3745
J. Phys. Chem. 1988, 92, 3745-3751 whereas this is impossible with the respective spectra of 2,6-DMADMA. This latter finding is consistent with the results from IEOEM discussed in the previous section. This means that the results for TM-ADMA are consistent with a two-state model, while both the results from IEOEM and S,-S, absorption spectra indicate a more complex structural behavior of 2,6-DM-ADMA. The facts that with TM-ADMA two fluorescence bands can be seen in medium polar solvents and that the long-wavelength band behaves quite “normally”, quite different from 2,6-DM-ADMA, indicate that the sterical hindrance of the rotation around the anthryl-dimethylaniline bond is important for the behavior of this class of molecules, which already had been deduced from kinetic measurements with other compounds of this
Concluding Remarks TM-ADMA has been used as a model compound that shows two distinct and spectrally well-resolved fluorescence bands. The results of a standard IEOEM measuring procedure optically integrating over both bands could be separated into the effect of the fluorescence from a polar state A and a nonpolar state B. The polarizabilities of the state A could be shown to give negligible amounts to its dipole moment effective in solutions. This as well as the fact that there are two well-resolved fluorescence bands with TM-ADMA in certain solvents gives a very strong hint that there are also (at least) two fluorescence bands with other molecules of this class, although they are not spectrally resolved. As a consequence, the interpretation of the behavior of ADMA and
other compounds on the basis of a one-state model where strong reaction-field-induced terms are importantg cannot be kept further. The results presented here with TM-ADMA come from a molecule where the charge transfer might be assumed to occur between the two parallel anthryl and dimethylamino subunits with an orthogonal phenyl plane in between. Then the experimental C m in polar solvents would be equivalent value for pe = 60 X to a transfer either of a fraction of one elementary charge over these 0.7 nm or of one elementary charge over about 0.38 nm, which would be considerably less than the distance between the charge center of a postulated anthracene anion and the positive nitrogen center. Hence, a simple one n-electron transfer picture between the dimethylamino group and the anthryl group is not applicable. It may be possible that, although the phenyl plane is orthogonal to the anthryl and to the dimethylamino subunit’s n-electron plane, a small amount of partial charge transfer between these subunits and the phenyl ring will be possible, thus leading to the smaller observed dipole moment than that expected from the dimethylamino anthryl one a-electron transfer.
-
Acknowledgment. The Mainz-group gratefully acknowledges financial support by the Fonds der Chemischen Industrie. The present work also was partly supported by Grants-in-Aid (No. 5843003, 60040059, 61040046, 61 123008) from the Japanese Ministry of Education, Science and Culture to N.M. Registry No. TM-ADMA, 109432-41-9; 2,6-DM-ADMA, 10943240-8.
Electrochemistry and Spectroelectrochemistry of Nitroxyl Free Radicals Judith R. Fish, Steven G. Swarts, Michael D. Sevilla, and Tadeusz Malinski* Department of Chemistry, Oakland University, Rochester, Michigan 48309-4401 (Received: September 1 1 , 1987; In Final Form: January 15, 1988)
This work reports electrochemical and spectroelectrochemical studies of the two nitroxyl radicals 2,2,6,6-tetramethyl- 1piperidinyloxy (TEMPO) and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-l-yloxy (3-carbamoyl-PROXYL). Oxidation and reduction reactions have been observed in aqueous media over the pH range 2-12 in the potential range -0.8 to +0.8 V by differential pulse voltammetry, cyclic voltammery, and thin-layer UV-visible spectroelectrochemistry,and the reaction products have been characterized by IR, NMR, and ESR spectrometry. At pH values less than 10, characteristic electrochemical behavior is observed to be analogous for both radicals, and the products from electron transfer compare quite favorably with those found by pulse radiolysis of aqueous solutions of nitroxyl radicals. At pH 2-9, a stable cation from a reversible oxidation and hydroxylamine following an irreversible reduction, as well as hydroxylated cation at pH higher than 9, are the same as those obtained in pulse radiolysis experiments. Spectroscopic evidence indicates that behavior following reduction at high pH differs for the two radicals. At pH 12, reduced TEMPO may undergo structural changes leading to the formation of a new radical consisting of a seven-membered ring.
Introduction in the study of bioNitroxyl spin labels have extensive molecules and biomembranes.1-3 combination with ESR spectroscopy, the labels can give information important to the structure, conformation, and motion of the biological entities. Recently, the reactions of free nitroxyl spin probes with free radicals induced by the radiation of water and biomolecules have been investigated.4-6 This work has shown that the radiation(1) Berliner, L. J. Spin Labeling Theory and Applications; Academic:
New York, 1976. (2) Likhtenshtein, G. I. Spin Labeling Methods in Molecular Biology; Wiley: New York, 1976. (3) Bobst, A. M.; Kao, S.; Toppin, R. C.; Ireland, J. C.; Thomas, I. E. J. Mol. Biol. 1984, 173, 63. (4) Nigam, S.; Asmus, K.-D.; Willson, R. L. J. Chem. Soc., Faraday Trans. I 1976, 72, 2324. (5) Swarts, S . ; Sevilla, M. D. Radiat. Res. 1987, 112, 21.
