Tryptophan Oxidation by Singlet Molecular Oxygen [O2 (1Îg

Chem. Res. Toxicol. 2008, 21, 1271–1283

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Tryptophan Oxidation by Singlet Molecular Oxygen [O2 (1∆g)]: Mechanistic Studies Using 18O-Labeled Hydroperoxides, Mass Spectrometry, and Light Emission Measurements Graziella E. Ronsein, Mauricio C. B. Oliveira, Sayuri Miyamoto, Marisa H. G. Medeiros, and Paolo Di Mascio* Departamento de Bioquı´mica, Instituto de Quı´mica, UniVersidade de Saõ Paulo, Saõ Paulo, SP, Brazil ReceiVed January 16, 2008

Proteins have been considered important targets for reactive oxygen species. Indeed, tryptophan (W) has been shown to be a highly susceptible amino acid to many oxidizing agents, including singlet molecular oxygen [O2 (1∆g)]. In this study, two cis- and trans-tryptophan hydroperoxide (WOOH) isomers were completely characterized by HPLC/mass spectrometry and NMR analyses as the major W-oxidation photoproducts. These photoproducts underwent thermal decay into the corresponding alcohols. Additionally, WOOHs were shown to decompose under heating or basification, leading to the formation of N-formylkynurenine (FMK). Using 18O-labeled hydroperoxides (W18O18OH), it was possible to confirm the formation of two oxygen-labeled FMK molecules derived from W18O18OH decomposition. This result demonstrates that both oxygen atoms in FMK are derived from the hydroperoxide group. In addition, these reactions are chemiluminescent (CL), indicating a dioxetane cleavage pathway. This mechanism was confirmed since the CL spectrum of the WOOH decomposition matched the FMK fluorescence spectrum, unequivocally identifying FMK as the emitting species. Introduction 1

Singlet molecular oxygen [O2 ( ∆g)] has been shown to be implicated in many biological events such as enzymatic processes of peroxidases (1, 2), reaction of ozone with biomolecules (3), and phagocytosis (4). Also, this highly reactive oxygen species has been shown to be involved in the genotoxic effects of UVA radiation (5) and in the lipoperoxidation process (6, 7). In addition, photodynamic effects mediated by light, molecular oxygen, and photosensitizers can be valuable tools in photodynamic therapy (8) or be implicated in diseases such as porphyria (9) and cataracts (10). Proteins have been considered the main targets for reactive oxygen species (11–13). Indeed, tryptophan (W)1 has been shown to be a highly susceptible amino acid to many oxidizing agents (13–15). For example, at physiological levels of pH, O2 (1∆g) has been shown to selectively oxidize W and four other amino acid residues: histidine, methionine, cysteine, and tyrosine (16). The reaction of O2 (1∆g) with W has long been a matter of concern (17–20) and has recently attracted considerably more attention because W-derived oxidation products such as Nformylkynurenine (FMK) and kynurenine (Kn) have been associated with some pathological conditions such as the development of cataracts (21, 22) and the formation of covalent aggregates of superoxide dismutase (23–25). Despite the intense interest in the mechanism of W oxidation, there are a lot of gaps that remain to be elucidated. Thus, it is proposed that W photooxidation gives rise to an unstable 3-hydroperoxyindolenine (1), the primary intermediate of which * To whom correspondence should be addressed. Tel: (55)(11)3091-3815 ext. 224. Fax: (55)(11)38155579. E-mail: [email protected]. 1 Abbreviations: O2 (1∆g), singlet molecular oxygen; W, tryptophan; WOOH, tryptophan hydroperoxide; WOH, tryptophan alcohol; FMK, N-formylkynurenine; W18O18OH, 18O-labeled hydroperoxides.

