Original Contribution

DEDIAZONIATION OF p-HYDROXYBENZENEDIAZONIUM ION IN ANEUTRAL AQUEOUS MEDIUM

BARTOLOME QUINTERO,* JOSE JORGE MORALES,* M IGUEL QUIROS,† MARıA ISABEL MARTıNEZ-PUENTEDURA,*and MARıA DEL CARMEN CABEZA*

*Department of Physical Chemistry, University of Granada, Granada, Spain; and†Department of Inorganic Chemistry, Universityof Granada, Granada, Spain

(Received14 December1999;Revised16 March 2000;Accepted5 May 2000)

Abstract—The dediazoniation of p-hydroxybenzenediazonium ion (PDQ) in a neutral aqueous medium has beenstudied under controlled experimental conditions to prevent photochemical and/or heterolytic side-reactions. Oxygenincreased the dediazoniation rate of PDQ and caused the appearance of quinone and hydroquinone. An accumulation ofquinone deriving from the reduction of PDQ by hydroquinone was also observed. In ESR analyses with different spintraps, the most stable signal was identified as belonging to the adduct of the p-hydroxyphenyl radical, even in thepresence of dimethylsulfoxide or ethanol. A general scheme is proposed including three pathways for the homolyticdediazoniation of PDQ. Pathway 1 represents dediazoniation induced by a hydroxyl ion, a slow process at neutral pHand an even slower one with deaerated samples; a favored quinoid structure is put forward to explain these results. Inpathway 2, the formation of a semiquinone radical via the reaction of an aryl radical with oxygen is put forward to justifythe increase in the dediazoniation rate in the presence of oxygen. In pathway 3, hydroquinone, produced by semiquinonedismutation, may act as a reducing agent. © 2000 Elsevier Science Inc.

Keywords—p-hydroxybenzenediazonium ion, Homolytic dediazoniation, p-hydroxyphenyl radical, Quinone, Hydro-quinone, Oxygen influence, Free radicals

INTRODUCTION

Reactions involving arenediazonium ions are widely em-ployed in organic chemistry [1]. Some arenediazoniumions may be formed by the reaction in an acid medium ofsodium nitrite with an appropriate substrate such as afood component [2] or a clinical drug [3–5]. This fact isimportant in that the reaction might well occur withcomponents provided in a diet or therapeutical treatment,i.e., the compounds may be formed in vivo. It is alsoknown that edible mushrooms contain arenediazoniumions and arylhydrazines, which can be metabolized intoarenediazonium ions [6]. As far as biological and relatedareas are concerned, it is well known that arenediazo-nium ions are mutagenic and may cause tumors in ani-mals [6–8] although the genotoxic agent has not beenclearly identified yet. Some studies suggest that the aryl

radical is the ultimate agent in causing mutagenic andcarcinogenic effects [9–12], although other publicationspoint to the genotoxicity of the aryl cation [13], diazenylradical [14], and arenediazonium ions as a whole [15,16],or a possible combination of the arenediazonium ion andthe aryl radical [17–19].

The mutagenic and carcinogenic capacity of aryl rad-icals revolves around their capacity to form C8-aryl-derivatives from purine bases and to degrade the pyrim-idine bases, converting them into nonabsorbentultraviolet products [9]. Furthermore, experimental evi-dence exists to implicate aryl radicals in strand breaks ofDNA [12]. This mutagenic and carcinogenic capacitymay be supported by the presence of electron donorssuch as ascorbate, NADH, p-hydroquinone, or catecholsin the cell or tissue medium, which are able to participatein the reduction of the arenediazonium compounds andthe subsequent appearance of aryl radicals.

Although it might be supposed that dediazoniationprocesses lead exclusively to carbon-centered radicals,some ESR analyses of the dediazoniation of several

Address correspondence to: Dr. Bartolome´ Quintero, Campus Uni-versitario de Cartuja s/n, Departamento de Quı´mica Fısica, Facultad deFarmacia, 18071 Granada, Spain; Tel:134 958 249071; Fax:134 958244090; E-Mail: bqosso@platon.ugr.es.

Free Radical Biology & Medicine, Vol. 29, No. 5, pp. 464–479, 2000Copyright © 2000 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/00/$–see front matter

PII S0891-5849(00)00321-X

464

benzenediazonium ions indicate the unexpected appear-ance of DMPO/•OH adducts. Thus, Reszka and Chignell[20], by ESR analysis of several arenediazonium ions,p-X-C6 H4-N2

1 (X 5 CH3-O2; Cl2; Br2; O2N

2; (Et)2N2), found the characteristic signals of DMPO/•OH inthose derivatives with substituents such as Cl2, Br2, orO2N

2. One tentative explanation for these results is thatDMPO/•OH might be formed from a silent hydroxyl-amine DMPOH-O-N5N-Ar, produced after the nucleo-philic addition of Ar-N5NO (H) to DMPO. On the otherhand, a DMPO/•OH adduct [17,18] has also been ob-served in a similar analysis using CH-, HOCH-, andHCOCH-substituted arenediazonium ions or arenediazo-nium precursors as arylhydrazines; but in this case thehydroxyl radical is thought to be formed from the break-down of diazohydroxide [21].

Thus, a detailed knowledge of the mechanisms asso-ciated with the degradation process of arenediazoniumions would appear to be essential to understand thegenotoxic properties of this kind of compound, particu-larly when the homolytic pathway may be competingwith the heterolytic one in producing potentially geno-toxic agents.

The arenediazonium compound analyzed in this studyis a tetrafluoroborate salt of p-hydroxybenzenediazoniumion (PDQ). This diazonium salt might be considered asbeing a suitable model for xenobiotics related to a widegroup of phenolic compounds, some of which have rec-ognized biological significance.

Apart from this, PDQ has a good electron-releasinggroup in para position as well as an electron-attractingdiazonium group; thus, an effective electron transferfrom the donor group to the acceptor might be expected.These structural characteristics, which lead to a differentexperimental behavior from that shown by otherarenediazonium ions with electron-withdrawing substitu-ents, are the reason that the dediazoniation processes forPDQ have not yet been fully described. Consequently,we have focused our attention on finding out more aboutPDQ degradation in a nearly neutral aqueous medium.The prime interest of this subject lies in the fact that thedediazoniation process may well generate species re-sponsible for attacking nucleobases and phosphodiesterbonds in DNA, an activity that is currently considered tobe one of the reasons behind the mutagenic capacity ofarenediazonium ions.

