Pulse-Radiolytic Investigations of Adrenalone
reactions 3 (R = CH3),4 (R = CHJ, and 5 (R = CH3), are in good agreement with the statistical mechanical values1s5 given in Table I. This suggests that the premise on which our analyses are based (i.e., the division of the excess enthalpies of exchange systems into “chemical” and “physical” contributions and the estimation of the latter from the excess enthalpies of corresponding no-exchange systems) is a reasonable one. Of the two approaches based on this, we have a slight preference for method B; the results in Tables I and IV suggest that the assumption of values for K, and AH,is more reasonable than the assumption of equilibrium constants for two independent exchange reactions. We regard the thermodynamic properties in Table IV as the “best” values to be obtained from our and earlier’~~,~ excess enthalpy measurements. Acknowledgment. We thank the New Zealand Universities Grants Committee and the University of Otago for financial support and Professor G. L. Bertrand for helpful discussions during the 33rd Calorimetry Conference, Logan, Utah, August, 1978.
The Journal of Physical Chemistry, Vol. 83, No.
19, 1979 2447
References and Notes (1) D. V. Fenby and A. Chand, Aust. J . Chem., 31, 241 (1978). (2) D. V. Fenby, Aust. J. Chem., 30, 2371 (1977). (3) D. V. Fenby and A. Chand, J. Chem. SOC.,Faraday Trans. I, 74, 1768 (1978). (4) A. Chand and D. V. Fenby, J. Chem. Thermodyn., 10, 997 (1978). (5) J. R. Khurma and D. V. Fenby, Aust. J. Chem., 32, 465 (1979). (6) W. C. Duer and G. L. Bertrand, J. Am. Chem. Soc., 97, 3894 (1975). (7) G.L. Bertrandand T. E. Burchfield, J. mys. Chem., 79, 1547 (1975). (8) M. Wolfsberg, Adv. Chem. Ser., No. 89, 185 (1969). (9) M. Wolfsberg, J. Chem. Phys., 50, 1484 (1969). (10) M. Wolfsberg, A. A. Massa, and J. W. Pyper, J . Chem. Phys., 53, 3138 (1970). (11) J. Bron and M. Wolfsberg, J . Chem. Phys., 57, 2862 (1972). (12) M. Wolfsberg, Acc. Chem. Res., 5, 225 (1972). (13) A. Chand and D. V. Fenby, J . Chem. Eng. Data, 22, 289 (1977). (14) E. Bose, Z. Phys. Chem., 58, 585 (1907). (15) F. Frignet, M. Ratouis, and M. DodB, Bull. SOC.Chim. Fr., 7, 2458 (1967). (16) S.R. Goodwin and D. M. T. Newsham, J . Chem. Thermodyn., 3, 325 (1971). (17) G. C. Benson, Int. Data Ser., Sel. Data Mixtures, Ser. A , 77 (1973). (18) R. S. Ramalho and M. Ruel, Can. J . Chem. Eng., 46, 456 (1968). (19) G. C. Benson, Int. Data Ser., Sel. Data Mixtures, Ser. A, 79 (1973). (20) F. W. Hobden, E. F. Johnston, L. H. P. Weldon, and C. L. Wilson, J. Chem. Soc., 61 (1939).
Pulse-Radiolytic Investigations of Catechols and Catecholamines. 3. Adrenalonet Wolf Bors;
Manfred Saran, and Christa Michel
Abteilung fur Strahlenblologie, Institut fur Biologie, Gesellschaft fur Strahlen- und Umweltforschung (GSF), 8042 Neuherberg bei Munchen, Federal Republic of Germany (Received December 15, 1978; Revised Manuscript Received June 12, 1979) Publication costs assisted by Gesellschaft fur Strahlen- und Umweltforschung (GSF)
Transient spectra, decay kinetics, rate constants with hydroxyl radicals (OH) and superoxide anions (OF),and the identity of unstable intermediates are reported for adrenalone. They were obtained after pulse radiolysis in various oxidizing and reducing aqueous systems. Both rate constants €or the reaction with .OHand 0, of 1.0 X 1O1O and 2.3 X 10’ M-l s-l, respectively, are quite high. In the case of .OHattack several products could be separated by high-pressure liquid chromatography whereas with 02no stable products are formed. A mechanism is put forward, proposing formation of both o- and p-semiquinones after water elimination from the initial .OHadduct, and respectively, the reversible formation of o-semiquinone in systems containing OZ-. Reduction by eaq-, the rate depending on the pH, resulted in the formation of the ketyl radical. Even though this species decays by ,a second-order process, we were unable to detect the putative dismutation product adrenaline. Side-chain cleavage to form 3,4-dihydroxyacetophenoneand methylamine occurs in alkaline solutions.
