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0022-3654/89/2093-0155$01.50/0 already described, the thiyl radical formed in reaction 1 may also react with dissolved oxygen. For cysteine, glutathio...
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J . Phys. Chem. 1989, 93, 155-161

155

An Electron Spin Resonance Investigation of the Structure and Formation of Sulfinyl Radicals: Reaction of Peroxyl Radicals with Thiols Steven G. Swarts, David Becker, Steven DeBolt, and Michael D. Sevilla* Department of Chemistry, Oakland University, Rochester, Michigan 48309 (Received: May 3, 1988)

In this work we present an electron spin resonance investigation of the irradiation of the alkyl mercaptans methyl, n-butyl, and rert-butyl mercaptan and the radioprotective thiols cysteamine and dithiothreitol in a number of aqueous and organic matrices in the presence of oxygen. Matrix peroxyl radicals (ROO') are formed after the irradiation of organic matrices in the presence of oxygen at 77 K. Upon annealing, these react with added thiols to form sulfinyl radicals (RSO'). Evidence for a thiol peroxyl radical (RSOO') intermediate is found. Hyperfine couplings and g values are reported for the sulfinyl radicals formed from the five thiols investigated. The incorporation of 170-labeled oxygen into the RSO' radical confirms that molecular oxygen is the source of the oxygen atom in the radical. The isotropic and parallel anisotropic I7O couplings indicate slightly less than 0.5 spin density on the oxygen. The couplings and spin densities are compared to those predicted from ab initio molecular orbital calculations for CH3SO'. Calculations for the sulfur-peroxyl intermediate, CH3SOO', predict that it will have similar ESR parameters as those predicted for the carbon-centered peroxyl radical, CH300'.

Introduction The ability of thiols to donate a hydrogen atom to repair free-radical damage on biomolecules is an important radioprotective property.'-3 Donation of the thiol hydrogen to a carbon-centered free radical on a biomolecule (R') results in restoration of the biomolecule to its original s t r u c t ~ r e ~as- ~well as formation of a relatively more stable thiyl radical (R'S') (reaction 1). In the presence of oxygen a competitive process occurs, in which the carbon-centered radical reacts with dissolved oxygen Pulse radiolysis to form peroxyl radical, ROO' (reaction 2)."' R'

+ R'SH R'

-

+ 02

R'S'

+

+ RH

ROO'

(1)

(2)

experiments in aqueous solutions at ambient temperatures on model systems have determined that the rate constants for reaction 1I2-l4 are typically 1-3 orders of magnitude smaller than the rate constants for reaction 2.l5-I7 As a consequence, irradiated biological and organic systems are expected to form significant quantities of peroxyl radicals, even in the presence of thiols. Thus, an understanding of the reactions of thiols with peroxyl radicals becomes important to understanding the radioprotective effect of thiols in the presence of oxygen. In addition to the reactions (1) B a q , 2.M.; Alexander, P. Fundamentals of Radiology; Butterworth: London, 1955. (2) Howard-Flanders, P. Nature 1960, 186, 485. (3) Scholes, G.; Weiss, J. Radiat. Res. Supp. 1 1959, 177. (4) Adams, G. E.; Armstrong, R. C.; Charlesby, A.; Michael, B. D.; Willson, R. L. Trans. Faraday SOC.1969, 65, 732. (5) Redpath, J. L. Radiat. Res. 1973, 54, 364. (6) Wolfenden, B. S.; Willson, R. L. J. Chem. Soc.,Perkin Trans. 2 1982,

80s. (7) Bonif6ci6, M.; Asmus, K.-D. J . Phys. Chem. 1984, 88, 6286. (8) Akhlaq, M. S . ; Schuchmann, H.-P.; von Sonntag, C. I n f . J . Radiat. Biol. 1987, 51, 91. (9) Born, W., Saran, M., Tait, D., Eds. Oxygen Radicals in Chemistry and Biology, Proceedings Third International Conference; de Grupter: Berlin, 1984. (10) von Sonntag, C. The Chemical Basis ofRadiarion Biology; Taylor and Francis: London, 1987. (1 1) Yanez, J.; Sevilla, C. L.; Becker, D.; Sevilla, M. D. J . Phys. Chem. 1907,91,487. (12) Barton, J. P.; Packer, J. E. Int. J . Radiat. Phys. Chem. 1970, 2, 159. (13) Quintiliani, R.; Badiello, R.; Tamba, M.; Esfandi, A.; Gorin, G. Int. J. Radiat. Biol. 1977, 32, 195. (14) SchBfer, K.;Bonif6ci6, M.; Bahnemann, D.; Asmus, K.-D. J. Phys. Chem. 1978,82,2777. (15) Adams, G. E.; Willson, R. L.; Trans. Faraday SOC.1969, 65, 2981. (16) Willson, R. L. In?. J. Radial. Biol. 1970, 17, 349. (17) Adams, G . E.; McNaughton, G. S.; Michael, B. D. Trans. Faraday SOC.1968, 64, 902.

already described, the thiyl radical formed in reaction 1 may also react with dissolved oxygen. For cysteine, glutathione, and penicillamine, the first radical product clearly detectable by electron spin resonance (ESR) spectroscopy in concentrated frozen matrices is the sulfinyl radical, RSO*'sa,b(reaction 3). The R'S'

-

+ O2

R'SOO'

-

R'SO'

+ other products

(3)

identity of the sulfinyl radical has been confirmed with ESR studies employing I70-labeled molecular oxygen.Is Very recent ESR work in dilute aqueous solutions has confirmed the presence of the expected thiol peroxyl intermediate, R'S00'.'9 The RSOO' radical was proposed as early as 1963 to be the result of the reaction of a thiyl radical with oxygen.20 Recent pulse radiolysis studies of the reactions of glutathione in the presence of oxygen report the observation of thiyl peroxyl radicals.2'-22 In this work we present an ESR investigation of the radiation-induced reaction of three alkanethiols and two radioprotectors in oxygenated systems. Our results show that the reaction of peroxyl radicals with thiols results in sulfinyl radicals probably through a thiol peroxyl intermediate.19 Further we present molecular orbital (MO) calculations which show that the ESR parameters of thiol peroxyl radicals (RSOO') are similar to those of carbon-centered peroxyl radicals (ROO'), and that thiol peroxyl radicals will therefore be difficult to distinguish from carboncentered peroxyl radicals.