produced reducing radicals such as the hydrated electron often result in the formation of hydroxylamine, whereas the attack of oxidizing radicals such as the hydroxyl radical often results in the formation of the cation. Interest has also been generated in their application to cancer therapy because of the ability of nitroxides to radiosensitize hypoxic cells by their reactions with radicals formed on DNA.’-’ It is proposed that the nitroxides undergo redox as well as coupling reactions with DNA radical^.^*^^'^'* One (6) Cadet, J.; Berger, M.; Ulrich, J. Can. J . Chem. 1984, 62, 6 . (7) Cadet, J.; Teoule, R. Free Radicals and Cancer; Marcel Dekker: New York, 1982; pp 183-200. (8) Adams, G. E.; Cooke, M. S. Int. J. Radiat. Biol. 1969, 15, 457. (9) Greenstock, C. L.; Adams, G. E.; Willson, R. L. Radiation Protection and Sensitization; Barnes and Noble: New York, 1970; pp 65-71. (IO) Willson, R. L.; Emmerson, P. T. Radiation Protecfion and Sensitization; Barnes and Noble: New York, 1970; pp 73-79. (1 1) Brustad, T.; Bugge, H.; Jones, W. B. G.; Wold, E. Int. J. Radiat. Biol. Relat. Stud. Phys., Chem., Med. 1972, 22, 115.
0022-3654/88/2092-3745$01.50/0 0 1988 American Chemical Society
3746
The Journal of Physical Chemistry, Vol. 92, No. 13, 1988 0
Fish et al.
i ,
0
1U2
I
’
0=6 -NH, Figure 1. Structural formulas for nitroxyl radicals TEMPO (2,2,6,6tetramethyl-1-piperidinyloxy, left) and 3-carbamoyl-PROXYL (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-l-xyloxy, right).
problem with the use of nitroxyl spin labels and free spin probes in living systems is that nitroxides have a relatively short half-life due to metabolic redox processes. Our interest in these radicals developed from their use as a means to monitor the radiation damage to DNA;5 however, to understand fully reactions between DNA radicals and nitroxides requires an understanding of the redox processes involved. This work reports electrochemical and spectroelectrochemical studies of the two nitroxide radicals 2,2,6,6-tetramethyl- 1-piperidinyloxy 1-yloxy (TEMPO) and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin(3-carbamoyl-PROXYL). The structures of the radicals investigated are shown in Figure 1. Production and reactions of the oxidized nitroxide (cation) and reduced nitroxide (anion) are observed by differential pulse voltammetry, cyclic voltammetry, and UV-visible spectroelectrochemistry. Reaction products have been characterized by IR, N M R , and ESR spectrometry.
Experimental Section Reagents. 2,2,6,6-Tetramethyl- 1-piperidinyloxy free radical and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-l-yloxy free radical were obtained from Aldrich. All electrochemical experiments were performed in H 2 0 containing 0.08 M tetraethylammonium perchlorate (TEAP, Eastman Kodak) as supporting electrolyte. TEAP was recrystallized and dried in vacuo prior to use. Standard buffer solutions comprised of tartaric acid, phosphoric acid, and potassium biphthalate, pH 2, potassium acid phthalate, pH 4, sodium and potassium phosphate, pH 8, potassium phosphate and sodium borate, pH 10, and tri- and disodium phosphate, pH 12, were obtained from Sargent Welch. Solution concentrations for cyclic and differential pulse voltammetry were 0.8 mM for TEMPO and 1.3 mM for 3-carbamoyl-PROXYL. Concentrations of 1.7 m M TEMPO and 1.6 mM 3-carbamoyl-PROXYL were used in spectroelectrochemical experiments. Instrumentation. A platinum button and a platinum wire served as the working and auxiliary electrodes, respectively, for cyclic and differential pulse voltammetric measurements made with a conventional three-electrode configuration and an IBM Model E C 225 voltammetric analyzer. A saturated calomel electrode (SCE), separated from the bulk of the solution by a fritted glass disk, was used as the reference electrode. A Princeton Applied Research Model 173 potentiostat with Model 179 digital coulometer was used for controlled-potential electrolysis (CPE) and coulometry. All potentials are reported versus aqueous SCE. Spectroelectrochemistry was performed in a thin-layer cell that followed the design of Fajer et aI.l3 The SCE was separated from the rest of the solution by a fritted bridge containing supporting electrolyte and solvent. UV-visible spectra were obtained with a Tracor Northern optical multichannel analysis system composed of a Tracor Northern 6050 spectrometer containing a crossed Czerny-Turner spectrograph in conjunction with the Tracor Northern 17 10 multichannel analyzer. Each spectrum resulted from signal averaging of 100 single, 19C-490 nm, 2.5-ms spectral acquisitions simultaneously recorded by a double-array detector with a resolution of 0.6 nm/channel. Proton N M R and IR spectra were acquired with Varian T60 and Beckman IR 33 spectrophotometers, respectively. A Varian (12) Brustad, T.; Jones, W. B. G.; Wold, E. Int. J . Radiat. B i d . Relat. Stud. Phys., Chem., Med. 1973, 24, 33. (13) Fajer, J.; Borg, D. C.; Forman, A,; Dolphin, D.; Felton, R.H . J . Am. Chem. SOC1970, 92, 345 1,
1
-0.4
( “v:s.
