Reactivity of acrylamide as an alkylating agent: A kinetic approach

Article abstract of DOI:10.1002/poc.1600

Acrylamide (AA), an industrially produced reactive molecule, is used worldwide. The US EPA and IARC have classified this molecule as a probable human carcinogen. In this work, the alkylating potential of AA was investigated kinetically. The conclusions drawn are as follows: (i) AA shows alkylating ability on the nucleophile 4-(p-nitrobenzyl) pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to those of DNA bases; (ii) the rate equation for the NBP-AA adduct formation is as follows: r = k_alk[AA][NBP]; (ii) the thermoentropic term, Tδ? Sθ, is the main term responsible for the lower reactivity of AA as an alkylating agent; (iii) the value of the Gibbs energy of activation, D?Gθ, for the NBP alkylation reaction by AA is consistent with the conclusions of epidemiologic studies concerning the carcinogenicity of this substance; (iv) the results obtained here may be useful when working with hydrophilic/lipophilic media, such as in Food Science, since the dielectric constant of the medium, where alkylation occurs, influences the reaction rate, and alkylation can be inhibited by lowering the dielectric constant of the medium. Copyright

Full text of DOI:10.1002/poc.1600

Research Article
Received: 4 May 2009,
Revised: 22 June 2009,
Accepted: 23 June 2009,
Published online in Wiley InterScience: 3 August 2009
(www.interscience.wiley.com) DOI 10.1002/poc.1600
Reactivity of acrylamide as an alkylating
agent: a kinetic approach
a
a
a
´
´
´
Isaac F. Cespedes-Camacho , Jose A. Manso , M. Teresa Perez-Prior ,
a
a
a
´
´
´
Rafael Gomez-Bombarelli , Marina Gonzalez-Perez , Emilio Calle
and Julio Casadoa
*
Acrylamide (AA), an industrially produced reactive molecule, is used worldwide. The US EPA and IARC have classified
this molecule as a probable human carcinogen. In this work, the alkylating potential of AA was investigated
kinetically. The conclusions drawn are as follows: (i) AA shows alkylating ability on the nucleophile 4-(p-nitroben-
zyl)pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to those of DNA bases; (ii) the
rate equation for the NBP–AA adduct formation is as follows: r ¼ kalk½AAꢀ½NBPꢀ; (ii) the thermoentropic term, TDzSu, is
the main term responsible for the lower reactivity of AA as an alkylating agent; (iii) the value of the Gibbs energy of
activation, DzGu, for the NBP alkylation reaction by AA is consistent with the conclusions of epidemiologic studies
concerning the carcinogenicity of this substance; (iv) the results obtained here may be useful when working with
hydrophilic/lipophilic media, such as in Food Science, since the dielectric constant of the medium, where alkylation
occurs, influences the reaction rate, and alkylation can be inhibited by lowering the dielectric constant of the medium.
Copyright ß 2009 John Wiley & Sons, Ltd.
Keywords: acrylamide; alkylation reactions
There are many biological and chemical methods available for
INTRODUCTION
determining the alkylating potential of mutagenic/carcinogenic
substances, although they usually have some limitations such as
high cost, use of animals, etc. Accordingly, in vitro experiments
are now acquiring increased importance.
4-(p-Nitrobenzyl)pyridine (NBP), a trap for alkylating agents[26]
with nucleophilic characteristics similar to those of DNA bases,[27]
was previously used by us to measure the alkylating potential and
the mechanism of the reaction, not only of highly electrophilic
agents such as some lactones[28–30] and N-alkyl-N-nitrosour-
eas,[31] but also of low electrophilic agents such as sorbic acid[32]
and its salts.[33] A correlation between the alkylating potential
and carcinogenicity was found.
Acrylamide (AA), an industrially produced reactive molecule, is
used worldwide to synthesize polyacrylamide. The monomeric
form is widely used as an alkylating agent for the selective
modification of protein SH groups and in fluorescence studies of
tryptophan residues in proteins.
AA has two reactive sites: the amide group and the conjugated
double bond (Fig. 1). The vinyl group is able to participate in
nucleophilic reactions with active-hydrogen-bearing functional
groups.[1] These include the SH of glutathione, the a-NH2 of free
amino acids, or the N-terminal amino acid residues of proteins
and DNA.[2–4] Because of its low molecular weight and high water
solubility, AA can easily pass through several biological
membranes. In recent years, it has been discovered that AA is
metabolized by cytochrome P450 2E1 to the epoxide glycida-
mide, which also possesses electrophilic reactivity.[5,6]
AlthoughmanyaspectsrelatedtoAAanalysishavebeenveryhot
topics,[34] to the best of our knowledge, the alkylating potential of
this molecule has not been investigated in quantitative kinetic
terms. Here we were prompted to address this issue.
On the basis of the studies in rats and mice,[7,8] the IARC and
EPA have classified AA as a probable human carcinogen.[9,10]
Many authors have examined the neurotoxicity, genotoxicity, and
developmental and reproductive effects of AA in humans and
animals,[11–15] and have demonstrated that this molecule is
neurotoxic and also has a low degree of genotoxicity. Regarding
the carcinogenic potential of AA and its alkylating capacity, a
report published in 2002 sparked a public debate about the
correlation between human exposure to AA and cancer risk.
Tareke et al. reported unexpectedly high levels of AA in a variety
of baked and fried foods.[16] Since that time, many researchers
have investigated the formation and elimination reactions of AA
in several model reaction systems.[17–19] Moreover, the Maillard
reaction (the main route of AA formation in foods),[20–23] and
the main factors affecting that formation[24,25] have been
investigated.
EXPERIMENTAL
To monitor the alkylation reaction of NBP by AA (Scheme 1),
2.4 ml aliquots of the alkylation mixture (AAþNBP) were removed
at different times and added to a cuvette containing 0.6 ml of
triethylamine reagent to stop the alkylation process, after which
*
Correspondence to: J. Casado, Departamento de Qu´ımica f´ısica, Universidad
de Salamanca, E-37008 Salamanca, Spain.
E-mail: jucali@usal.es
´ ´ ´
I. F. Cespedes-Camacho, J. A. Manso, M. T. Perez-Prior, R. Gomez-Bombarelli,
a
´
´
M. Gonzalez-Perez, E. Calle, J. Casado
Departamento de Qu´ımica f´ısica, Universidad de Salamanca, E-37008
Salamanca, Spain
J. Phys. Org. Chem. 2010, 23 171–175
Copyright ß 2009 John Wiley & Sons, Ltd.