is postulated to be able to rearrange into FMK (7) (17–20). However, at least three paths have been suggested for the transformation of this hydroperoxide into FMK (Scheme 1). First, a hydrated indolenine (2) and its rearranged product (3) were suggested as the intermediates (26, 27). This mechanism is proposed to involve the heterolysis of the O-O hydroperoxide bond, followed by alkyl migration to yield 3, which subsequently breaks down into FMK. Another suggested pathway is decomposition occurring through homolysis or heterolysis of the O-O bond of the tricyclic hydroperoxide (4), giving rise to an eightmembered hydroxyketone intermediate (5), which then decomposes into FMK (28). Finally, a dioxetane (6) derived from a ring chain tautomerism between 1 and 4 is also postulated as a likely intermediate (17, 18). Besides the oxidative pathway leading to FMK, an alcohol (8) has also been reported as a concurrent decomposition product derived from the reduction of the 3-hydroperoyindolenine (1) or its ring tautomer (4) (17, 18). In this context, the current study was undertaken to investigate the chemical basis involved in W oxidation by O2 (1∆g). We are concerned about the chemistry of the initially formed hydroperoxides, their stability, further reactions, and the mechanism leading to FMK conversion. In addressing this issue, each W-derived photoproduct was fully characterized by HPLC/MS/ MS and NMR analyses. Indeed, the mechanism of hydroperoxide decomposition was investigated by measuring the formation of 18O-labeled products and by investigating the chemiluminescence of these reactions.

Materials and Methods Materials. L-W, deuterium oxide (D2O), H218O (97%), Rose Bengal, and xylenol orange were supplied by Sigma-Aldrich (St. Louis MO). An 18O2 gas cylinder (99% 18O) came from Isotec-Sigma (St. Louis, MO). Iron(II) sulfate heptahydrate was

10.1021/tx800026g CCC: $40.75  2008 American Chemical Society Published on Web 05/06/2008

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Scheme 1. Proposed Reaction Pathways for O2 (1∆g)-Mediated Oxidation of W

purchased from Merck (Darmstadt, Germany). Hydrogen peroxide was supplied by Pero´xidos do Brazil (PR, Brazil). All other solvents were of HPLC grade and were purchased from Merck (Rio de Janeiro, Brazil). The water used in the experiments was treated with the Nanopure Water System (Barnstead, Dubuque, IA). Photosensitization. Twenty millimolar L-W was dissolved in D2O containing 10 µM Rose Bengal and irradiated using light from a tungsten lamp (500 W) filtered through a 360 nm cutoff filter. Irradiation was applied for 3 h in an ice bath under a continuous flux of oxygen. The Rose Bengal was removed by passage through an Acrodisc Syringe Filter (Pall Gelman, Ann Arbor, MI) with a 0.2 µm Supor membrane. Photosensitization in an 18O2-Saturated Atmosphere. Photooxidation in an 18O2-saturated atmosphere was carried out as described in previous work (6, 29, 30). Briefly, the same procedure described above was followed, but the oxygen contained in the system was removed by successive freezing and thawing under vacuum. This procedure was repeated at least five times to ensure complete removal of 16O2. Thereafter, the whole system was connected to an 18O2 gas cylinder under 0.5 atm. Photoproducts Analysis and Purification by HPLC. The reaction products were analyzed using a Shimadzu HPLC system (Shimadzu, Tokyo, Japan). For analytical purposes, the system was equipped with a 250 mm × 4.60 mm (particle size 5 µm) C18 reverse-phase column (Phenomenex). The mobile phase consisted of 4% acetonitrile in 0.05% formic acid at a flow rate of 1.0 mL/min. The eluant was monitored using a photodiode array detector at 215 nm. To isolate the photoproducts, a semipreparative C18 reverse-phase column (250 mm × 10 mm, particle size 10 µm, Phenomenex) was used at a flow rate of 4.6 mL/min. The separation was carried out with a gradient of water-acetonitrile, using isocratic elution with 2% acetonitrile for 8 min, then a linear gradient from 2 to 6% in 1 min, maintaining 6% up to 15 min, then reaching 40% in the next 3 min, followed by washing of the column for 3 min, and then returning to 2% in 3 min, followed by column stabilization for another 5 min at 2%. The product peak fractions were collected and immediately lyophilized. HPLC/Mass Spectrometry Analysis. HPLC/MS/MS analysis was carried out in a Shimadzu HPLC system (Tokyo, Japan) coupled to a Quattro II mass spectrometer (Micromass, Manches-

