Mechanistic Studies of the Reactions of Nitrone Spin Trap PBN with Glutathiyl Radical

Dmitriy N. Polovyanenko,† Victor F. Plyusnin,‡,§ Vladimir A. Reznikov, §,|

Valery V. Khramtsov,⊥ and Elena G. Bagryanskaya*,†

International Tomography Center SB RAS, NoVosibirsk 630090, Russia, Institute of Chemical Kinetics &Combustion, NoVosibirsk 630090, Russia, NoVosibirsk State UniVersity, NoVosibirsk 630090, Russia,NoVosibirsk Institute of Organic Chemistry SB RAS, NoVosibirsk 630090, Russia, and Dorothy M. DaVis Heart& Lung Research Institute, The Ohio State UniVersity, Columbus, Ohio 43210

ReceiVed: December 7, 2007; In Final Form: February 1, 2008

We performed mechanistic studies of the reaction of PBN with the physiologically relevant glutathiyl radical,GS•, formed upon oxidation of the intracellular antioxidant, glutathione, GSH. The scavenging rate constantof GS• by PBN has been measured directly by laser flash photolysis and indirectly by competitive EPR of thespin adduct of PBN and another spin trap, DMPO (5,5-dimethyl-1-pyrrolineN-oxide), and was found to be6.7× 107 M-1 s-1. Reverse decomposition of the paramagnetic PBN-glutathiyl radical adduct to the nitroneand thiyl radical was observed for the first time. The rate constant for the reaction of the monomoleculardecomposition of the radical adduct was found to be 1.7 s-1. Diamagnetic, EPR-invisible products of PBNadduct degradation were studied by1H NMR and 19F NMR using newly synthesized fluorine-substitutedPBN.

Introduction

Chemical mechanisms of pharmacological action of nitronesin different biological models are poorly understood. PBN,N-tert-butyl-R-phenylnitrone, is a widely used spin trap fordetection of multiple short-lived radicals. PBN has been shownto demonstrate neuroprotective, anti-inflammatory, and anti-aging properties.1-10 Nevertheless the mechanism of therapeuticactivity of PBN is still unclear. The primary role of the radicaltrapping mechanism in nitrone therapeutics is considereddoubtful particularly taking into account the extremely lowtrapping efficiency of the superoxide radical.11

Glutathione, GSH, is considered to be a major intracellularantioxidant.12 However, the product of one-electron oxidationof GSH, glutathiyl radical, GS•, may further contribute inoxidative damage processes, e.g., via formation of the highlyreducing glutathione disulfide radical anion.13 Previously weand others reported high rates of GS• scavenging by cyclicnitrones 5-diethoxyphosphoryl-5-methyl-1-pyrrolineN-oxide(DEPMPO)14 and 5,5-dimethyl-1-pyrrolineN-oxide (DMPO).15

For the first time we also observed monomolecular decomposi-tion of the paramagnetic adduct, ST/GS•, back to GS• and theparent nitrone14 and decomposition of the corresponding reduceddiamagnetic adduct, ST/GSH, back to GSH and parent nitrone.14

These latter mechanisms result in recycling of both nitrone spintrap and glutathione and, in combination with the high trappingefficiency of GS•, may contribute to the biological activities ofthe nitrones. In our opinion, these mechanisms should beespecially taken into account when pharmacological activity ofthe hydrophobic membrane-permeable PBN is discussed. How-ever, neither the rate constant of GS• scavenging by PBN nor

mechanisms of decomposition of the corresponding adducts havebeen reported. In this work we measured the scavenging rateconstant of GS• by PBN using direct detection of GS• by laserflash photolysis and indirectly by competitive EPR detectionof the spin adducts of PBN and another spin trap, DMPO (5,5-dimethyl-1-pyrrolineN-oxide). To complement the EPR, weemployed1H NMR and 19F NMR using a newly synthesized

* To whom correspondence should be addressed. E-mail: elena@tomo.nsc.ru.

† International Tomography Center SB RAS.‡ Institute of Chemical Kinetics & Combustion.§ Novosibirsk State University.| Novosibirsk Institute of Organic Chemistry SB RAS.⊥ The Ohio State University.