Experimental Section Thiols were commercially obtained and used without further purification. Thiols were dissolved in either nitrogen-, air-, or oxygen-saturated solvent and y-irradiated at 77 K for doses between 0.1 and 0.4 Mrad. A range of concentrations was employed to distinguish biomolecular solute-solute reactions from other reaction paths. The solvents used were methanol, from Aldrich (TCFE) Chemical Corp., and 1,1,2-trichloro-2,2,l-trifluorcethane from PCR Research Chemicals. Solutions of 50% (v/v) glycerol in water and 8 M NaC104 were also used as media. After irradiation, spectra were recorded with a variable-temperature Varian E-4560 dual cavity in conjunction with a Varian E-Line Century Series ESR spectrometer. All spectra are calibrated with reference (18) (a) Sevilla, M. D.; Becker, D.; Swarts, S.; Herrington, J. Biochem. Biophys. Res. Commun. 1987, 144, 1037. (b) Becker. D.; Swarts, S.; Champagne, M.; Sevilla, M. D. Int. J . Radiaf. Biol. 1988, 53, 767. (19) Sevilla, M. D.; Yan, M.; Becker, D. Biochem. Biophys. Res. Commun. 1988, 155,405. (20) Packer, J. E. J . Chem. Soc. 1963, 2320. (21) Tamba, M.; S h o n e , G.; Qunitiliani, M. I n f . J . Radiaf. Biol. 1986, 50, 595. (22) Monig, J.;Asmus, K.-D.; Forni, L.; Willson, R. L. In?. J. Radiaf.Biol. 1987, 52, 589.

0022-3654/89/2093-0155$01.50/00 1989 American Chemical Society

'

156 The Journal of Physical Chemistry, Vol. 93, No. 1 , 1989

Swarts et al.

TABLE I: Hyperfine Couplings and g Values for Sulfinyl Radicals compound matrix gz gY gx methyl mercaptan 8 M NaCIO4 2.0021 2.0010 2.022 TCFE 2.0021 2.0094 2.020 g,,, = 2.0107 TCFE dimethyl sulfoxide' single crystal 2.003 2.011 2.023 tert-butyl mercaptan

n-butyl mercaptan cysteamine dithiothreitol

methanol 8 M NaCIO, 1: 1 glycerol/H20 TCFE TCFE methanol TCFE TCFE methanol 1:l glycerol/H,O 6 M H2S04 methanol

2.0025 2.0095 2.0084 2.0035 2.01 1 2.003 1 2.0029 2.0088 giso = 2.0106 (135 2.0032 2.01 1 2.0029 2.0090 gino= 2.0104 2.0027 2.0093 2.009 2.003 2.009 2.003 2.0027 2.0093

A, G 12 (3 H ) 13 (3 H ) Aiso = 11.4 (3 H) 11.6 (3 H) 59, -21, -14 (33S)b

2.0201 2.021 2.020 2.020

A , = 56 ( 1 7 0 ) Aiso= 15.5 (170) 15 (1 H) 15 (1 H) Aiso = 15 (1 H), 2.5 (1 H) 15 (1 H ) 16 (1 H ) 14 (1 H ) 15.5 (1 H)

K) 2.02 1 2.021 2.0209 2.020 2.020 2.021

temp, K 101 102 125 173 77 143 173 100 140 98 95 124 113 173 157 113

'Reference 29. b A , , A,, A ,

A

II

C

11

Y

E

/VI

F i p e 1. First derivative ESR spectra found after irradiation of a sample of 0.2 M fert-butyl mercaptan in TCFE under O2at 77 K. Spectra A-D were observed after annealing to the temperatures indicated and spectrum E after recooling to 77 K. The series of spectra illustrates the conversion of a matrix peroxyl radical, ROO' (A, B), to a sulfinyl radical, RSO' (C, D, E). The changes in spectra observed for C, D, and E are due to averaging of anisotropic couplings in RSO' since at 135 K the matrix is soft and allows for rotation. The small low-field component in C and E does not arise from the sulfinyl radical. The three markers are centered at g = 2.0056 with 13.09 G between individual components.

to Fremy's salt (g = 2.0056, aN = 13.06 G). Results Alkanethiols. Irradiation of dilute solutions of alkanethiols in TCFE results in the production of the matrix radicals (e.g., CC12FCF2'and CCIF2CFCl') by dissociative electron attachment and solute cation radicals (RSH') by hole ~ a p t u r e . ~ In ~ -the ~~ (23) Wang, J. T.; Williams, F. J . Phys. Chem. 1980, 84, 3156. (24) Shida, T.; Kubodera, H.; Egawa, Y. Chem. Phys. Lett. 1981, 79, 179.