Our results indicate that under our experimental con-ditions PDQ dediazoniation is a homolytic process, butthat special care must be taken to avoid photochemicaldecomposition, which could provoke serious interfer-ences. Several authors have already reported noxiousbiological effects, caused presumably by a carbon-cen-tered radical derived from PDQ, but until now no one hasput forward a scheme for the homolytic dediazoniation

of PDQ. We propose that this homolytic degradation istriggered by the hydroxyl anion, but the primary productsthen produce an autocatalyzed reaction in which aryl,hydroxyphenylperoxyl, and semiquinone radicals appearto be involved. Of noteworthy importance is the involve-ment in the dediazoniation process of quinone-semiqui-none-hydroquinone redox equilibria, a complex systemrelated to various biological problems [22,23], the reac-tivity of which has recently been reexamined [24]. Wehave not detected the hydroxyl radical adduct in the PDQdediazoniation; nonetheless, mechanistic ways of form-ing reactive oxygen species from the previously men-tioned radicals could well exist.

MATERIALS AND METHODS

Chemicals and solutions

Chemicals of the highest available purity (Merck;Darmstadt, Germany; and Aldrich Chemical Co.; Mil-waukee, WI, USA) were used to obtain PDQ tetraflu-oroborate. Diethylenetriaminepentaacetic acid (DTPA)were bought from Sigma Chemical Co. (St. Louis, MO,USA). 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), N-t-butyl-a-phenylnitrone (PBN), anda-(4-pyridil-1-oxide)-N-t-butylnitrone (4-POBN) (Sigma) were used as spintraps. Hydroquinone (Merck) was used to synthethizequinone [m.p.5 115°C;e245 (phosphate buffer, pH 7)521,5006 400 M21 cm21)]

Synthesis and spectroscopic characterization of PDQ

PDQ tetrafluoroborate was synthesized according tothe procedure described by Daneˆk et al. [25], with slightmodifications. The solid obtained was stored in darknessat below218°C. The synthesized solid was subject toelemental analysis in a Fisons-Carlo Erba EA1108CHNS-0 Elemental Analyzer (Scientific InstrumentCentre [SIC], University of Granada). The PDQ massspectra were obtained in a Fisons Platform II low-reso-lution mass spectrophotometer (SIC, University ofGranada) using the positive chemical ionization tech-nique (CI1). A Fisons triple sector Autospec-Q high-resolution mass spectrometer (EBE) was also used, em-ploying the LSIMS technique (bombing with Cs1), witha warm up to 2mA, an accelerating voltage of 25 kV, anda glycerol matrix. IR spectra were registered at roomtemperature with an FT-IR Nicolet 20SXB spectropho-tometer (SIC, University of Granada) within the range of400 to 4000 cm21, with a resolution of 2 cm21. ABrucker RFS 100/S spectrometer equipped with a Nd:YAG laser with excitation wavelength at 1064 nm(Technical Services Unit, University of Jae´n), with aFourier FT-Raman was also used. Perkin-Elmer Lambda

465Dediazoniation of PDQ

5 and Lambda 16 spectrophotometers were used for thespectroscopic analysis of PDQ tetrafluoroborate. TheUV-Vis absorption spectra were obtained at a scan speedof 480 nm/min. Merck-Hitachi equipment for HPLC,including a Merck L-6220 biocompatible pump and aMerck L-4500 diode array detector, were used. Aqueousmedia were filtered by Millipore HA filters (Millipore;Bedford, MA, USA) with a pore size of around 0.45mm.The column was a Spherisorb ODS-2 (4.6 mm3 200mm) with a particle size of 5mm. The mobile phaseswere deaereated with a Selecta P 150 W ultrasoundgenerator, producing waves of 40 kHz. Mobile phasemethanol/ammonium formiate 0.1 M (1:39) with a flowof 0.7 mL/min was routinely used. The possible appear-ance of hydroquinone was monitored by steady statefluorescence using a Shimadzu RF-5001 PC spectrofluo-rimeter. A Radiometer pH M64 potentiometer with aGK2401C mixed electrode was used whenever calledfor. The calibrations were carried out with Crison bufferreferences (pH 4 and pH 7).

Stability preliminary tests

We checked for any effects caused on PDQ tetraflu-oroborate degradation by either environmental light orapparatus light sources. PDQ tetrafluorborate solutionsamples were exposed to laboratory light for up to 5 minand the results showed that irradiation by environmentallight for periods of less than 2 min caused no significantalterations to the absorption spectra. Irradiation from thelight sources in UV-Vis spectrophotometers did not alterthe spectrum recorded after seven consecutive scans at480 nm/min. Nevertheless, irradiation by the spec-trofluorimeter’s xenon lamp did cause fast decomposi-tion. These results were taken into account when design-ing the methodology for the spectrophotometric andspectrofluorimetric measurements. All measurementswere routinely made with aliquots taken from a stocksolution kept in darkness. The influence of oxygen dis-solved in the solution upon the PDQ decomposition ratewas noted at an early stage in our experiments; thus,whenever deaereated samples were required the oxygenwas purged by bubbling with argon for at least 10 min.

ESR assays

For ESR measurements, a Bruker ESP 300E spec-trometer, capable of obtaining spectra in the X band (9 to10 GHz), was used. Among the preliminary tests, ESRspectra of DMPO were obtained in an aqueous mediumbuffered with 0.2 M phosphate at pH 7 at a concentrationof 110 mM. After running between 10 and 20 scans at again of 1.25 106, we observed weak signals, probably

caused by paramagnetic contaminants. The main signalsshowed aN 5 15.6 G and aH 5 22.5 G, which lowered inintensity in the presence of DTPA 0.5 mM. We alsofound a group of four signals of relative intensity 1:2:2:1,with an aH 5 aN ' 15 G of lower intensity than theprevious ones. Nevertheless, these signals, which wereevident under high-gain instrumental conditions andmultiple scans, were not detected under the instrumentalconditions used to analyze the PDQ samples.

To obtain the ESR spectra, we used 0.5 mL of PDQtetrafluoroborate solution (160 mM), with an added 0.5mL of a solution of DMPO 220.8 mM, in 0.2 M phos-phate buffer, pH 7. Other experiments were also carriedout with different concentrations of spin traps and chang-ing the order of the addition of reagents, spin trap,scavenger, and PDQ. The spectra were obtained 18 min,1 h, and 24 h after mixing the reagents. We also analyzedthe influence of the oxygen present in the ESR spectra. Inthe experiments in which a scavenger was employed, thePDQ concentration in the final solution was the same asin the previous cases (80 mM) in a buffered medium ofphosphates (0.1 M). The concentrations of spin trap andscavenger employed in the different cases were: 100mM/2.8 M for DMPO/DMSO, 50 mM/1.4 M for PBN/DMSO, and 50 mM/1.4 M for 4-POBN/DMSO. In theexperiments in which ethanol was used as scavenger, theproportions were: 100 mM/2.0 M for DMPO/EtOH, 50mM/2.0 M for PBN/EtOH, and 50 mM/2.0 M for4-POBN/EtOH.