ter, United Kingdom) with a Z-spray source. The samples were separated by HPLC, as previously described, and a small fraction of the eluant was directed into the mass spectrometer at a flow rate of 100 µL/min. MS/MS analyses were done with the electrospray ionizer in the positive ion mode. The source and desolvation temperature were kept at 100 and 150 °C, respectively. The optimal flow rates of the drying and nebulizing gas were found to be 300 and 15 L/h, respectively. The cone voltage was set at 10 V, and the collision energy was at 10 eV. Fullscan data were acquired over a mass range of 100-500 m/z. NMR Spectroscopy. NMR analyses (1H, 13C, COSY, and HETCORR) were performed on a Bruker Avance DRX 500 spectrometer (Bruker-Biospin, Germany). Aliquots containing each isolated photoproduct were dissolved in D2O and analyzed. The specific experiments conducted for each photoproduct are described in the Results. Hydroperoxide Measurements. After the purification step, each fraction containing hydroperoxide was dissolved in water or D2O, and the hydroperoxide concentration was evaluated by the ferric-xylenol orange method (FOX), using H2O2 as a standard (31). Hydroperoxide Stability. The thermal stability was investigated by incubating a 3 mM concentration of the purified solution of each hydroperoxide (final pH, 6, dissolved in water) at 4, 25, and 37 °C for 8 h. The decomposition by metals was evaluated by incubating a 3 mM concentration of the hydroperoxide solution (pH 6, dissolved in water) with 100 µM FeSO4 or CuCl2 for 8 h at 37 °C. Decomposition under different pH values was examined by incubating the hydroperoxides in three different pH values, 5.5 (sodium acetate buffer, 25 mM), 7.4, and 8.5 (tris buffer, 25 mM), for 8 h at 37 °C. The hydroperoxides’ stability to a reducing agent was investigated through incubation with 100 mM NaBH4 for 1 h at 4 °C. All of the reactions were carried out in the absence of light and under stirring. For each analysis, aliquots were removed at the stated time points, and residual peroxide concentrations were determined by the FOX measurement. The results represent the average and SD of at least three independent determinations. In addition, HPLC or HPLC/MS/MS analyses (as previously described) were done to investigate the identities of the formed decomposition products. Decomposition of Labeled WOOH to FMK in Water. For this experiment, a 1.5 mM concentration of isolated trans and

Tryptophan Oxidation by O2 (14g)

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Figure 1. HPLC-ESI/MS analyses obtained after 3 h of W photolysis. (A) Chromatogram with UV detection (λ ) 215 nm). (B) Mass spectrum of 8 min product peak. (C) Mass spectrum of 10 min product peak. Insets: UV-vis spectra of each photoproduct; A.U., arbitrary units.

cis 18O-labeled hydroperoxides was incubated at pH 8.5 (tris buffer) for up to 3 h. Samples were maintained at 37 °C, protected from light and under stirring. Aliquots were taken at 30 min intervals, and the solutions containing the formed FMK were analyzed by HPLC/MS. Decomposition of Labeled WOOH to FMK in Dried Ethanol. Isolated trans (425 µM) and cis (295 µM) W18O18OH were dissolved in dried ethanol and heated to 70 °C for 30 min. Samples were protected from light and kept under stirring. A 100 µM concentration of W was used as an internal standard. After the stated time points (0, 5, and 30 min), the products were quantified by HPLC/MS/MS in the multiple reaction monitoring (MRM) mode. The transitions applied were as follows: 237 to 220 for unlabeled FMK; 239 to 222 and 241 to 224, respectively, for FMK labeled with one or two oxygen atoms; 211 to 194 for Kn; 241 to 148 for W18O18OH; and 205 to 188 for the internal standard (W). Oxygen Exchange Experiments. A 1 mM concentration of an unlabeled, purified FMK was dissolved in 97% H218O and incubated at 37 °C under stirring for up to 4 h. The labeled (one or two oxygen atoms, m/z 239 and 241, respectively) and unlabeled (m/z 237) FMK contents were analyzed by HPLC/ MS/MS immediately after their dissolution and after 2 and 4 h of incubation. Chemiluminescence Studies. Light emission was measured with a photocounting device consisting of a red-sensitive photomultiplier tube (9203BM Thor EMI Electron tubes) cooled to -24 °C with a thermoelectric cooler (FACT 50 MKIII; EMI Gencom, Plainview, NY). The potential applied to the photomultiplier was -1100 V. The phototube was connected to an amplifier discriminator (model 1121; Princeton Instruments, NJ), which transmitted the signal to a computer. Light emissions at specified wavelength intervals were obtained with cutoff filters (Melles Griot Inc., Carlsbad, CA) placed between the cuvette and the photomultiplier. The first experiment consisted of a mixture of the WOOH (3 mM) placed in a cuvette in front of the photomultiplier and stabilized at 37 °C. After 1 min, the photomultiplier shutter was opened, and after 2 min, a NaOH solution (final concentration, 20 mM) was injected into the cuvette. A control reaction was carried out, replacing the NaOH solution by water. Another experiment involved placing a 3 mM concentration of a WOOH mixture in a cuvette in front of the