, I

135K

CH3S0

"I/ Figure 2. Isotropic ESR spectra of the sulfinyl radicals obtained from the irradiation of (A) 0.2 M tert-butyl, (B) 0.2 M n-butyl, and (C) 0.4 M methyl mercaptans in oxygenated TCFE at 77 K. These spectra were observed after annealing samples initially showing the matrix peroxyl radicals to 135, 124, and 125 K, respectively, temperatures at which the matrix is soft and the radicals can tumble freely on an ESR time scale.

presence of dissolved oxygen the matrix radicals form their respective peroxyl radicals at 77 K. For example, Figure 1A shows the spectrum of the matrix peroxyl radical(s) after irradiation of a sample of 2-methyl-2-propanethiol (tert-butyl mercaptan) in TCFE; other radical species (RSH"?) interfere in the central portion of spectrum (g = 2.004). The observed low-field g value of 2.038 is reasonable for a peroxyl r a d i ~ a l . ' ~ ~Warming ~ ' - ~ ~ the sample t o 135 K results in decay of the peroxyl radical spectrum and concomitant formation of a sharp singlet at g = 2.0106. (Figure 1B-D). This sharp resonance is assigned to a motionally averaged sulfinyl radical. This assignment agrees with earlier observations of sulfinyl radical^,'*^^^ including the tert-butylsuifinyl r a d i ~ a l . ~ ~Cooling , ~ ' the sample to 77 K slows the tumbling motion (25) Toriyama, K.; Numoni, K.; Iwasaki, M. J . Chem. Phys. 1982, 77, 5891. (26) Symons, M. C. R. Chem. Phys. Lett. 1980,69, 1980. (27) Kevan, L.; Schlick, S . J . Phys. Chem. 1986, 90, 1998. (28) Becker, D.; Yanez, J.; Sevilla, M. D.; Alonso-Amigo, M. G.;Schlick, S . J . J . Phys. Chem. 1987, 91, 492. (29) Nishikida, K.; Williams, F. J . Am. Chem. SOC.1974, 96, 4781. (30) Kawamura, T.; Krusic, P. J.; Kochi, J. K. Tetruhedron Lett. 1972, 4075.

Structure and Formation of Sulfinyl Radicals EXPERIMENTAL

SIMULATION

Figure 3. Experimental and computer simulated anisotropic ESR spectra for alkylsulfinyl radicals. The experimental spectra were found after irradiation of (A) 0.4 M tert-butyl mercaptan in methanol, (B) 0.4 M n-butyl mercaptan in methanol, and (C) saturated methyl mercaptan in 8 M NaC104. The experimental spectra were acquired after samples under O2were warmed to 114 K, 110 and 179 K, respectively and then recooled to temperatures in the figure. Parameters used in the computer simulated spectra are given in Table I.

of the radical and produces a spectrum (Figure 1E) which exhibits g-value anisotropy. The principal values of the g tensor observed are in excellent agreement with earlier ESR investigations of the penicillamine, cysteine, and glutathione sulfinyl radicals.'* These results suggest an overall radical conversion of matrix peroxyl radical(s) to the solute sulfinyl radical (CH3)3CSO' (Table I). A similar conversion of matrix peroxyl radical(s) to a solute alkylsulfinyl radical is observed for n-butyl mercaptan and methyl mercaptan in TCFE. Figure 2A-C shows; the spectra of each of the three alkanethiol sulfinyl radicals at temperatures at which the TCFE matrix has softened appreciably to allow for molecular motion. In all three spectra, little g anisotropy is observed, indicating that the radicals are tumbling. The observed g values are given in Table I. When samples of methyl mercaptan and n-butyl mercaptan are cooled to about 100 K, anisotropic spectra are observed as found for tert-butyl mercaptan (Table I). The isotropic n-butylsulfinyl radical spectrum (Figure 2B) consists of a principal 15-G doublet which shows indications of further splitting by a second proton at 2.5 G. These two couplings are assigned to the two @methylene protons in CH3CH2CH2CH2SO' which are rigidly held at different orientations relative to the sulfur p-orbital that contains unpaired spin density. In contrast, the spectrum of the methylsulfinyl radical (Figure 2C) shows an 11.4-G 1:3:3:1 quartet originating from three equivalent protons on the methyl group, indicating that the methyl group is rotating internally. This hyperfine coupling is in excellent agreement with the 11.6 G obtained from an earlier single-crystal investigation of the methylsulfinyl radical.29 In order to further investigate the structures of these sulfinyl radicals we recorded their spectra at low temperatures where molecular tumbling is slow on a ESR time scale. The spectra in TCFE show the anisotropy expected for sulfinyl radicals; however, the resolution of the anisotropic features was found to be improved in methanol and 8 M NaC10,. Figure 3 shows the spectra obtained at 77 K for samples of terr-butyl mercaptan in methanol (part 3A), n-butyl mercaptan in methanol (part 3B), and methyl mercaptan in 8 M NaC10, (31) Gilbert, B. C.; Laue, H. A. H.; Norman, R. 0. C.; Sealy, R. C. J . Chem. SOC.,Perkin Trans. 2 1974, 892.

