Nitrosation of phenolic compounds: Effects of alkyl substituents and solvent

Article abstract of DOI:10.1007/s706-002-8245-0

Nitrosation reactions of phenol, o-cresol, 2,6-dimethylphenol, o-tert-butylphenol, 2-hydroxyacetophenone, and 2-allylphenol in water and water/acetonitrile were studied. Kinetic monitoring of the reactions was accomplished by spectrophotometric analysis of the nitrosated products at 345 nm. The dominant reaction was C-nitrosation via a mechanism consisting of an attack on the nitrosatable substrate by NO⁺/NO₂H₂⁺ followed by a slow proton transfer. The values of the rate constants of phenolic C-nitrosation were increased by electron donating substituents, and a good Hammett correlation was observed with ρ=-6.1. The results also revealed the strong effect of pH and the permitivity of the reaction medium on the rate constant, whose maximum values were observed for pH≈3, decreasing strongly for higher pH values. The study in water/acetonitrile with up to 25% acetonitrile showed that it is possible to inhibit the reaction strongly by increasing the percentage of the organic component. The conclusions drawn show that (i) it is possible to predict the rate of nitrosation of phenolics as a function of the meta-substituents on the phenol ring and (ii) the nitrosation of phenolics can be strongly inhibited by increasing the pH of the reaction medium as well as by lowering its dielectric constant.

Full text of DOI:10.1007/s706-002-8245-0

Monatshefte fuÈr Chemie 133, 157±166 ꢀ2002)
Nitrosation of Phenolic Compounds:
Effects of Alkyl Substituents and Solvent
1
1
2
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Samuel Gonzalez-Mancebo , Javier Lacadena , Yolanda Garcõa-Alonso ,
Jesus Hernandez-Benito , Emilio Calle , and Julio Casado
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2
2
2;Ã
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1
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Facultad de Biologõa, Universidad SEK, E-40003 Segovia, Spain
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2
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Departamento de Quõmica Fõsica, Facultad de Quõmica, Universidad de Salamanca,
E-37008 Salamanca, Spain
Summary. Nitrosation reactions of phenol, o-cresol, 2,6-dimethylphenol, o-tert-butylphenol, 2-hy-
droxyacetophenone, and 2-allylphenol in water and water=acetonitrile were studied. Kinetic monito-
ringof the reactions was accomplished by spectrophotometric analysis of the nitrosated products at
345 nm. The dominant reaction was C-nitrosation via a mechanism consistingof an attack on the
‡
‡
nitrosatable substrate by NO =NO2H2 followed by a slow proton transfer. The values of the rate
constants of phenolic C-nitrosation were increased by electron donatingsubstituents, and a good
Hammett correlation was observed with ꢀ ˆ À6.1. The results also revealed the strongeffect of pH
and the permitivity of the reaction medium on the rate constant, whose maximum values were ob-
served for pH ꢀ 3, decreasing strongly for higher pH values. The study in water=acetonitrile with up
to 25% acetonitrile showed that it is possible to inhibit the reaction strongly by increasing the
percentage of the organic component. The conclusions drawn show that ꢀi) it is possible to predict
the rate of nitrosation of phenolics as a function of the meta-substituents on the phenol ringand ꢀ ii)
the nitrosation of phenolics can be strongly inhibited by increasing the pH of the reaction medium as
well as by loweringits dielectric constant.
Keywords. Kinetics; Reaction mechanisms; Nitrosation; Phenolics.
Introduction
Considerable attention has been paid to the possibility of suppressingthe formation
of nitroso compounds, endogenously and in commercial materials, through the use
of inhibitors of nitrosation. Biologists have been mainly interested in the use of
these compounds as models for producinga broad range of cancers [1±4], whereas
chemists have been more interested ®rst in the mechanisms of formation of nitroso
compounds [5±10], and second in blockingor inhibitingthe formation of these
species [11±14].
Whereas aromatic C-nitration is a well-known reaction and is now well
understood [15, 16], aromatic C-nitrosation has been studied mechanistically in
relatively few cases, even though certain nitrosatable substrates are of considerable
interest due to the pathogenic properties of the reaction products. This is the case
Ã
Correspondingauthor. E-mail: jucali@usal.es