´
I. F. CESPEDES-CAMACHO ET AL.
Figure 1. Structure of acrylamide
the absorbances at the wavelength of maximum absorption of
the adduct (AD) were followed (l ¼ 570 nm). To render NBP
soluble, the alkylation mixtures were prepared at three different
water/dioxane ratios: 7:3, 6:4, and 5:5 (vol). Detailed reaction
conditions are given in the figure and table legends.
A Shimadzu UV-2401-PC spectrophotometer with a thermo-
electric six-cell holder temperature control system (ꢁ0.1 8C) was
used.
Figure 2. Spectrograms showing the formation of the acrylamide–NBP
adductwithtimeina7:3 water/dioxanemedium, over the2–40day interval.
[NBP]o ¼ 3 ꢃ 10ꢄ4 M; [AA]o ¼ 0.1M; T ¼ 37.5 8C
Reaction temperatures were kept constant (ꢁ0.05 8C) with a
The initial rate (vo) can be written as follows:
Lauda Ecoline RE120 thermostat.
Positive-mode electrospray ionization mass spectra were
recorded on a Waters ZQ4000 spectrometer, by direct injection
with methanol as solvent.
Water was deionized with a MilliQ-Gradient (Millipore). AA was
obtained from Fluka (ꢂ 98%); dioxane (99%) was purchased from
Panreac. Triethylamine (99%) and NBP (98%) were from Sigma
Aldrich.
All kinetic runs were performed in triplicate. Numerical
treatment of the data was performed, using SigmaPlot software,
Version 10.0.
1
DA
vo ¼
(2)
ð"lÞ Dt
where e, l, and DA represent the molar absorption coefficient of
the adduct, the light path, and the variation of absorbance with
time, respectively.
From Eqs (1) and (2), the alkylation rate constant can be written
as follows:
vo
kalk
¼
(3)
½AAꢀ½NBPꢀ
Table 1 presents the values of kalk in different water/dioxane
media at several temperatures.
RESULTS AND DISCUSSION
To follow the formation of the AA–NBP adduct kinetically, its
absorption coefficient (l ¼ 570 nm) must be known, and hence it
was determined. Several experiments were performed using
[AA]o ¼ 0.1 M and six NBP concentrations in the (0.5–5.0) ꢃ 10ꢄ4 M
range. Whenabsorbancereaches a plateau, itmay beassumedthat
thereactionofNBPwithAAiscompleted. Figure4andTable2show
the results obtained for different media.
Kinetics of the alkylation reaction
The blue AA–NBP adduct (AD) shows maximum absorption at
l ¼ 570 nm. To check its structure, a positive-mode electrospray
ionization mass spectrum was obtained with a mass/charge
ratio ¼ 286.2, which is coherent with the suggested structure for
the adduct (Scheme 1).
Figure 2 shows the increase in absorption caused by the
formation of the adduct with time. As AA was in excess compared
to NBP, it may be assumed that the total amount of NBP was
converted into adduct.
Figure 3 shows a typical kinetic run for the alkylation of NBP by
AA. As the time for reaching the plateau was very long, the initial
rate method (IRM) was used.[35] In this a way, some possible
reactions different from NBP alkylation were also circumvented
(approximately two months after the initiation of the NBP
alkylation reaction, a second reaction emerged).
Figure 5 shows the good fit of the kalk values to the Eyring–
Wynne-Jones equation:[36]
z
RT
z z
D Hu D Su
D Gu
ꢄ
ꢄ
RT
kT
kT
RT
e
k ¼
e
¼
e
(4)
h
h
Formation of the NBP–AA adduct is according to the rate
Eqn (1).
d½ADꢀ
r ¼
¼ kalk½AAꢀ½NBPꢀ
(1)
dt
Figure 3. Formation of the acrylamide–NBP adduct in a 7:3 water/diox-
ane medium; variation in absorbance (l ¼ 570 nm) with time.
[NBP]o ¼ 1.5 ꢃ 10ꢄ4 M; [AA]o ¼ 0.1 M; T ¼ 37.5 8C
Scheme 1. Method for monitoring the NBP alkylation by acrylamide
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Copyright ß 2009 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2010, 23 171–175