Introduction The oxidation sequence of adrenaline (epinephrine) t o adrenochrome has repeatedly been postulated to proceed via adrenalinequinone and its cyclization product leucoadreno~hrome,l-~ but these hypothetical compounds have never been isolated in neutral or alkaline solution^.^ During pulse-radiolytic investigations of the oxidation sequence of adrenaline,6 we deemed it necessary to examine isoelectronic adrenalone for a possible tautomeric interrelationship with intermediates of the adrenaline oxidation. The paper presents transient spectra, kinetic data, and discusses the identity of the transient species of adrenalone after reducing and oxidizing pulse radiolysis in neutral and alkaline aqueous solutions. A subsequent paper7 will deal with the model catechol compounds A preliminary report was presented at the 10th International Congress for Biochemistry, Hamburg, 1976, Abstract 16-8-259.
3,4-dihydroxybenzaldehyde and 3,4-dihydroxyacetophenone. Materials and Methods Adrenalone (Fluka) was used without further purification. Solutions were prepared with triply distilled and pyrolyzed water. The strong spectral changes around the pK value of 6.8 required that the pulse-radiolytic experiments be performed at such pH values where a slight deviation in the pH would not result in gross changes of the absorption spectra. As all experiments were run in the absence of buffers to avoid complexation of the catechol moiety (ref 8; the pH was adjusted by addition of NaOH or HC104),such changes did occur. Absorption spectra and spectroscopic determinations of dissociation constants were done on Unicam SP 800 and Perkin-Elmer Model 575 spectrometers. High-pressure liquid chromatography (LC; Waters APG 204, complete set of wavelength filters) was used to check
0022-3654/79/2083-2447$0 1.00/0 0 1979 American Chemical Society
2440
The Journal of Physical Chemistry, Vol. 83, No. 19, 1979
80
t
W. Bors, M. Saran, and C. Michel 40
,
.,."*,.
o
i
'Ot
I
200
300
400
500
600
h[nml
Flgure 1. Correction of transient spectra of adrenalone with eaq-: (dashed line) uncorrected spectrum; (solid line) corrected spectrum, hi = 3.4, concentration 21.5 p M ; (dotted line) solute depleted at the same concentration. Adrenalone: 0.1 mM; ted-butyl alcohol, 10 mM; N P , pH 9.3; initial transient at 5 ps after the pulse, dose normalized with D = 6.114 krd.
the purity of the compounds and to determine product formation after pulse-radiolytic reduction and oxidation. Optimal resolution was obtained on reversed phase columns (pC18 Bondapak) with water-acetonitrileacetic acid (9O:lO:l) as solvent. The pulse-radiolytic experiments were performed with a Febetron 707 (Field Emission Corp.) with 40-ns pulses of 1.8-MeV electrons. The dose rate range was 0.7-8 krdlpulse. Data acquisition and evaluation were done with a Datalab DL 905 transient recorderldigitizer and a Wang 2200 B computer.
Results Bleaching of the strong absorption peak at 350 nm could conceivably allow one to calculate the net depletion of the solute. However, in each case the calculated G values are considerably lower than the corresponding theoretical G values. This strongly suggests that formation of products absorbing at 350 nm has already taken place less than 1 ps after the pulse. All spectral data are presented as corrected and dose-normalized time-dependent transient spectra, obtained with the theoretical initial G values and the corresponding doses of each experiment. This causes a shift of the absorption maxima of the transient species as exemplified in Figure 1. Reduction. The reduction of adrenalone (0.1 mM) by hydrated electrons in nitrogenated aqueous solutions containing either 10 or 100 mM tert-butyl alcohol resulted in identical transient spectra, therefore all .OH radicals have been scavenged (Figure 2). Aside from the fact that isosbestic points at 241, 261, 322, and 420 nm were observed only in neutral solutions, comparison of corrected and uncorrected spectra as well as in neutral vs. alkaline solutions reveal the following (the corrections were based on the yields of ea;- of 2.8 in neutral and 3.4 in alkaline solutions as only a t high pH H atoms are converted into eaq;; ref 9): (1) A short-wavelength peak at 230 nm in neutral solutions shifts to 245 nm in alkaline solutions. (ii) The major peak at 275 nm in neutral solution (Figure 2) gives similar wavelength maximum and molar absorptivity (12300 M-l cm-l) as the reduced spectral transients of dihydroxybenzaldehyde and dihydroxyacetophenone.7 (iii) The smaller peak (pH 7.5) respective shoulder (pH 9.5) appears at the same wavelength (300-310 nm) as for benzaldehydelO and acetophenone.'l-13 They are the most prominent absorption bands in uncorrected spectra and
Figure 2. Time-dependent transient spectra of adrenalone after reduction by hydrated electrons. Spectra corrected with G = 2.8 and dose normalized for D = 3.493 krd: (-) 5 ps, (- -) 9.2 ps, (-.-) 17 ks,(-*e-) 58 ps, 190 ;us. Adrenalone, 0.12 mM; fer?-butyl alcohol, 0.1 M,N, pH 7.5. (..e.)
