10279
J. Am. Chem. SOC.1992,114, 10279-10288
4-Carboxybenzophenone-SensitizedPhotooxidation of Sulfur-Containing Amino Acids. Nanosecond Laser Flash Photolysis and Pulse Radiolysis Studies Krzysztof Bobrowski,* Bronislaw Marciniak,+and Gordon L.Hug* Contribution from the Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556. Received June 30, I992
Abstract: Sulfur-containing amino acids were oxidized via photosensitizationby 4-carboxybenzophenone(CB) in neutral aqueous solutions. The mechanism of this reaction was investigated with flash photolysis and pulse radiolysis techniques. The rate constants were determined for the quenching of the CB triplet state by 12 amino acids (with variable relative location and number of terminal functions COOH and NH2 with respect to the sulfur atom) and were found to be 1.8 X 108-2.9 X lo9 M-l s-I. Time-resolved transient spectra accompanying the quenching events were assigned to the triplet state, the ketyl radical of CB, the radical anion of CB, and the (S;.S)+ radical cations of some of the amino acids. The presence of the radical ions showed the nature of the quenching process to be electron-transferin character. Two temporally distinct processes were observed for ketyl radical formation. A fast component occurred on a nanosecond time scale. It is ascribed to electron transfer from the sulfur atom to the triplet state of CB followed by (1) the diffusion apart of the charge-transfer (CT) complex and (2) the intramolecular proton transfer within CT complex. The first process was the more efficient one and led to the formation of sulfur-centered radical cations and ketyl radical anions which undergo fast protonation. A slower formation of ketyl radicals occurred on a microsecond time scale and is characterized by pseudo-first-order rate constants, which depend linearly on the CB ground-state concentration (k lo9 M-l s-l). This dark reaction is assigned to the one-electron reduction of CB by the a-aminoalkyl radicals produced from the free-radical cation of the amino acids as a result of intramolecular electron transfer from the carboxyl group to the sulfur-centered radical cation followed by decarboxylation. Yields were determined for some of the processes: the limiting overall quantum yields of ketyl radical (0.4-1.4), the quantum yields of ketyl radical formed in the primary photochemical process and in the dark reaction, and quantum yields of ketyl radical anion. A detailed mechanism for the CB-sensitized photooxidation of sulfur-containing amino acids is proposed and discussed.
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Introduction Electron- and hydrogen-transfer processes in the quenching of aromatic carbonyl triplets by a large variety of organic substrates have been the subject of many photochemical inve~tigations.l-~ There have also been several studies concerning photochemical generation of radical centers in amino acid^,^-^ peptide^,^ and proteins.I0J These two sets of studies come together in biological systems since excited carbonyl triplets can be produced 'in v i v ~ " , ~either ~ J ~by direct light absorption or by a variety of dark processes, both en~ymatically'~ and nonenzymati~ally.~~ Owing to their electrophilic character, the nr* triplet states of aromatic ketones can be used as typical one-electron oxidant^.^^^ The similarity between the behavior of nr* carbonyl triplets and oxygenated radicals is well-known,I6 and the analogy makes this excited state a suitable model for radical reactions that result in the damage of cell components. To the best of our knowledge, there has not been a systematic, quantitative study that gives a full understanding of the nature of the photooxidation mechanism of the reactions of nr* carbonyl triplets with this interesting class of biological compounds. Moreover, in the past few years, a growing interest in oxidation processes in biological systems has stimulated several investigations of sulfur-containing amino a ~ i d s . ~ * ~Thicether ,~J' groups located in the side chain of these amino acids participate in the maintenance of the structure of protein molecules, form coordination bonds with metal ions, are part of substrate binding sites, and also take part in the transfer of electrons.I8 The nucleophilic sulfur atom is very susceptible to oxidation. Therefore sulfur-centered radicals are expected to play a key role in the migration of unpaired electrons over long distances through the peptide and protein matrix.I9 Considering all the information gathered so far, the oxidation mechanism of sulfur-containing amino acids and peptides has revealed a very important irreversible route, namely, decarb o ~ y l a t i o n . ~ @The ~ ~ immediate chemical consequence of decarboxylation is the formation of a-aminoalkyl radicals, which 'Fulbright Scholar, on leave from the Faculty of Chemistry, A. Mickiewicz University, 60-780 Poznan, Poland. 'On leave of absence from the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-532 Warsaw, Poland.