SCE )
-0.6
-0.8
Figure 2. Differential pulse voltammograms of (a) TEMPO (8 X IO” M) and (b) 3-carbamoyl-PROXYL (1.3 X M) in H 2 0 (0.08 M TEAP; scan rate 2 mV s-l, amplitude = 20 mV).
A
I
0.8
0.6
0.4 0.2 0.0 -0.2 -0.4 -0.6 - 0 . 8 POTENTIAL ( V v s . SCE
Figure 3. Cyclic voltammograms of (a) TEMPO (8 X M) and (b) 3-carbamoyl-PROXYL(1.0 X lo-’ M) in H,O (0.08 M TEAP scan rate 0.1 v s-’).
Century E-Line ESR spectrometer with dual cavity and variable-temperature capability was employed in this work. ESR parameters were measured versus Fremy’s salt (A(N) = 13.09 G and g = 2.0056).
Results and Discussion Oxidation and Reduction of Neutral TEMPO. Figure 2a illustrates a typical differential pulse voltammogram (DPV) for TEMPO obtained in aqueous solution at pH 7. When potentials are scanned from zero toward the positive direction, the DPV shows a clearly defined, symmetric peak at E, = 0.48 V with peak width at half-peak current equal to 94 mV. The definition and symmetry of the peak and the peak width at half-peak current are an indication of a one-electron transfer controlled by mass transport to the electrode. The DPV obtained when potentials are scanned from zero in the negative direction shows one peak at E , = -0.72 V. This peak is close to the limit of the range of the solvent, of lower current, broad, and poorly defined, which suggests irreversibility of the electrochemical process. Figure 3a illustrates a typical cyclic voltammogram (CV) of TEMPO obtained at pH 7. An anodic peak (Ia) is observed at E,, = 0.52 V, and upon scan reversal, a cathodic peak (IC) at E , = 0.46 V, which gives a half-wave potential = 0.49 V. A linear plot of i , or ,i versus L ‘ ‘ / ~ which , passes through the origin, and a constant value for with different rates of potential scan indicate that both processes are faradaic. The peak separation,
The Journal of Physical Chemistry, Vol. 92, No. 13, 1988 3747
Electrochemistry of Nitroxyl Free Radicals TABLE I: Electrochemical Parameters for Redox Reactions of Nitroxyl Radicals in H20(0.08 M TEAP, Scan Rate 0.1 V s-') peak E l j z , current, V pA
compound TEMPO (8 X M) wave I wave I1 3-carbamoyl-PROXYL (1.3 x 10-3 M) wave I wave I1
diffusion coeff D, cm2s-'
heterogeneous electron-transfer rate constant k , cm s-I
TABLE II: Absorption Maxima and Molar Absorptivities of Oxidized and Reduced Nitroxyl Radicals in H20(0.08 M TEAP) Am*,, nm ( i ~ ~ - ~ e ) compound neutral oxidn I redn I redn I1 TEMPO 210 (0.28)
+0.49 -0.62' f0.62 -0.65"
4.3
5.1
X
PH 7
250 (1.63) 250 (1.56) 250 (1.63) 258"(1.44) 258" (1.41) 258" (1.44) 280" (1.19)
pH 12
249 (2.45) 249 (3.0) 258" (2.20) 270 (3.0) 290 (3.0)
3.1 X
3.3 5.0 3.0
212 (0.29) 2.1 x 10-5
2.3 x 10-3
reoxidn I1 249 (2.2) 258" (2.0) 280" (1.7)
OCathodic peak potential.
1
reredn I1 249 (2.3) 258" (2.0) 280" (1 .O)
3-carbamoylPROXYL 211 (0.37)
1
PH 7
244 (2.25) 244 (1.88) 244 (2.25) 254* (1.76) 254b (1.76) 254b (1.76) 268b (1.53)
pH 12
243 (2.60)
216 (0.44)
0
222 (1.5) 0
reoxidn I1 222 (1.5)
r
X W
"Shoulder. bIsosbestic point.
2
1
200
250
300
350
WAVELENGTH (nm)
Figure 4. Electronic absorption spectra of (a and b) TEMPO (1.7 X lo4 M ) and (c and d) 3-carbamoyl-PROXYL (1.6 X lo4 M) in H z O (0.08 M TEAP). Spectra taken during (a and c) oxidation at applied potential of +0.75 V and (b and d) reduction at -0.80 V.
E,, - E,, of 60 f 5 mV for scan rates less than or equal to 0.1 V s-l and oxidation peak current equal to the accompanying reduction peak current confirm diffusion-controlled, reversible, one-electron transfer. Characteristic peak I parameters are listed in Table I. The number of faradays transferred was determined by coulometry carried out at potentials 200 mV more positive than the peak potential of Ia. Coulometry verified a transfer of 1 faraday for both the oxidation and reverse process. Peaks Ia and ICcan be assigned to the oxidation-reduction processes of TEMPO according to the reaction
A similar redox process for TEMPO at platinum electrodes in nonaqueous media occurring at E l j z = 0.46 and 0.50 V in acetonitrile and propylene carbonate, respectively, has been suggested previ0us1y.l~ cm2 s-l was calculated by A diffusion coefficient of 5.1 X using the Randles-Sevcik equation for potential scan rates lower than 0.1 V s-l. A heterogeneous electron-transfer rate constant of 3.1 X cm s-I was calculated for potential scan rates higher than 0.1 V s-l according to the method described by Nicholson and Shain.15 (14) Mairanovskii, S. G. Dokl. Akad. Nuuk. SSSR 1962, 142, 1120. (15) Nicholson, R. S.; Shain, I. Anal. Chem. 1964, 36, 706. (16) Neiman, M. B.; Mairanovski, S . G.; Korvarska, B. M.; Rozantser, E. G.; Gintsberg, E. G. Isu. Akad. Nauk. SSSR, Ser. Khim. 1964, 8, 1518; Chem. Abstr. 1966, 64, 14065e.