photomultiplier and heating the solution to 70 °C. The photomultiplier shutter was opened after 1 min of analysis. Light emissions at specified wavelength intervals were obtained for each isolated isomer (trans or cis, 3 mM) by heating the solutions to 70 °C. The spectra thus obtained were compared with the FMK fluorescence spectrum, which was recorded in a SPEX Fluorolog 1681 fluorometer.

Results Photoproduct Formation. After 3 h of photolysis, the reaction products were analyzed by HPLC with UV-vis detection. Separation of the photoproducts gave rise to two major product peaks eluting at retention times of 8 and 10 min, along with a peak from the parent amino acid at 23 min (Figure 1). UV-vis spectra of these two photoproducts showed the same absorption maxima at 234 and 296 nm. Similarly, the mass spectra of these compounds were identical (molecular ion at m/z 237) and consistent with the addition of two oxygen atoms to the W molecule. These results are in agreement with previous reports of two isomeric hydroperoxides (WOOH) as the main photoproducts in W photooxidation (17, 18, 28). Incubation of the solution containing the photoproducts with NaBH4 for 1 h at 4 °C led to a complete loss of 8 and 10 min product peaks, along with the appearance of two new product peaks at 5 and 7 min (Figure 2). These new product peaks had identical UV-vis (absorption maxima at 235 and 292 nm) and mass spectra (m/z 221). The mass spectra of these new compounds showed a loss of one oxygen atom when compared with 8 and 10 min product peaks. These results are consistent with the reduction of the previously formed hydroperoxides (two oxygen atoms) to the corresponding alcohols (WOH, one oxygen atom). Characterization of W Photoproducts by HPLC/MS/MS and Isotopic Labeling. Seeking to come up with further information about the structure and fragmentation of the photoproducts, photosensitization was carried out in an 18O2saturated atmosphere, and the photoproducts were isolated and analyzed by HPLC/MS/MS. Figure 3 compares the mass spectra of 8 min product peak photosensitized in 16O2 (Figure 3A) and 18 O2-saturated atmospheres (Figure 3B). The isomer eluted for 10 min showed the same profile (data not shown). As mentioned

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Figure 2. HPLC-ESI/MS analyses obtained after incubation of the solution containing the photoproducts with NaBH4 for 1 h at 4 °C. (A) Chromatogram with UV detection (λ ) 215 nm). (B) Mass spectrum of 5 min product peak. (C) Mass spectrum of 7 min product peak. Insets: UV-vis spectra of each photoproduct; A.U., arbitrary units.

Figure 3. HPLC/MS/MS analyses obtained after isolation of the 8 min product peak. (A) Unlabeled (WOOH) and (B) hydroperoxide. The isomer that eluted for 10 min showed the same profile.

earlier, under 16O2-saturated atmosphere, the hydroperoxides display a molecular ion at m/z 237. MS/MS analysis of W16O16OH showed mainly the ions at m/z 220, 203, and 146, which correspond, respectively, to the loss of the OH, H2O2, and [OH + CH(NH2)COOH] groups. These data were further supported with the results of the 18O-labeled hydroperoxide. W18O18OH exhibits a molecular ion at m/z 241, consistent with the increase by four atomic mass units (amu) as compared with the unlabeled molecule. This result indicates that two atoms of 18 O2 were successfully incorporated into the hydroperoxide. In addition, MS/MS analysis of the W18O18OH confirms the previously attributed fragmentations, showing losses of 222, 203, and 148 relative to the 18OH, H218O2, and [18OH + CH(NH2)COOH] groups, respectively. NMR Analyses. The data obtained with the HPLC/UV-vis, isotopic labeling, and HPLC/MS/MS techniques were confirmed by NMR studies of the isolated photoproducts. 1H, COSY, and 13 C NMR experiments of each isolated WOH (2-carboxy-3ahydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole) were consistent with proposed structures and the literature (18, 19) (Table 1). The stereochemistry of each product was inferred by comparison with Nakagawa et. al (18). In that study, the structure of 3ahydroxy-1,2-dimethoxycarbonyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3b]indole was determined by X-ray analysis. This molecule was a carbamate ester analogous to the more polar alcohol isomer and was shown to have the trans configuration with regard to the

