J . Phys. Chem. 1991, 95,6481-6493 run resembled those of reference 19 rather than of reference 17 where, in each case, histograms were published for clusters of Lennard-Jones spheres. Inverse Laplace transformations of the autocorrelation functions would give single, sharp peaks if single phases consistent with eq 16 were present, and multiple peaks if two phases coexisted. Such transforms are notoriously unstable, however, and have not been investigated in detail. Concluding Remarks
Several procedures have been shown to be useful in the processing of data for molecular clusters derived from Monte Carlo runs. They reduce effects of spurious noise and sharpen the ability
6487
to monitor phase changes. They are characterized explicitly in the foregoing to serve as a basis for analyzing a large body of information acquired to date and to define quantities to be introduced in subsequent publications describing results for clusters of polyatomic molecules. Acknowledgment. This research was supported by a grant from the National Science Foundation. Numerical calculations were made possible by a generous allocation of computing time from the Michigan Computing Center. We gratefully acknowledge the awarding of a Regents' Fellowship to F.J.D. Registry No. TeF,, 7783-80-4; benzene, 71-43-2.
Electron Spin Resonance Evidence for Intra- and Intermolecular QQ* Bonding In Methionine Radicals: Relative Stabilities of S-CI, S-Br, S-N, and S-S Three-Electron Bonds Mark H. Champagne, Melisa W. Mullins, AMY-mile Colson, and Michael D. Sevilla* Department of Chemistry, Oakland University, Rochester, Michigan 48309 (Received: February 28, 1991) An electron spin resonance investigation of threeelectron uu* bond formation in methionine, methionine peptides, and model structures in frozen aqueous solutions is presented. Irradiation of 12 M LiCl or 6.3 M LiBr solutions at 77 K results in the C1;- and Br2*-radicals, respectively. These species are found to attack the methionine sulfur atom to form C l S < and B r a < adducts. In basic media both these species are found to react on further annealing to form the cyclic intramolecular adduct, S I N + . Further annealing results in the more stable intermolecularly bonded disulfide radical adduct, S a < . Experiments performed to compare the stabilities of the various three-electron bonded species show the following order in increasing stability: >SIO+SICI, >SIBr, >S'+NH,, >S'+cS%NH,, and >S'+c< bonding are presented. The calculations clearly show the localized uu* nature of the bonding and further predict that the S I N species is stable toward deprotonation from the amine group.
Introduction
The loss of an electron from an ion or molecule often results in a free radical with the potential for the formation of an additional intra- or intermolecular three-electron bond.'-IO One of the simplest of such species is CI2'-, which is produced by the attack of a chlorine atom on chloride ion.' Such radical species possess a relatively localized bond with $u*' electronic structure Electron spin resonance work has and a bond order of I/,. identified a variety of such species and has established the criteria for mr* bonding, Le., a localized initial radical site on a single ~
(1) Castner, T. 0.;Kanzig, W. J . Phys. Chem. Solids 1957. 3, 178.
(2) bcker, D.;Plank, K.; Sevilla, M. D. J. Phys. Chem. 1983,87, 1648. ( 3 ) Rideout, J.; Symons, M. C. R.; Swarts, S.; Besler, B.; Sevilla, M. D. J . Phys. Chem. 1985,89, 5251. (4) Abu-Raqabah, A.; Symons, M. C. R. J . Chem. Soc., Faraday Trans. 1990,86, 3293. (5) Gilbert, B. C.; Hodgeman, D. K. C.; Norman, R. 0.C. J. Chem. Soc., Perkin Trans. I1 1973, 1748. (6) Petersen, R. L.;Nelson, D. J.; Symons, M. C. R. J. Chem. Soc.. Perkin Trans. I1 1978. 225. (7) Symons. M. C. R.; Peterson, R. L. J. Chem. Soc., Faraday Trans. 411 1978, 210. (8) Hiller, K.-0.; Masloch, B.;Gobl, M.; Asmus, K.-D. J. Am. Chem. Soc. 1981, 103, 2734. (9) Asmus. K.-D.; Gobl, M.; Hiller, K.-0.; Mahling, S.;Monig, J. J. Chem. soc. 1985,641. (10) Hiller, K.-0.; Asmus, K.-D. J . Phys. Chem. 1983, 87, 3682.
