peak area at a retention time of
tR = 28 min (eqn (9)) as well as the increase in that of pNSG (tR =
20 min) (eqn (10)) over time:
Fig. 4 a) Variation in the pNSO hydrolysis rate constant with the acidity
of the medium in water/dioxane media 10 : 0 ( ), 9 : 1( ) and 8 : 2 (᭢);
᭹
᭺
T = 37.5 ◦C. b) Influence of [OH-] on kAD
.
hyd
Spontaneous hydrolysis reaction
The pNSO hydrolysis rate constant for the spontaneous reaction
(khyd = kohyd ; pH = 4.5–7) was calculated from the parameter b of
eqn (4), such that khyd = (6.3 0.6) ¥ 10-5 min-1 (T = 37.5 ◦C).
t
AreapNSG = AreapNSG•(1 - e-k
)
(9)
hyd
As a consequence, a value of kH = (1.6
0.2) ¥ 10-6 M-1
AreapNSO = AreapNSOoe-k
t
O
hyd
2
(10)
min-1 (T = 37.5 ◦C; [H2O] = 38.9 M) was obtained. Activation
parameters were provided by the fit of kohyd at different temperatures
to the Arrhenius and Eyring-Wynne-Jones equations respectively
(Table 3). The negative entropies of activation are consistent with
a bimolecular nucleophilic substitution (SN2-type mechanism),
as expected for a styrene oxide with an electron-withdrawing
substituent in the ring.17–21
Since in acid media the variation in absorbance along time in
7 : 3 water/dioxane is too small to be followed spectrophotomet-
rically, and since the reaction rate (T = 37.5 ◦C) is too fast to
be followed by HPLC separation and UV detection, the pNSO
hydrolysis reaction rate constants were obtained indirectly.
The hydrolysis catalytic coefficient in 7 : 3 water/dioxane, kH+
=
(3.2 0.1) ¥ 10-1 M-1 min-1 (T = 37.5 ◦C), was determined by
extrapolation from its values in media with higher water ratios
(Table 4).
Efficiency of the pNSO as an alkylating agent
The alkylating capacity of pNSO can be defined as the fraction (f )
of initial pNSO forming the adduct (eqn (12)):
kalk NBP
[
]
f =
(12)
k
NBP + k
]
hyd
[
(
)
alk
The f values found at different temperatures and in media with
different acidities are shown in Tables 2 and 5, respectively.
Hydrolysis of NBP-pNSO adduct
The hydrolysis of the NBP-pNSO adduct is base-catalyzed (Fig.
4b). The values of the hydrolysis rate constants were calculated
from the parameter c of eqn (4). Since eqn (13) gives
Fig. 3 a) Chromatogram of the temporal evolution of acid-catalyzed
hydrolysis, l = 310 nm. b) Kinetic profiles obtained by following peak area
variation for pNSO and its hydrolysis product over time, l = 254 nm. T =
25.0 ◦C; pH = 2.99; [pNSO]o = 4.93 ¥ 10-5 M.
AD
AD
hyd
−
AD
⎡
⎤
OH + khyd,o
⎥
c= k = k
(13)
hyd
⎢
⎣
−
⎦
OH
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Table 5 Influence of pH on f and AL; T = 37.5 ◦
C
Table 6 Alkylating capacity of pNSO compared with that of other
alkylating agents
pH
f
103AL (min)
Alkylating agent
f
Mutagenicityg
LD50 (M)h
4.50
4.97
5.42
5.91
6.11
6.29
7.17
0.73
0.76
0.77
0.75
0.74
0.86
0.84
216
72
39
22
15
19
3.5
b -Propiolactonea
b-Butyrolactonea
Alkyldiazonium ionsb
Diketenec
0.76
0.62
0.6–0.9
1
0.13
0.82
(b) 0.55
(a) 0.06
4.5
n.a.
0.32–11
~ 0
Acetonitrile oxided
p-Nitrostyrene oxidee
2.00 ¥ 10-3
5.70 ¥ 10-3
Styrene oxide f
0.22
[NBP] = 1.49 ¥ 10-2 M. The errors associated with f and AL values are
about 10%.
7 : 3 water/dioxane medium, [NBP]o = 2.0 ¥ 10-2 M.a Values taken from ref.
34. b Ref. 35. c Ref. 27. d Ref. 36. T = 35.0 ◦C. e This work; pH = 7.17; T =
37.5 ◦C. f Unpublished. pH = 6,99; T = 37.5 ◦C. g Ref. 37. Values expressed
as the increase in the number of 6-thioguanine-resistant mutants per 106
viable cells over the control values per mg ml-1. h Ref. 14 Mutagenicity in
Salmonella typhimurium TA100.
the slope and intercept of the k /[OH-] plot allow the values of
AD
hyd
AD
hyd
AD
hyd
k
= (1.53 0.07) ¥ 103 M-1 min-1 and k = (2.2 0.5) ¥ 10-5
-
OH
min-1 to be determined.
Discussion
that of b-propiolactone: the most carcinogenic lactone (BPL, see
Table 6).34
The presence of the nitro group in the para position of styrene
oxide governs the reactivity of pNSO. Whereas very little difference
between the energy barriers for the b- and a-transition states in
the reaction with pyridine (Table 1) were obtained in silico for
styrene oxide in the case of p-nitrostyrene oxide, this difference
is as large as that due to propylene oxide (PO). The difference
of ~10 kJ mol-1 (ca. a hundred-fold decrease in the reaction rate)
suggests that the attack on the b-position is favoured. This helps to
understand the fact that alkylation reactions with SO usually yield
a mixture of both adducts,19,30,31 whereas PO only reacts through
the b position31,32 and it also suggests the negligible formation of
an a-adduct by pNSO. Several experimental findings have also
shown that the NBP alkylation reaction by pNSO occurs through
the b position.
