Nitrosation kinetics of phenolic components of foods and beverages

Article abstract of DOI:10.1002/(sici)1097-4601(1997)29:2<119::aid-kin5>3.0.co;2-x

The kinetics of the reactions between sodium nitrite and phenol or m-, o-, or p-cresol in potassium hydrogen phthalate buffers of pH 2.5-5.7 were determined by integration of the monitored absorbance of the C-nitroso reaction products. At pH > 3, the dominant reaction was C-nitrosation through a mechanism that appears to consist of a diffusion-controlled attack on the nitrosatable substrate by NO⁺/NO₂H₂⁺ ions followed by a slow proton transfer step; the latter step is supported by the observation of basic catalysis by the buffer which does not form alternative nitrosating agents as nitrosyl compounds. The catalytic coefficients of both anionic forms of the buffer have been determined. The observed order of substrate reactivities (o-cresol ≈ m-cresol > phenol ? p-cresol) is explained by the hyperconjugative effect of the methyl group in o- and m-cresol, and by its blocking the para position in p-cresol. Analysis of a plot of ΔH^# against ΔS^# shows that the reaction with p-cresol differs from those with o- and m-cresol as regards the formation and decomposition of the transition state. The genotoxicity of nitrosatable phenols is compared with their reactivity with NO⁺/NO₂H₂⁺.

Full text of DOI:10.1002/(sici)1097-4601(1997)29:2<119::aid-kin5>3.0.co;2-x

Nitrosation Kinetics of
Phenolic Components of
Foods and Beverages
M. PAZ FERNÁNDEZ-LIENCRES,* EMILIO CALLE, SAMUEL GONZÁLEZ-MANCEBO,
and JULIO CASADO†
Departamento de Química física, Facultad de Química, Universidad de Salamanca, E-37008, Salamanca, España
BARTOLOMÉ QUINTERO
Departamento de Química física, Facultad de Farmacia, Universidad de Granada, E-18071, Granada, España
ABSTRACT
The kinetics of the reactions between sodium nitrite and phenol or m-, o-, or p-cresol in
potassium hydrogen phthalate buffers of pH 2.5–5.7 were determined by integration of the
monitored absorbance of the C-nitroso reaction products. At pH Ͼ 3, the dominant reaction
was C-nitrosation through a mechanism that appears to consist of a diffusion-controlled at-
ϩ
ϩ
tack on the nitrosatable substrate by NO /NO H ions followed by a slow proton transfer
2
2
step; the latter step is supported by the observation of basic catalysis by the buffer which
does not form alternative nitrosating agents as nitrosyl compounds. The catalytic coefficients
of both anionic forms of the buffer have been determined. The observed order of substrate re-
activities (o-cresol Ϸ m-cresol Ͼ phenol ϾϾ p-cresol) is explained by the hyperconjugative ef-
fect of the methyl group in o- and m-cresol, and by its blocking the para position in p-cresol.
#
#
Analysis of a plot of ⌬H against ⌬S shows that the reaction with p-cresol differs from those
with o- and m-cresol as regards the formation and decomposition of the transition state. The
ϩ
ϩ
genotoxicity of nitrosatable phenols is compared with their reactivity with NO /NO H
.
2
2
©
1997 John Wiley & Sons, Inc.
INTRODUCTION
derstood [3]. The nitrosation of certain aromatics is
nevertheless of considerable biomedical interest in
view of the proven carcinogenic or mutagenic proper-
ties of the resulting products. This is the case of phe-
nol and its derivatives [4–7]: the phenol derivative
tyramine, which occurs in cheese, meat extract, beer,
and soybean products [8–10] has been identified as
one of the precursors largely responsible for the mu-
tagenic activity of certain Japanese soy sauces treated
with nitrite [11,12]; and bamethan [1-(4-hydroxy-
phenyl)-2-butylaminoethan-ol], a phenolic drug used
for long-term oral treatment of cardiovascular dis-
ease, is both nitrosatable and a directly acting muta-
gen [13]. In view of their status as nitrosatable pre-
ϩ
14
The nitrosonium ion NO being some 10 times less
reactive than the nitronium ion NO2 [1,2] much less
attention has been paid to the C-nitrosation of aro-
matics than to their nitration, which is now well un-
ϩ
Received May 8, 1995; accepted March 5, 1996
*
On leave from Departamento de Química física y
Analítica, Facultad de Ciencias Experimentales, Universi-
dad, E-23071, Jaén, Spain.
†
Author to whom correspondence should be addressed.
International Journal of Chemical Kinetics, Vol. 29, 119–125 (1997)
©
1997 John Wiley & Sons, Inc. CCC 0538-8066/97/020119-07