atom in a molecule, followed by bonding to an unshared pair on another atom with similar ele~tronegativity.~-' A considerable amount of work employing pulse radiolysis has been reported by Asmus and co-workers which describes the formation of R 2 S I X (X-halide), R,SIOR, R2SINR,+, and R2SLSR2+three electron bonded species from thiol ethers and analogues.8-'5 For methionine these workers have reported intramolecular S I 0 and S L N bonds and intermolecular SIS bonds."" Recently, Bobrowski and Holcman reported such intramolecular bonding in methionine peptides.16J7 This previous work shows that such intermediates are likely formed in the radiation damage to biological structures. As a consequence, their formation and chemistry are of considerable interest. Although S I X , S O , SIN,and S I S uu* bonding in me(1 1) Gobl, M.; Bonifacic, M.; Asmus, K.-D. J. Am. Chem.Sac. 1984,106, 5984. (12) Asmus, K.-D. Acc. Chem. Res. 1979, 12, 436. (13) Mahling, S.;Asmus. K.-D.; Glass, R. S.; Hojiatie, M.; Wilson, G.S. J . Org. Chem. 1987, 52, 3717. (14) Bonifacic, M.; Asmus, K.-D. J. Org. Chem. 1986, 51, 1216. (15) Monig, J.; Goslich, R.; Asmus, K.-D. Ber. Bunsen-Ges.Phys. Chem. 1986, 90. 1 IS. (16) Bobrowski, K.; Holcman, J. J . Phys. Chem. 1989, 93,6381. (17) Bobrowski, K.; Holcman, J. Inr. J . Radiaf. Biol. 1987, 52, 139.
0022-3654191I2095-6487%02.50/0 0 1991 American Chemical Societv
Champagne et al.
6488 The Journal of Physical Chemistry, Vol. 95, No. 17, 1991
TABLE I: Hyperfine CoupuasS sad g Values for Mcthioaine u2u*l Radicals g values
compound
solvent
pH
radical
81
gav
nuc
T,K 1 IO
SI+N H~-
2.009 2.005 2.009
>sa< Brs
s*NH~-
>S-< CI3
she methioninol 4-(methylthio)- I-butanol peptides N-acetyl-L-methionine
L-methionyl-L-methionine
LiCl LiCl LiCl
N N N
> S a < 2.000
>Sa< LiCl LiCl
N and B
N
CI-ISC
2.009 2.000
>w< CI3
S a < glycyl-L-methionine
LiCl
N and B
Cl3
S a
s'NH,Cl3
9, A, = 390 nm (150 K). (D) The S%S radical, found in basic or neutral pH, A, = 475 nm (160 K).
Since this reaction was not observed in methionine at lower pH, clearly deprotonation of the amine group potentiates the formation of intramolecular S I N bonds. Above 160 K a singlet at 2.0090 (Figure 3C) appears showing that the S I N radical reacts with a second neutral molecule to form the usual aa+-bonded disulfide cation radical as observed at neutral pH. UV-VisSpectra of the S X I , and S 2 3 Radical Intermediates in 12 M LiCl at 77 K. In order to compare our results to the previous pulse radiolysis work,*-** which employed UV-vis spectroscopy, we have recorded the UV-vis spectra of each of the radical intermediates identified in this work for methionine. Samples in 12 M LiCl were prepared as for the ESR experiments except that the electron scavenger K3Fe(CN)6 was deleted since it absorbs in the vis-UV range. This did not present a difficulty since carbon-centered radicals formed by electron attachment have lower extinction coefficients and absorb a t shorter wavelengths than the aa*-bonded species. All spectra were taken at 77 K after annealing. In Figure 4A is shown the UV-vis spectrum of CI2'-, ,A, = 345 nm, and a weak absorption due the trapped electron a t longer wavelengths. On annealing to ca. 140 K,C12*-attacks methionine to form the C l 3 adduct (Figure 4B, & = 390 nm). On further annealing to 148 K in basic solution, this species converts to the intramolecularly bonded S L N species (Figure 4C, A, = 390 nm). Subsequent annealing to 160 K in basic or neutral solutions results in the disulfide cation, S 3 + (Figure 4D, A,, = 475 nm). The wavelengths of maximum absorption for the species identified by ESR are within 10 nm of those reported for the same species in aqueous solutions at room temperature.s-" As a consequence, our results confirm the previous pulse radiolysis work; however, we note that while the S L N and SLCl bonded