Product(s) of anodic and cathodic processes of peaks Ia and IC were characterized by in situ thin-layer spectroelectrochemistry. Figure 4a shows the electronic absorption spectra obtained during CPE of TEMPO at 0.8 V. Wavelengths of absorption maxima and molar absorptivities are presented in Table 11. The original compound, TEMPO, shows a single band at Amax = 250 nm and a shoulder at & = 258 nm. During the oxidation, both decreased slightly, and a broad shoulder at A, = 280 nm appeared and increased. Following exhaustive oxidation of the neutral radical, the spectrum was monitored at zero current. No spectral changes were observed during an observation period of 3 h. After reduction of the electrolyzed solution at 0.35 V, the original spectrum of TEMPO was obtained, indicating that the cation produced in the oxidation is stable in the interval of time monitored and corroborating reversibility of the electron-transfer process. The scan of the negative potential region shows one cathodic peak (IIc) at E, = -0.62 V (Figure 3a). As with DPV, cathodic peak IIc is poorly defined. There is no accompanying anodic peak, which suggests the reduction is electrochemically irreversible in the time scale of cyclic voltammetry or the electrochemical process is followed by a chemical reaction (EC mechanism). Due to the proximity of the reduction potential of peak I1 to the limit of the range of the solvent, CPE was carried out at a potential 100 mV more negative than the peak potential. Coulometry verified a 1 faraday charge transfer for the reduction process. Thin-layer absorption spectra obtained during the reduction process are shown in Figure 4b. The original 250- and 258-nm bands decreased and disappeared. No other bands appeared in the region monitored, and the solution remained colorless. Attempts to reoxidize the product of this electrolysis by applying a potential 200 mV more positive than the reduction peak were unsuccessful, and there were no further changes in the spectra during or after the attempted electrolysis. Failure to regenerate the original spectrum and the lack of an accompanying reoxidation current corroborate process irreversibility. The one-electron reduction process of TEMPO can be described as follows:
>y-O.
+ e-
IIC
>J&O-
(2)
It has been postulated that initial electron transfer can be followed
3748 The Journal of Physical Chemistry, Vol. 92, No. 13, 1988
Fish et al.
by protonation of the anion produced4J7in acidic or neutral media leading to the production of the hydroxylamine as shown in eq 2-4. Macroscale electrolysis of TEMPO was performed at pH
>N-OI11
+ H+(aq)
-
>N-OH IX
(3)
7 at a potential of -0.82 V. The product of reduction was isolated and identified as hydroxylamine by the presence of peaks at 3320 cm-' and 4.5 ppm, attributable to OH stretch, which are not present in the parent molecule in IR and N M R spectroscopy, respectively. In acidic media, a second protonation step should be expected: ,N-OH \
IV
+
H+(aq)
-
'N+H
I'
(4)
I-
z w
a a 3 0
I
OH V
Formation of 'NH-OH has been reported in pulse radiolysis e~periments;~ however, the electrochemical reduction of TEMPO in strongly acidic media does not occur since the reduction of hydrogen ion occurs at more positive potentials than does the reduction of TEMPO (see following discussion). Oxidation and Reduction of Neutral 3-Carbamoyl-PROXYL. Characteristic electrochemical behavior observed for neutral 3-carbamoyl-PROXYL is analogous to that observed for neutral TEMPO. In differential pulse voltammetry, process I, the reversible oxidation, is indicated by a clearly defined, symmetric peak at E p = 0.62 V with a peak width at half-peak current equal to 100 mV. Process 11, the irreversible reduction, is indicated by a poorly defined peak at E , = -0.75 V (peak IC, Figure 2b). In cyclic voltammetry, anodic peak Ia appears at E,, = 0.65 V, and cathodic peak ICat E* = 0.59 V. The half-wave potential is 0.62 V. Poorly defined, peak IIc appears at E , = -0.65 V with no accompanying anodic peak (Figure 3b). Diagnostic parameters for the process and results of coulometric experiments are as discussed for TEMPO. A diffusion coefficient of 2.1 X lob5cm2 sW1and a heterogeneous electron-transfer rate constant of 2.3 X cm s-l for process I were calculated as described (Table I). This electron-transfer rate constant is somewhat smaller than that measured for TEMPO. Electronic absorption spectra obtained during in situ spectroelectrochemistry of 3-carbamoyl-PROXYL show the same pattern of response observed with neutral TEMPO (Figure 4c,d). The original compound, 3-carbamoyl-PROXYL, showed a single = 244 nm, which decreased during oxidation at the band at A, plateau of wave Ia, while a broad shoulder a t A,, = 268 nm = 254 appeared and increased. An isosbestic point occurs at nm (Figure 4c). Results of stability studies, performed as described for TEMPO, again corroborate cation stability up to 3 h and process I reversibility. Figure 4d shows the electronic absorption spectra monitored during CPE of reduction process 11. During the reduction, the original 244-nm band decreased and disappeared. As with TEMPO, process I1 irreversibility was confirmed by lack of current during attempts to reoxidize the product of the electrolysis and absence of further spectral changes. pH-Dependent Oxidation and Reduction of TEMPO and 3Carbamoyl-PROXYL. To assess the effect of hydrogen ion on the redox processes observed and to assess the effect of hydroxide ion on the stability of the cation produced during the oxidation, we performed pH-dependent cyclic voltammetry and spectroelectrochemistry using standard buffer solutions. Typical cyclic voltammograms of TEMPO and 3-carbamoylPROXYL obtained at pH 2 and 12 are shown in Figure 5. At pH 2, the faradaic, diffusion-controlled, one-electron, reversible transfer process is indicated by redox couples at E I l 2= 0.48 and 0.62 V for TEMPO and 3-carbamoyl-PROXYL, respectively (17) Asmus, K.-D.; Nigam, S.; Willson, R. L. Int. J . Radiaf. B i d . 1976, 29, 211.