Figure 1. EPR spectra obtained during continuous irradiation of themixture of 40 mM PBN, 10 mM S-nitrosoglutathione, 0.5 mM DTPAin 0.1 M phosphate buffer, pH 7.0. Spectrometer settings were asfollows: microwave power, 2.017 mW; modulation amplitude, 1 G;sweep time, 20.97 s; number of scans, 1.

4841J. Phys. Chem. B2008,112,4841-4847

10.1021/jp711548x CCC: $40.75 © 2008 American Chemical SocietyPublished on Web 03/26/2008

fluorinated PBN analogue (fPBN) to detect diamagnetic, EPR-invisible products of PBN adduct degradation. The results arediscussed particularly in relation to possible contribution of thereactions of PBN with GS• to the antioxidant activity of thisnitrone.

Materials and Methods

Chemicals.DMPO, PBN, glutathione disulfide (GSSG), andglutathione (GSH) were obtained from Sigma. PBN and DMPOwere distilled in vacuum.S-nitrosoglutathione (GSNO) wassynthesized as described in the literature16 and stored at-15Co. Freshly prepared GSNO and deionized water were used.19F labeled PBN was synthesized by preparing a solution of2,6-difluorobenzaldehyde (1 g, 7 mmol),N-(tert-butyl)hydroxy-lamine hydrochloride (1.23 g, 9.8 mmol), and ammonium acetate(1.1 g, 14 mmol) in methanol (20 mL) and kept for 24 h atroom temperature and then evaporated at reduced pressure. Theresidue was diluted with 20 mL of saturated brine and theprecipitate of tert-butyl(2,6-difluorobenzylidene)azane oxide(fPBN2) was filtered off and washed with brine and water andthen air-dried and recrystallized from hexane. The yield was1.4 g (94%), mp 65-67 °C, containing, %: C 61.92; H 6.07;N 6.50. Calculated for C11H13F2NO, %: C 61.96; H 6.15; N6.57. Horseradish peroxidase17 and hydrogen peroxide wereobtained from Sigma.

NMR Measurements.NMR spectra were measured using aBruker DRX-200 spectrometer and 5 mm tubes. The1H and19F NMR experiments were preformed in 0.1 M buffer, namelyKD2HPO4 was added to D2O and the solution was then titratedto pHobs 7.0 which corresponds to pD 7.4.18 A Nd:YAG laser(wavelength 355 nm, power 3 mJ per pulse, frequency 10 Hz)was used as a light source.19F and1H NMR spectra of a mixtureof GSNO and PBN or fPBN in buffer solution were measuredbefore and after 25 min of irradiation. A standardπ/2 pulsesequence was used with1H decoupling in19F NMR measure-ments.

EPR Measurements.EPR measurements were carried outusing a Bruker EMX spectrometer with a quartz flat cell, 0.25mm thickness (optical path). The spectrometer settings were asfollows: modulation amplitude, 1.0 G; sweep width, 60 G forboth DMPO and PBN experiments; frequency, 9.76 GHz;modulation frequency, 100 kHz; microwave power, 5.21 mW;time constant, 10.24 ms; sweep time, 10.48 s; and number ofscans, 5 (PBN experiments) or 16 (fPBN experiments). A 500

W high-pressure mercury lamp was used for photolysis of themixtures of GSNO directly in the resonator of the EPRspectrometer. The wave length (340-360 nm) was selectedusing light filters. Radical adduct concentration was determinedrelative to the integral signal intensity of the stable nitroxylradical, 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-yloxy, mea-sured under the same experimental conditions.

Laser Flash Photolysis (LFP).A detailed description of theLFP equipment has been published previously.19 Solutions in arectangular cell (10 mm× 10 mm) were irradiated though thediaphragm (diameter 2 mm) with a LOTIS TII laser (355 nm,6 ns, 30 mJ per pulse). The experiments were carried out atroom temperature in water pH 7.0. Argon bubbling wasperformed for 10 min before and throughout the experiments.Thirty scans were preformed for each kinetic analysis. Burningout of the sample was monitored by HP 8354 Agilent spectro-photometer and was less than 5% during kinetic measurements.Burning out of the sample per one laser pulse in irradiatedvolume was less than 18%. Stationary photolysis of GSNO wasperformed using the same laser.