Kinetic assays

We first checked the spectral response as a function ofthe concentration of the PDQ solutions (from 2.5mM to0.5 mM) and then made spectrophotometric measure-ments using 10mM PDQ solutions in HCl (pH 1.97),acetic/acetate buffer (pH 4.69), and phosphate buffer (pH7.00). For the analysis of anaerobic samples, we usedappropriate cuvettes prepared to avoid the reentry ofoxygen. HPLC chromatograms were obtained using re-verse phase and an isocratic regime. The mobile phasewas methanol/ammonium formiate 0.1 M (1:39). PDQsolutions in phosphate buffer (0.05 M, pH 6.97) at con-centrations of 12 mM and 10mM were used. The chro-matograms were made at 25°C and 37°C with solutionsprepared 24 h previously. We also studied the effects,either separately or combined, of O2 and DTPA (0.5mM) upon 10mM samples.

RESULTS

Synthesis and characterization of PDQ

PDQ tetrafluoroborate is a yellowish crystalline solid(m.p.5 135°C to 140°C with decomposition probably

466 B. QUINTERO et al.

due to a concomitant irreversible thermal reaction takingplace alongside the physical process). The results of theelemental analysis were C: 42.8%,; H: 2.76% and N:17.05% coincide well with the stoichiometric formulaproposed by Daneˆk et al. [25]:

Nevertheless, the results given by high-resolutionmass spectrometry agree with the calculated molarweight for a monomer (found mass5 121.0400460.00015 uma; calculated mass5 121.040188uma),while in the low-resolution mass spectrum the molecularion appeared with an m/z relationship equal to 121. Ourfailure to obtain the mass of the unaffected dimer may bedue either to the polar nature of the matrix (glycerol)used in the high-resolution experiment or to the experi-mental conditions used to obtain the low-resolution massspectrum. The IR spectrum showed the presence of aband at 2189 cm21, which may be put down to N[Nstretching (2110 cm21 for diazophenol [26]; 2075.8cm21 for p-benzoquinone diazide at 10K [27]). Thisband is located at 2150 cm21 (pH 7) and at 2239 cm21

(pH 1) for PDQ solutions, indicating that a dissociationprocess in the hydroxyl group appears related to anabsorption shift to a lower frequency.

Another strong absorption band recorded for solidPDQ is found at 1591 cm21, which seems to correlatewith the band at 1574 cm21, present for PDQ in aqueousmedia both at pH 1 and pH 7, suggesting that absorptionis not involved in the dissociation of the hydroxyl group.Thus, we might make a tentative assignment to an aro-matic group. The Raman spectrum for solid PDQ prac-tically matches the IR spectrum, but the IR signal at 1591cm21 is now split into three signals in the range from1500 to 1700 cm21.

PDQ analysis in an aqueous solution

There are no changes in the shape of the recordedabsorption spectra from aqueous solutions of PDQ atconcentrations of up to 500mM (phosphate buffer, pH6.96), which allows us to assume that no molecularaggregates form and neither does azocoupling takeplace below this concentration. PDQ solutions at neu-tral and basic pH values show absorption bands withmaxima at 348 nm and 251 nm. In an acid medium, theabsorption is also characterized by two bands, with

maxima at 316 nm and 229 nm. The influence of pH onthe PDQ absorption spectra may be interpreted inaccordance with the following equilibrium:

The protonated species is known as p-hydroxyben-zene-diazonium or paradiazophenol (PDF). In theequilibrium shown above, this compound appears inthe usual Lewis notation, although a new model hasrecently been proposed, which implies a dative bond-ing of a nearly neutral N2 group to a carbenium ion[28 –30]. The nonprotonated species, PDQ, is basicallya resonance hybrid between at least the two canonicforms shown (four resonance structures are consideredin [27]). The yellow color observed in the PDF/PDQsystem may therefore be considered to be the result ofthe contribution of a quinoid species to the resonancehybrid, making the solution colorless at acid pH due tooxygen protonation, which creates a typical diazoniumstructure and thus decreases the double-bonding con-jugation. This circumstance may well explain the IRabsorption shift mentioned above. Therefore, it mightbe considered that oxygen protonation reduces theelectron-donor properties of the hydroxyl group, re-sulting in a shift of the absorption band to a higherfrequency compared to the location of the band cor-responding to the unprotonated quinoid molecule.

The form of the absorption spectrum does notchange at pH. 7 and so the absorption maximum at348 nm might be considered to be a spectral charac-teristic of PDQ. A value ofe 5 41,9906 220 M21

cm21 was obtained at 348 by using solutions bufferedto pH 6.96 in the concentration range of 2.5mM to20.1 mM. Via measurements made at the above-men-tioned wavelength, we obtained a value of pK53.35 6 0.02 for the acid-dissociation equilibrium at25°C. Chromatographic HPLC analysis of newly pre-pared PDQ aqueous solutions (phosphate buffer, pH6.96) showed only one signal, with a retention time of6.66 min, associated with a spectrum with a maximumat 348 nm.

467Dediazoniation of PDQ

Analysis of PDQ dediazoniation in an aqueoussolution

The difference between the absorption spectra mea-sured using aliquots taken from a reaction solution keptin darkness and those made using the same solution forall the other measurements are shown in Fig. 1. A highscan speed was used for both, so for the latter case thetotal irradiation time at the absorption wavelength of300–400 nm was around 3 min throughout the 24 hanalysis. Moreover, the irradiation of PDQ at 348 nmgenerated by the spectrofluorimeter’s xenon lamp causedhydroquinone to appear almost immediately (Fig. 1C).This was revealed by its emission spectrum (maximum ataround 327 nm) and also by changes in the UV-Vis

absorption spectrum, where a significant decrease inabsorbance in the PDQ band could be seen, together witha simultaneous increase at 280 nm, where hydroquinoneabsorbs (Fig. 1D). As mentioned above, these results ledus to keep the solutions in darkness as routine procedure;aliquots were taken when needed, thus reducing theinfluence of environmental light and the accumulativeeffects of instrumental irradiation.