1
20
FERNÁNDEZ-LIENCRES ET AL.
cursors of genotoxic substances, it is of some concern
that phenol, catechol, vanillin, and other phenolics
have been detected in smoked fish and meats [14]. It
has also been reported that the presence of phenolic
compounds can block the N-nitrosation reactions of
other substrates [15,16], and although this has been
questioned on the grounds that N-nitrosation is cat-
alyzed by monohydroxy phenols [17] (but not by all
polyhydroxyphenols [15]), it should be borne in mind
that catalytic activity is only observed when the con-
centration of nitrosating agent significantly exceeds
the concentration of phenol, which rarely occurs in
vivo or in environmentally significant situations.
In spite of the biomedical significance of the nitro-
sation of phenolics, little research has been done on
the mechanisms of these reactions [18]. However, ac-
cording to Challis and co-workers [1,19–21] the C-
nitrosation of simple phenols in perchloric media
generally involves the formation of a dienone inter-
mediate that loses a proton from its nitrosated carbon
to yield the nitrosophenol in equilibrium with its
oxime form. p-Alkylphenols yield the correspond-
ing 2-nitrosocompounds, whereas p-bromophenol
is transformed by a fast substitution reaction into
p-nitrosophenol, although in both cases the nitroso-
compound is rapidly oxidized to the corresponding
nitrocompound.
nol and of m-, o-, and p-cresol by sodium nitrite in
potassium hydrogen phthalate buffers that did not
form any potential nitrosating agent.
EXPERIMENTAL
Phenol and m-, o-, and p-cresol were Aldrich p.a.
products (Steinheim, Germany). Solutions of NaNO2 ,
HClO , and NaOH (all Merck p.a. products, Darm-
4
stadt, Germany) and of potassium hydrogen phthalate
(Panreac p.a., Barcelona, Spain) were made up by
weight (NaNO solutions after desiccation for 2 h at
2
110ЊC). Reaction mixtures were made up in potas-
sium hydrogen phthalate buffers of pH 2.5–5.7. pH
was measured with a Radiometer M64 pH-meter
equipped with a GK2401B combined electrode.
Reaction kinetics were followed in a Shimadzu
2101PC Vis-UV spectrophotometer by monitoring
absorbance by the nitrosated product at 345 nm, an
isosbestic point at which there is no interference by
3
the reagents (Fig. 1). To 3 cm of substrate solution in
3
a cell thermostatted at 25 Ϯ 0.1ЊC was added 0.1 cm
of sodium nitrite solution to a concentration of 1 10
Ϫ4
M (very much less than the initial substrate concen-
Ϫ3
trations: 0.030–0.200 M for phenol, between 5 10
Ϫ2
and 6 10 M for o- and m-cresol, and 0.040–0.125
In the case of the nitrosation in perchloric media
the 2-nitrosocompounds are considered to be rapidly
oxidized to the corresponding nitrocompound. How-
ever, the course of the reaction between nitrite and
p-methoxyphenol appears to depend on the concen-
tration of perchloric acid in the medium: 4-methoxy-
M for p-cresol). The absorbance-time data were
processed by the integration method; all reactions
were followed to at least 70% completion.
RESULTS AND DISCUSSION
2
-nitrophenol is only formed if the perchloric acid
concentration is low (pH 2–3), and even then it is not
the major product; if perchloric acid concentration is
high, benzoquinone is formed in quantitative yield
Spectrum of p-nitrosophenol at various pH values is
shown in Figure 1. It can be observed the appearance
[20]. Similar results for phenol and 4-phenoxyphenol
as well as 4-methoxyphenol were obtained by
Moodie and co-workers [22–24] in studies that, like
others on the nitrosation of certain phenolic drugs
[13,25–28], also showed that the course of these re-
actions can depend on the concentration of dissolved
oxygen in the medium and on whether the concen-
tration of nitrite is greater or less than that of the
substrate.
While different studies have been carried out in
buffered media by buffers such as acetic-acetate or
monochloroacetic-monochloroacetate ([29]; Challis
et al., loc. cit.), practically no work has been done
with buffers which do not form alternative nitrosating
agents in the form of nitrosyl compounds.
Figure 1 UV Spectra of p-nitrosophenol (1.38 ϫ 10Ϫ4 M,
T ϭ 298 K, and I ϭ 0.20 M) under various pH conditions:
(1) pH ϭ 12.16; (2) pH ϭ 10.77; (3) pH ϭ 7.49; (4) pH ϭ
In this work we investigated the nitrosation of phe-
6.49; (5) pH ϭ 5.78; and (6) pH ϭ 2.53.