18

O-labeled (W18O18OH)

relative position of the hydroxyl and ester groups. After that, aiming to confirm the alcohols’ stereochemistry, the analogous carbamate ester was subjected to alkaline hydrolysis, resulting in the more polar alcohol. Thus, this alcohol was assigned as the trans-3ahydroxyhexahydropyrroloindole. Therefore, the other isomer was identified as the cis-alcohol. Accordingly, in our study, the WOH with the faster mobility in HPLC analysis (5 min product peak, more polar isomer) was assigned as the trans isomer, and the WOH with the slower mobility (7 min product peak) was assigned as the cis-alcohol. The hydroperoxides (2-carboxy-3a-hydroperoxy-1,2,3,3a,8,8ahexahydropyrrolo[2,3-b]indole) were also fully characterized by 1 H, 13C, COSY, and HETCORR NMR experiments (Table 2). The data were consistent with proposed structures and the literature (18). The stereochemical assignments for each isolated hydroperoxide were established by comparison with the previously identified WOH. Thus, the hydroperoxide with the faster mobility in HPLC analysis (8 min product peak, more polar isomer) was shown to have the trans configuration in relation to the carboxylic acid group, since it was reduced to the alcohol that elutes at 5 min. Similarly, the hydroperoxide that elutes at 10 min was assigned as the cis isomer, since it was reduced to the alcohol at 7 min (the above assigned cis isomer). Stability of the WOOH. Once the major photoproducts of W photooxidation were fully characterized as two isomeric hydroperoxides, the work then focused on the stability of these

Tryptophan Oxidation by O2 (14g) Table 1. 1H and

a

C NMR Chemical Shifts of the trans- and cis-2-Carboxy-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo [2,3-b]indole in D2O

Chemical shift, ppm. b Mult., multiplicity. c Jxy, coupling constant (protons, x and y), Hz.

Table 2. 1H and

a

13


13

C NMR Chemical Shifts of the trans- and cis-2-Carboxy-3a-hydroperoxy-1,2,3,3a,8,8a-hexahydropyrrolo [2,3-b]indole in D2O

Chemical shift, ppm. b Mult., multiplicity. c Jxy, coupling constant (protons, x and y), Hz.

compounds. Indeed, we were also interested in the decomposition products of these hydroperoxides. Thus, as indicated by the FOX assay, both hydroperoxides were relatively stable upon incubation at 4, 25, and 37 °C for 8 h (Figures 4 and 5A1, respectively, for the trans and cis isomers). These results were

further underpinned by an analysis of the HPLC profile of the reactions, since after 4 h of incubation at the aforementioned temperatures, no significant loss was observed in the hydroperoxide product peaks (Figures 4 and 5A2, respectively, for the trans and cis isomers).

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Figure 4. Decomposition of trans-WOOH. (A) Thermal stability: (A1) FOX measurement after the incubation of 3 mM trans-WOOH at 4 ((), 25 (9), or 37 °C (2) for 8 h; (A2) HPLC analyses after the incubation of trans-WOOH for 4 h at 4, 25, or 37 °C. (B) Metal-catalyzed decomposition of trans-WOOH: (B1) FOX measurement after the incubation of 3 mM trans-WOOH at 37 °C ((, control reaction) or at 37 °C in the presence of Cu(II) (9) or Fe(II) (2); (B2) HPLC analyses after the incubation of trans-WOOH for 4 h at 37 °C or at 37 °C in the presence of Cu(II) or Fe(II). (C) pH-dependent decomposition of trans-WOOH: (C1) FOX measurement after the incubation of 3 mM trans-WOOH at 37 °C in pH 5.5 ((), 7.4 (9), or 8.5 (2); (C2) HPLC profile after the incubation of trans-WOOH for 4 h at 37 °C in pH 5.5, 7.4, or 8.5. Results of the FOX assay represent the average and SD of at least three independent determinations.