could therefore be analyzed kinetically. In neutral solutions the absorption peak disappears in a second-order process with a decay rate of 2k = (5.7 f 0.4) X lo9 M-l s-l; in alkaline solutions we obtain (1.9 i 0.3) X lo9 M-l s-l. (iv) The peak at 360 (neutral pH) and 340 nm (alkaline pH) could reflect both adrenalone regenerated in a dismutation reaction or fragmentation to dihydroxyacetophenone absorbing at the same wavelength. The latter reaction, corroborated by high pressure LC, occurred more readily in alkaline solutions. For further elucidation of these spectra, experiments with COT as a reducing agent (solutions contained formate and N 2 0 to convert both ea; and .OH to COT) were performed. They yielded transients at 250 and 345 nm and none at 300 nm, also none were found above 400 nm (data not shown). This is in line with the failure of COT to produce ketyl radicals from carbonyl compounds.14 High pressure LC was used to determine the products after reduction to adrenalone. Even though the bimolecular decay of the transient is indicative of dismutation, no trace of adrenaline as putative product could be found. The product of side-chain cleavage according to Gohn et al.,153,4-dihydroxyacetophenone, appears only above pH 6.8. Oxidation. The transient spectra after pulse-radiolytic oxidation of adrenalone by .OH radicals are quite similar, even though they were obtained under different conditions (Figure 3). The results given are for neutral solutions in which eaq- were converted to -OH radicals either in N20-saturated solutions (Figure 3a), in nitrogenated solutions containing H20z (Figure 3 4 , or for .OH in the presence of 02-in oxygenated solutions (Figure 3b). The spectra in alkaline solutions correspond closely to those at neutral pH, except that the peak at 275 nm is missing (data not shown). Differences are also apparent for the oxidation in the presence of H 2 0 z (Figure 3c). The presence of a transient absorption at 235 nm instead of 250 nm somewhat resembles the spectrum after oxidation by 0 2 - . Turning to oxygenated solutions containing forma+- we find a high similarity for the spectra obtained with 3 2 alone and in the additional presence of catalase (lo0nM), as shown in Figure 4. Comparison of these data with the oxidation by -OH radicals reveals identical transient species a t 270-285 nm, a more or less regenerated absorption at 350 nm (and 250 nm) and, in the case of 02- attack, no absorption whatsoever at 450 nm. A feature exclusive to the oxidation by 02-is the fact that the original spectrum is completely restored. Such a reversible reaction with 02is also supported by the presence of three isosbestic points
The Journal of Physical Chemistry, Vol. 83, No. 19, 1979 2449
Pulse-Radiolytic Investigations of Adrenalone
TABLE I: Spectral Data of Oxidized Initial Transient Species of Adrenalone oxidized species
I. generating radicals *OH
pH
N2O
.OH
.OH
H2OaINa t
02-
M-' cm-I
e,
system
0,
0 2
-
HCOO- /O,
0 2
-
HCOO-/CAT/O,
7.5 9.3 7.4 7.5 9.5 7.3e 9.5 7.4e
h,
nm
255 250 235' 250d 250d 230 250d 230
I1 erc," G h , nm
11400 13 300
10100 10 500 10400 1 2 300 1 2 500
11 1 0 0
5.6 5.6 6.4 6.4 6.4 6.4 6.4 6.4
E,
IV
M-'
cm-I
erc, IIIb G h,nm h,nm
275
20200
2.8
270 275
16000
3.2
11 300
4.8
280
10900
6.4
285
7 700
6.4
355 350 350 350 350 350 350 345
465 450 450 465 470d
M-'
cm-'
erc, G
5400 5400 3200 5 000 5 700
2.8 2.8 3.2 1.4 1.4
E,
G value) based on 1:l stoichiometry of 0-andp-semiquinone formation from .OH adduct, and respectively, from exclusive generation of the o-semiquinone by 0 2 - attack (see Scheme I). Maximal absorption a t intermediate Regenerated original absorption, no initial species can be observed. ' Shoulder. observation times. e Isosbestic points at 241-242, 257-258, and 3 1 6 nm. a erc is the effective radical concentration (yield expressed as
100-
A
I
100
100
hhrnl
250
300
t
350
h [n ml
400
1
/"\
1
100
t
200
~
300
400
h [nml
500
200
600