v
, , , , , , , 0.8 0.6 0.4 0.2 0.0 -0.2 -0.4 -0.6 -0.8
POTENTIAL ( V vs. SCE )
Figure 5. Cyclic voltammograms for (a and b) TEMPO and (c and d) 3-carbamoyl-PROXYLin H 2 0 (0.08 M TEAP) at (a and c) pH 2 and (b and d) pH 12 (scan rate 0.1 V s-I).
(Figure 5a,c). The acidic environment has a slight influence on the oxidation of both TEMPO and 3-carbamoyl-PROXYL. The oxidation process of TEMPO is easier, as indicated by the IO-mV shift of E l / 2in the negative direction. Half-wave potential for 3-carbamoyl-PROXYL appears to be the same at pH 2 and pH 7. Negative potential scan shows no peaks prior to the reduction of protons at -0.35 V. For TEMPO at pH 12, when the potential is scanned from zero toward positive potentials, a poorly defined anodic peak, Ia, is observed at E , = 0.56 V, a positive shift of 10 mV, indicating a more difficult oxidation process. Unlike at pH 7, there is no accompanying cathodic peak. Peak IIc is observed at E , = -0.72 V and, as at pH 7, is without an accompanying anodic peak. However, the potential of the reduction is slightly shifted 10 mV in the negative direction. For 3-carbamoyl-PROXYL at pH 12, the same scanning procedures reveal a small shoulder around 0.3 V due to nonfaradaic adsorption/desorption (seen only in the first scan), a well-defined anodic peak, Ia, at E , = 0.59 V, a negative shift of 10 mV, and no accompanying cathodic peak. Peak IIc is observed at E , = -0.75 V without an accompanying anodic peak. The oxidation peak, Ia, current observed following a negative potential scan is approximately twice as high as the reduction peak, II,, current. As with TEMPO, the reduction potential is shifted 10 mV toward the negative direction, indicating that the reduction process is more difficult at p H 12 for both radicals. Figure 6 shows the electronic absorption spectra of TEMPO (a and c) and 3-carbamoyl-PROXYL (b and d), obtained during exhaustive electrolysis at pH 12. Before electrolysis, the spectra of both radicals are similar to their spectra at pH 7; however, molar absorptivity is higher at pH 12 (Figure 6a; Table 11). Electronic absorption spectra obtained during spectroelectrochemistry at pH 12 show differences in the pattern of response both from that observed with the neutral radicals and from each other. Initially, during controlled-potential oxidation of TEMPO at = 250 nm and the shoulder at A,, 0.75 V, both the peak at ,A, = 258 nm appear to decrease, parallel to observations obtained with neutral TEMPO. Several seconds into the electrolysis, the spectra begin to show marked differences from those observed previously. The peak increases and the shoulder shifts and broadens. Another shoulder appears a t A, = 280 nm, which shifts and increases. The final spectrum shows three highly overlapped peaks at 250, 270, and 290 nm. Attempts to reduce the
The Journal of Physical Chemistry, Vol. 92, No. 13, 1988 3149
Electrochemistry of Nitroxyl Free Radicals 3.
2. i-
z
U
1.
C K 3
0 0 r.
X 0
2.
0.8
0.4
0.0
-0.4
-0.8
POTENTIAL ( V vs. SCE)
1.