The Journal of Physical Chemistry, Vol. 93, No. I, 1989 157 (part 3C). Dissolved O2was present in each sample. Each sample was irradiated at 77 K and annealed to produce the respective sulfinyl radical before the spectra were recorded. At the high concentrations (3 M) used here for tert-butyl and n-butyl mercaptans, only a small fraction of the sulfinyl radicals originate with matrix or solute peroxyl radicals. Most are formed directly from the thiyl radicals produced initially after irradiation.18 For the methyl mercaptan in aqueous NaC104 a solute peroxyl radical (RSOO' or ROO') converts to a solute sulfinyl radical as the solvent cannot easily form peroxyl radicals. The low-temperature spectrum of tert-butylsulfinyl radical in methanol (Figure 3A) displays g-value anisotropy, indicating that the radical is not freely tumbling in the matrix. Since there are no &protons present on the carbon adjacent to the radical site, no hyperfine coupling is observed. The observed principal g values (2.0025, 2.0095, 2.0201) are similar to those observed for other sulfinyl radicals, and the isotropic g value calculated from the anisotropic values (2.0107) closely matches the isotropic g value observed for terr-butyl mercaptan in TCFE (2.0106). In addition, the g values are, within experimental limits, the same as those for the tert-butylsulfinyl radical in TCFE (2.0029, 2.0088, 2.020). The low-temperature spectrum of the n-butylsulfinyl radical in methanol (Figure 3B) also exhibits g-value anisotropy, indicating that the radical is also rigidly held in the matrix. In addition, there is present a nearly isotropic hydrogen hyperfine splitting of approximately 15 G from a single hydrogen which is due to one of the two methylene protons adjacent to the S-0 group. This splitting agrees with that observed for the same radical in TCFE. However, the small coupling from the second methylene proton, which is resolved in TCFE, is obscured by the broad line widths in methanol. The spectrum obtained for the methylsulfinyl radical in 8 M NaC10, (Figure 3C) shows g anisotropy and an isotropic hyperfine coupling of 12 G from three equivalent hydrogens. The observed hyperfine pattern indicates that motional averaging of the methyl hydrogens occurs, as was observed in for the same radical in TCFE. However, the fact that g anisotropy persists indicates that the radical, as a whole, is not tumbling. Anisotropic computer s i m ~ l a t i o n swere ~ ~ performed to verify the analysis of the spectra (Figure 3 simulations). The g values used for each simulation are given in Table I. For the tert-butylsulfinyl radical simulation a 4.0-G line width was used. For the n-butylsulfinyl radical, the use of a slightly anisotropic hydrogen coupling (1 H; A, = 14 G, A, = 16.5 G, and A , = 14 G) along with a 4.5-G line width gave the best fit to the experimental spectrum. Finally, for the methylsulfinyl radical simulation, three isotropic hydrogen couplings of 12.4 G were used with a 2.5-G line width to enhance the resolution. The simulated spectra match the corresponding experimental spectra well. Comparison of the spectrum of the rigid n-butylsulfinyl radical in methanol (Figure 3B) with the motionally averaged spectrum in TCFE (Figure 2B) provides some insight into the motion that occurs in the TCFE. The complete motional averaging of the g tensor for the radical in TCFE indicates that it is tumbling freely in the matrix. However, this motionally averaged spectrum still exhibits a single 15-G hydrogen coupling and shows evidence for a second coupling of 3 G. If rotation around the C-S bond was occurring as part of the motion, one would expect to observe two equal hyperfine couplings from the two rotationally averaged methylene hydrogens. Thus, even at temperatures which allow the sulfinyl radical to freely tumble in TCFE, the methylene group remains fixed relative to the S-0 group on an ESR time scale. ''0, Labeling Studies. In this series of experiments, dilute solutions of the alkanethiols in TCFE and methanol were irradiated at 77 K after saturation with I7O2(50 atom %). For tert-butyl mercaptan in TCFE, warming the sample to 151 K converts the matrix peroxyl radical to tert-butylsulfinyl radical. The spectrum of the sulfinyl radical at 151 K is shown in Figure 4A. The six hyperfine line components expected from a single I7O nucleus with a nuclear spin of 5 / 2 are clearly visible and correspond to a hyperfine splitting of 15.5 G. This spectrum was (32) Lefebvre, R.; Maruani, J. J . Chem. Phys. 1965, 42, 1480.

158

The Journal of Physical Chemistry, Vol. 93, No. 1 , 1989

B

-512

Swarts et al.

I

+5/2

Figure 4. ESR spectra found after irradiation of a sample of 0.2 M terr-butyl mercaptan in TCFE under I7O2. The sample was annealed to (A) 151 K and then recooled to (B) 100 K. The spectrum in (A) shows the centrally located single line from the '60-labeled terr-butyl sulfinyl radical and isotropic I7O couplings (aim= 15.5 G) from the '70-labeled

RSO. The spectrum in (B) contains the respective anisotropic spectra for these radicals showing the 5 6 - G anisotropic 170hyperfine coupling (A,) for the 170-labeledrert-butylsulfinyl radical (centered at g, =

2.003). Small line components due to a interfering peroxyl intermediate (RSOO' or ROO') are also present; the arrow indicates one such component. recorded at a relatively high temperature at which the radical is rapidly tumbling. Therefore, the observed spectrum is essentially isotropic, and the 15.5-G splitting is presumed to correspond to the 170isotropic hyperfine coupling for this radical. A scan of the wings at higher gain showed no other lines beyond those already observed. The singlet which dominates the central portion of the spectrum is the motionally averaged spectrum qf the I60substituted radical. Cooling of the sample to 100 K freezes the radical motion and, as expected, results in a highly anisotropic spectrum (Figure 4B). The anisotropic All 170splitting, centered at g = 2.003, is 56 G. Since the splittings between the individual hyperfine lines vary slightly, the overall 56-G splitting is calculated by measuring the distance, in gauss, between the I = + 5 / 2 line and the I = -$/* line, and dividing by five. Table I summarizes these results. Line components not attributable to a sulfinyl radical, one at g = 2.034 (arrow, Figure 4B) and other smaller components, are also present in the low-temperature spectrum of tert-butyl mercaptan in TCFE. The positions of these lines are characteristic of a rigid peroxyl radical. We can conclude that the peroxyl radical present is not a TCFE matrix peroxyl radical, since we have observed that the TCFE peroxyl radical consistently gives a sharp singlet at g = 2.01604 above 125 K, and no such singlet is observed at 151 K (Figure 4A). Thus, the peroxyl radical present must originate with the tert-butyl mercaptan solute and is either a carbon-centered species, ROO', or more likely a sulfur peroxyl radical, RSOO'. A possible ambiguity in using TCFE as a solvent is that, as mentioned above, irradiation of a thiol dissolved in TCFE results in the formation of thiol cation radicals as well as matrix peroxyl radicals. These cation radicals may, by deprotonation, form thiyl radicals which, in the presence of oxygen, can react to form sulfinyl radicals (reaction 3). In order to show that peroxyl radicals are a source of sulfinyl radicals, other solvent systems (methanol, glycerol-water 50:50 v:v, aqueous NaC104) were employed in which cation radicals are not formed. Results for these other solvents were reported above and in Table I. Below we describe results in more dilute solutions in methanol which clarify the mechanism of production of the sulfinyl radicals.