Figure 3. Time-dependent transient spectra of neutral solutions (pH 7.5) of adrenalone oxidized by hydroxyl radicals. (a) Solutions of 0.1 mM adrenalone saturated with N,O. Correction with G = 5.6 resulted in solute depletion of 37.1 pM; dose normalized for 6.41 krd; (-) 10 ps, (---) 100 ps, 10 ms. (b) Solutions of 0.1 mM adrenalone saturated with O2 Correction with G = 6.4 for the sum of G.OH G ,resulted in c = 42.2 pM, dose normalized for 6.41 krd; (-) 10 ps, (---) 100 ps, (-+ 10 ms. (c) Solutions of 0.1 mM adrenalone and 10 mM H,02 saturated with N., Correction with G = 6.4 gave a concentration of 15.0 pM at a dose of 2.268 krd; (-) 5 us, (---) 85 ps, (---) 1.5 ms, (---) 25 ms. (-e-)
+
at 242, 257, and 316 nm in the time-dependent spectra of both Figure 4a and 4b (no isosbestic points are observed in alkaline solutions). The involvement of HzOz in the reversal of semiquinone formation was tested by (i) converting ea¶-to .OH in the presence of Hz02(Figure 3c) and (ii) by scavenging excess H20zwith catalase (Figure 4b). As can be seen, the presence of HzOz results in a decrease of the molar absorptivity (decreased yield) of the 440-460-nm transient species and a shifting from 250 to
250
300
350
A [nml
400
450
Figure 4. Timedependent transient spectra after oxidation of neutral solutions of adrenalone by superoxide anions. (a) Oxygenated solutions of 0.1 mM adrenalone and 10 mM sodium formate, pH 7.3; solute depleted with c = 35.2 pM (G= 6.4, D = 5.309 krd); (-) 10 ps, (---) 100 ps, (--) 1 ms, 10 ms, (.-e) 50 ms. (b) Oxygenated solutions of 0.1 mM adrenalone and 10 mM sodium formate, pH 7.4. Catalase (100 nM) was added to scavenge H202. Solute depleted wlth c = 21.8 pM (G = 6.4, D = 3.406 krd); (-) 50 ps, (---) 11 ms, (--) 42 ms, (---) 161 ms. (--e-)
235 nm. The addition of catalase basically shows a faster recurrence of the original absorption. A compilation of the spectral data pertaining to the oxidized species after attack of .OH and/or 02is shown in Table I. The rate constants with the primary radicals -OH,OF, and e,; were mainly determined by competition experiments and are given in Table 11. The rate constants with ea¶-were extremely high, yet competition with nitrate ( h = 1.05 X 1O1O M-l s-l, ref 16) and kinetic evaluation of the decay of e, - a t 670 nm both gave the same value. Radical (1.3 an%4.6 pM) and substrate (0.5-2 pM) con-
2450
The Journal of Physical Chemistry, Vol. 83,
TABLE 11: Rate Constants with Primary Radicals in Aqueous Solutions radical pH
rate constant,
eaB- 7.5
0.14) 0.10) 0.46) 0.14)
competitor ( h , M-I s-')
M-I s - l
(2.71 ?r 7.5 (2.70 ?r 9.5 (2.85 i .OH 7.5 (1.03 k
X
10"
x 10" x 10" X
10"
02-7.0 (2.34 f 0.31) x 10'
W. Bors, M. Saran, and C. Michel
No. 19, 1979
a
NO,(1.05 x 10'') DCIP (4.0 i 0.29) x 10'' DCIP (2.14 k 0.05) x l o 8
a Direct observation of the decay of eaq- at 670 nm (for further explanation, see text),
centration were a t a minimum, while ethanol as .OH scavenger was 5 mM. In preliminary experiments to determine the rate constant with .OH radicals, we used tert-butyl alcohol as a competitive scavenger (5.2 X lo8 M-l s-l, ref 17). This meant that the observation of the species at 450 nm, which according to our mechanism (see Scheme I) is a secondary product, does not reflect the initial .OH attack. To avoid this potential source of error, the redox dye 2,6-dichlorophenolindophenol (DCIP) was used instead. Evaluation of the bleaching at 600 nm did not interfere with any transient absorption of the catechol compound. The reference rate constant of DCIP with -OH radicals (4.0 X 10" M-' s-l) was determined with reference to the rate constant of mannitol, a nonabsorbing -OH scavenger (unpublished results), which is quite often used in biology for this purpose. Its rate constant with .OH, generally assumed to be around 1 X lo9 M-l S-','~J~ was determined in our laboratory and indeed gave this value. p-Benzoquinone could not be used as competitor for Oz-20as it reacted directly with the catechol. We again used DCIP, after redetermining its rate constant with 02by competition with superoxide dismutase. We obtained (2.14 f 0.05) X lo8 M-' s-' at pH 7.0, based on the enzymatic dismutation rate of 3.7 X lo9 M-l s-' ,21 as compared to 1.7 X lo8 M-I s-l given by Greenstock and Ruddockz0 High Pressure LC Experiments. Additional evidence for various reactions was obtained by preliminary product analysis with reversed phase and cation exchange high pressure