NITROSATION KINETICS OF PHENOLIC COMPONENTS
121
of an isosbestic point at 345 nm attributed to an equi-
librium between the tautomeric forms (benzoquinone
mono-oxime and p-nitrosophenol) and the p-ni-
trosophenolate anion, pK ϭ 6.5 [30].
a
Kinetic experiments were carried out in potassium
hydrogen phthalate buffers, which were found not to
react with the nitrosating agent. Plots of ln(A Ϫ
ϱ
A )/(A Ϫ A ) against time were linear, showing the
0
ϱ
t
reactions to be of first-order with respect to nitrite:
v ϭ k1exp [NaNO2]
(1)
Figure 2 Influence of the concentration of substrate upon
the pseudo-first-order rate constant of its nitrosation. pH ϭ
3.5; [nitrite] ϭ 10Ϫ4 M; T ϭ 310 K (T ϭ 298 K for phe-
nol); I ϭ 0.10 M; (a) phenol; (b) o-cresol; (c) m-cresol; and
It must be pointed out that the first-order respect to
nitrite indicates that the nitrosation by N O is not in-
2
3
volved in this mechanism. Reaction is believed to
occur by the attack of nitrosatable substrate by
(d) p-cresol.
ϩ
ϩ
NO H /NO ions kinetically indistinguishable.
2
2
The corresponding first-order pseudoconstants
were calculated from the slopes of these plots. Their
logarithms depended linearly on the logarithm of sub-
strate concentration (Fig. 2), allowing the second-
order coefficients k2exp to be calculated:
the observed data are explicable in terms of aromatic
electrophilic substitution by nitrosonium or nitrous
acidium ions (Scheme I), a mechanism previously
suggested by other studies of the nitrosation of phe-
nols by nitrous acid [19,33], and which parallels the
mechanism proposed for the nitrosation of phenols
rate ϭ k2exp [nitrite] [substrate]
(2)
[
[
34] and species such as sulphamic acid and cysteine
35] by alkyl nitrites in aqueous acid solution. This
These k2exp were then transformed into corrected val-
ues k2corr that took into account the dissociation of ni-
trous acid (pK 3.15 [31]):
mechanism implies the rate equation
ka KNO
ϩ
[nitrite][phenol][Hϩ]2
v ϭ
(4)
ϩ
kϪa
k2corr ϭ k2exp (1 ϩ KHNO2 /[H ])
(3)
ϩ
ϩ
(
K ϩ [H ]) 1 ϩ
[H ]
N
΂
΃
Kkb
There was no need to consider dissociation of the
substrates, all of which have pK values very much
higher than the pH range used in this work: pK (phe-
nol) ϭ 10.00, pK(o-cresol) ϭ 10.29, pK(m-cresol) ϭ
The slow step in Scheme I is hypothesized, following
Challis [1], to involve protonation of the nitroso oxy-
gen in the transition state (Scheme II). Since this im-
plies susceptibility to basic catalysis by organic
bases, Schemes I and II are supported by the finding
that, for phenol, k2corr depends linearly on buffer con-
centration, [B]T :
1
0.09, and pK(p-cresol) ϭ 10.26 [32].
The fact that for all the substrates k2corr peaked at
an acidity of about pH 3.5 (Table I) suggests that the
reaction mechanism was the same in all cases. In fact,
Table I Values of k2corr ϫ 10 , M sϪ1 in the Nitrosation of Phenol and Related Compounds
2
Ϫ1
Ϫ4
in Solutions of Hydrogen Phthalate Buffers: [Nitrite] ϭ 10 M; [Phenol] ϭ
0
.20 M; [o- and m-Cresol] ϭ 0.06 M; [p-Cresol] ϭ 0.125 M; and T ϭ 310 K
pH
Phenol
pH
o-Cresol
pH
m-Cresol
pH
p-Cresol
2
2
3
4
4
5
5
.59
.99
.75
.01
.55
.10
.53
3.44
3.99
5.43
5.39
3.22
2.29
1.09
2.58
2.99
3.51
3.71
4.00
4.48
5.07
20.2
23.9
26.7
24.7
22.8
21.1
20.2
2.59
3.00
3.51
3.72
3.97
4.49
5.07
16.6
18.0
22.1
20.9
20.0
17.1
9.10
2.56
2.99
3.51
3.74
3.97
4.45
5.04
0.700
1.03
1.66
2.03
1.80
1.27
1.09