H2C

,CH2S0.

'c

I

ti-C

1

H

OH OH

Figure 5. ESR spectra found after irradiation of a sample of 0.2 M dithiothreitol in methanol under oxygen at 77 K. The spectra shown were obtained after annealing the sample to (A) 103 K, (B) 108 K,(C) 113 K, and (D) 113 K (5 min later). This sequence shows the conversion of the methanol peroxyl radical HOCH200' (A) through a possible RSOO' intermediate (B) to the sulfinyl radical of dithiothreitol (C, D). The computer simulation in E was generated using the g values and couplings in Table I and shows a good fit to the experimental spectrum in D.

103K

'J

Figure 6. ESR spectra obtained from a frozen solution of 0.2 M cysteamine in methanol irradiated under oxygen, annealed to (A) 103 K, (B) 113 K, (C) 117 K, and recooled to (D) 103 K. The spectra show the

conversion of a matrix peroxyl radical (A) to other peroxyl intermediates (arrows 1 and 3 in B) and finally to the cysteamine sulfinyl radical (C). Recooling to 103 K increases the intensity of the spectrum of peroxyl radical (3). Peroxyl radicals 1 and 3 originate with the cysteamine solute and possibly represent two different forms of sulfur peroxyl radicals. Cysteamine and Dithiothreitol. Figure 5A-C shows the spectra obtained by annealing a sample of dithiothreitol in CH,OH containing dissolved O2after y-irradiation at 77 K. The initial methanol peroxyl radical found at 103 K (Figure 5A) shows a low-field component with g, = 2.035. At 108 K this species converts to another more highly resolved peroxyl radical species with g, = 2.039 and substantial line intensity at gy = 2.008 and g, = 2.003 (Figure 5B). This species may be the dithiothreitol

The Journal of Physical Chemistry, Vol. 93, No. 1, 1989 159

Structure and Formation of Sulfinyl Radicals

n 0-s

A

\ V L H

A

H

CH3CH2CH2

B

Figure 7. Illustration depicting the conformation of the n-butylsulfinyl radical suggested from its &proton hyperfine couplings. The sulfinyl radical is viewed along the S-C bond. The carbon is hidden behind the sulfur atom. The dihedral angles are given relative to the major axis of the p-orbital on the sulfur atom which contains the unpaired electron.

thiyl peroxyl radical (RSOO'). On annealing to 113 K it readily converts to the dithiothreitol sulfinyl radical (Figure 5, C and D). A computer simulation3* (Figure 5E) of the sulfinyl radical spectrum found at 113 K closely matches the experimental spectrum. The parameters in Table I were employed in the simulation. Figure 6A-C indicates a similar sequence of events for a sample of cysteamine in oxygenated methanol. y-Irradiation produces methanol peroxyl radical (Figure 6A). Annealing to 113 K results in three low-field resonances due to peroxy radicals. The sharp line component at 2.039 (labeled 1) is the same as found in dithiothreitol and was associated with an possible RSOO' radical. The second is at 2.035 and is associated with remaining methanol peroxyl radical. The third component at 2.032 is discussed further below. At 117 K peroxyl radicals 1 and 2 convert to the cysteamine sulfinyl radical. Recooling of the sample to 103 K results in the spectrum shown in Figure 6D, in which three line components of peroxyl radical 3 at g = 2.032, ca. 2.008 and ca. 2.004, are superimposed on the sulfinyl radical spectrum. Evidently, at 117 K this peroxyl radical undergoes a motion which tends to broaden the lines in the spectrum, but cooling to 103 K slows down or stops the motion. Since the methanol peroxyl radical has no line component at g = 2.032, the peroxyl radical observed in Figure 6D must originate with the cysteamine solute. Although a carbon-centered peroxyl radical (ROO') could reproduce the g values, the sharp resolution of the components is unusual for an ROO' radical and points to another, perhaps less reactive form, of a sulfur peroxyl r a d i ~ a 1 . I ~ Conformation of the n-Butylsulfnyl Radical. The @-proton hyperfine couplings (15 and 2.5 G) found for the n-butylsulfinyl radical show that the SO group remains fixed on an ESR time scale in relation to the adjacent methylene group. Since the @-protoncoupling for methylsulfinyl radical is also known (1 1.4 G), an estimate of the conformation at the radical site can be made on the basis of the approximate relation between @-protoncoupling and dihedral angle (e) as given in the equations 15 G = Bp" cos2 6

(4)

11.4 G = Bp"/2

(5)

Solution results in Bp" = 22.8 G, and the free radical conformation shown in Figure 7. The smaller coupling (2.5 G ) is not used in this analysis since it appears at higher temperatures. At room temperature Gilbert et aL3I report that sulfinyl radicals show equivalent @-protons. At the intermediate temperatures used here, partial rotational averaging is expected and the smaller coupling is therefore not reflective of the static conformation. Ab Initio Molecular Orbital Calculations: Spin Density Distribution. In order to understand the structure and spin density distribution in sulfinyl radicals we have performed ab initio calculations for the simplest alkyl sulfinyl radical, CH3SO', using the GAUSSIAN 82 set of program^.^^,^^ The basis set size was varied (33) Binkley, J. S.; Whiteside, R. A,; Raghavachari, K.; Seger, R.; Defrees, D. J.; Schegel, H. B.; Topial, S.; Kahn, L. R.; Frisch, M. J.; Fuder, E. M.; Pople, J. A. GAUSSIAN 82; Carnegie Mellon University: Pittsburgh, PA.

1082

c

1 11.804

'6

H

Figure 8. Ab initio MO structures predicted after full optimization at the 6-31G(d) level for (A) CH,SOO' and (B) CH3SO'.