LC. (i) Despite a second-order decay of the initial ea; adduct, evidence for the formation of adrenaline, a possible dismutation product, was negative. Side-chain cleavage resulting in dihydroxyacetophenone and probably methylamine, a reaction similar to the one proposed for the reduction of adrenaline in acidic solutions,15 occurs only in alkaline solutions. (ii) In the case of the oxidation by -OH radicals, up to six products could be separated. As they were also formed in the presence of oxygen, hydrogen peroxide, and/or light alone, their identification as genuine pulse-radiolytic products remains inconclusive. Fortunately, under these same conditions, the yield of some of the products of pulse radiolysis could be increased considerably, thus enabling the separation of some of the compounds for further chemical analysis. The results shall be published elsewhere. (iii) No products could be detected after exclusive oxidation by 02-, supporting our hypothesis of a completely reversible reaction with this radical species. Discussion Reduction. The transient species of adrenalone, absorbing at 275 nm in neutral solutions and decaying by a
second-order process, evidently is the ea; adduct, with the structure of a ketyl radi~a1.l~ Rao and HayonZ2report a wavelength maximum at 295 nm at higher concentrations of tert-butyl alcohol. Their nomenclature "semiquinone", however, seems to be as questionable as in the case of the "adrenalin semiquinone". Both compounds are catechol derivatives and therefore cannot be reduced to semiquinones. We suggest the following structure for the ketyl radical, in agreement with the reduced transient species of benzaldehyde as proposed by Lilie and Henglein'O and the acetophenone ketyl radical as discussed by both Adams et a1.I2 and Hayon et
Both species have their major absorption peak recorded at 300 nm and, respectively, at 310 nm. The bimolecular decay rate in neutral solutions of 2k = 5.7 X lo9 M-I s-l for our 300-nm species is higher than the value reported for acetophenone of 9 X lo8 M-' s-l,13 but similar to those of the electron adduct of both catechol model compounds (5.5 X lo9 and 3.6 X lo9 M-I s-' ). The alternative structure of the ea; adduct, advanced by Gohn et would be addition of ea; at the protonated secondary amine group. The subsequent side-chain cleavage to form dihydroxyacetophenone and methylamine was not expressed by changes in the transient spectrum. High pressure LC experiments demonstrated this reaction (eq 2) only in alkaline solutions where such a protonation
H+h HO
IHj
H HO
O
dcn2
is considered unlikely. However, no alternate mechanism could be devised, even though different mesomeric structures of adrenalone, based on the keto, enol, and quinhydrin structures can be envisaged:
The enol form may be slightly favored due to the additional hydrogen bond, whereas the quinhydrin structure would be the least aromatized form, yet containing the same hydrogen bond which may also be formed upon reduction of the carbonyl function. No tautomeric equilibrium with the isoelectronic actual adrenaline semiquinone absorbing at 245 nm6 could be observed. The rate constants for the attack of ea< in excess of 1011 M-' s-l are problematic. During competition experiments the participation of spur scavenging may result in such high values. Yet, directly observing the decay of ea< should result in correct values, except for the chance reaction with other solutes. As the selection of the proper concentrations of radical, substrate, and scavenger was based on the known rate constants of ea; with itself, ethanol, and carbonyl corn pound^,^^ we are presently unable to explain the values obtained by this method. We hope that observations at faster time scales will resolve this problem.24 Alternatively, the search for better scavengers of .OH radicals which do not react with ea; is presently being pursued in our laboratory. Oxidation by .OH Radicals. While adrenalone behaves like a carbonyl compound during reduction, oxidation reactions are primarily governed by the catechol functional group. The pertinent reactions which have to be considered for the pulse-radiolytic oxidation of adrenalone