Results and Discussion

EPR Studies of Glutathiyl Radical Scavenging by PBNSpin Trap. To study scavenging of the GS• radical by PBNand its fluorinated analog, fPBN, we performed the photolysisof GSNO in the presence of the nitrones and analyzed the EPRspectra of the adducts. The formation of PBN/GS• and fPBN/GS• adducts during photolysis of the mixture of 40 mM PBN,10 mMS-nitrosoglutathione, 0.5 mM DTPA in 0.1 M phosphatebuffer, pH 7.0, was observed (Figure 1, panels a and b,respectively). The hyperfine constants of the EPR spectrumshown in Figure 1a are equal toaN ) 1.540( 0.007 mT andaH ) 0.315 ( 0.004 mT and are in good agreement withliterature for the spin adduct of glutathiyl radical with PBN.20

The hyperfine coupling constants of the fPBN/GS• adduct werefound to be equal toaN ) 1.555( 0.008 mT andaH ) 0.634( 0.006 mT.

Figure 2 shows the kinetics of PBN spin adduct decay withthe initial concentration of PBN/GS• adduct varied dependingon light intensity and irradiation time of the sample.

Fitting PBN/GS• decay kinetics (Figure 3) with the first-orderdecay described by eq 1, is in good agreement with the

Figure 2. Kinetics of the PBN/GS• spin adduct decay measured usingEPR after irradiation of the solution containing 10 mM GSNO, 40 mMPBN, 0.5 mM DTPA in 0.1 M phosphate buffer, pH 7.0 (irradiationtime,b, 2 s;0, 1.5 s;O, 1 s). Solid lines are calculated monoexponentswith parameterkobs ) (1.2( 0.1) s-1 (see eq 1). Insert: kinetics of thePBN/GS• spin adduct decay on logarithmic scale. Solid lines are a linearapproximation of the experimental data.

Figure 3. Dependence of thekIobs value on the termk10[GSNO]/(

kscPBN[PBN] + k10[GSNO]). Kinetics of the PBN/GS• spin adduct

decay were measured using EPR after irradiation of the solutioncontaining different concentrations of GSNO and PBN in 0.1 Mphosphate buffer, 0.5 mM DTPA, pH 7.0; (O) [PBN] ) 40 mM, andGSNO concentrations vary from 0.5 mM to 20 mM; (9) [GSNO] )10 mM, and PBN concentrations vary from 20 to 80 mM. Theexperimental kinetics were fitted to (eq 2) yielding the value ofkI

PBN

) (1.7 ( 0.2) s-1.

4842 J. Phys. Chem. B, Vol. 112, No. 15, 2008 Polovyanenko et al.

experimental data yielding the observed rate constant of adductdecay,kobs ) 1.2 s-1.

Similar to the previously observed decomposition of spinadducts of glutathiyl radicals with DMPO and DEPMPO,14 thereversibility of the decomposition of the PBN spin adduct backto the parent nitrone and GS• can be proposed. Taking intoaccount that GS• radical beyond scavenging by PBN predomi-nantly decays in reaction with GSNO with rate constantk10 ) (1.5 ( 0.3) × 109 M-1 s-1 (measured in this workby laser flash photolysis, see below), the following equationfor the observed rate constant of spin adduct decay can beobtained

wherekscPBN and kI

PBN are the rate constants for scavenging ofGS• radical by PBN and dissociation of PBN/GS• adduct,respectively. This prediction is in good agreement with experi-mental data (Figure 3) which support the linear dependence ofkobs on termk10[GSNO]/ksc

PBN[PBN] + k10[GSNO]. Note that atlow GSNO concentrations eq 2 is not correct and the otherpathways of GS• radical decay may become significant andshould be taken into account (eqs 11-13). The fitting ofexperimental data to eq 2 usingksc

PBN ) (6.7 ( 1.5)× 107 M-1

s-1 (measured in this work by laser flash photolysis, see below)yields the value of the rate constant of PBN/GS• decompositionequal tokI

PBN ) (1.7 ( 0.2) s-1.To measure the rate constants of glutathiyl radical scavenging

by PBN and fPBN a competitive EPR approach was used.