The PDQ and PDF species belonging to the protolyticequilibrium had different stabilities in an aqueous solu-tion. Absorption spectra were registered every 1.5 h byusing 10mM solutions in a hydrochloric-acid medium,pH 1.97; in acetate buffer 0.2 M, pH 4.69; and in phos-phate buffer, pH 7.00, all of which were kept in darkness

Fig. 1. (A) Absorption spectra of 10mM PDQ in phosphate buffer (pH 6.93) under aerobic conditions at 25°C using aliquots takenfrom a solution kept in darkness. (B) The same as (A) but using the same sample for all of the measurements. (C) Fluorescence spectra(lexc 5 280 nm) of 10mM PDQ in phosphate buffer (pH 6.93) kept in darkness under aerobic conditions at 25°C (nonirradiated) andthe same solution after 15 min of irradiation at 348 nm provided by the spectrofluorimeter’s xenon lamp. (D) UV-Vis spectra of thesame solutions.

468 B. QUINTERO et al.

at 25°C under aerobic conditions. Only at neutral pH didsignificant destruction of PDQ occur after 12 h.

We also analyzed any possible influence of molecularoxygen dissolved in the solution by comparing absor-bance changes vs. time between unpurged solutions andthose previously purged of oxygen by bubbling withargon for 10 min. The average percentages (results of atleast four different measurements) for PDQ destructionin aqueous solutions (phosphate buffer, pH 7) were cal-culated from the decrease in absorbtion at 348 nm reg-istered at 24 h and 96 h after their preparation (Table 1).The solutions were always kept in darkness at 25°C. Ascan be seen, the reaction rate increased in the presence ofoxygen and decreased in the presence of DTPA. Theemission spectra of the same solutions used in spectro-photometry were registered (Fig. 2). Hydroquinoneemission was noticeable in those cases where there wasa significant percentage of degradation after 24 h. A PDQsolution in an acetate, pH 4.6, kept for 24 h in darknessat 25°C, showed no emission band in fluorescence (dot-ted line in Fig. 2). We furthermore ascertained that thepresence of a chelating agent of potentially reducingmetal ions, DTPA (0.5 mM), did not affect the spectralcharacteristics of PDQ.

HPLC analysis of PDQ gave the chromatogramshown in Fig. 3. The chromatogram is greatly enlarged toreveal the appearance of signals belonging to very minorcompounds. A signal is evident with a retention time of10.79 min, which can be put down to hydroquinonebecause the retention time of pure hydroquinone is 10.64min and the absorption spectrum associated with thissignal coincides with the hydroquinone spectrum. Ofequal interest is the chromatographic signal that appearsat 30.13 min; the retention time and absorption spectrumassociated with this chromatographic signal may wellindicate the presence of quinone (the retention time forpure quinone is 29.6 min). The chromatogram of thesample kept under similar conditions but previously

purged with argon (anaerobic conditions) shows no sig-nal for either hydroquinone or quinone.

PDQ reduction by hydroquinone

Experiments were made using PDQ/H2Q ratios of1:100, 1:25, and 1:1 in neutral (pH 7.0) and acid (pH 4.6)aqueous-buffered media. From the kinetic curves ob-tained by measuring the absorbance values at 348 nmduring different reaction, it can clearly be seen that H2Qreduces PDQ, which disappears more rapidly either asthe pH becomes more acidic or the concentration of H2Qincreases (data not shown). The results of spectrofluoro-metric analysis carried out on the solution containingPDQ/H2Q (1:1) seem to indicate, however, that hydro-quinone is not consumed during the PDQ reduction pro-cess (Fig. 4). It is apparent from these curves that nosignificant changes occur in relative fluorescence inten-sity vs. time in the hydroquinone spectra registered in thepresence of PDQ. This fact is more evident at the red endof the emission band (. 360 nm), where it coincides inintensity with the emission of pure hydroquinone. It mustbe pointed out that the difference between the hydroqui-none spectrum and those spectra registered in the pres-ence of PDQ can be attributed to reabsorption phenom-

Fig. 2. Emission spectra (lexc 5 280 nm) obtained with 10mM PDQin phosphate-buffered solutions (pH 7) at 25°C kept in darkness. FO5sample without DTPA under aerobic conditions; DO5 sample withDTPA under aerobic conditions; FA5 sample without DTPA underanaerobic conditions; DA5 sample with DTPA under anaerobicconditions; dotted line5 0.16 mM PDQ solution in 0.1 M acetatebuffer (pH 4.6) kept in darkness for 24 h at 25°C.

Table 1. Influence of Dissolved Molecular Oxygen and theChelating Agent DTPA (0.5 mM) in Average Percentages (Result ofat Least Four Different Measurements) of Degradation for 10mM

PDQ in Neutral Aqueous Solutions (Phosphate Buffer pH 7) Kept inDarkness at 25°C

time (h) FO DO FA DA

24 58.10 30.07 1.30 3.696 97.33 79.08 54.88 20.67

Data calculated from the decrease in absorbance at 348 nm recorded24 h and 96 h after preparation.

FO 5 aerated solution of PDQ in the absence of DTPA; DO5aerated solution of PDQ in the presence of DTPA; FA5 deaeratedsolution of PDQ in the absence of DTPA; DA5 deaerated solution ofPDQ in the presence of DTPA.

469Dediazoniation of PDQ

ena, since the excitation wavelength (280 nm) overlapsthe PDQ absorption spectrum (molar absorptivity around103 M21 cm21).

The PDQ degradation observed does not appear tocorrelate with the changes in H2Q concentration, but itshould be emphasized that the region around 288.5 nm,where hydroquinone has an absorption band, is the least-

modified part of the spectrum. These results were ob-tained again in an analysis of reaction mixtures contain-ing PDQ:H2Q ratios of 1:100 at pH 7 and 4.6 and 1:25 atpH 7, all under aerobic conditions (Fig. 5). In Fig. 5 itcan be seen that the process takes place with a simulta-neous increase in absorbance at about 250 nm, the pointat which the characteristic absorption maximum of qui-none is located.

We made a kinetic analysis taking the absorbance datameasured with a PDQ:H2Q ratio of 1:25 at 245 nm vs.time. It is important to bear in mind that 245 nm is notonly the point where an absorption maximum for qui-none may be located but also the absorption wavelengthfor PDQ and H2Q, and that both PDQ and quinone havehigh molar extinction coefficients. Therefore the exper-imental data have been corrected to take into account thedecrease in PDQ concentration, according to the follow-ing equation:

A cor245 5 A245 2

ePDQ245

ePDQ348 APDQ

348 (1)

where Acor245 is the corrected absorbance at 245 nm, A245

is the uncorrected absorbance at 245 nm,ePDQ245 is the

PDQ molar extinction coefficient at 245 nm (4000 M21

cm21), ePDQ348 is the molar extinction coefficient at 348 nm

(41,990 M21 cm21), and APDQ348 is PDQ absorbance at its

absorption maximum. One further correction was there-fore made to eliminate the background absorption causedby hydroquinone and the kinetic data related to the

Fig. 3. Chromatogram corresponding to a 12 mM PDQ phosphate-buffered solution (pH 7) kept in darkness for 24 h at 25°C underaerobic conditions.