Pulse-Radiolytic Investigations of Adrenalone
have previously been discussed in great detail for the case of adrenaline.6 Using the same rationalization, we conclude that the transient a t 270-285 nm, present in all systems generating either .OH or OF, represents the o-semiquinone. This is supported by the fact that both dihydroxybenzaldehyde and dihydroxyacetophenone exhibit a similar transient species under the same conditions.7 The additional absorption peak at 230-255 nm most likely also represents the semiquinone. The 440-460-nm transient species, appearing only in systems containing -OH radicals, showed a higher stability and delayed formation in oxygenated solutions. On the basis of similar absorption maxima for p-semiquinones (ref 26 and 27; see also the compilation in ref 28), we conclude that water elimination from the initial .OH adductBJOleads to both isomeric semiquinones. This is also supported by preliminary findings of in situ radiolysis/EPR experim e n t ~ indicating ,~~ the presence of two similar organic radicals. The .OH adduct itself, formed in a diffusioncontrolled reaction (k = 1.0 X 1O1O M-l SI), is assumed to absorb at 330-340 nm, analogous to the model comp o u n d ~ .It~ is extremely unstable and its spectrum is very rapidly superimposed by a regeneration of the original absorption a t 350 nm. The decay kinetics of the oxidized transient species were difficult to evaluate. As all semiquinones disappear by second-order processes, except for the first-order decay of the 275-nm transient (3 X lo3s-l) in the presence of oxygen (pH 7.5), no quantitation was possible due to the unknown yields and consequently unknown molar absorptivities. We did, however, make such an evaluation for the model compounds on the assumption that the proposed semiquinone formation proceeds with 1:1 st~ichiometry.~ Reversible Oxidation by 02-.Superoxide anions react quite rapidly with adrenalone (2.3 X lo7 M-l s-l at pH 7) as compared to adrenaline itself (4.0 X lo4 M-l s-l , ref 32, corrected for the influence of ionic strength 5.6 X lo4 M-l s-l, ref 33). On the basis of complete reversal of the initial change in absorption and on the presence in neutral solutions of three isosbestic points in the time-dependent spectra (Figure 4),we propose a reversible reaction with 02-.No evidence was found for a nucleophilic addition of 0; as proposed for the reaction of K O p with carbonyl compounds in organic solvents.34 Subsequent experiments either including supplementary amounts of HzOz(Figure 34, scavenging the generated HzO2by catalase (Figure 4b), as well as the absence of LC separable products of the reaction with 02-all supported the hypothesis that 02generates the o-semiquinone, which is re-reduced by Hz02 and/or 02-.H202 is present in excess of the stoichiometric amount as it is formed both by the radiolysis of water, as a coproduct during the generation of the semiquinone (eq 3) and by a limited spontaneous dismutation. A less than
quantitative reaction with 02-is implied by the observation of the theoretical yield of 02-in the initial absorption spectra of dihydroxybenzaldehyde and dihydroxyacetophenone7 even though the rate constants are similar to adrenalone. On the basis of these data, we are able to present a scheme for the initial reactions after pulse-radiolytic oxidation of adrenalone (Scheme I). Except for the still to be determined stable products after oxidation by .OH radicals (to be published elsewhere), the scheme proposes two novel features, which have not been previously reported for the pulse radiolysis of dihydroxylated aromatic
The Journal of Physical Chemistry, Vol. 83, No. 19, 1979 2451
Scheme I: O x i d a t i o n Mechanism of Adrenalone by .OWa n d 0 2 -
L
o-semiquinone 260-275 n m
1
products
p-semi quinone 410-450 nw
1
products
compounds: (i) water elimination from the initial .OH adduct of a catechol derivative results in both the 0-and p-semiquinones, and (ii) the reaction with 0, is completely reversible, as the semiquinone is re-reduced by the coproduct Hz02and/or 0, itself. Whether these particular reactions are limited to catechol compounds with an acarbonyl function in C4position shall be of further interest.