Scheme I
lkq
\
C=O
/
+
\ CH-S-R /
are known to be strongly reducing species.24 Consequently the damage caused by carbonyl triplets in such biological units may (1) Hoshino, M.; Shizuka, H. In Photoinduced Electron Transfer. Part C Organic Subsfrafes;Fox, M. A., Chanon, M., Eds.; Elsevier: New York, 1988; Chapter 4.5, pp 313-371 and references cited therein. (2) Turro, N. J. Modern Molecular Photochemistry; Benjamin/Cummings: Menlo Park, CA, 1978; pp 362-413 and references cited therein. (3) Scaiano, J. C. J. Photochem. 1973/74, 2, 81-118. (4) Pienta, N. J. In Photoinduced Electron Transfer. Part C: Organic Subsfrafes;Fox, M. A., Chanon, M., Eds.; Elsevier: New York, 1988; Chapter 4.7, pp 421-86 and references cited therein. ( 5 ) Bhattacharyya, S. N.; Das, P. K. J. Chem. SOC.,Faraday Trans. 2 1984.80, 1107-1 116. (6) Encinas, M. V.; Lissi, E. A,; Olea, A. F. Photochem. Phofobiol. 1985, 42, 347-352. (7) Seki, H.; Takematsu, A.; Arai, S. J. Phys. Chem. 1987, 91, 176-179. (8) Cohen, S. G.; Ojanpera, S. J. Am. Chem. SOC.1975, 97, 5633-5634. (9) Elad, D. Isr. J. Chem. 1970, 8, 253-257.
0002-7863192115 14-10279$03.00/0 0 1992 American Chemical Society
10280 J . Am. Chem. SOC.,Vol. 114, No. 26, 1992 appear at positions different from the initial site of attack. This may result in a change of the redox properties of the system and/or in the loss of enzymatic activity accompanied by a change of the native conformation of the protein. It is thus of great interest to know where and via which mechanism carbonyl triplets preferentially react at specific sites of a sulfur-containing amino acid, and how such reactions are influenced by the local chemical environment of the amino- and carboxyl functional groups. In this paper we shall address some of these questions with respect to the mechanism proposed by Inbar et al.25*26 and by &hen and Ojanpera6 for the quenching of excited triplet states of benzophenone by thioether and by methionine, respectively. According to this mechanism (Scheme I), the initial step is the formation of a charge-transfer complex (CTC) between benzophenone triplet and thioether ( k ). This is followed either by formation of ketyl and a-(alkylthic$alkyl radicals as a result of hydrogen transfer from the carbon ( a to a sulfur atom) to the carbonyl oxygen ( k H )or by spin inversion and back electron transfer to regenerate the reactants (kbET).This mechanism has generally been accepted, and it satisfactorily explains some of the experimental observations (high k,, 107-109 M-’ s-I, generally higher in acetonitrile than in benzene, indicating a polar contribution to the mechanism). Low quantum yields (0.05-0.2) for photoreduction of benzophenone by thioethers are explained in terms of favorable competition between the hydrogen transfer ( k H ) and the reversion to the ground state (kbET).For methionine two CT complexes have been proposed:* CTC-S [>C-O--.+SC-O--.+’NI]. These two forms exist in equilibrium, and only CTC-N may lose C 0 2 directly to form a-aminoalkyl radicals. It is of significance to note that no evidence for the formation of sulfur-centered radicals was found in these systems. In this paper we present the results of nanosecond laser flash photolysis studies of the benzophenone-sensitized photooxidation of sulfur-containing amino acids in aqueous solutions. These amino acids have different numbers of the terminal functions, COOH and NHz, and have varying relative locations of these groups with respect to the sulfur atom. The aim of this work is not only to describe the kinetic aspects of electron transfer to the excited triplet and ground states of 4-carboxybenzophenone, but also to present data regarding transient absorption spectra of various intermediates, including an intermolecular S:.S-bonded radical cation and a ketyl radical anion. The presence of these species is direct proof of dissociation of the CTC-S (involving transfer of a full unit of charge) into separated ions (a process not included either in the Inbar et al.25mechanism for sulfides or in the Cohen and (10) Encinas, M. V.; Lissi, E. A,; Vasquez, M.; Olea, A. F.; Silva, E. Photochem. Phoiobiol. 1989, 49, 557-563. (1 1) Hill, R. R.; Coyle, J. D.; Birch, D.; Dawe, E.; Jeffs, G. E.; Randall, D.; Stec, I.; Stevenson, T. M. J . A m . Chem. SOC.1991, 113, 1805-1817. (12) Cilento, G. Pure Appl. Chem. 1984, 56, 1179-1190. (1 3) Cilento, G. In Chemical and Biological Generation of Excired Srafes; Adam, W., Cilento, G., Eds.; Academic Press: New York, 1982; pp 277-307. (14) Durin, N.; Haun, M.; De Toledo, S. M.; Cilento, G.; Silva, E. Photochem. Phorobiol. 1983, 37, 247-250. (15) Sugioka, K.; Nakano, M. Biochim. Biophys. Acra 1976, 423, 203-216. (16) Encinas, M. V.; Lissi, E. A.; Soto, H. J . Phorochem. 