1
22
FERNÁNDEZ-LIENCRES ET AL.
Table II Catalysis by Hydrogen Phthalate Buffers and
Rate Constants of the Nitrosation of Phenola
kB , MϪ2 sϪ1
k2H2O 10 , M sϪ1
3
Ϫ1
pH
2
.46
.88
.29
.52
.76
.22
.55
.81
.24
.64
0.109
0.133
0.151
0.181
0.162
0.179
0.171
0.179
0.159
0.156
10.7
10.8
10.1
9.99
9.37
7.76
6.66
5.36
2.78
1.73
2
3
3
3
4
4
4
5
5
a
Phenol] ϭ 0.20 M; [Nitrite] ϭ 10Ϫ4 M; I ϭ 0.20
M
[
(
NaClO ); and T ϭ 298 K.
4
therefore confirm the suggestion of Castro et al. [29]
on the basis of results obtained in acetic acid/acetate
buffers. The values of k
ious pH are listed in Table II.
and k determined at var-
Scheme I
2H O
2
B
Identification of eqs. (2) and (4) implies
k2corr ϭ k2H2O ϩ k [B]
(5)
B
T
ϩ
ka KNO
ϩ
[H ]
k2H2O
ϭ
(6)
where k2
H O
is the rate constant for the uncatalyzed
k
Ϫa
2
ϩ
΂1
ϩ
[H ]
΃
reaction, k the buffer catalytic constant, and [B] ϭ
B
T
Kkb
Ϫ
ϭ
[
BH ] ϩ [BH ] ϩ [B ].
2
The increase in reaction rate with buffer concen-
Fitting eq. (6) to the experimental data by means of
tration was indeed due to general basic catalysis
rather than to the formation of more effective ni-
trosating agents in the form of nitrosyl compounds,
because no nitrosyl compounds were detected
whereas in other media they have been detected and
the equilibrium constants of their formation and hy-
drolysis have been measured [36,37]). Our findings
an optimization algorithm [38,39] (see Fig. 3) yields
Ϫ2 Ϫ1
the value k K
NOϩ
ϭ 661 Ϯ 48 M s and k /Kk ϭ
a
Ϫa b
Ϫ1
6
3.183 Ϯ 5.000 M .
Similar pH dependence has been reported for the
nitrosation of 2-naphtol [21] (those experiments were
carried out in dilute HClO , but since ClO does not
catalyze the reaction, the observed pH dependence is
(
Ϫ
4
4
comparable with the pH dependence of k2
in our
H O
2
work).
Since KNO
ϩ
ϭ 3 ϫ 10Ϫ7 MϪ1 [40], k value has
a
9
been calculated to be ka ϭ (2.2 Ϯ 0.2) ϫ 10
Ϫ1 Ϫ1
M s . That means the aromatic electrophilic substi-
tution shown in Scheme I is diffusion-controlled like
ϩ
ϩ
for other nitrosation reactions by NO H /NO
2
2
[41–44].
Having taken into account that, in the presence of
the phthalate buffer
Ϫ
ϭ
Ϫ
Bϭ
k2corr ϭ k2H2O (1 ϩ kBH [BH ] ϩ k [B ]) (7)
it is easy to demonstrate that
k2corr ϭ k2H2O
ϩ
kBH
Ϫ
K [H ] ϩ k K K
Bϭ
1
1
2
Ά1 ϩ
΃[B]T· (8)
΂
ϩ 2
ϩ
Scheme II
[H ] ϩ K [H ] ϩ K K
1 1 2