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S. Gonzalez-Mancebo et al.
158
for phenol and its derivatives [17±20]. Phenolic compounds are commonly used as
antioxidants, ¯avourings, and spices in food. Foods containing signi®cant quantities
of phenolics include green tea ꢀ>10% of its dry weight are polyphenols) and instant
coffee [21].
The relevance of polyphenols in current chemistry and chemical technology
should be stressed [22±24]. The biosynthetic pathways leadingto tannins, antho-
cyanins, ¯avonoids, etc., as well as the role of plant polyphenols in ¯avouring,
colouring, leather tanning, and herbal medicines are aspects of the interest in the
®eld of food science from the point of view of determiningtheir potential capacity
as nitrosatable substrates. In addition, some polyphenols are able to interact with
chlorine or nitrite to yield products of greater mutagenic potential than the original
compounds themselves [25].
As part of our ongoing research on the inhibition=blockingof nitrosation
reactions, and prompted by earlier results [26], in a recent study on the nitrosation
reactions of phenolic compounds [27] the reactivity of these substrates has been
found to depend on three main intrinsic factors: ꢀi) the preferred para-orientation
of the hydroxyl group for electrophilic attack by nitrosating agents, ꢀii) the hyper-
conjugative effect of the methyl substituent, which causes electronic charge to ¯ow
into the aromatic nucleus, as well as the opposite electronic withdrawingeffect of
the halogen substituents, and ꢀiii) the steric hindrance of alkyl substituents ¯anking
the site of nitrosation which reduces or even prevents nitrosation.
In order to gain deeper insight into the in¯uence of alkyl substituents in the
phenolic ringas well as that of pH and the nature of the reaction medium we in-
vestigated the nitrosation reactions of phenol ꢀ1), o-cresol ꢀ2), 2,6-dimethylphenol
ꢀ3), o-tert-butylphenol ꢀ4), 2-hydroxyacetophenone ꢀ5), and 2-allylphenol ꢀ6) by
sodium nitrite in potassium hydrogen phthalate buffer.
Results and Discussion
Figure 1 offers a schematic representation of the reactions studied, Fig. 2 shows a
typical kinetic run. All kinetic runs were performed in triplicate.
On workingwith water and water =acetonitrile mixtures with up to ꢁ 25% of the
latter, the rate equation was always ®rst order in nitrite ꢀFig. 3):
rate ˆ k1‰nitriteŠ
ꢂ1†
‡
‡
This result suggests that the nitrosating species is NO or NO2H2 , which are
kinetically indistinguishable.
Fig. 1. Nitrosation reactions of phenolic compounds

Nitrosation of Phenolic Compounds
159
Fig. 2. Variation in the absorbance of the nitrosated product at 345 nm with time; [6] ˆ 3.95 Á 10À2 M;
[NaNO2] ˆ 1 Á 10À4 M; pH ˆ 3.03; I ˆ 0.2 M; T ˆ 298 K
Fig. 3. First-order reaction with respect to the nitrite concentration; [6] ˆ 3.95 Á 10À2 M; pH ˆ 3.03;
I ˆ 0.2 M; T ˆ 298 K; [NaNO2] ˆ 10À4 M; At, Ao, and A are the absorbances at any time t, at the
1
beginning ꢀt ˆ 0), and upon completion of the reaction, respectively
Experiments carried out with different concentrations of substrate turned out to
be ®rst order in substrate ꢀFig. 4), allowing the second-order rate constant k2 to be
calculated as
rate ˆ k2‰nitriteŠ‰substrateŠ
The true ꢀcorrected) value of the rate constant, k2corr, can be expressed as
ꢂ2†
k2corr ˆ k2H O ‡ kB‰phthalateŠ;
ꢂ3†
k2H O beingthe rate constant in the absence of buffer and kB the rate constant in its
2
2
presence.
Since Eq. ꢀ3) allows one to obtain k2H O by extrapolatingto [phthalate] ˆ 0 and
2
takinginto account the dissociation of nitrous acid ꢀ pKa ˆ 3.148 [28]) it is possible
to express k2corr as a function of k2:
‡
k2corr ˆ k2H O ˆ k2ꢂ1 ‡ KN=‰H Š†
ꢂ4†
2

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S. Gonzalez-Mancebo et al.
160
Fig. 4. Effect of the concentration of o-cresol on the pseudo-®rst-order rate constant of nitrosation;
[NaNO2] ˆ 1 Á 10À4 M; I ˆ 0.2 M; T ˆ 298 K; ꢃ: pH ˆ 2.87; 9: pH ˆ 5.10
The reaction mechanism can be explained in terms of aromatic electrophilic
substitution by nitrosonium or nitrous acidium ions ꢀScheme 1), a mechanism
previously suggested in other studies [29, 30] and handled by us earlier [26, 27].
Scheme 1

