Effects of ascorbic acid on arenediazonium salts reactivity: Kinetics and mechanism of the reaction

Article abstract of DOI:10.1002/1522-2675(20010321)84:3<632::AID-HLCA632>3.0.CO;2-0

We have examined the kinetics and mechanism of dediazoniation of o-, m- and p-methylbenzenediazonium (ArN₂^-) tetrafluoroborate in the presence of ascorbic acid (H₂A) at different pHs by combining spectophotometric (VIS-UV), high performance liquid chromatography (HPLC), and polarographic measurements. Kinetic data show that, at low pH, observed rate constants increase linearly with increasing ascorbic acid concentration, but the saturation kinetics observed at higher pH suggest the formation of a transient diazo-ether complex preceding the slow step of the reaction. Experimental evidence for the formation of such a complex was obtained from a competitive coupling reaction with the Na salt of '2-naphthol-6-sulfonic acid' and by titration of ascorbic acid (H₂A) with the arenediazonium ions (electrochemical measurements). HPLC Analysis of dediazoniation products indicates that, in the absence of H₂A, only the heterolytic phenol derivative, ArOH, is formed quantitatively, in keeping with the predictions of the D_N+A_N mechanism. In the pH 2-4 range and in the presence of H₂A, reduction products (ArH) are obtained in addition to heterolytic products (ArOH), corroborating that certain biological reducing agents like ascorbate (HA^-) are capable of inducing reductive fragmentation of ArN₂^- into aryl radicals. All evidence is consistent with two competitive reaction pathways, the thermal decomposition of ArN₂⁺, and a rate-limiting decomposition of the transient diazo ether 'complex', formed during the reaction of ArN₂⁺ with HA^- in a rapid pre-equilibrium step.

Full text of DOI:10.1002/1522-2675(20010321)84:3<632::AID-HLCA632>3.0.CO;2-0

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Effects of Ascorbic Acid on Arenediazonium Salts Reactivity:
Kinetics and Mechanism of the Reaction
by Ugo Costas-Costasa), Elisa Gonzalez-Romerob), and Carlos Bravo-Diaza)*
a) Facultad de Ciencias, Departamento de Quimica Fisica y Quimica Organica, Universidad de Vigo,
E-36200 Vigo-Pontevedra (fax: ‡‡ 34-986-812382; e-mail cbravo@uvigo.es)
b) Facultad de Ciencias, Departamento de Quimica Analitica y Alimentaria, Universidad de Vigo,
E-36200 Vigo-Pontevedra
We have examined the kinetics and mechanism of dediazoniation of o-, m- and p-methylbenzenediazonium
‡
(ArN2 ) tetrafluoroborate in the presence of ascorbic acid (H2A) at different pHs by combining spectophoto-
metric (VIS-UV), high performance liquid chromatography (HPLC), and polarographic measurements.
Kinetic data show that, at low pH, observed rate constants increase linearly with increasing ascorbic acid
concentration, but the saturation kinetics observed at higher pH suggest the formation of a transient diazo-ether
complex preceding the slow step of the reaction. Experimental evidence for the formation of such a complex was
obtained from a competitive coupling reaction with the Na salt of ꢀ 2-naphthol-6-sulfonic acidꢁ and by titration of
ascorbic acid (H2A) with the arenediazonium ions (electrochemical measurements). HPLC Analysis of
dediazoniation products indicates that, in the absence of H2A, only the heterolytic phenol derivative, ArOH, is
formed quantitatively, in keeping with the predictions of the DN ‡ AN mechanism. In the pH 2 ± 4 range and in
the presence of H2A, reduction products (ArH) are obtained in addition to heterolytic products (ArOH),
corroborating that certain biological reducing agents like ascorbate (HA ) are capable of inducing reductive
‡
fragmentation of ArN2 into aryl radicals. All evidence is consistent with two competitive reaction pathways, the
‡
thermal decomposition of ArN2 , and a rate-limiting decomposition of the transient diazo ether ꢀcomplexꢁ,
‡
formed during the reaction of ArN2 with HA in a rapid pre-equilibrium step.
1. Introduction. ± N-Nitroso compounds may play a significant role in human
carcinogenesis because the ubiquity of their precursor compounds leads to the ready
formation of N-nitrosamines and other N-nitroso compounds in the human micro and
macroenvironment [1][2]. N-Nitroso compounds can be classified in two broad
categories, N-nitrosamine type and N-nitrosamide type [2]. Of these, N-nitrosamines,
which can be considered amides of nitrous acid, are the most stable and are formally
derived from the reaction of a secondary amine with nitrous acid. The N-nitrosamide-
type substances have a carbonyl group attached to the N-atom bearing the NO group
and include N-nitrosamides, N-nitrosocarbamates, and N-nitrosoureas. The instability
of these N-nitrosamide-type compounds results from having two very electropositive
functional groups (NNO and CO) joined and, under appropriate conditions, these
compounds rearrange to give diazonium ion intermediates.
By far the best-known and most studied nitrosation reactions are the N-nitrosations
of amines and related compounds [2]. Both aromatic and aliphatic amines react with a
range of nitrosating agents to give, finally, products of deamination. Primary amines,
Scheme 1, yield primary nitrosamines, which are not stable, and, in a series of rapid
reactions that include proton transfer and loss of H2O, yield diazonium ions that can
eventually react further to give dediazoniation (Scheme 1,a) or nucleophilic addition