Acknowledgment. We appreciate the expert technical assistance by Mr. A. Kruse.
References and Notes Harley-Mason, J. J . Chem. SOC.1950, 1276-1282. Harrison, W. H. Arch. Biochem. Biophys. 1963, 707, 116-130. Hawley, M. D.; Tatawawadi, S. V.; Piekarski, S.; Adams, R. N. J . Am. Chem. SOC. 1967, 89, 447-450. Harrison, W. H.; Whisler, W. W.; Hill, B. J. Biochemistry 1968, 7 , 3089-3094. Heacock, R. A.; Powell, W. S. In "Progress in Medicinal Chemistry"; Ellis, G. P.;West, G. B. Eds.; NortbHolland PublishingCo.: Amsterdam, 1933; VOI. 9, pp 275-339, Bors, W.; Saran, M.; Michel, C.; Lengfelder, E.; Fuchs, C.; Spottl, R. Int. J . Radiat. Biol., 1975, 28, 353-371. Submitted for publication. Chaix, P.; Morin, G.-A,, Jezequel, J. Biochim. Biophys. Acta 1950, 5,472-488. Czapski, G. Adv. Chem. Ser. 1968, No. 87, 106-130. Lilie, J.; Henglein, A. Ber. Bunsenges. Phys. Chem. 1969, 73, 170-1 76. Chutny, B. Nature (London), 1967, 273,593-594. Adams, G. E.; Michael, B. D.;Willson, R. L. Adv. Chem. Ser. 1968, 81 . 289-308. Hayon, E.; Ibata, T.; Lichtin, N. N.; Simic, M. J. Phys. Chem. 1972, 76,2072-2078. Faraggi, M.; Redpath, J. L.; Tal, Y. Radiat. Res. 1975, 64,452-466. Gohn, M.; Getoff, N.; Bjergbakke, E. Int. J. Radiat. Phys. Chem. 1975. 8.533-538. . ... Peled, E:; Czapski, G. J . Phys. Chem. 1970, 74, 2903-2911. Willson, R. L.; Greenstock, C. L.; Adams, G. E.; Wageman, R.; Dorfman, L. M. Int. J. Radiaf. Phys. Chem. 1971, 3, 211-216. Halliwell, 6. FEES-Lett. 1978, 92,321-326. Cederbaum, A. I.; Dicker, E.; Cohen, G. Biochemistry 1976, 77, 3058-3064. Greenstock, C. L.; Ruddock, G. W. Int. J. Radiat. Phys. Chem. 1976, 8,367-369. McAdam, M. E. Biochem. J. 1977, 767, 697-699. Rao, P. S.;Hayon, E. J . Phys. Chem. 1973, 77, 2274-2276. Anbar, M.;Bambenek, M.; Ross, A. B. Natl. Stand. Ref. Data Ser., Nafl. Bur. Stand. 1973, No. 43. After completion of this manuscript an article appeared," in which similarly high rate constants for photochemicallygenerated eaq-were reported. Whether the proposed pairwise recombinatlon of eaq-also plays a role in our radiolytic systems is open to question and awalts picosecond pulse radiolysis studies. Lee, J. Y.;Baugher, J. F.;Grossweiner, L. I. Photochem. Photobiol. 1979, 29, 867-874. (26) Chibisav, A. K.; Kuz'min, V. A.; Vlnogradov, A. P. Dokl. Akad. Nauk SSSR 1969, 187, 142-146. (27) Land, E. J.; Swallow, A. J. J. Biol. Chem. 1970, 245, 1890-1894.
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M. V. George, Ch. V. Kurnar, and J. C. Scaiano
The Journal of Physical Chemistry, Vol. 83, No. 19, 1979
Strahlenchemie, MPI fur Kohlenforschung, Mulheim/Ruhr, West Germany. (32) Asada, K.; Kanematsu, S. Agric. Bo/. Chem., 1976, 40, 1891-1892. (33) Bors. W.: Michel. C.: Saran. M.: Lenafelder, E. 2. Naturforsch. C 1978, 33, 891-896. (34) Le Berre, A,; Berguer, Y. C. R. Acad. Sci. 1965, 260, 1995-1998.