Fig. 4. Fluorescence spectra (lexc 5 280 nm) recorded every 5 minduring 1 h with aliquots of the reaction mixture PDQ/H2Q (10mM:10mM) (A) pH 7, kept at 25°C in darkness under aerobic conditions anda solution of hydroquinone 10mM (B).

470 B. QUINTERO et al.

appearance of quinone were then analyzed according tothe integrated equation:

lnA`

245

~A`245 2 At

245 2 AH245!

5 kt

where A245 is the absorbance at 245 nm at infinite time,for which we accepted a value of 0.252 (correspondingas this does to the total transformation of PDQ intoquinone), AH

245 is the absorbance at the same wavelengthdue to the presence of hydroquinone (which is presumed

to be constant, with a value of 0.06 obtained from theordinate in the plot of the corrected data using Eqn. 1),and At

245 is the corrected absorbance at 245 nm. Usingthis equation, a straight line is obtained for the appear-ance of quinone with a slope of 4.3 1023 min21 (Fig. 6,right-hand ordinate). In addition, considering the reduc-tion process during its very early phase (up to 45 min forPDQ), the kinetic data for the disappearance of PDQ canbe adjusted to the integrated equation for a first-orderreaction, obtaining a straight line with a slope of 3.61023 min21 (Fig. 6, left-hand ordinate).

Fig. 5. UV-Vis absorption spectra using reaction mixtures of 10mM PDQ and H2Q. Phosphate-buffered solution (pH 7) at a ratioPDQ:H2Q (1:100) (curve A); the same ratio in acetic/acetate-buffered solution (pH 4.6) (curve B) and phosphate-buffered solution pH7 at a ratio PDQ:H2Q (1:25) (curve C). The samples were always kept in darkness at 25°C under aerobic conditions.

471Dediazoniation of PDQ

ESR analysis of PDQ dediazoniation in an aqueousmedium

Initially it was observed that with all the different spintraps the PDQ solutions showed no changes in eithercolor or transparency after being kept for several days atroom temperature. Nevertheless, the reference sample,kept under the same conditions but with no spin trap,underwent considerable alteration, including a darkeningin color and the appearance of a solid. The ESR spectrain Fig. 7 show the evolution of the PDQ/DMPO systemunder aerobic conditions at room temperature, where anincrease in signal intensity can be seen over time. Fur-thermore, the signals in the first spectrum (Fig. 7A)appear to be split, probably due to the existence of a spinadduct mixture. Nevertheless, the ESR spectrum regis-tered after 24 h (Fig. 7B) shows no split signal. Thus, itmay be supposed that the system contained at least twomain adducts, one of which disappeared after 24 h.

By subtracting the spectrum registered after 24 h fromthat registered after only 1 h, we were able to obtain thespectrum of the adduct that disappeared (Fig. 7C), whichturns out to have values of aN 5 15.4 G and aH 5 23.0G. The ESR spectrum obtained from the PDQ/DMPOsystem after being kept for 24 h under anaerobic condi-tions at room temperature is shown in Fig. 7 B2. Theinfluence of oxygen is revealed mainly in the generaldecrease in signal intensity registered in the ESR spectrain its presence. The presence of DTPA also produced asignificant decrease in the intensity of the ESR signals. Inaddition to the experiments just described, we analyzedthe ESR spectra to discover the effect of including a spintrap at different times after the addition of PDQ solution

to the buffered medium. The results were no differentfrom those found when the trap was added before thePDQ solution was made up.

ESR analysis of PDQ dediazoniation in an aqueousmedium in the presence of scavengers

The spectra for the DMPO/DMSO/PDQ, PBN/DMSO/PDQ, and 4-POBN/DMSO/PDQ systems wereobtained at different times after the preparation of themixture (,18 min, 16 h, and 24 h). Figure 8 shows thespectrum registered after 24 h. The same results werefound in the spectra recorded after shorter times. Thevalues for the hyperfine coupling constants were DMPO/DMSO/PDQ: aN 5 15.9 G, aH 5 24.5 G; PBN/DMSO/PDQ: aN 5 15.9 G, aH 5 4.1 G; and 4-POBN/DMSO/PDQ: aN 5 15.6 G, aH 5 3.2 G. We also recorded theESR spectra for the PDQ system containing differentspin traps and ethanol as scavenger. The spectra werealways very similar to those obtained in the presence ofDMSO under both aerobic and anaerobic conditions. Itmust be pointed out that the spectra recorded with thePDQ/EtOH/DMPO system showed a signal for only oneadduct and not two, as seemed to be the case in thePDQ/DMPO system. We also made an ESR analysis ofPDQ solutions treated with hydroquinone in the presenceof DTPA. Once again the spectrum had the characteristicprofile of an adduct with aH 5 24.9 G and aN 5 15.9 G.

DISCUSSION

The species derived from the protolytic equilibriumPDF/PDQ are not equally stable in solution. Within the

Fig. 6. Plots of the experimental data corresponding to the decrease in absorbance at 348 nm (left-hand ordinate) and the increase inabsorbance at 245 nm (right-hand ordinate). See details in text.

472 B. QUINTERO et al.

pH range assayed only neutral solutions of PDF/PDQunderwent any degradation process, which took placewith an absorbance loss at the PDQ absorption maxi-mum. According to the value found for pKa (3.35), bothPDQ and PDF exist at neutral pH, with PDQ beingpredominant. It is known that in weakly alkaline aqueousmedia dediazoniation of diazonium ions could take placeprior to the formation of an intermediate Ar-N5N-Rcomplex, which then decomposes to give the diazoateand diazenyl radicals [31]. A diazenyl radical has usuallybeen put forward as an intermediate compound in theprocess of homolytic dediazoniation since experimentalevidence of its formation has been found during homo-

lytic dediazoniation in nonaqueous media [32]. Dreher etal. [33] have pointed out that this radical is probably alsoformed in an aqueous medium, but so far it has beenundetectable, due almost certainly to its great instability.The diazoate radical has been characterized by Cadoganet al. [34].