TABLE 11: Ab Initio MO Calculations for CH3SO' PI spin density on' S 0

STO-3G 0.11 0.89

basis set 6-31G(d) 6-31++G(3d) 0.3 1 0.44 0.67 0.58

expt 0.59b 0.41

6-3 1++G(3d) Calculated Couplings (G) anisotropic nucleus isotropicc 13C -6.8 "S 12.0 (8) I7O -36.3 (-16) 'H 10.7 (17) 'H 10.7 (17) 'H -0.8 (0)

Ad 0.13 29.7 (51) -66.1 (-42) 2.0 2.0 1.98

B -0.09 -12.8 (-22) 32.9 (21) -0.7 -0.7 -0.7

C -0.04 -17.0 (-29) 33.3 (21) -1.3 -1.3 -1.3

'Values are the sum of the spin densities in the pz orbitals on each atom. For oxygen the major spin density is in the 2p, orbital while for S it is in 3p, orbital as expected. bValues calculated from experimental hyperfine couplings after correction for charge. Experimental values are in parentheses. 33.Sand 'H values are from Nishikida and Will i a m ~ and , ~ ~170value are from this work for tert-butyl SO and may not be directly comparable. dThe principal axis for I7O and ' ) S have A along the pz orbital axis.

from small (ST03-G) to large (6-31++G(3d)) with better results found for the larger basis sets. The structure was optimized at the 6-31G(d) level; the results of this optimization are shown in Figure 8B. The predicted S-O bond length of 1.524 A suggests considerable double bond character. Optimization at the 63 1G(2d,p) level gave nearly identical results for bond lengths except that the S-0 bond length was decreased to 1.499 A. In Table I1 we report the isotropic and anisotropic hyperfine couplings and approximate spin densities predicted by the calculations for various basis sets at the optimized geometry found for the 631G(d) calculation. A comparison of the predicted couplings and the experimental values shows that the values for oxygen are predicted to be too large while the anisotropic values for sulfur are predicted to be too small. The unpaired spin is essentially confined to the sulfur and oxygen p, orbitals: however, the calculations overemphasize the density on oxygen. Increasing the basis set has the effect of reducing the oxygen spin density and increasing that on sulfur; the largest basis set employed (631++G(3d)) gives a reasonable fit to the charge-corrected spin densities calculated from experimental couplings (see Table I11 and Discussion). Increasing the basis set size also has the effect (34) Baler, B. H.; Sevilla, M. D.; MacNeille, P. J. Phys. Chem. 1986, 90, 6446.

160 The Journal of Physical Chemistry. Vol. 93, No. 1, 1989

Swarts et al.

TABLE 111: Ab Initio MO Calculations for CHgOO. and CH30W at 6-31G(d) Level PI :Pin

density on'

CH3SOO'

CH,OO'

S 0 0

0.01 0.15 0.84

0.14 0.86

Calculated Couplings ( G ) anisotropic nucleus "C 3s ' 170 170

" 'H 'H

isotropic CHSSOO' CHS00' -4.4

-0.7 -1.2 -19.2 -41.5 -0.2 -0.2 -1.2

-18.0 -40.7 3.3 3.3 -1.7

CH3SOO' A -0.16 -1.9 -26.0 -89.9

-0.7 -0.7 -0.4

CH300'

B

C

Ab

B

-0.08 0.8 9.2 44.5 -0.5 -0.5 -0.2

0.23 1.1 16.8 45.4 1.2 1.2 0.6

-0.69

-0.22

-25.3 -89.8 -1.9 -1.9 -1.4

9.8 44.4 -1.5 -1.5 -1 .O

C 0.91 15.5 45.4 3.4 3.4 2.4

"Values are the sum of the spin densities in the pz orbitals on each atom. bThe principal axis for I7O has A along the pr orbital axis of increasing the predicted S-0 bond polarity. For example the 6-31++G(3d) basis set predicts a charge of +0.98 on sulfur and 0.82 on oxygen, while for 6-31G(d) we find +0.54 and -0.48. Since the nature of the thiyl peroxyl radical is of great importance to the understanding of radiation induced thiol peroxidation,'0J+2' and since it may be an intermediate in this work, we have also performed an analogous set of calculations for the CH3SOO*radical. Its geometry was optimized at the 6-3 1G(d) level and isotropic as well as anisotropic hyperfine couplings calculated at various levels. These results are reported in Figure 8 and Table 111. For comparison we have also included in the table the results of the calculation for the methylperoxyl radical. The calculation was performed at the 6-31G(d) basis set at the geometry optimized at this level and reported in our previous calculations for alkylperoxyl radicals.34 The results in Table 111 clearly show that the thiyl peroxyl radical is predicted to have ESR parameters very similar to those for an alkylperoxyl radical. The oxygen spin densities, as well as the 170isotropic and anisotropic hyperfine couplings are predicted to be virtually identical in the two radicals. The small spin density on sulfur in Table I11 for CH,SOO' suggests that g values for this species will be like those of a typical peroxyl radical. Experimental values for the 170couplings in carbon-centered peroxyl radicals are smaller for the terminal oxygen and larger for the inner oxygen than the calculated couplings. Experimental values are consistent with a 0.7/0.3 division of the unpaired electron. The calculation thus only gives an approximate prediction of these values. Spin Density. The spin density on the oxygen in the tert-butylsulfinyl radical can be estimated from the experimental values of the I7Oanisotropic hyperfine coupling, All (56 G), and the I7O isotropic hyperfine coupling, ah (15.5 g). For the type of coupling found on I7O,the following relations hold.