(28) Habersbergerova, A.; Janovsky, I.; Kourim, P. Radiat. Res. Rev. 1972, 4, 123-231. (29) Adams, G. E.; Michael, B. D. Trans. Faraday SOC. 1967, 63, 1171-1180. (30) Neta, P.; Fessenden, R. W. J . Phys. Chem. 1974, 78, 523-529. (31) The experiments were performed by S. Steenken at the Institut fur
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Photochemistry and Photooxidation of Tetraphenyl-p-dioxin M. V. George,' Ch. Vijaya Kumar,lb and J. C. Scaiano*la Radiation Laboratory,",* University of Notre Dame, Notre Dame, Indiana 46556 (Received April 2, 1979) Publication costs assisted by the U.S. Department of Energy
Laser flash photolysis studies of tetraphenyl-p-dioxin have led to the characterization of its triplet state. The T-T absorption spectra shows maxima at 350 and 545 nm; the triplet has a lifetime of 535 ns in methanol and can be quenched by di-tert-butyl nitroxide, paraquat dications, oxygen, and di-tert-butyl selenoketone. The interaction of the triplet with oxygen leads to the formation of singlet oxygen which in turn reacts with the title compound to yield benzil. Introduction In our earlier studies3on the thermal and photochemical transformation of tetraphenyl-p-dioxin ( l ) , we have suggested that the transformations of 1 involve the biradical intermediate 2, which then leads to benzil (3), and minor amounts of tolan (4), Scheme I. We have now examined the photochemistry of 1 in the absence and presence of oxygen by using a combination of quantum yield and nanosecond flash photolysis studies. Combined, these techniques have allowed us to establish the mechanism and kinetics of the photoprocesses involved. We have also been able to characterize the triplet state of 1 from its phosphorescence and T-T absorption spectra. We find that 1 is quite photostable under anaerobic conditions, and that the generation of benzil takes place via the intermediacy of singlet oxygen.
Results This section has been divided according to the experimental technique employed. Laser Flash Photolysis. Excitation of solutions of 1 with the pulses (337.1 nm, -3 mJ, 8 ns) from a nitrogen laser leads to transient absorptions which decay with clean first-order kinetics and a lifetime of 535 ns in methanol and 630 ns in benzene. The lifetimes are concentration independent (0.004-0.0004 M range), indicating that self-quenching is not important, The transient spectrum, observed 10 ns after the laser pulse, is almost identical in both solvents and is shown in Figure 1. Figure 2 shows a typical decay trace and the first-order fitting of the data in the two solvents used. We attribute the observed transient absorptions to the triplet state of 1, on the basis of its behavior toward a variety of molecules (see below). A. and A are the transient absorptions due to triplet 1 immediately after the laser pulse and at time t, respectively. The signals observed can be quenched by di-tert-butyl nitroxide, oxygen, di-tert-butyl selenoketone, and paraquat dications; they cannot be quenched by moderate concentrations of 2,3-dimethyl-2-butene or 2,5-dimethyl2,4-hexadiene. Figure 3 shows the corresponding kinetic plots according to eq 1,where kexptis the first-order rate (1) kexpt - h'expt = k,[Q1 constant associated with the decay of the triplet state, 0022-3654/79/2083-2452$0 1.OO/O
1
0
3
5
TABLE I : Kinetics of Triplet Quenching by Various Substrates at Room Temperature substrate
solvent
2,5-dimethyl-2,4-hexadiene methanol 2,3-dimethy1-2-butene benzene di-tert-butyl nitroxide benzene di-tert-butyl selenoketone benzene paraquat dications methanol benzene oxygen
~ Q / M - s'- '
< 1 X lo6
< 5 X lo4 5 X lo7 1.8 X l o 8 4.9 x lo9 1 . 7 X lo9
koexpt,the same parameter in the absence of quencher (Le.,
= q - l ) , and k,, the rate constant for quenching by Q. kinetic data are summarized in Table I. In the case of paraquat dications, 6, the reaction (eq 2) KOex
1*
t H 3 c - t N i ~ - C H a
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6
with the triplet state results in the formation of the radical 0 1979 Arnerlcan Chemical Society