1981, 16,43-49. (17) von Sonntag, C. In The Chemical Basis of Radiation Biology; Taylor/Francis: New York, 1987; pp 353-374. (18) Torchinsky, Yu. M. In Sulfur in Proteins; Metzler, D., Ed.; Pergamon Press, Oxford, 1979. (19) Priitz, W. A. In Sulfur-Centered Reacriue Inrermediares in Chemistry and Biologv; Chatgilialoglu, C., Asmus, K.-D., Eds.; Plenum Press, New York, 1990; pp 389-399. (20) Asmus, K.-D.; Gobl, M.; Hiller, K.-0.; Mahling, S.; Monig, J. J . Chem. SOC.,Perkin Trans. 2 1985, 641-646. (21) Steffen, L. K.; Glass, R. S.; Sabahi, M.; Wilson, G. S.; Schoneich, J . Am. Chem. SOC.1991, 113, 2141-2145. Ch.; Mahling, S.; Asmus, K.-D. (22) Bobrowski, K.; Schoneich, Ch.; Holcman, J.; Asmus, K.-D. J. Chem. SOC.,Perkin Trans. 2 1991, 353-362. (23) Bobrowski, K.; Schoneich, Ch.; Holcman, J.; Asmus, K.-D. J . Chem. Soe., Perkin Trans. 2 1991,975-980. (24) Hiller, K.-0.; Asmus, K.-D. J . Phys. Chem. 1983, 87, 3682-3688. (25) Inbar, S.;Linschitz, H.; Cohen, S.G. J . Am. Chem. Soc. 1982, 104, 1679-1682. (26) Guttenplan, J. B.; Cohen, S. G. J . Org. Chem. 1973,38,2001-2007.
Bobrowski et al. Ojanpera* mechanism for methionine). Over a longer time scale (microseconds), a second process was observed for ketyl radical generation. This process is ascribed to the one-electron reduction of the benzophenone ground state by the a-aminoalkyl radicals that were formed earlier as a result of an intramolecular electron transfer from the carboxyl group to the sulfur-centered radical cation followed by decarboxylation. Further evidence to support the proposed reaction mechanism of photoinduced electron transfer to the triplet state by sulfur-containing amino acids is provided by the generation of the appropriate a-aminoalkyl-type radicals and kinetic studies in complementary pulse radiolysis experiments. On the basis of the above observations, a modified reaction scheme (Scheme 11, vide infra) has been proposed for the photoreduction of benzophenone by sulfur-containing amino acids in aqueous solutions.
Experimental Section Materials. 4-Carboxybenzophenone (Aldrich) and the sulfur-containing amino acids were obtained from Sigma as the best available grades and were used without further purification. A sample of homomethionine was a generous gift from Professor K.-D.Asmus (HMI, Germany). Water was purified by a Millipore Milli-Q system. Solutions. The concentrations of sulfur-containing amino acids in the laser flash photolysis experiments were in the range 8 X 10-5-10-3 M (in the quenching experiments) and 2 X M (when recording spectra in the time range following full quenching of the 4-carboxybenzophenone triplet). 4-Carboxybenzophenone concentration of 2 X lo-’ M in water was employed in quenching experiments. During monitoring of the secondary process, its concentration was in the range of 2.5 X 104-2 X lo-’ M. All solutions were deoxygenated by bubbling high-purity argon through them. They were buffered in the presence of NaH2P0,/ N a 2 H P 0 , (0.025 M). Pulse radiolysis experiments were performed in order to measure rate constants of the reaction of a-aminoalkyl radicals (derived from various sulfur-containing amino acids) with 4-carboxybenzophenone. Solutions were generally prepared with =2 X M sulfur-containing amino acid and with four to six concentrations of 4carboxybenzophenone ranging from 2 X to 2 X lo-’ M. Subsequently, all solutions were saturated with N 2 0 , in order to convert hydrated electrons into hydroxyl radicals via e;, N 2 0 ‘OH OHN2. Laser Flash Photolysis. The nanosecond laser flash photolysis apparatus has been described in detail elsewhere2’ with the exception that the computer analysis was in this case performed on a VAX-l1/780. Laser excitation at 337.1 nm ( ~ 1 - 3mJ, pulse width = 8 ns) from a Molectron UV-400 nitrogen laser was used in a right-angle geometry. Rectangular quartz cells (0.5 X 1 cm) with a path length of 0.5 cm for the monitoring beam were used. The transient absorbances at preselected wavelengths were monitored by a detection system consisting of a monochromator, a photomultiplier tube (PMT), and a 1-kW pulsed xenon lamp as the monitoring source. The signal from the P M T was processed by a 79 12 A D Tektronix transient digitizer controlled by an LSI 11/2 microcomputer. A typical experiment consisted of signal-averaging a series of 5-15 decay traces from reproducible shots from the laser. Cutoff filters were used to avoid spurious response to second-order scattering from the monochromator grating. pulse Radiolysis. All pulse radiolysis experiments were performed by applying 10-ns pulses of high-energy electrons from the Notre Dame 7 MeV A R C 0 LP-7 linear accelerator. Absorbed doses were on the order of 4-6 Gy (1 Gy = 1 J/kg). A description of the pulse radiolysis setup and data collection system is reported e l s e ~ h e r e . ~The ~ . ~experiments ~ were carried out with a continuous flow of the sample solution.