Figure 7. Cyclic voltammograms for TEMPO in H 2 0 (0.08 M TEAP) pH 12 (a) after electrolysis at -0.8 V and (b) followed by oxidation at 0.65 V. 200 250 300 350 200 250 3no WAVELENGTH (nm)
Figure 6. Electronic absorption spectra for (a and b) TEMPO and (c and d) 3-carbamoyl-PROXYL in HzO (0.08 M TEAP) at pH 12. Spectra taken during (a and c) oxidation at applied potential of +0.75 V and (b and d) reduction at -0.80 V. species formed by applying potentials both of 500 mV more negative than peak Ia and a t the plateau of peak IIc were unsuccessful as no current is observed and the spectrum remains unchanged. Cyclic voltammograms of the electrochemically oxidized solution show no peaks in the potential range -0.8 to +0.8 V. During controlled-potential oxidation of 3-carbamoylPROXYL, the 250-nm peak decreases and shifts. The final spectrum shows a single narrow peak at A, = 222 nm with decreased molar absorptivity (e = 1600). This species was also irreducible in the range of potential available, and the electrochemically oxidized solution showed no peaks in the potential range -0.8 to +0.8 V during cyclic voltammetry. Thus, it would appear that oxidation of both radicals at pH 12 produces an electrochemically inert species probably by a following chemical reaction with hydroxide ion. Instability of the positive ion (species 11) produced by the hydroxyl radical at high pH has been noted.I7 Conductivity data from pulse radiolysis experiments suggest that the mechanism for this process is neutralization of the positive ion:
+
‘N=O+ /
OH-
-
\/
HO-N=O
(5)
VI
I1
followed by dissociation according to an acid-base equilibrium:”
\/
HO-N=O V I
+
OH-
-
\/
O--N=O
(6)
VI1
Figure 6d shows the electronic absorption spectra of 3-carbamoyl-PROXYL at pW 12 monitored during CPE at the plateau of peak IIc. During the reduction, the 243-nm peak decreases and disappears. No other bands appear in the region monitored, and the solution remains colorless. Cyclic voltammograms of the electrolyzed solution show no peaks when the potential is scanned from 0.0 to -0.8 V. Following the negative potential scan, when potentials are scanned from zero in the positive direction, an anodic peak is observed at E,, = 0.58 V. When the potential scan is reversed, no cathodic peak is observed in the range +0.8 to -0.8 V. This suggests that the pH 12 reduction product of 3-carbamoyl-PROXYL, unlike its analogue at p H 7, is oxidizable at positive potentials but the oxidation product itself is not reducible.
Electronic absorption spectra obtained for the oxidation of this reduction product during exhaustive electrolysis at 0.8 V show a peak, shifted slightly from A, = 222 nm, which appears and = 250 nm. The peak increases as does a broad shoulder at A, = 222 nm reaches a maximum at e = 1600, while the at A, shoulder at A,, = 250 nm decreases and disappears. The final spectrum is the same as that produced by the original oxidation of 3-carbamoyl-PROXYL at pH 12. Cyclic voltammograms of the reoxidized solution show no peaks in the potential region -0.8 to +0.8 V, and no further spectral changes are observed even when potentials as negative as -0.8 V are applied. It seems safe to assume that the reoxidation of reduced 3carbamoyl-PROXYL proceeds by an EEC mechanism involving a two-electron transfer from species 111 to 11:
>N-OI11
-,>N=O+ I1
+ 2e-
(7)
followed by the previously noted acid-base equilibria to produce VI and VI1 (eq 5 and 6). Inasmuch as the irreversibility of the reduction at lower pH values is thought to be due to protonation leading to the hydroxylamine, reversibility at high pH seems a logical mechanism. Figure 6b shows the electronic absorption spectra of TEMPO at pH 12 obtained during CPE at the plateau of peak IIc. During the reduction, the 250-nm peak and the shoulder at A, = 258 nm decrease and disappear. Cyclic voltammograms of the electrolyzed solution show no reduction peaks in the negative potential scan. When potentials are scanned from zero in the positive direction, an anodic peak is observed at E p = 0.38 V (Figure 7a). When the potential scan is reversed, a cathodic peak is observed at E , = 0.28 V. While this suggests that the pH 12 reduction product of TEMPO is oxidizable at positive potentials, the presence of a new quasireversible couple and the magnitude of the shift in half-wave potential suggest the species undergoes rearrangement and/or chemical reaction. The overall mechanism for the redox reactions of TEMPO and 3-carbamoyl-PROXYL at different pH is given in Figure 10 (Scheme I). Both radicals exhibit a reversible oxidation in the range of pH 2-9 forming a stable cation (wave Ia,c) and an irreversible reduction (wave IIc) leading to the formation of hydroxylamine. At high pH, the oxidation is irreversible due to successive acid-base equilibria with OH-, and the reduction product is oxidizable at positive potentials. The reoxidation, however, is different for the two radicals. 3-Carbamoyl-PROXYL can be reoxidized to the unstable cation (11), whereas TEMPO appears to form a new species (see discussion below). These results from electrochemistry compare quite favorably with those found by pulse radiolysis of aqueous solutions of ni-. t r ~ x i d e s . ~The J ~ hydrated electron, hydrogen atom, and hydroxyl
3750
The Journal of Physical Chemistry, Vol. 92, No. 13, 1988
Fish et al. pH 1-14
O
awe
I a.c
0 w a v e / I c 0-
pH 9-14
200
250
300
350 200 250 WAVELENGTH (nm)
300
350
--
u
Figure 8. Electronic absorption spectra for TEMPO in H20 (0.08 M TEAP) at pH 12. Spectra taken following reduction at -0.80 V during (a) oxidation at applied potential of +0.65 V and (b) reduction at -0.80 V.