NITROSATION KINETICS OF PHENOLIC COMPONENTS
123
hyperconjugative effect of ortho and meta methyl
groups [46], which may be expected to facilitate the
transfer of a proton from water to the 9NO group of
the dienone intermediate (Scheme II). The poor reac-
tivity of p-cresol may be attributed fundamentally to
the occupation of the preferred para position by the
methyl group, which forces nitrosation to occur at the
less reactive ortho position.
At first sight, the above order of reactivities seems
to contradict the findings of Rosenkranz et al. [47],
who in their studies of the genotoxicity of nitrosat-
able phenols and their derivatives found confirmation
of the order p-cresol Ͼ phenol Ͼ o-cresol Ϸ
m-cresol predicted by an artificial intelligence system
for the investigation of structure-activity relation-
ships. It should be borne in mind, however, that in the
conditions considered by Rosenkranz et al. (presence
of excess nitrite, which as mentioned in the Introduc-
tion is in any case an unlikely natural situation) the
intermediate reaction products are phenyldiazoniums
rather than, as in our work, nitrosocompounds. The
order of genotoxic activities resulting of the study of
Rosenkranz et al. reflects the readiness of the phenols
to form phenyldiazoniums rather than their reactivi-
Figure 3 Variation of the second-order rate constant with
pH for the nitrosation of phenol. The curve represents the
dependence on pH calculated from eq. (6) and the points
represent the observed rate constants (extrapolated to zero
phthalate buffer concentration at pH ϭ 2.5–5.7). Experi-
mental conditions in Table II.
where
Ϫ
ϩ
K ϭ [BH ][H ]/[BH ]
1
2
(9)
ϭ
ϩ
Ϫ
K ϭ [B ][H ]/[BH ]
2
Comparing eqs. (5) and (8) it results
ϩ
ϩ
ties with NO /NO H .
2
2
Table III lists the activation parameters calculated
from the results of experiments to determine the in-
fluence of temperature over the range 289–310 K for
constant reagent concentrations and acidity (pH 3.5).
Plotting ⌬H against ⌬S (Fig. 5) shows that the
reaction with p-cresol differs from others in that it
fails to exhibit a compensation effect [48], i.e., that
the para methyl group which thwarts the tendency of
the nitrosonium ion to react at the para position of
even activated substrates [49], substantially alters
the thermodynamic balance of the formation of the
transition state. The contiguity of the O" and
ϩ
ϩ
k
2H O
[H ]/K ϩ 1 ϩ K /[H ]
2
1
2
ϭ
(10)
ϩ
ϩ
k [H ]
B
Bϭ
kBHϪ[H ] ϩ k K2
Figure 4 shows the experimental data fit well to eq.
8). Given that K ϭ 1.3 ϫ 10
#
#
Ϫ3
(
1
and K ϭ 3.9 ϫ
1
2
Ϫ6
0
[45] catalytic coefficients can be calculated,
Ϫ1
Ϫ1
kBHϪ ϭ 13 Ϯ 6 M and kBϭ ϭ 132 Ϯ 13 M .
Almost throughout the pH range used in this work,
the substrate reactivities indicated by the observed
values of k2corr exhibited the order o-cresol Ϸ
m-cresol Ͼ phenol ϾϾ p-cresol. That o- and m-cresol
are more reactive than phenol is attributable to the
H
N ϭ O groups in the intermediate formed in
the p-cresol reaction (Scheme II), suggests that this
effect of the para methyl group may be due to the rate
of proton transfer, being slowed by the formation of
an intramolecular hydrogen bond.
Table III Activation Parameters for the Nitrosation of
Phenol and Related Compounds. Temperature Range
2
89–310 K and pH ϭ 3.50
⌬H , kJ molϪ1
#
Ϫ⌬S , J mol KϪ1
#
Ϫ1
Substrate
Phenol
43 Ϯ 1
31 Ϯ 1
32 Ϯ 1
35 Ϯ 1
132 Ϯ 7
o-Cresol
m-Cresol
p-Cresol
157 Ϯ 12
156 Ϯ 12
169 Ϯ 16
ϩ
ϩ
Figure 4 Dependence of k2 /k [H ] on [H ] in the ni-
H O
B
2
trosation of phenol at 298 K and I ϭ 0.20 M in the pres-
ence of potassium hydrogen phthalate buffer.

1
24
FERNÁNDEZ-LIENCRES ET AL.
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CONCLUSION
The kinetics of the reactions between sodium ni-
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#
#
Analysis of the plot ⌬H /⌬S shows that the reaction
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1
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ϩ
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The authors thank the Commission of the European Com-
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as part of a project on the nitrosatable molecules present in
foodstuffs (Contract No. 93CVVF1-610-0). Thanks are also
due to the Spanish Comisión Interministerial de Ciencia y
Tecnología (CICYT) for its support of research on precur-
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