ACRYLAMIDE AS AN ALKYLATING AGENT
Table 1. Rate constants of NBP alkylation by AA as a function of temperature and medium composition
106 kalk (Mꢄ1 sꢄ1 a
)
Water/dioxane (v/v)
25.0 8C
27.5 8C
30.0 8C
32.5 8C
35.0 8C
37.5 8C
7:3
6:4
5:5
6.10
1.83
0.70
7.20
2.05
0.81
9.00
2.60
0.94
10.1
3.17
1.30
11.0
3.83
1.55
13.7
4.69
1.98
a Values of the rate constants are reproducible to 5%.
Table 3. Initial rate of formation of the NBP–AA adduct as a
function of the composition of the medium
Water/dioxane (v/v)
vo (ꢃ109 M minꢄ1 a
)
7:3
6:4
5:5
9.80 ꢁ 0.40
3.90 ꢁ 0.06
1.20 ꢁ 0.04
a Values are given within a 95% confidence interval.
Figure 4. Determination of the mean absorption coefficient value of
the AA–NBP adduct in a 7:3 water/dioxane medium. [AA]o ¼ 0.1 M;
[NBP]o ¼ 0.4 ꢃ 10ꢄ4 M (1), [NBP]o ¼ 0.8 ꢃ 10ꢄ4
M
(2), [NBP]o ¼ 1.6 ꢃ
10ꢄ4 M (3), [NBP]o ¼ 2.4 ꢃ 10ꢄ4 M (4), [NBP]o ¼ 3.2 ꢃ 10ꢄ4 M (5),
[NBP]o ¼ 4.0 ꢃ 10ꢄ4 M (6); l ¼ 570 nm; T ¼ 37.5 8C
Table 2. Absorption coefficients of the AA–NBP adduct in
different water/dioxane media
Water/dioxane (vol)
e570 (ꢃ10ꢄ3 Mꢄ1 cmꢄ1 a
)
7:3
6:4
5:5
5.4 ꢁ 0.2
5.8 ꢁ 0.2
5.9 ꢁ 0.3
Figure 6. Variation in the initial rate of formation of the NBP–AA adduct
(AD) in different water/dioxane media: 7:3 (*), 6:4 (D), 5:5 (&) (vol)
a Values are given within a 95% confidence interval.
Table3andFig.6showthevariationintheinitialrateofformation
of the NBP–AA adduct in different water/dioxane media.
Table 4 reports the values of the activation parameters for NBP
alkylation.
Table 4. Activation parameters for NBP alkylation by AA in
different water/dioxane media
Water/dioxane (v/v)
Activation parameter
7:3
6:4
5:5
DzHu (kJ molꢄ1 a
)
45 ꢁ 2
193 ꢁ 9
104 ꢁ 2
57 ꢁ 2
163 ꢁ 8
107 ꢁ 2
65 ꢁ 2
145 ꢁ 7
110 ꢁ 2
ꢄDzSu (J molꢄ1 Kꢄ1 a
)
DzGu (35 8C) (kJ molꢄ1 a
a Values are given within a 95% confidence interval.
)
Figure 5. Eyring plot for the alkylation of NBP by acrylamide in different
water/dioxane media: 7:3 (*), 6:4 (D), 5:5 (&) (vol)
J. Phys. Org. Chem. 2010, 23 171–175
Copyright ß 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/poc