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Scheme 1. Simplified Representation for the Nitrosation of a Primary Amine To Yield an Arenediazonium Ion,
and Further Decomposition (a) dediazoniation; b) nucleophilic addition)
(Scheme 1,b) products. This behavior contrasts with that shown by the secondary
amines, which yield stable nitrosamines, since there are no a-H-atoms available for the
necessary proton transfer [2].
The carcinogenic properties of N-nitrosamines and other N-nitroso compounds
have been known for over 30 years [1][3]. While compounds of the N-nitrosamide-type
are known to be direct mutagenic agents requiring no biological activation, N-
nitrosamines must be metabolized in order to elicit their mutagenic or carcinogenic
properties [1][3]. Although the mode of carcinogenic biochemical activation is not
known for all nitrosamines, it is believed that many of them are activated through the
process of a-hydroxylation, resulting in formation of an unstable a-hydroxynitros-
amine, which decomposes readily to a diazonium ion, which, in turn, is an aggressive
alkylating agents due to their ability to generate aryl radicals [4][5].
‡
The reactions of arenediazonium ions, ArN2 (Scheme 1) have attracted consid-
erable attention [6 ± 8], from both synthetic and mechanistic points of view, because of
their extraordinary sensitivity to environmental changes [9][10]. In spite of there being
a substantial body of knowledge available about their reactions, some of the
mechanisms are not completely understood. Electron-density analysis of a number of
diazonium ions has shown that both aliphatic and aromatic diazonium ions are best
thought of as carbenium ions closely associated with an internally polarized N2
molecule [11], which implies a dative C N bond (N ! C s donation and C ! N p
back-donation, designated with an arrow in all schemes). Among others, arenediazo-
nium ions are currently being employed as chemical probes to determine interfacial
concentrations in micellar systems [12 ± 16], to study topologies and orientations of
aggregate-bound polypeptides [17], as aryl radical sources for synthetic precursors
[18], and their role in DNA arylations [19][20] and DNA deaminations [21] is being
explored.
‡
Arenediazonium compounds, ArN2 , are strong oxidizing agents that undergo
homolytic fragmentation to produce aryl radicals upon reacting with certain electron
donors [10][22], and there is good evidence [23 ± 26] that the ability of arenediazonium