Nitrosation of Phenolic Compounds
161
Since the slow step is a proton transfer ꢀsee Scheme 1) and since, under the work-
ingconditions, ‰nitriteŠ ˆ ‰NOÀ2 Š ‡ ‰HNO2Š, the rate equation is readily achieved:
2
‡
‡
kaKNO ‰nitriteŠ‰substrateŠ‰H Š
rate ˆ
ꢂ5†
kÀa
‡
‡
ꢂKN ‡ ‰H Š†ꢂ1 ‡ ‰H Š†
Kkb
Comparison of Eqs. ꢀ2) ꢀexperimental) and ꢀ5) ꢀmechanistic) and takinginto
account Eq. ꢀ4) affords
‡
‡
kaKNO ‰H Š
k2H O
ˆ
ꢂ6†
2
kÀa
‡
ꢂ1 ‡ ‰H Š†
Kkb
‡
Figure 5 shows the variation in k2H O with [H ]; Eq. ꢀ6) ®ts the experimental
2
data.
The denominator of Eq. ꢀ6) carries a sum of terms which permits the con-
sideration of two limitingcases. When the second term in the denominator is much
larger than 1, the rate constant does not depend on the [H ]. This will be most likely
‡
‡
true at high [H ] as can be seen from Fig. 5. When the second term in the de-
nominator is signi®cantly less than one, the rate constant should be ®rst order in
[H ], as also shown in Fig. 5.
This variation in k2H O with [H ] is of biological relevance. As shown in Fig. 5,
‡
‡
2
in more acid media ꢀpH ꢄ 3) the values of k2H O vary slowly, fallingsharply when
2
the acidity of the medium decreases. This suggests that attention should be focused
on the effects of compounds that increase the acidity of the stomach such as those
containinghydrochloric acid used to relieve the symptoms of hypochlorhydria.
Equation ꢀ6) permits to make some simplistic considerations aimed at ration-
alizingthe experimental values of k2H O. It seems logical that any factor that con-
tributes to an increase in negative charge density in the para-position with respect to
2
the ÀOH group of the nitrosatable substrate should increase its reactivity ꢂk2H O†,
2
both by increasingthe ka value and by favouringproton transfer from the solvent
ꢀwater) to the ÀNO group of the intermediate dienone ꢀScheme 1).
‡
Fig. 5. Effect of [H ] on the rate constant of nitrosation; 9: [1] ˆ 0.200 M; [NaNO2] ˆ 1 Á 10À4 M;
I ˆ 0.2 M; T ˆ 298 K; ~: [2] ˆ 0.200 M; [NaNO2] ˆ 1 Á 10À4 M; I ˆ 0.2 M; T ˆ 298 K

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S. Gonzalez-Mancebo et al.
162
In order to test this, the Hammett correlation given below ꢀEq. ꢀ7) [31]) was
used:
log ꢂk=ko† ˆ ꢁm ꢀ
ꢂ7†
In Eq. ꢀ7), ko and k denote the kinetic constants for the reference reaction ꢀhy-
drogen as substituent, 1 in this case) and for reactions with other substituents in
meta-orientation with respect to the ®nal ÀNO position ꢀScheme 1).
The substituent constant ꢁm is of general signi®cance, exceeding the range of
validity of the Hammett equation, i.e. derivatives of benzene. If the substituent
effects are classi®ed as inductive, mesomeric, and steric, only the ®rst two are in-
volved in ꢁm. If the sum is electron withdrawing, the sign of ꢁm is positive; if it
is electron releasing, it is negative; the corresponding values have been tabulated
[32].
In the case of polysubstituted substrates ꢀe.g. 3), the ꢁ values have been shown
to be mostly equal to the sum of the values of individual substituents [33, 34].
The slope ꢀ, termed `reaction constant', is a function of the reaction series
studied. Table 1 lists the values of the substrate reactivities indicated by the values
of k2H O
.
2
Figure 6 shows the good ®tting of the results to the Hammett equation ꢀR ˆ
0.984). The slope of ꢀ ˆ À6.1 indicates an electrophilic attack of nitrosatable
phenolic substrates and is consistent with the positive charge of the nitrosating
‡
‡
agents NO =NO2H2 ꢀsee Scheme 1).
Additionally, on the basis of the proposed reaction mechanism ꢀScheme 1),
with a slow step consistingof proton transfer from water to the ÀNO group of
the intermediate dienone, one might speculate that the progressive substitution of
the water of the reaction medium by a solvent with a lower dielectric constant
would hinder proton transfer, thus hamperingthe nitrosation. Figure 7 shows the
variation in k2H O with the composition of the medium consistingof water =aceto-
nitrile mixtures.
2
In conclusion, the kinetic study of the nitrosation of phenols reveals that the
reactivity of these C-nitrosatable substrates appears to depend on both structural
and external factors such as pH and the permittivity of the medium. The values of
the experimental rate constants ®t the Hammett correlation. This enables one to
make predictions about the reactivity of more complex phenolics.
In view of the foregoing, attention should be paid to the possible enhancement
of the nitrosation of certain potentially C-nitrosatable substances as a consequence
of the presence of electron releasingsubstituents. This could be the case for some
polyphenols present in tea, which have some strongly nucleophilic carbon atoms,
as well as the polyphenol quercetin present in wines ꢀparticularly in red varieties)
which has been demonstrated to be genotoxic upon nitrosation [35]. Despite this, it
should be stressed that even if quercetin and other polyphenols do seem to be major
mutagens in wines before or after their nitrosation, it is also known that this ¯a-
vonol and other ¯avonoids may display antimutagenic and anticarcinogenic prop-
erties [36].
The results also point to the strongin¯uence of pH and the permittivity of
the reaction medium on the nitrosation rate constant. Maximum values for the