´
I. F. CESPEDES-CAMACHO ET AL.
Table 5. Chemical reactivity of some alkylating agents
Alkylating agent
DzHu (kJ molꢄ1 a
)
ꢄDzSu (J molꢄ1 Kꢄ1 a
)
DzGu (35 8C) (kJ molꢄ1 a
)
b-Propiolactone (7/3)b
b-Butyrolactone (7/3)b
Potassium sorbate (7/3)c
Acrylamide (7/3)d
41 ꢁ 2
47 ꢁ 2
78 ꢁ 7
45 ꢁ 2
148 ꢁ 6
148 ꢁ 6
70 ꢁ 12
193 ꢁ 9
87 ꢁ 2
93 ꢁ 2
99 ꢁ 6
104 ꢁ 2
a Values are given within the 95% confidence interval.
b Values are taken from Reference[28]
.
.
c Values are taken from Reference[32]
d This work.
Since comparison of the Gibbs energy of activation, DzGu, for
NBP alkylation by different alkylating agents can be useful, these
values were also calculated. Table 5 reports the values obtained
for some alkylating agents studied by us previously. These values
reveal the following:
(iv) The values of the Gibbs energy of activation, DzGu, or NBP
alkylation reactions by different alkylating agents reveal the
existence of some correlation between chemical reactivity
and carcinogenic potential. The high value of DzGu in the case
of AA is consistent with the conclusions of several epide-
miological studies addressing the carcinogenicity of this
substance.
(v) The results obtained may be useful when working with
hydrophilic/lipophilic media, as is frequent in Food Science.
Since the dielectric constant of the medium, where NBP
alkylation occurs, influences the reaction rate, the alkylation
reaction can be strongly inhibited by lowering the dielectric
constant of the medium. Thus, the simultaneous presence of
mixtures of alcoholic spirits and foods containing vegetable
oils, such as salads or tinned foods, could inhibit the alkylat-
ing potential of the AA ingested with them.
(1) The existence of a correlation between chemical reactivity
and carcinogenic potential: while b-propiolactone and b-
butyrolactone are classified as possible human carcinogens,
sorbate has a low genotoxic capacity. The higher value of
DzGu in the case of AA permits its low carcinogenic capacity to
be understood.
(2) While the DzHu value for the alkylation reaction by AA is
almost the same as that of b-propiolactone (one of the most
potent carcinogens), the thermoentropic term, TDzSu, seems
to be the main term responsible for the lower reactivity of AA
as an alkylating agent.
The values of the activation parameters (Table 4) help to
rationalize the fact that NBP alkylation by AA in different water/
dioxane media is inhibited by the increase in organic component
in the reaction medium (Table 1). The rise – in absolute value – of
the activation entropy due to an increase in the amount of water
in the reaction medium and the simultaneous decrease in DzHu
suggest an active polar complex, similar to the resulting
adduct.[37]
As an increase in the dioxane percentage in the reaction
medium implies a decrease in its dielectric constant (e ¼ 48.54 in
7:3 water/dioxane),[38] it should be possible to inhibit the
alkylating capacity of AA via a decrease in the dielectric constant
of the reaction medium.
Acknowledgements
´
´
We thank the Spanish Ministerio de Ciencia e Innovacion
(CTQ2007-63263), as well as the Spanish Junta de Castilla y Leon
(Grant SA040 A08) for supporting the research reported in this
article. I.F.C.C. thanks the Spanish Ministerio de Asuntos Exteriores
´
y de Cooperacion (MAEC-AECID) for Ph.D. grant. R.G.B. thanks the
Ministerio de Ciencia e Innovacion and M.G.P. thanks the Junta de
´
´
Castilla y Leon for Ph.D. grants. We also thank the referees for their
valuable comments.
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J. Phys. Org. Chem. 2010, 23 171–175

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