The activation parameters and the reaction orders revealed
that spontaneous pNSO hydrolysis and alkylation reactions occur
through SN2 mechanisms, respectively, as expected. The reaction
between styrene oxide and guanosine at position N7 also takes
place through this mechanism.33 In the pH 4.5–7 range, these
reactions are not influenced by the acidity of the medium (Fig. 5)
and, consequently, neither is the alkylating capacity of pNSO:
f . The value of this parameter, only slightly influenced by
temperature, (Table 2), indicates that under all the conditions
studied 80–90% of the initial amount of pNSO participated in the
alkylation reaction, which points to the strong alkylating capacity
of pNSO. It should be noted that pNSO is a strong alkylating
agent, whose f value for NBP alkylation reaction is similar to
Unlike that lactone, pNSO forms an unstable adduct with the
alkylation substrate. This makes it necessary to evaluate the net
effectiveness of pNSO as an alkylating agent; that is, to take
into account the adduct hydrolysis reaction, of the same order
of magnitude as the alkylation reaction at cellular pH (Fig. 5). To
do so, we defined adduct life (AL) as the total amount of adduct
present along the progress of the reaction per unit of alkylating
agent concentration, such that (eqn (14)):
∞
AD dt
[
]
∞
AD
hyd
⎛
⎞
⎟
⎠
∫
′
−k
t
′
kalk
− k
(
+k
alk hyd
0
⎟
⎜
AL =
=
e
t −e
)
⎟dt
⎟
⎜
AD
⎜
∫
′
pNSO
kalk + khyd −khyd
⎝
[
]
0
o
′
kalk
f
=
=
AD
k
ka' lk + khyd k AD
(
)
hyd
hyd
(14)
NBP-pNSO adduct hydrolysis varies over two orders of mag-
nitude from pH 4.5 to 7, reducing the NBP-pNSO adduct life
considerably from 150 days to 55 h (Table 5). In spite of this
alkylating capacity, the effectiveness of pNSO as an alkylating
agent is low in comparison with that of other alkylating agents
investigated previously.36
The results provided by previous studies14,16,38,39 focused on
the search for a correlation between the alkylating capacity and
chemical reactivity of styrene oxides indicated that the mutagenic
effect of styrene oxides depends on the structure of the adducts,
but is not correlated with the amount of them formed. In some
of these works, it was suggested that mutagenicity would only be
correlated with the reactivity on the benzylic side, with a-adduct
formation.14,38,40 Since styrene oxides that form only b-adducts,
such as pNSO or a-methylstyrene oxide derivatives, have also
been shown to have mutagenic activity,14,41 these explanations are
not totally satisfactory. According to our results, such findings
could be explained (Table 6) in terms of the high stability of
a-adducts, which should provide a greater—but not exclusive—
contribution to carcinogenicity than unstable b-adducts. The
longer the persistence of adducts over time, the higher the
probability that mutagenic effects will appear. Since for several
compounds a lack of carcinogenicity has previously been shown to
Fig. 5 Influence of acidity on the reactions in the NBP alkylation reaction
by pNSO in a 7 : 3 water/dioxane medium: ( )kalk, ( ) khyd , (᭢) kAD; T =
᭹
᭺
hyd
37.5 ◦C; [NBP]o = 1.56 ¥ 10-2 M.
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be correlated with the stability and instability of the corresponding
adducts, f and AL could help to find a correlation between the
reactivity and biological activity of styrene oxides.
correlating the chemical reactivity and mutagenicity of styrene
oxides.
(v) A pNSO-guanosine adduct was detected.
The mutagenicity shown by BPL > pNSO > SO could be
explained in these terms (Table 6). The higher the ratio and stability
of the adduct formed, the higher the mutagenicity.
Acknowledgements
We thank the Spanish Ministerio de Ciencia e Innovacio´n and
Fondos FEDER (Project CTQ2010-18999) for supporting the
research reported in this article. M.G.P. thanks the Junta de
Castilla y Leo´n for Ph. D. grant. R.G.B. thanks the Spanish
Ministerio de Educacio´n, and I.F.C. the Spanish Ministerio de
Asuntos Exteriores y de Cooperacio´n (MAEC-AECI Grant) for
PhD grants.
Final remarks: pNSO-guanosine adduct formation
Since, stated above, NBP is a model nucleophile for the N7 position
of guanine in DNA,42–45 the efficiency of pNSO as an alkylating
agent of NBP has been investigated,46 the last step of the present
work was to check the pNSO alkylating capacity on a biomimetic
substrate such as the guanosine molecule (Guo). This is in contrast
with a previous work,15 in which no evidence of its formation
was found. The mass spectra of the aliquots eluted at a retention
time of 8.83 min revealed the formation of a pNSO-guanosine
adduct in the reaction of guanosine and pNSO (Fig. 6). This is in
contrast with the results from earlier work,15 in which no evidence
of its formation was found. Currently, experiments to investigate
in depth the formation mechanism of the pNSO-Guo adduct are
being carried out.
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