Taking into account the first-order spin-adduct decay and usingthe approximation of a quasistationary condition of photolysis,the following equations can be obtained:

wherekscDMPO andkI

DMPO are the rate constants of scavenging ofGS• radical by DMPO and decay of DMPO/GS• adduct,respectively, and [PBN/GS•]S and [DMPO/GS•]S are concentra-tions of corresponding spin adducts in a quasistationary condi-tion. Assuming negligible changes in PBN and DMPO con-centration during the experiment, the measured ratio ofconcentrations of radical spin adducts allows us to obtain thescavenging rate constant for PBN according to the followingequation:

EPR spectra of two adducts with variations of DMPO concen-tration are presented in Figure 4. Two methods of glutathiylradical generation were used: (i) GSNO photolysis and (ii)oxidation of GSH by hydrogen peroxide (H2O2) and horseradishperoxidase (HRP).17

The ratio of PBN/GS• and DMPO/GS• adduct concentrationscalculated from the integrals of corresponding EPR spectra arelisted in Table 1. The rate constant of glutathiyl radical

Figure 4. EPR spectra obtained during irradiation of 0.1 M phosphate buffer solution, pH 7.0, containing 0.5 mM DTPA (a) 40 mM PBN, 3-12mM DMPO and 10 mM GSNO; (b) 40 mM PBN, 3-12 mM DMPO, 10 mM GSH, 0.2 mM mM H2O2 and 0.1 mg/mL HRP. The spectra weremeasured under steady-state conditions which were confirmed by the plateau for spin adducts concentrations.

TABLE 1: Ratio of PBN/GS• and DMPO/GS• Adduct Concentrations Obtained by Calculation of the Double Integral of theCorresponding EPR Lines (Figures 4 and 5)

[DMPO],mM

[PBN],mM

[DMPO/GS•]/[PBN/GS•](GSNO photolysis)

[DMPO/GS•]/[PBN/GS•](GSH oxidation)

[DMPO/GS•]/[fPBN/GS•](GSH oxidation)

3 40.5 (1.6( 0.2) (1.4( 0.1) (1.2( 0.2)5.9 41.5 (3.1( 0.5) (3.2( 0.2) (2.5( 0.3)

11.8 40.5 (4.8( 0.3) (7.0( 0.2) (4.6( 0.8)

d[ST/GS•]dt

) -kobs[ST/GS•] (1)

kobs) kIPBN

k10[GSNO]

kscPBN[PBN] + k10[GSNO]

(2)

d[PBN/GS•]dt

)

kscPBN[PBN][GS•] - kI

PBN[PBN/GS•]S ) 0 (3)

d[DMPO/GS•]dt

)

kscDMPO[DMPO][GS•] - kI

DMPO[PBN/GS•]S ) 0 (4)

kscPBN ) kI

PBN[DMPO]

[PBN]

[PBN/GS•]S

[DMPO/GS•]S

kscDMPO

kIDMPO

(5)

Reaction of PBN with Glutathiyl Radical J. Phys. Chem. B, Vol. 112, No. 15, 20084843

scavenging by PBN was calculated using eq 5 and was equalto ksc

PBN ) (7.0 ( 1.6) × 107 M-1 s-1. The value forkscDMPO )

(1.1 ( 0.5) × 108 M-1 s-1, was measured using laser flashphotolysis (see below), and the rate constant for DMPO/GS•

decomposition,kIDMPO ) (0.14 ( 0.04) s-1, which was ob-

tained from dependence of observed decay rate constants ofDMPO/GS• on GSNO and DMPO concentrations.14

The rate constant of GS• radical scavenging by fPBN wasmeasured using a similar competitive method (see Figure 5 andTable 1). The rate constant of the fPBN/GS• spin adduct decaywas obtained by fitting EPR kinetics measured after photolysisof a mixture of 10 mM GSNO and 40 mM fPBN (data notshown) and waskI

fPBN ) (0.9 ( 0.2) s-1. The obtainedscavenging rate constant of glutathiyl radicalksc

fPBN was (4.3(0.8) × 107 M-1 s-1.