At this juncture it must be remembered that arenedia-zonium compounds are generally very unstable and thatthey become more stable according to the nature of thesubstituents, especially if they are electron donors [35].Hydroxyl substitution contributes to the strength of theAr-N2

1 bond by means of a resonance mechanism,mainly as the OH2 group is deprotonated. Even PDF is

Fig. 7. ESR spectra obtained with a PDQ/DMPO system in phosphate-buffered solutions (pH 7). Final concentrations were 80 mMPDQ, 110 mM DMPO, and 0.1 M phosphate. (A) 1 h after adding the spin trap; (B) 24 h after adding the spin trap. All of the sampleswere kept in darkness at room temperature under aerobic conditions. (B2) ESR spectrum obtained from a sample equivalent to that usedfor spectrum shows in (B), but kept in anaerobic conditions; (C) resulting spectrum obtained after subtracting the experimental ESRspectrum (B) from spectrum (A).

473Dediazoniation of PDQ

presumed to exert less oxidant power in comparison toother arenediazonium ions, also due to the hydroxilicsubstituent. Hence, we may take it that both PDQ andPDF have structural features that do not favor the deg-radation process. In addition, the concentration of thehydroxyl anion is low at neutral pH. So, if we supposethat there is no reducing agent in the medium, the lowreactivity of PDQ/PDF species together with the lowconcentration of OH2 might explain the slow degrada-tion rate observed for PDQ in neutral aqueous solutions.

The process of PDQ degradation in such solutions isclearly influenced by the presence of dissolved molecularoxygen, as revealed by both spectrophotometric andspectrofluorometric measurements. As far as the spec-trofluorometric data are concerned, it is worth noting thata comparison of the emission band recorded under aer-obic conditions with that of pure hydroquinone leads usto assign the former to hydroquinone with considerableassurance. The appearance of this band was influencedboth by the presence of molecular oxygen and by pH.Some interference could also be observed when DTPAwas present in the solution. From these results a connec-tion between the degradation of PDQ in aerobic solutionsat neutral pH and the appearance of hydroquinone mightreasonably be inferred. Moreover, our chromatographicexperiments indicated the formation of hydroquinoneand quinone in aerobic samples, although these com-pounds were not found in samples prepared under anaer-obic conditions. It would appear, therefore, that hydro-quinone is involved in a redox side-process to originatequinone.

It is important to remember that we controlled all ourexperiments to protect all our samples from being ex-posed to light and in this way we avoided the formationof carbene from PDQ via a photochemical pathway [36]and, consequently, any possible reaction between car-bene and the nucleophilic solvent capable of forminghydroquinone. It seems that we were successful in pre-

venting this reaction because spectrofluorometric analy-sis at pH 4.6 showed no signal arising from the formationof hydroquinone. Hydroquinone can also originate fromthe nucleophilic reaction of water with the phenylcationresulting from a heterolytic dediazoniation, as has beenreported in the case of hydroxy- and chloro-dediazonia-tion of some arenediazonium ions in acid media [37,38].Nevertheless, we could not detect the presence of hydro-quinone at pH 4.6 either by spectrofluorometric analysis(irradiated samples, as mentioned above) or by HPLC.The absence of phenol and other typical products of ahomolytic reaction, such as Ar-Ar or Ar-N5N-Ar, coin-cides with results reported elsewhere [10].

Reducing metal ions may also be involved in thedegradation of PDQ, bearing in mind that the presence inthe sample of a chelating agent such as DTPA decreasesthe reaction rate. Nevertheless, the quantity of reducingions, mainly Fe21, in the buffers used is very low[around 0.17 ppm for phosphate buffer (0.05 M), 0.35ppm for acetate buffer (0.2 M), and 0.003 ppm for HClsolution at pH 1]. Thus, it would seem rather improbablethat PDQ should be reduced by means of these ions viaan independent degradation pathway. Moreover, degra-dation rates in the experiments made in the absence ofoxygen and DTPA were very low. Although an indirectpathway involving metal ions might be put forward toexplain the effects observed in the presence of DTPA,i.e., a chelate-Fe21-O2 complex [39], which would re-duce the amount of oxygen available to form the hy-droxyphenylperoxyl radical included in the scheme pro-posed below, we found no evidence for such a pathwayin our experiments. We confine ourselves therefore to theobservation that some sort of interference is observedwhen DTPA is present, but as yet we have found nosatisfactory explanation for the results obtained.

On the basis of the fact that hydroquinone is a stableproduct in the PDQ degradation process, it seemed to usworthwhile investigating whether hydroquinone might

Fig. 8. ESR spectra obtained with a /DMSO/PDQ spin trap system after 24 h at room temperature under aerobic conditions. From theleft, DMPO/DMSO/PDQ, PBN/DMSO/PDQ, and 4-POBN/DMSO/PDQ.

474 B. QUINTERO et al.

not act as a reducing agent for PDQ in a secondaryprocess. Our experimental results indicated that hydro-quinone provokes a faster degradation of PDQ than thatinduced by hydroxyl ions, the process at pH 4.6 beingeven faster than that at pH 7.0. It is apparent that thereduction of PDQ at pH 4.6 is enhanced by the presenceof the more highly oxidant species PDF at a proportionof around 5%. At pH 4.6 and pH 7.0, hydroquinone is inprotonated form since the pKa value for the first proto-lytic equilibrium H2Q/HQ2 is 9.70 [40,41].

The most notable aspects of the kinetic analysis of thereduction of PDQ by H2Q were that the decrease inabsorbance observed in the absorption maximum of PDQwas not paralleled by a concomitant decrease in theregion where the absorption maximum of H2Q occursnor in the fluorescence emission by H2Q. A numericalcheck can be made by taking the molar absorptivities at288 nm of PDQ (565 M21cm21) and H2Q (2513M21cm21) and calculating the degradation ratio of PDQfrom the absorbance measurements made at 348 nm.Simultaneously, the PDQ reduction process is followedby an increasing absorption in the region in which qui-none absorbs.

The values obtained from the slopes of the curvesplotted in Fig. 6 might be considered as being the resultof a mol-to-mol conversion of PDQ into quinone, in spiteof the probable reduction reaction formulated as:

2PDQ1 H2Q3 2PDQ•2 1 Q 1 2H1

where PDQ•2 represents a diazenyl radical. This meansthat the experimental data during the initial steps of theprocess do not seem to fit very closely to the rate equa-tion set out below:

2 d@PDQ#

dt5 2

d@Q#

dt5 k@PDQ#@H2Q#

since, according to this equation, the quinone productionrate should be double that of the degradation of PDQ.Furthermore, the slight variation shown in the absorptionmaximum of hydroquinone during the first step of thereaction would accord more readily with a steady statefor this compound, i.e., its acting as a intermediate ratherthan a reactant as described in the above reduction reac-tion.