I Q-
0
0
W a v e Ill a.c
Wave lVa,c
Figure 10. Electrochemical pathway for redox precesses of TEMPO and 3-carbamoyl-PROXYL: scheme I (top), general pathway, pH 1-14; scheme I1 (bottom), structural changes of TEMPO following reduction process at pH 9-14 and subsequent oxidation.
i,
Figure 9. ESR spectra in D 2 0 of (a) original TEMPO and (b) product of reduction at -0.8 V followed by oxidation at +0.65 V.
radical formed by radiation are found to produce the same species found following electroreduction and -oxidation of the TEMPO and 3-carbamoyl-PROXYL. For pH less than 7, the hydrated electron reacts to form the nitroxide anion, which subsequently forms the hydroxylamine (V) by protonation. The same sequence is inferred from data obtained by electroreduction. In irradiated solutions, the hydrogen atom also forms the hydroxylamine (V) by direct attack on the nitroxide. Reaction of the nitroxide with hydroxyl radical leads to the nitroxide cation (11) and, at high pH, the hydroxylated cations (VI and VII) as found in this work. The only species not observed in the electrochemical experiment that has been observed by pulse radiolysis is hydroperoxide (= NOOH) formed from hydroxyl addition to the nitroxyl group. Inasmuch as the redox mechanisms are identical for TEMPO and 3-carbamoyl-PROXYL except for the reoxidation of TEMPO a t pH 12, additional 0.65 were undertaken to clarify the latter process. Reoxidation of TEMPO at p H 12. Electronic absorption spectra were obtained for the oxidation (at 0.65 V) of the reduction product of TEMPO (at -0.8 V, Figure 8a). The final spectrum = 250 nm, a shoulder at A,, = 258 consists of a peak at A, = 280 nm, markedly different nm, and a broad shoulder at A, from that obtained from oxidation at pH 12 before reduction. Cyclic voltammograms obtained following reoxidation show a quasireversible couple at E,, = 0.66 V and E , = 0.56 V and no peaks in the negative potential region from 0.0 to -0.8 V (Figure 7b). Macroscale electrolysis of TEMPO was performed at pH 12, and the product(s) of reduction at -0.8 V followed by reoxidation at 0.65 V were isolated and examined by IR, N M R , and ESR spectroscopy. The reoxidation product is not stable under conditions of IR analysis, but spectra give evidence of C=C vinyl stretch in the region 1640-1660 cm-'.No signal was seen in N M R spectra, indicating that the product is a radical. The ESR spectrum obtained in D 2 0 is shown in Figure 9. Analysis of this spectrum yields a nitrogen coupling at 16.3 G and two equivalent hydrogen couplings of 12.3 G.
Differences in molecular configuration regarding steric hindrance of the NO. group have been proposed as an explanation for different reactivities of RNO. compounds.** In this case, we propose that the proximity of the methyl groups, which causes inaccessibility of the reactive site in DNA base transients to the N O group of some six-membered ring RNO. compounds," results in ring cleavage (eq 8) of the anionic (unprotonated) reduction 0
0-H
I11
VI11
IX
product at high pH forming a species with a terminal double bond (VIII), which rather than hydrolyzing to form hydroxylamine, would be expected to undergo a chemical reaction involving a loss of proton or acid-base equilibrium at high pH to form an anion (IX). This species could be the molecule that is observed to oxidize more easily than the original molecule forming the quasireversible couple observed in cyclic voltammetry at El/*= 0.33 V following reduction at -0.8 V (eq 9).
The data observed in the ESR spectrum of the reoxidized product can be explained by a ring-closure reaction of X, forming a seven-membered ring as shown in eq 10. This species (XI) 0
0
I. (10)
X
XI
would be expected to show nitrogen coupling and two equivalent hydrogen couplings in the range observed. Also, species XI would be expected to undergo similar electronic transitions to origind TEMPO, accounting for the similarity in UV spectra. The quasireversible couple at El,* = 0.61 V could be attributed to the (18) Emmerson. P.T.:Fielden. E. M.: Johansen. I. I n t . J . Radiat. Biol. 1971, 19, 229.
J. Phys. Chem. 1988, 92, 3751-3760 oxidation-reduction reaction of the radical XI to the cation XII:
-
IVa
R-N-O R-N=O+ (1 1) XI IVC XI1 The previously electrolyzed solution (-0.8 V; 0.65 V) was reduced at 0.0 v. Electronic absorption spectra obtained during = 249 nm increases this reduction again show the peak at A,, while the shoulder at A, = 258 nm decreases slightly and the shoulder at 280 nm decreases and disappears (Figure 8b). Cyclic voltammograms of the electrolyzed solution again show the quasireversible couple at E , = 0.38 V and EP = 0.28 V identical with that shown in Figure 7a. No other peaks are observed in the potential Range 0.0 to -0.8 V. It was possible to regain the spectrum of the oxidation product and the reduction product by repetitive electrolyses at 0.65 and 0.0 V, respectively. Redox processes that occur for TEMPO alone at pH 12 are proposed in Scheme I1 of Figure 10.