Laser Flash Photolysis Studies of the Reaction of the GS•

Radical with DMPO and PBN. The photolysis of GSNO wasused for generation of thiyl radicals.S-Nitrosoglutathioneabsorption spectrum has maxima at 340 nm (absorption coef-ficient ε ) 922 M-1 cm-1) and 550 nm (ε ) 15 M-1 cm-1),21

whereas glutathione exhibits little absorption above 250 nm.Therefore, we used laser excitation at 355 nm (GSNOε ) 770M-1 cm-1) and monitored transient absorption changes from275 to 600 nm. The mechanism of the photolysis has beenstudied previously in detail.22 Reactions occurring during GSNOirradiation in the presence of spin trap (ST) are shown inreactions 6-13. The photolysis of nitrosothiol leads to theformation of glutathiyl radicals (GS·) via homolytic cleavageof the S-N bond22,23 (reaction 6). In the presence of a spintrap (ST), the glutathiyl radical is effectively scavenged withthe formation of the radical adduct (ST/GS•; reaction 7). Underthe experimental conditions, namely low GS• radical concentra-tions, only reaction 10 significantly contributes to GS• decay(see reactions 6-13). The insignificant difference between thekinetics of glutathiyl radical decay measured upon GSNOphotolysis in air-saturated and argon-bubbled solutions (datanot shown) indicates the negligible role of reaction 12. Inagreement with second-order dependence on the concentrationof generated radical species, the performed numerical calcula-tions confirm the negligible contribution of reactions 11 and

13, even for diffusion-controlled values of the rate constantsk11 andk13.

Figure 6 shows transient absorption spectra at multiple timepoints after laser pulse during photolysis of GSNO. Directexcitation of GSNO led to bleaching of its ground state and toreduction in absorption of GSNO. Glutathiyl radical’s absorptionspectrum is similar to the spectrum of GSNO and has anabsorption maximum at 330 nm (ε ) 580 M-1 cm-1).23-25

Therefore, decrease of the optical density at 330 nm is relatedto glutathiyl radical decay. Note that PBN has an absorptionmaxima at 285 nm (ε ) 18 400 M-1 cm-1), whereas DMPOabsorption above 280 nm is negligible.

The time dependence of the transient signals was differentwhen measured at different detection wavelengths. Kinetics ofglutathiyl radical decay measured at 340 nm in the presence orabsence of spin traps (PBN or DMPO) are shown in Figure 6b.

Figure 5. EPR spectra obtained during continuous generation ofglutathiyl radicals in horseradish peroxidase enzymatic system, 10 mMGSH, 0.5 mM H2O2 and 0.5 mg/mL HRP in 0.1 M phosphate buffer,pH 7.0, 0.5 mM DTPA, in the presence of 40 mM fPBN and DMPO(a) 0, (b) 3, (c) 5.9, and (d) 11.8 mM.

Figure 6. Flash photolysis of 0.25 mM GSNO in argon-bubbled water,pH 7.0. (a) Transient absorption spectra at different time delays afterlaser pulse following excitation at 355 nm. (b) Kinetic traces measuredat 340 nm in the absence of the spin trap and in the presence of 10mM PBN (solid lines are the first-order fittings).

GSNO98hV

GS• + NO• (6)

GS• + ST98kST

ST/GS• (7)

ST/GS• 98kI

products (8)

2(ST/GS•) 98kII

products (9)

GS• + GSNO98k10

GSSG+ NO• (10)

2GS• 98k11

GSSG (11)

GS• + O298k12

GSOO• (12)

GS• + NO• 98k13

GSNO (13)

4844 J. Phys. Chem. B, Vol. 112, No. 15, 2008 Polovyanenko et al.

In the absence of spin traps, the GS• decay is determined byreaction 10 and was found to be equal tok10 ) (1.5 ( 0.3) ×109 M-1 s-1, in good agreement with literature data, 1.7× 109