In general, uncatalyzed dediazoniation reactions inaqueous media in the presence of oxygen are thought toproceed via the formation of a highly reactive interme-diate aryl cation [1]. ESR analysis, however, confirmsthe homolytic nature of the PDQ degradation processunder our experimental conditions. In principle, as wasto be expected in a free radical reaction, the spin trapinterfered with the degradation of PDQ, maintaining the

original color and transparency of the medium afterseveral days at room temperature, while the referencesample, kept under the same conditions but containing nospin trap, darkened and a solid substance was formed.

The ESR spectra obtained from the PDQ/DMPO sys-tem point to the existence of at least two radical species.One of them gives a stable adduct both in the samplecontaining dissolved oxygen and in that previously bub-bled with argon, and the signals from this adduct can stillbe recorded 24 h after preparing the solution. The hy-perfine coupling-constant values for this adduct, aN 516.0 G and aH 5 25.0 G, can be related to those of ap-hydroxyphenylic adduct according to the data found inthe literature [42], which indicate values for DMPO/•Arwithin the ranges aN 5 15.8–16.0 G and aH 5 24.3–24.8G. These results also coincide quite well with thosereported by Kikugawa et al. (aN 5 15.9 G, aH 5 24.8 G)[10] for the same system and the same time of 24 hpostpreparation. Kikugawa et al. make no mention, how-ever, of a second unstable adduct that eventually disap-pears both from the aerobic and anaerobic samples. Theassignment of this adduct is not easy within the tenets ofthe general dediazoniation scheme described by Dreheret al. [33]. Dediazoniation induced by hydroxyl ionsproduces primary radicals such as the diazenyl radical(2O-C6H4-N5N•) or the diazoate radical (2O-C6H4-N5N-O•). In the former case, the corresponding adductis unlikely to arise from such an unstable radical and,anyway, the shape of the spectrum would be much morecomplex than the sextet actually found. Another possi-bility would be an adduct formed from the diazoateradical; but, in general, the values listed in Buettner’stables [42] give hyperfine coupling constants for oxygen-centered radicals of aN ' aH or aN . aH. The identity ofthis adduct remains unclear.

The influence of oxygen is most evident in thedecrease in intensity of the ESR signals, while itapparently has no influence upon the intensity corre-sponding to the short-lived adduct signal. Both mightbe explicable in the light of the fact that oxygen trapsp-hydroxyphenylic radicals to give corresponding per-oxides; therefore, the proportion of aryl radical to betrapped by DMPO is less. Thus, the relationship inintensity between the signals would be favorable to theunstable radical in the presence of oxygen. DTPA alsocaused a significant decrease in the intensity of theESR signals. This may be due to the observed abilityof the iron (III) complex of DTPA to react directlywith DMPO [43]; but, as indicated above, DTPAinterferes with the degradation rate of PDQ, and there-fore a side-reaction, interfering with the formation ofthe aryl radical, can not be completely ruled out.

The possible formation of hydroxyl radicals in thedediazoniation of some salts of benzenediazonium ions

475Dediazoniation of PDQ

has been reported [17,18,20]. Under our experimentalconditions, we were unable to detect the characteristicquadruplet of a DMPO/•OH adduct. In fact, the use in allof the experiments of a relatively high concentration ofDMPO should have favored the formation of hydroxyl-amine DMPOH-O-NN5N-Ar, as proposed by Reszkaand Chignell [20] in their tentative explanation as to howa DMPO/•OH adduct might be formed. Neither do ourresults seem to fit in with the idea of a homolytic break-down into diazohydroxide p-X-C6H4-N2-OH [17,18].One plausible explanation may be the small contributionof the induced hydroxyl anion pathway to the overalldediazoniation process; if even a low concentration ofdiazohydroxide were to be present, the competitive for-mation of a diazoate anion may well prevent any exten-sive homolytic reaction.

We also made ESR analyses using spin traps such asDMPO, PBN, and 4-POBN in combination with a hy-droxyl radical scavenger such as DMSO or ethanol todiscriminate between carbon- and oxygen-centered rad-icals [44]. When DMSO was used as scavenger andDMPO as spin trap, only one signal remained stable inthe different spectra, and that could be identified as thearyl radical adduct. When PBN was used as spin trap, theresults for hyperfine coupling constants aH 5 4.0 G andaN 5 15.8 G were consistent with the values accepted forPBN/•Ar [9,39,45,46]. With regard to the hyperfine cou-pling-constant values found for the 4-POBN adduct, asignificant deviation of about 25% was calculated for theconstant corresponding tob-hydrogen compared to thevalues cited in the literature for the 4-POBN/•CH3 adduct[42]. These data indicate that the combination of thethree spin traps and DMSO prevent the detection of anyadduct from the methyl radical, which might be expectedif a hydroxyl radical were formed. Identical results wereobtained in the experiments made with samples purgedby argon. The spectra recorded using ethanol as scaven-ger were always very similar to those obtained in thepresence of DMSO under both aerobic and anaerobicconditions. Likewise, in the ESR spectra recorded withsamples containing PDQ and the reducing agent hydro-quinone, the only detectable signals were identified asbelonging to a p-hydroxyphenyl radical adduct. The re-sults of these analyses seem to indicate that no hydroxylradical is formed during PDQ dediazoniation under ourexperimental conditions.

In summary, since we controlled the occurrence ofheterolytic and photochemical reactions under our ex-perimental conditions, the degradation of PDQ in aneutral aqueous medium would seem to be basically ahomolytic dediazoniation process. The characteristicsof a radical process are supported by the presence of ap-hydroxyphenyl radical, detected by using differentspin traps. These results agree satisfactorily enough

with those already described in the literature for PDQand other benzenediazonium ions. Nevertheless, we doprovide here some hitherto unreported data, in partic-ular the influence of dissolved molecular oxygen uponthe increase in the degradation rate of PDQ. Theevidence obtained from aerobic degradation concern-ing the formation of hydroquinone and quinone asmajor final products and their absence in anaerobicconditions, together with the accumulation of quinonefrom the reduction of PDQ by hydroquinone, all sug-gest that dediazoniation induced by a hydroxyl anionis not the only active pathway in the system understudy. Consequently, we propose the followingscheme (Fig. 9) to explain our experimental results,which includes three pathways.