Conc1usion This study has confirmed that two nitroxyl radicals commonly used as spin labels and free spin probes undergo redox reactions in aqueous media over the pH range 2-12 in the potential range
3751
available through metabolic processes. The redox products at pH 2-9, a stable cation from a reversible oxidation and hydroxylamine following an irreversible reduction, as well as hydroxylated cation at high - PH, - are the same as those obtained in pulse radiolysis experiments. Spectroscopic evidence indicates that behaiior following reduction at high pH differs for the two radicals. Reduced 3-carbamoyl-PROXYL can be reoxidized to the cation, which again undergoes successive acid-base equilibria with OH-. At pH 12, reduced TEMPO may undergo structural changes and, upon reoxidation, may form a new radical consisting of a sevenmembered ring.
Acknowledgment. We express our gratitude to P. A. Pappalardo for important suggestions concerning the ring-opening mechanism of TEMPO and to J. Mielcarek for technical assistance in preparing the figures accompanying this paper. This research was partially supported by the Office of Health and Environmental Research of the U.S. Department of Energy. Registry No. TEMPO (species I), 2564-83-2;TEMPO (species V), 110880-85-8;TEMPO (species VII), 72384-88-4;TEMPO (species XII), 114446-62-7;3-carbamoyl-PROXYL (species I), 4399-80-8; 3-carbamoyl-PROXYL (species V), 61514-99-6; 3-carbamoyl-PROXYL (species VII), 114446-61-6;TEAP, 2567-83-1; Pt, 7440-06-4.
Vibrational Relaxation Induced by Very Low Energy Collisions in the S1('B2J State of Naphthalene: A Search for Resonance Enhancement of the Cross Section Desmond J. Muller, Ruth I. McKay, Geoffrey B. Edwards, Warren D. Lawrance, Judith P. Hardy, Andrew B. Rock, Kevin J. Selway, Scott H. Kable, and Alan E. W. Knight* School of Science, G r i f f h University, Brisbane, Australia (Received: September 21, 1987; In Final Form: January 21, 1988)
Vibrational relaxation rate coefficients of naphthalene under collisional assault by argon in a supersonic free jet are reported for the temperature range 1.6-15 K. The rate coefficients are for V-Trelaxation from the u4: = 1 level (enb = 435 cm-I) in electronically excited (IB2J naphthalene, to the field of destination states that lie within energetic reach, Le., in the range tVlb5 450 cm-I. These data represent the first direct measurement in an SIpolyatomic of the temperature dependence of the absolute magnitude of the vibrational relaxation rate coefficient under conditions of very low energy collisions. The rate coefficient is found to be weakly temperature dependent in the temperature range studied. The temperature dependence of the rate coefficient follows the temperature dependence of the elastic collision rate calculated for particles whose interaction is characterized by the Lennard-Jones 12-6 potential. The vibrational relaxation efficiency, deduced by comparison of the observed relaxation rate with the Lennard-Jones elastic rate, is ~ 0 . 0 8at these low temperatures. This efficiency is consistent with the expectations based on room temperature data for similar large polyatomics. We conclude that the efficiency of low energy collision induced vibrational relaxation can be explained without invoking assumptions concerning the existence of low-energy resonances in the vibrational relaxation cross section.
Introduction The temperature dependence of vibrational energy transfer has attracted considerable attention in the past. Yardley' has reviewed the state of the field, at least up until 1980, with special reference to the theoretical methods that have been used to assist in the interpretation of vibrational relaxation data. The basis for these theories, however, commonly arises from experiments carried out at near room temperature or above (sometimes considerably above, e.g., in shock tubes). Molecular beams have recently opened up an avenue for investigating the low to very low energy collision regime where the local temperature of the interaction is only a few kelvine2 It is significant that, in Levy's 1980 review on spectroscopy in supersonic expansion^,^ only one reference was made to vibrational
relaxation measured under free jet conditions. A more recent review by Rice4 highlights the recent work in this area. There are now several molecules for which vibrational relaxation has been studied under supersonic free jet conditions, including Br2,9 BrC1,'O aniline," toluene,I2 fluorobenzene,I2 g l y ~ x a l , ' ~ (3) Levy, D. H. Annu. Rev. Phys. Chem. 1980, 31, 197. (4) Rice, S. A. J . Phys. Chem. 1986, 90, 3063. (5) Sulkes, M.; Tusa, J.; Rice, S. A. J . Chem. Phys. 1980, 72, 5733. (6) Van Zoeren, C.; Rock, A. B.; Kable, S. H.; Edwards, G. B.; Knight, A. E. W., submitted for publication in J . Chem. Phys. (7) Baba, H.; Sakurai, K. J . Chem. Phys. 1985, 82, 4977. (8) McClelland, G.M.; Saenger, K. L.; Valentini, J . J.; Herschbach, D. R. J . Phys. Chem. 1979, 83, 947. (9) Bullman, S . J.; Farthing, J. W.; Whitehead, J. C. Mol. Phys. 1981, 44, 97. . .
(1) Yardley, J. T. Introduction to Molecular Energy Transfer: Academic:
New York, 1980. (2) Anderson, J. B. In Molecular Beams and Low Density Gas Dynamics; Wegener, P. P., Ed.; Dekker: New York, 1974.
0022-365418812092-3751$01SO10
(10) Farthing, J. W.; Fletcher, I. W.; Whitehead, J. C. Mol. Phys. 1983, 48,
1067.
(11) Tusa, J.; Sulkes, M.: D. A.; Rice, S. A,; Jouvet, C. J . Chem. Phys. 1982, 76, 3513.
0 1988 American Chemical Society