M-1 s-1 22.Kinetics measured at 340 nm at different concentrations of

spin traps, PBN or DMPO, were well described by first-orderdecay. The dependencies of observed pseudo-first-order rateconstant (k1

GS) on concentrations of PBN (O) and DMPO (0)spin traps were fit by linear regression (Figure 7). The rateconstants of glutathiyl radical scavenging derived from theslopes of the linear regression (Figure 7) were found to be equalto ksc

PBN ) (6.7 ( 1.5) × 107 M-1 s-1 for PBN andkscDMPO )

(1.1( 0.5)× 108 M-1 s-1 for DMPO. The observed scavengingrate constant of glutathiyl radical by DMPO is about two timessmaller than the value previously reported from the measure-ments using the EPR competitive approach.26

Changes in optical density were also observed at 380 nm.The same signals were detected previously by Wood et al.22

and were attributed to unidentified transient species that absorbat 380 nm but not at 320-340 nm. Kinetics at 380 nm werenot dependent on concentration of PBN or DMPO spin traps inagreement with the formation of the previously reportedunidentified product with absorption at 380 nm. Stationaryphotolysis of GSNO (0.25 mM, OD) 0.2) at 355 nm resultedin an increase of absorption in the region of about 260 nm (datanot shown) which is characteristic of the disulfide formation.Similarly, photolysis of 0.25 mM GSNO in the presence of 25mM PBN or 25 mM DMPO resulted in GSSG formation only.The absorption coefficient for GSSG at 260 nm is equal to 290M-1 cm-1. No new absorption peaks were observed in the regionfrom 330 to 550 nm.

NMR Studies of the Reaction of PBN and its FluorinatedAnalogue fPBN with GS•. NMR spectra measured before andafter photolysis of GSNO solutions in phosphate buffer in thepresence of PBN are shown in Figure 8, panels a and b,respectively. The appearance of the new lines (four doubletswith chemical shifts in the range of 2.8-3.4 ppm, Figure 8b)indicates the formation of the main reaction product, GSSG.NMR lines with chemical shift in the range of 1.1-1.4 ppmcorrespond to the products of light-induced PBN hydrolysis27

followed by formation of aldehyde (NMR line at 9.9 ppm). Thesame NMR lines are observed during photolysis of the PBNsolution without GSNO. The estimated concentration of alde-hyde formed is 0.5 mM.

NMR spectra of the irradiated PBN solution in the presenceof GSNO did not reveal additional diamagnetic products of PBNadduct decay. Specifically, the formation of the reducedparamagnetic product of PBN/GS•, hydroxylamine PBN/GSH,was not observed. Hydroxylamine formation would lead to theappearance of an NMR signal near 7.9 ppm, which is charac-teristic of a proton at the double bond. Also, in the case ofhydroxylamine formation, the ratio between integrals of the

Figure 7. Dependence of observed first-order rate constant of glutathiylradical decay (k1

GS) on concentrations of spin traps, PBN (O) andDMPO (0), measured at 340 nm after photolysis (355 nm) of 0.25mM GSNO in water, pH 7.0.

Figure 8. 1H NMR spectrum of the solution of 20 mM PBN and 1 mM GSNO in 0.1 M deuterated phosphate buffer, pHobs7.0, before (a) and after(b) laser irradiation, Peaks corresponding to the products of light-induced PBN hydrolysis are marked by an asterisk (*).

Reaction of PBN with Glutathiyl Radical J. Phys. Chem. B, Vol. 112, No. 15, 20084845

NMR lines of protons III and IV (Scheme 1 and Figure 8) shouldbe affected, while it remains unchanged during the experimentbeing equal to 1:9. Note that there is a small probability that1H NMR signals of the product are hidden in the much moreintensive NMR lines of the observed spectra. To overcome thisdifficulty, we employed19F substituted fPBN and19F NMR,expecting enhanced differences between the chemical shifts ofthe fPBN trap and the corresponding products, hydroxylamine,fPBN/GSH, and nitrone, fPBN/GS. Note that the differencebetween chemical shifts of the19F NMR signal of fluorinatedDMPO and corresponding hydroxylamine formed from the spinadduct with C-centered radicals can be as large as 10 ppm.28

1H NMR spectra obtained after photolysis of GSNO and fPBNwere similar to that for GSNO photolysis in the presence ofPBN and showed the accumulation of GSSG and formation ofproducts of light-induced fPBN hydrolysis only. In the corre-sponding19F NMR spectra, only the peaks of the fPBN trapand products of its hydrolysis were observed (data not shown).