+ 2B

All = aiso

A , = aiso- B

(11)

Our experiment yields the magnitude of the hyperfine coupling, but not the sign. However, since the nuclear magnetic moment of oxygen is negative, am and E must both be negative. Equations I and I1 and our experimental data (IAlll = 56 G, laix,l= 15.5 G) lead to A , = + 5 G and Boxygen = -20.5 G. Experimentally, the line components from the I7OA , couplings are all obscured by the centrally located I60-substituted sulfinyl radical spectrum, allowing us to put an experimental upper limit of approximately 3-4 G for the magnitude of A , , in reasonable agreement with this calculation. The spin density on oxygen in the tert-butylsulfinyl radical was calculated as suggested by G ~ r d y . ~This ~ method includes (35) Gordy, W. Theory and Applications of Electron Spin Resonance, Wiley: New York, 1980; pp 256-259.

corrections for charge and for screening of the relevant orbitals. The charge used was obtained from the 6-31++G(3d) ab initio molecular orbital calculation for the methylsulfinyl radical (see above), which gave -0.82 for the charge on oxygen. A revised calculated value of Bo (the value of E for unit spin density on = -60.10 G36(rather than the older -51.4 G) oxygen) of EooxY*en and nuclear screening constant of 0.25 for the oxygen p-orbital were used." A value of 0.41 for the spin density on oxygen results. We also calculated the spin density on sulfur in the methylsulfinyl radical in the same way, using Nishikida's and Williams'29 experimental value of Bsulfur= +25.5 G. The charge on sulfur of +0.98, nuclear screening constant of 0.20, and new BPlfur= +35.87 G (rather than the older +27.8 G) were obtained from the same sources as given above for oxygen. A value of 0.59 was obtained for the spin density on sulfur in the methylsulfinyl radical. Even though these spin density calculations are based on experimental values of E for two different sulfinyl radicals, and a theoretically calculated charge for both sulfur and oxygen in the methylsulfinyl radical, the fact that they add up to 1.00 indicates a consistency in the A spin density distributions for the two. In addition, the observed nearly identical g values for the methylsulfinyl radical and tert-butylsulfinyl radical supports the assumption that the spin density distributions and electronic structure for both are similar.

Discussion The production of sulfinyl radicals by the radiolysis of thiols in oxygenated systems has only been recently reported.I8J9 In this previous work irradiation of cysteine in a oxygenated water ice produced the cysteine thiyl radical which then efficiently reacted with molecular oxygen to produce the sulfinyl radical.'* In our present work, we have shown that production of a sulfinyl radical can be initiated by the reaction of a peroxyl radical with a thiol. In TCFE and methanol matrices, the conversion of the respective matrix peroxyl radicals to sulfinyl radicals is highly efficient, often resulting in a near one-to-one conversion. There are two possible mechanisms for this conversion. The first involves the formation of thiyl and thiyl peroxyl radicals as intermediates, reactions 6-8. Our tentative observation of the RSOO' species ROO'

+ R'SH R'S'

R'SOO'

-

+0 2

+ R'SH

-+

ROOH

+ R'S'

R'SOO' R'SO'

+ R'SOH

(6) (7)

(8)

in tert-butyl mercaptan, dithiothreitol, and cysteamine adds some support to this mechanism. However, the fact that even with low oxygen concentrations we find a high conversion of peroxyl radicals to sulfinyl radicals suggests another perhaps competing mechanism which involves a direct bimolecular reaction (36) Morton, J. R.;Preston, K. F. J . Magn. Reson. 1987,30, 577

+ R’SH

-

-

J . Phys. Chem. 1989, 93, 161-164

+ ROH

161

Reaction 9 is very similar in nature to reaction 8 and it is thus difficult to eliminate (9) as a competing mechanism. Further, the intermediate proposed is analogous to the intermediate [RSSS(H)R] suggested for the reaction of RSS’ with RSH.37 Using bond energies of 170 kJ (0-0),435 kJ (0-H), 380 kJ (S-H), and 360 ld (S-O), we predict reaction 9 to be exothermic by approximately 245 kJ.38 Gilbert et al.” found that sulfinyl radicals resulted from reaction of thiols with the Ti3+-H202couple at pH 1-2 and their g values and hyperfine couplings (allowing for differences in rotational averaging) correlate quite well with those reported here. Although the authors reported that the

radicals produced were a result of *OHattack, their system results in HO; as well as ‘OH. Consequently reactions similar to those proposed here may have taken place. Our molecular orbital calculations and spin density distributions indicate the S-O bond in RSO’ is highly polar with substantial double bond character. We suggest that in sulfinyl radicals, the a-spin density on oxygen is 0.4 while that on sulfur is 0.6. The sulfur peroxyl radical, RSOO’, is predicted to be similar to other peroxyl radicals in its ESR parameters. Our recent results show that the cysteinethiol peroxyl radical19 has g values similar to carbon-centered peroxyl radicals but also a more equivalent distribution of the spin density between the two oxygens than found for carbon-centered peroxyl radicals39or predicted by MO theory.

(37) Nelson, D. J.; Peterson, R. L.; Symons, M. C. R. J . Chem. SOC., Perkin Trans. 2 1977, 2005. (38) The first three bond energies used here are standard values (Handbook of Chemistry and Physics, 56th ed.;CRC: 1975; p F224). The sulfinyl radical S-0 bond energy is estimated as follows. Our molecular orbital calculations yield a bond length of approximately 150 ppm for this bond. This is near the typical length of 149 pm for the S-0 bond in alkyl sulfoxides (Kucsman, A,; Kapovits I. In Bernardi, F., Csizmadia, I. G., Mangini, A,, Eds. Organic Sulfur Chemistry; Elsevier: New York, 1985; pp 192-195), which have a average S-0 bond energy of 360 kJ/mol (Oae, S., In Bernardi, F., Csizmadia, I. G., Mangini, A,, Eds. Organic Sulfur Chemistry; Elsevier: New York 1985; p 30). Thus, we estimate 360 kJ for the S-0 bond energy in sulfinyl radicals. (39) Sevilla, M. D.; Champagne, M.; Becker, D. J . Phys. Chem., in press.