species are not distinguishable by UV-vis spectroscopy, they are easily distinguished in the ESR spectra. Attempts To Form the S-0 Adduct. The results found for methionine implicate only the amine and thio groups in inter- and intramolecular bonding. In an attempt to observe the formation the S O intramolecular complex two methionine derivatives were investigated, methioninol and 5-(methy1thio)- 1-butanol. Annealing an irradiated solution of methioninol (10 pL/mL) in 10 M LiCI/D20 with 10 mg/mL K3Fe(CN)6from 100 to 140 K results in the expected sequence of reactions from CI, to SLCI, to S-I-N+,and finally to S S + as found for methionine in Figure 3. Methioninol differs from methionine by the replacement of the carboxyl group with a hydroxy group. Since the sequence of spectra for methioninol are identical with those for methionine, at higher pH, it is clear that the carboxyl group in methionine and the hydroxy group in methioninol play no important role in intramolecular bonding in our system. The spectra of the S'N+ displays the characteristic triplet with A , of 46 G. The parameters for all species are reported in Table I. Annealing an irradiated solution of 4-(methylthio)-l-butanol (10 pL) in 12 M LiCl/D20 with 10 mg/mL K&(CN)6 resulted in the conversion of Clz- to S X l (reaction 1). The direct conversion to the bimolecular disulfide radical cation ( S S + )proceeds through reaction 2. The SLCl quartet has All = 69 G, and the unresolved singlet of S S + has a g value of 2.0092. Both values are comparable to values found for methionine. No evidence for an SLO intermediate was found for either of these compounds. Peptides and Analogues. A number of methionine-containing peptides, glycylmethionine, methionylglycine, methionyl-
The Journal of Physical Chemistry, Vol. 95, No. 17, 1991 6491
uu* Bonding in Methionine Radicals
TABLE II: STO-X;* Calculated Spin Dehpities and Hypertine Couplings hyperfine couplings, G (expt) atomic spin radical X densities S s x S X A b ALX, A,, A,,, A h , A,, >S-l-CI
0.44 (0.64
0.56 0.36)'
56, 13, 16
>S'+p
s+NH,-
0.74
0.26
74, 4, 7
>S: 'NH-
0.0
1.o
o,o, 0
isotropic proton couplings a 5.2, 6 H (8) 6.2, 12 H (S BrLS< Br(4)
+
-
+
The signal due to the B r l S adduct has an intense component at g = 2.13 which is characteristic of this species. For this radical the line width is reduced and the bromine isotopic splittings are resolved. The single bromine A,(*IBr) coupling in the adduct is 428 G with A, of ca. 100 G (Figure 6B, Table 11). A computer simulation (see Figure 6C)using the anisotropic parameters given in the figure legend gives a good fit to the overall spectrum and shows that the intense component at 2.13 is a combination of parallel and perpendicular components (Figure 6C). Symons and Peterson report similar Br couplings for several B r S ' uu* bonded species? Further annealing of the sample to 146 K results in the formation of the usual disulfide cation radical by reaction 5 . In
Champagne et al.
6492 The Journal of Physical Chemistry, Vol. 95, No. 17, 1991
C
Figure 8. ESR spectra found after y-irradiation of 3 mg of methionine in a basic 6.3 M LiBr solution with K3Fe(CN)6as an electron scavenger at 77 K. The sequence (A) to (B)to (C) shows the gradual conversion of the B r s radical singlet to the S S N adduct radical (triplet).