Thus, NMR measurements did not show new diamagneticproducts during GS• generation in the present of PBN and fPBNdespite the effective scavenging of GS• radical by the traps andfast decay of the corresponding paramagnetic adducts. Note thatpreviously we observed the formation of a single diamagneticproduct upon photolysis of GSNO in the presence of DEPMPOspin trap,14 which was assigned to the corresponding nitroneproduct (ST/GS). This product is formed upon bimoleculardecomposition of the ST/GS• adduct which significantly con-tributed to the decay of the DEPMPO/GS• adduct. Contrary toDEPMPO/GS•, the decay of the PBN/GS• adduct is pure first-order (Figures 2 and 3) which implies that there is negligiblecontribution of bimolecular disproportionation to the decay. Thelower contribution of the bimolecular disproportionation in thedecay of the PBN/GS• adduct compared with that for DEPMPO/GS• might be related to steric hindrance due to the presence ofa bulky phenyl group in the PBN structure. Therefore, theabsence of additional NMR signals during GSNO photolysisin the presence of the PBN and fPBN spin traps is in agreementwith the observed first-order decay of the paramagnetic adduct,and support the reversibility of the reaction of GS• with PBNand fPBN.

Conclusion

In the present work, we performed mechanistic studies ofthe reaction of PBN spin trap with glutathiyl radical. The rateconstants of scavenging of glutathiyl radical by PBN and itsfluorinated analogue, fPBN, have been measured for the firsttime and were found to be equal (6.7( 1.5)× 107 and (4.3(0.8)× 107 M-1 s-1, respectively. The reverse reactions, releaseof GS• radical from the PBN/GS• and fPBN/GS• adducts, havebeen unambiguously confirmed by the analysis of EPR andNMR spectra of reaction products. The proposed kinetic schemeallows for quantitative descriptions of all of the observedkinetics. The reciprocal proportionality of the pseudo-first-orderdecay rate constant,kI, to the concentration of spin trap, and itsdirect proportionality to the concentration

of the competitive reagent for GS•, is in excellent agreementwith this scheme. The rate constants of GS• release from thePBN/GS• and fPBN/GS• adducts were found to bekI

PBN ) (1.7( 0.2) s-1 and kI

fPBN ) (0.9 ( 0.2) s-1, respectively. Theobservations described here show another example of a funda-mental chemical behavior that applies to a variety of redoxreactions with nitrone spin traps, which must be consideredrelevant.

When considering the pharmacological activity of PBN, oneshould take into account that it is highly lipophilic and thereforepossesses membrane-permeable character. Being diffusibleinside cells, PBN reacts with the GS• radical with an extremelyhigh rate constant. At low physiological rates of GS• formation,the recombination of GS• radicals is less probable than its spintrapping. The corresponding antioxidant efficiency of PBN isdetermined by the significantly lower oxidizing potential of thePBN/GS• adduct when compared with that of the highlyoxidizing GS• radical. Note also that the competition betweenmonomolecular decomposition and reduction of the PBN/GS•

adduct might be significantly affected by the intracellular redoxstate which influences PBN functionality.

Acknowledgment. The financial support of Russian Foun-dation for Basic Research Grant Nos. 05-04-48632, 05-03-32474, 06-03-32110, and 07-02-91016-AF is gratefully ac-knowledged. D.N.P. thanks Zamaraev Foundation for financialsupport. This work was supported by a program of complexintegration projects of SB RAS-2006 (Grants 4.16 and 77).

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Reaction of PBN with Glutathiyl Radical J. Phys. Chem. B, Vol. 112, No. 15, 20084847