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Helvetica Chimica Acta ± Vol. 84 (2001)
ions to generate radicals may be responsible, to some extent, for the mutagenic and
carcinogenic properties of aromatic diazonium compounds [4][5][23 ± 27]. To prevent
nitrosation, a number of nitrosating scavengers are currently employed in the food [28]
and cosmetic [29] industries. Among them, natural antioxidants like ascorbic acid
(H2A), a-tocopherol, butylated hydroxytoluene, sodium bisulfite, etc. are in common
usage [1][28]. Although these nitrosamine inhibition reactions are relatively well-
known, little attention has been paid to the reactions of those natural antioxidants with
arenediazonium ions [9][10]. A number of studies concerning the effects of H2A on
dediazoniations have been reported [5][22][30][31], and there is some controversy
about its effect on the mechanism of the reaction.
‡
Ascorbate ion (HA ) has been used to promote dediazoniation of ArN2 in the
‡2
presence of intentionally added metal ions like Cu [32]. In these studies [22] the
‡
‡2
‡1
decomposition of ArN2 was attributed to its reduction by the Fe or the Cu
‡3
‡2
generated from the action of HA on the Fe or Cu metal ions, but the possibility
that HA can directly reduce ArN2 was not considered. Doyle et al. reported that H2A
‡
‡
reacts with 4-X C6H4 N2 (X ˆ NO2, Cl, or NHC6H5) to yield stable compounds
whose structures have been spectroscopically identified as 3-O-arenediazoascorbic
acids (diazo ethers) [31]. In contrast, Reszka et al., have recently found that the
‡
reaction of 4-X C6H4 N2 (X ˆ NO2, Cl, Br, OMe, or N(Et)2) with H2Aunder similar
conditions (pH ˆ 7) generates aryl radicals [5].
In recent work, we have reported a novel method to determine H2A in freshly
prepared natural orange juices [33]. The method is based on the accelerating effect of
H2A on the dediazoniation of 3-methylbenzenediazonium ion, but the mechanism of
such a reaction was not depicted. To expand our knowledge regarding the effects of
H2A on dediazoniations, we carried out a kinetic study of the reaction between ascorbic
acid and o-, m-, and p-methylbenzenediazonium ions (OMBD, MMBD, and PMBD
respectively) within the pH range 1 ± 4, of interest to the food industry. In addition to
the kinetics and mechanism of the reaction, the study may be pertinent to the food
industry because of the extensive use of H2A as a natural antioxidant. It can be also of
interest to biochemists due to the ready ability of aryl radicals to add to DNA bases and
to react with important physiologically relevant electron donors.
2. Results. ± 2.1. Effects of Citric Acid and Britton-Robinson Buffer on the Observed
Rate Constant and Product Yields in the Absence of H2A. The effects of citric acid (CA)
and acidity (HCl or Britton-Robinson (BR) buffer) on the thermal decomposition of
OMBD, MMBD, and PMBD in the absence of H2Awere studied by spectrophotometric,
polarographic, and chromatographic techniques. The protocols employed allowed measure-
‡
ment of rate constants for ArN2 decomposition and of product formation and distribution
at different acidities (see Exper. Part). Fig. 1 shows the data obtained for OMBD,
chosen as representative. Fig. 1,a shows the evolution of the first polarographic peak
(
50 mV) of OMBD with time and the corresponding first-order plot depicting
OMBD loss that yields an observed rate-constant kobs of 10.2 Â 10 4 s , in agreement
with published data [34][35]. A very similar value was obtained by spectrophoto-
metrically monitoring the disappearance of OMBD absorbance at l ˆ 314 nm.
Rate constants for dediazoniation product formation were obtained by chroma-
tography. Preliminary experiments show that only one dediazoniation product, o-
1

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Fig. 1. Representative kinetic plots for the decomposition at 358 of OMBD (ca. 2.00 Â 10 4 m) in the absence of
H2A. a) Evolution of the first polarographic peak ( 50 mV), b) variation in the yield of o-cresol, and c)
*
*
variation in the formation of the azo dye (l ˆ 490 nm) with time ( ) and first-order plot ( ).
cresol, is formed in significant yields. Fig. 1,b shows the variation in the yield of o-cresol
1
with time and the corresponding first-order plot, yielding a kobs of 10.5 Â 10 4 s . The
protocol employed (see Exper. Part) also allowed us to determine indirectly the kobs for