B Figure 7. ESR spectra found after y-irradiation of 2 pL of 4-(methylthio)-1-butanol in 1 mL of 6.3 M LiBr/D20 and K,Fe(CN), at 77 K. (A) Annealing to 145 K, Brf attack on the thioether forms the B r x radical which shows an intense singlet at g = 2.13. (B and C) These
spectra show the near-completeconversion of the B r s to S%3 radical with annealing. Identical results were found for methionine. Figure 7 we show the conversion of the Brls species at g = 2.13 to the disulfide cation singlet a t g = 2.009 for 4-(methylthio). 1-butanol in 6.3 M LiBr. Identical results were found for methionone. BrAS< + S< >S%< Br(5)
-
-
+
For irradiated frozen solutions of 27:l 12 M LiC1/D20:6.3 M LiBr/D20 containing methionine and K3Fe(CN)6,we find that a t 109 K the C1;- signal predominates owing to the 50:l ratio of CI- to Br- in the solution. At 157 K a mixture of the C I S adduct spectra and smaller amounts of B r l S singlet at 2.13 are found. However, by 162 K the C I l S converts to B r l S and by 166 K B r l S reacts to the disulfide cation. These results clearly suggest the reactions: Cl2.- + >s C F S C + Cl(7)
(22)
D CHI
+
Relative Stabilities of C l l s and B r l s . The relative stabilities of C I S and B r l s a d d u ~ t swere ~ ~ .investigated ~~ in a series of experiments in glasses made of mixtures of LiBr and LiCl solutions. For samples in a 5050 12 M LiC1/D20:6.3 M LiBr/D20 solution with methionine or other thiol ethers such as 4-(methy1thio)-butanol and K,Fe(CN),, we find, after y-irradiation at 77 K, that the initial signal showed Br2*-exclusively and heating produced the B r S adduct, again recognized by the 430-G bromine coupling and the intense component at 2.13. No C I S adduct was observed. These results suggest that the following reaction takes place before the production of Cl2'-: Cl' + 2BrBr2*- CI(6)
758.
C
--
CFS
S:CI.
-
>S'Br,
- stability
e*
>S-NH2,
.*
>S-Sc
These uu*-bonded species in methionine have been suggested previously from UV-visible spectra obtained during pulse radiolysis of solutions of methionine and analogues!*'5 However, the identification of the various species is somewhat more certain in this work since both the nuclear hyperfine couplings of the bonded nucleus (Cl, N, Br) and the g values are highly specific to each type of nucleus. Our UV-visible spectra of the species identified as those found by ESR show the same or nearly the same ,A, for the same species suggested by pulse radiolysis experiments. An intramolecularly bonded SLO radical was reported in pulse radiolysis work by Bobrowski et al.16J7in methionine peptides and by Asmus and co-workers in methionine and other structures as well.e11 We were unable to observe the SA0 adduct in LiCl solutions due to the preferential formation of the Z C l adducts. As a consequence, our results suggest the S I 0 species if formed would be less stable than the Z C l species. Asmus et al. report that the S I 0 bond is considerably weaker than the S I N in methionine in agreement with our workeq It has been observed by several groups that increasing differences in electronegativities in the bonding atoms generally lower the stability of the uu* bond.2*22Our results are in general accord with this 'rule of thumb" for S-X type bonding. Using the MO calculations of Clark25J6and experimental work, Asmus and co-workers have estimated the bond energy of the S'C species to be in the 4080-kJ rangeels From their work a good estimate for the sfs species in methionine would be 60 kJ/mol. The complete conversions from one species to another in our experiments suggest a significant difference in bond energies between individual species, perhaps 5-10 kJ. Formation of the SN ' species is highly sensitive to the state of protonation of the amine group. The conversion of the S C 1 adduct to the intramolecularly bonded S I N radical species is found to occur only at basic pH's where the amine group is not protonated. Molecular orbital calculations suggest that the intramolecular bond in S N species is only stable as a cation (Figure 9C) and is not stable as the deprotonated neutral species (Figure 9D). The molecular orbital calculations further suggest that the nature of the bondng is as expected for these species, Le., they possess a localized u* orbital which contains the unpaired electron. Surprisingly, we found little evidence for facile intramolecular disulfide bonds in the methionylmethionine dipeptide, whereas these were easily produced in pulse radiolysis e~periments.'4'~The disulfide cation uu* bond forms in our system but at temperatures at which intermolecular formation was also found. Acknowledgment. This investigation was supported by the National Cancer Institute of the DHHS and by the Office of Health and Environmental Research of the US.Department of Energy. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of M.H.C. as an undergraduate research fellow. M.W.M. and A.-O.C. were supported as undergraduate and graduate students, respectively. Registry No. LiCI, 7447-41-8; LiBr, 7550-35-8; Clz'-, 12595-89-0; Br2*-. 12595-70-9; L-ethionine, 13073-35-3; methioninol, 502-83-0; 4methylthio-1-butanol, 20582-85-8; N-acetyl-L-methionine, 65-82-7; Lmethionyl-L-methionine, 7349-78-2; glycyl-L-methionine, 554-94-9; Lmethionine, 10332-17-9.