Nitrosation of Phenolic Compounds
163
Table 1. Reactivities ꢀk2H O, Eq. ꢀ3)) of 1 and its derivatives for nitrosation reactions; pH ˆ 2.9;
2
I ˆ 0.2 M; T ˆ 298 K
Substrate
Structure
k2H O=MÀ1sÀ1
2
1
0.0131Æ0.0001
ꢀ0.010Æ0.001)a
2
3
4
0.071Æ0.002
ꢀ0.07Æ0.01)a
0.37Æ0.01
ꢀ0.53Æ0.09)a
0.0915Æ0.0002
5
0.00014Æ0.00003
0.037Æ0.002
6
a
Results obtained at pH ˆ 2.5 [27]
Fig. 6. Fittingof the reaction rate constants of nitrosation to the Hammett equation

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S. Gonzalez-Mancebo et al.
164
Fig. 7. Variation in the rate constant for the nitrosation of phenolic compounds with the percentage
of acetonitrile in the reaction medium; ^: [1] ˆ 4 Á 10À2 M; [NaNO2] ˆ 10À4 M; pH ˆ 2.9; I ˆ 0.2 M;
T ˆ 298 K; 9: [2] ˆ 4 Á 10À2 M; [NaNO2] ˆ 10À4 M; pH ˆ 2.9; I ˆ 0.2 M; T ˆ 298 K
rate constant were observed for pH ꢀ 3, decreasingsharply for higher pH values.
This is of interest in both food science and the medical ®eld, since the intake
of acid=antacid compounds may strongly affect the rate of nitrosation in the
stomach.
A kinetic study of the nitrosation reactions of phenolic compounds in water=
acetonitrile media shows that the reactions can be strongly inhibited by increasing
the percentage of the organic component. These results may be signi®cant in
cases of the presence of alcoholic spirits in the human stomach. Since the
dielectric constant of these mixtures is lower than that of water, a slowingdown
of the in vivo nitrosation of phenolics would be expected in the presence of these
substances.
Experimental
Compounds 1±6 were purchased from Aldrich ꢀp.a., 98% pure). Solutions of NaNO2, HClO4,
NaClO4 ꢀto adjust ionic strength), NaOH ꢀall Merck p.a. products), and potassium hydrogen phthalate
ꢀPanreac p.a.) were made up by weight ꢀNaNO2 after desiccation for 2 h at 110ꢅC). Reaction mixtures
were prepared in potassium hydrogen phthalate buffers of pH ˆ 2.5±5.5 ꢀthis buffer does not
generate any potential nitrosating agent); pH was measured with a Radiometer M64 pH-meter
equipped with a GK2401B combined electrode.
Reactions were monitored kinetically by spectrophotometric analysis of the nitrosated products
at 345 nm. A Shimadzu 2101PC double-beam apparatus with thermoelectric control was employed
to maintain the temperature within 0.1ꢅC. The absorbance vs. time data were processed by the inte-
gration method [37]. All reactions were followed to at least 70% completion.
Acknowledgements
Â
The authors thank the Spanish Ministerio de Ciencia y Tecnologõa for partially supportingthe
research reported in this article ꢀproject BQU2001-1934) as well as the University of Salamanca for
 Â
a Ph.D. Grant to J. Hernandez Benito. S. Gonzalez Mancebo and J. Lacadena thank the University

Nitrosation of Phenolic Compounds
165
SEK and the Junta de Castilla y Leꢀon for supportingthis research ꢀProject SEK 3 =00F). Thanks are
also due to the referees and editors for comments and suggestions.
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Received July 13, 2001. Accepted +revised) September 18, 2001