+
-
+
+
Results 1. Quenching of the CB Triplet by Sulfur-Containing Amino Acids. Quenching Rate Constants and Ketyl Radical Quantum Yields. Twelve sulfur-containing amino acids were used as quenchers of the 4-carboxybenzophenone, CB, triplet state in aqueous solution at pH = 6.8. Since only neutral solutions were studied, it will be understood that the CB triplet is always in its carboxylate anion (pK, = 4.5 for CB)31and that the amino (27) Nagarajan, V.; Fessenden, R. W. J. Phys. Chem. 1985, 89, 2330-2335,(28) Schuler, R. H. Chem. Educ. 1985, 2, 34-37. (29) Patterson, L. K.; Lilie, J. Inr. J . Radiar. Phys. Chem. 1974, 6, 129-14 I . (30) Inbar, S.; Linschitz, H.; Cohen. S. G. J . Am. Chem. SOC.1981,103, 7323-7328
J . Am. Chem. SOC.,Vol. 114, No. 26, 1992 10281
Photooxidation of Sulfur-Containing Amino Acids
- 3.0
-
+.
e
,.,[
/ P
X
I
Lanlhlonlna
0
n
*
0
5
I
I
I
2
3
4
I
5
6
[a] x
7
8
9
1
0
I
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F i 1. Plots according to eq 1 for 4carboxybenzophenone (CB) triplet quenching by (a) homomethionine, (b) thiaproline, (c) S-(carboxymethyl)cysteine, and (d) lanthionine in aqueous solution at pH = 6.8. Inserts: experimental traces for CB triplet decay at 540 nm in the presence of 0.32 m M homomethionine (upper insert) and 0.30 m M lanthionint (lower insert).
acids are in their zwitterionic form32with the exception of Nacetylmethionine, S-(carboxymethyl)cysteine, and S-(2carboxyethy1)cysteine. The quenching rate constants, k,, were obtained from the experimentally measured, pseudo-first-order rate constants, k,,, for the decay of the CB triplet by the formula
where TT is the lifetime of the CB triplet in the absence of a quencher. Typical experimental traces for triplet decay in the presence of about 0.3 X M homomethionine and lanthionine are presented in the insets of Figure 1. The long-lived absorption has been attributed to the formation of the 4carboxybenzophenone ketyl radical, K, that was established by comparison of the observed transient spectra with that from Inbar et aL30 (absorption maximum at 570 nm). Whenever the efficiency of ketyl formation was low, e.g. homomethionine, the triplets produced by flash excitation were monitored at their long-wavelength absorption maximum at 540 nm; for other quenchers, the triplets were monitored a t 480 nm. The higher ratio of molar absorption coefficients of triplet vs ketyl radical absorption a t 480 nm, compared to the corresponding ratio at 540 nm, made 480 nm a more favorable monitoring wavelength than the maximum of the triplet a t 540 nm. The peudc-fit order rate constants, kobs,were calculated using eq 2 taking into account concomitant, underlying growing in of the photoproduct absorption
where AO, A , and A" are the absorbance changes at times 0, t , and infinity, respectively. As it will be shown and discussed later (Scheme 11), the initial products of CB triplet quenching are the ketyl radical, K, and the ketyl radical anion, CB'-. The latter becomes protonated on a very short time scale, hundreds of nanoseconds (see for example Figure 5A). Fortunately, in the spectral region of 480 nm the values of molar absorption coefficients for K and CB'- are very similar (the isosbestic point for K/CB'- pairs appears at about (31) Hurley, J. K.; Linschitz, H.; Treinin, A. J. Phys. Chem. 1988, 92, 5 I5 1-5 159. (32) Lehninger, A. L. Biochemie; Verlag Chemie: Weinheim, Germany, 1985.
2
4
6
I / [Q] x
8
1 0 1 2
M''
Figure 2. Plots of l / ~ . k s f yvsl the reciprocal of concentration of (a) a-methylmethionine and (b) thiaproline (according to eq 12) in aqueous solution at pH = 6.8.
490 nm).30 Therefore, during monitoring of triplet decays, no significant corrections were necessary for the fast protonation of ketyl radical anions. Some typical plots based on eq 1 are presented in Figure 1, and the quenching rate constants obtained for all the sulfur-containing amino acids used are summarized in Table I. The k, a t pH 6.8 for alanine, an amino acid which does not contain sulfur, was