Acknowledgment. This investigation was supported by PHS Grant ROlCA45424-01 awarded by the National Cancer Institute, DHHS, and by the Office of Health and Environmental Research of the US.Department of Energy. Acknowledgment is also made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of S.D.B. as a P R F Undergraduate Summer Fellow. Registry No. Methyl mercaptan, 74-93-1; tert-butyl mercaptan, 7566-1; n-butyl mercaptan, 109-79-5;cysteamine, 60-23- 1; dithiothreitol, 3483-12-3; methylsulfinyl radical, 25683-64-1; tert-butylsulfinyl radical, 1 17020-91-4; n-butylsulfinyl radical, 117020-92-5;cysteamine sulfinyl radical, 117020-93-6;dithiothreitol sulfinyl radical, 117020-94-7.

ROO’

[ROOS(H)R’]

R’SO.

(9)

Lifetime and Electronic Spectra of p -Methoxybenzyl Radical in the Lowest Excited Doublet State in Solution Kunihiro Tokumura,* Tomomi Ozaki, Masahiro Udagawa,+and Michiya Itoh* Faculty of Pharmaceutical Sciences and Division of Life Sciences, Graduate School, Kanazawa University, Takara-machi, Kanazawa 920, Japan (Received: May 13, 1988)

-

-

The D, D1absorption (A, = 340 nm) and the D1 Dofluorescence (A, = 490 nm) spectra of the lowest doublet excited state (D1) of p-methoxybenzyl radical in solution were detected in the 248-nm KrF laser photolysis of p-(chloromethy1)anisole followed by the 308-nm XeCl laser pulse excitation. A long lifetime of 120 ns at room temperature and a slightly temperature-dependent nonradiative relaxation were confirmed for p-methoxybenzyl in the DI state. The rotation of the methoxy group seems to be responsible for the relaxation with an activation energy of 1.1 kcal mol-’.

-

-

perturbation of the p-methoxy group.22 In this study, both the D, D1 absorption and the D1 Do fluorescence spectra were

Introduction Since the detection of the fluorescence of benzyl radical in the vapor phase has been reported by Schiiler et al.,I numerous fluorescence spectroscopic studies of benzyl radical in the static vapor p h a ~ e and ~ - ~in the supersonic as well as in the rigid glass a t low were reported. Complicated fluorescence spectra were suggested to be attributable to the proximity of two lowest doublet excited states (DI and D2) with small oscillator strengths for the Dl-Do and D2-Do transitions. Fluorescence lifetimes of benzyl were determined to be 0.88 ps7 in the noncooled vapor phase and ca. 1.4 ps13 in 3-methylpentane at 77 K. On the other hand, the fluorescence spectra of benzyl itself2°,21and benzyls with p-methyl and p-chloro substituents2’ in fluid solution were recently detected by transient spectroscopy employing photolysis and probe laser pulses, and an extensively temperature-dependent fluorescence lifetime was confirmed for them. It was suggestedZ0J that the proximity of D, and D2 states is also responsible for the extensively temperature-dependent nonradiative relaxation. This suggestion led us to examine the excited-state relaxation of p-methoxybenzyl, the D, and D2 states of which are assumed to be no longer close lying owing to a strong

(1) Schiiler, H.; Reinbeck, L.; Koberle, R. Z . Naturforsch., A 1952, 7 A , 421, 428. (2) Walker, S.; Barrow, R. F. Trans. Faraday SOC.1954, 50, 541. (3) Schuler, H.;Stockburger, M. Spectrochim. Acta 1959, 13; 841. (4) Watts, A. T.; Walker, S . J . Chem. SOC.1962, 4323. (5) Cossart-Magos, C.; Leach, S . J. Chem. Phys. 1972, 56, 1534. (6) Cossart-Magos, C.; Leach, S . J . Chem. Phys. 1976, 64,4006. (7) Okamura, T.; Charlton, T. R.; Thrush, B. A. Chem. Phys. Lett. 1982, 88, 369. (8) Heaven, M.; Dimauro, L.; Miller, T. A. Chem. Phys. Lett. 1983,95, 347. (9) Fukushima, M.; Obi, K. Abstr. Jpn. Symp. Mol. Struct. Spectra 1986, 182; 1987, 582. (10) Johnson, P. M.; Albrecht, A. C. J . Chem. Phys. 1968, 48, 851. (11) Watmann-Grajcar, L. J . Chim. Phys. 1969, 66, 1023. (12) Friedrich, D. M.; Albrecht, A. C. J . Chem. Phys. 1973, 58, 4766. (13) Bromberg, A.; Friedrich, D. M.; Albrecht, A. C. Chem. Phys. 1974, 6, 353. (14) Friedrich, D. M.; Albrecht, A. C. Chem. Phys. 1974, 6, 366. (15) Okamura, T.; Obi, K.; Tanaka, I. Chem. Phys. Lett. 1974, 26, 218. (16) Laposa, J. D.; Morrison, V. Chem. Phys. Lett. 1974, 28, 270. (17) Okamura, T.; Tanaka, I. J . Phys. Chem. 1975, 79, 2728. (18) Hiratsuka, H.; Okamura, T.; Tanaka, I.; Tanizaki, Y. J . Phys. Chem. 1980, 84, 285. (19) Miller, J. H.; Andrews, L. J . Mol. Spectrosc. 1981, 90, 20. (20) Meisel, D.; Das, P. K.; Hug, G. L.; Bhattacharyya, K.; Fessenden, R. W. J . Am. Chem. SOC.1986, 108, 4706.

Present address: Chemicals Research Laboratory, Showa Denko, Ohgimachi 5-1, Kawasaki 210, Japan.

0022-3654/89 , ,/2093-0 16 lS01.50 /O 0 1989 American Chemical Societv I

-