Pathway 1 corresponds to induced dediazoniationby a hydroxyl anion, as has been outlined by Dreher etal. [33]. This pathway is relatively slow for PDQ at25°C and nearly neutral pH, and very slow if thesamples have been previously purged by argon. As wehave pointed out above, the donor nature of the2OHgroup may well go some way to explaining this ex-perimental behavior, since the formation of a quinoidspecies is favored at nearly neutral pH, thus reducingthe possibilities of a nucleophilic attack by hydroxylions. Apart from this, we can obtain comparative databy calculating the half-wave potential value (E1/2

° ) forthe electrodal reduction of PDQ and PDF according tothe equation [47]:

E°1/ 2 5 ~0,299sP 1 0,314! 6 ~0,024!

wheresP is obtained from Hansch et al. [48].sP for the2OH group is20.37 and for2O2 is 20.81, so thatE1/2

° is 0.229 V for PDF (protonated form) and 0.129 Vfor PDQ (unprotonated form). It can be verified in eithercase that the values are below those obtained for diazo-nium salts such as p-methyldiazonium (0.250 V), whichis not very oxidative.

Nevertheless, although the independent contribu-tion of pathway 1 is apparently not very effective atnearly neutral pH, a remarkable increase in the rate ofthe process is observed when molecular oxygen ispresent in the samples. Molecular oxygen generallyinterferes with radical processes and in this case thewell-known reaction of oxygen with aryl radicals (k'2.109 M21s21 [49]) might be taken into consideration,assuming the formation of the corresponding hydroxy-phenylperoxyl radical, which would not increase thereaction rate via pathway 1, in due accord with itsirreversible nature.

The detection of hydroquinone and quinone as a finalproduct in the process leads to the possible involvement

476 B. QUINTERO et al.

of a semiquinone radical as intermediate, formed fromthe dimerization of peroxyl radicals and the subsequentformation of tetroxide, which would then decompose togive oxygen and a semiquinone radical in a concertedreaction similar to those undergone by alkylperoxyl rad-icals [50]. This reaction has been claimed in the reduc-tion of the 4-methoxybenzenediazonium cation by cate-chol [31], where ESR signals corresponding to a Zn21-semiquinone complex were obtained. The reduction ofPDQ via a semiquinone radical (pathway 2) would con-tribute towards a significant increase in the global reac-tion rate found in the presence of oxygen.

Besides this, the dismutation of a semiquinone radicalwould generate hydroquinone and quinone. This possi-bility has been reported in the literature [31] and isconsistent with both our spectroscopic and chromato-graphic results. The dismutation process would generatea new reducing agent, hydroquinone, which may act viapathway 3 in our scheme. Our data have proven thatadded hydroquinone leads to a decrease in the absor-bance of PDQ and the appearance of quinone in themedium, suggesting a reduction process similar to thoseobserved with other arenediazonium salts in the presenceof hydroquinone at 25°C [51].

According to the scheme outlined above, the globalprocess could be considered as being the sum of thefollowing reactions, in which PDQ•2 represents a p-hydroxybenzenediazenyl radical.

PDQ 1 H2Q 3 PDQ•2 1 SQ•2 1 2H1

2PDQ•2 1 O2 3 2SQ•2 1 2N2

SQ•2 1 PDQ 3 Q 1 PDQ•2

2SQ•2 1 2H1 3 Q 1 H2Q

2PDQ 1 O2 3 2Q 1 2N2

It is important to bear in mind the relatively small con-tribution by pathway 1, and so we have not taken intoconsideration with the other reactions that of dediazonia-tion induced by a hydroxyl anion. In the overall process,it is presumed that p-hydroxyphenyl radicals disappearby means of a fast reaction with oxygen, which would beapproximately acceptable in the initial step of the reac-tion since during the later stages of the reaction otherpossible reaction pathways for the radical can not beruled out. If all these factors are taken into consideration,the overall process expressed by the equations set outabove would be compatible with the observed accumu-lation of quinone and the similarity between the apparentreaction rates for the degradation of PDQ and the ap-pearance of quinone. Moreover, it may be deduced fromthe overall process represented in the scheme that a netconsumption of hydroquinone is not to be expected, justas has been observed in the experiments made usinghydroquinone as a reducing agent. On the contrary, un-

Fig. 9. Proposed scheme for the dediazoniation of PDQ in a neutral aqueous medium.

477Dediazoniation of PDQ

der anaerobic conditions the pathway for the formationof the semiquinone radical and hydroquinone is inter-rupted, limiting the degradation process to the attack byhydroxyl anions, which, as has been observed, proceedsvery slowly.

Evidence has been put forward to suggest that 1,4benzoquinone might be able to photo-oxidize water, thusproducing hydroxyl radicals [52]; but we consider thisprocess to be somewhat unlikely under our experimentalconditions. On the other hand, it is worth noting thatsome species involved in pathways 2 and 3 might pro-duce hydrogen peroxide by means of a reduction ofmolecular oxygen by the semiquinone radical, whichcould then generate a hydroxyl radical in a Fenton reac-tion [53–55]. Moreover, hydrogen peroxide could also beformed by the heterolytic decomposition of tetroxide. Itmay be presumed that the appearance of a hydroxylradical is theoretically acceptable for our system in ad-dition to those possibilities pointed out in the literature[17,18,20,43]. Nevertheless, none of our experimentsusing a spin trap separately or spin trap and scavengercombinations resulted in the detection of the adduct froman •OH radical. We believe that a further complete in-spection of this issue seems reasonable to clarify such animportant mechanistic aspect of this kind of autocata-lyzed reaction, in which a recognized mutagenic/carci-nogenic substrate generates active reducing compounds,thus favoring the possible appearance of a genotoxicagent.

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ABBREVIATIONS

CIDNP—chemically induced dynamic nuclear polariza-tion

DMPO—5,5-dimethyl-1-pyrroline-N-oxideDTPA—diethylenetriaminepentaacetic acidESR—electron spin resonanceEtOH—ethanolFT-IR—fourier transform InfraredHPLC—high-performance liquid chromatographyH2Q—hydroquinoneLSIMS—liquid secondary ion mass spectroscopyNADH—reduced nicotinamide adenine dinucleotidePBN—N-tert-butyl-a -phenylnitronePDF—paradiazophenol (conjugated acid of PDQ)PDQ—p-hydroxybenzenediazonium ionPDQ•2—p-hydroxybenzenediazenyl radical4-POBN—a-(4-pyridil-1-oxide)-N-tert-butylnitrone

Q—quinoneSQ•2—semiquinone radical

479Dediazoniation of PDQ