In addition to thermal stability, we were also interested in the reactivity of hydroperoxides to biologically important metals. To address this issue, two metal ions were chosen as follows: Cu(II), an oxidizing species, and Fe(II), a reducing agent. Interestingly, cupric ions did not have a marked effect upon WOOH decomposition, showing a decrease of approximately 35% in the amount of both WOOH after 8 h of incubation (Figures 4 and 5B1, respectively). The decomposition products followed by HPLC chromatograms (Figures 4 and 5B2) were identified as the corresponding alcohols of each WOOH. However, it should be noted that, after 4 h of incubation, the intensity of the peaks of these byproducts was only slightly higher than that of the control samples incubated at 37 °C in the absence of copper. In contrast to experiments involving cupric species, ferrous ions react relatively rapidly with the two isomeric hydroperoxides, although the reaction rates appear to be different. For example, while 4 h of incubation with Fe(II) resulted in an almost complete depletion of the trans isomer, incubation with the cis isomer resulted in approximately 50% of decomposition (Figures 4 and 5B1). These results are confirmed by HPLC analyses since, after 4 h of incubation with Fe(II), the product peak corresponding to the trans-hydroper-

oxide disappeared completely, while the cis-hydroperoxide product peak was still visible (Figures 4 and 5B2). Despite the distinct Fe(II) reduction rates of the two isomers, the corresponding WOH was identified as the main product of decomposition of these reactions. The stability of WOOH was also evaluated in three different pH values: 5.5, 7.4, and 8.5. The results indicate that both hydroperoxides were relatively stable in pH 5.5 over a period of 6 h (Figures 4 and 5C1). However, the more the pH increased, the faster the decomposition of the hydroperoxides occurred. Thus, after 6 h of incubation at pH 7.4, the amount of transand cis-hydroperoxides decreased by 89 and 75%, respectively, when compared with control samples incubated in water at 37 °C (Figures 4 and 5C1). Indeed, a more pronounced decomposition was observed at pH 8.5, since an almost complete loss of both WOOH occurred after 6 h of incubation (Figures 4 and 5C1). The HPLC analyses support the FOX measurements, showing a decrease in both WOOH product peaks in response to the increase in the solution’s pH (Figures 4 and 5C2). However, contrary to experiments involving thermal stability or metal reactivity, the main product was not the corresponding WOH. For both WOOH, a new product peak appeared after 11



Figure 5. Decomposition of cis-WOOH. (A) Thermal stability: (A1) FOX measurement after the incubation of 3 mM cis-WOOH at 4 ((), 25 (9), or 37 °C (2) for 8 h; (A2) HPLC analyses after the incubation of cis-WOOH for 4 h at 4, 25, or 37 °C. (B) Metal-catalyzed decomposition of cis-WOOH: (B1) FOX measurement after the incubation of 3 mM cis-WOOH at 37 °C ((, control reaction) or at 37 °C in the presence of Cu(II) (9) or Fe(II) (2); (B2) HPLC analyses after the incubation of cis-WOOH for 4 h at 37 °C or at 37 °C in the presence of Cu(II) or Fe(II). (C) pH-dependent decomposition of cis-WOOH: (C1) FOX measurement after the incubation of 3 mM cis-WOOH at 37 °C in pH 5.5 ((), 7.4 (9), or 8.5 (2); (C2) HPLC profile after the incubation of cis-WOOH for 4 h at 37 °C in pH 5.5, 7.4, or 8.5. Results of the FOX assay represent the average and SD of at least three independent determinations.

min of elution. This product peak was completely characterized by UV-vis, HPLC/MS/MS, and NMR experiments as being the N-formylkynurenine (FMK, Figure 6), a well-known decomposition product of W under a variety of oxidizing conditions (14). N-Formylkynurenine was also obtained upon warming the WOOH at 70 °C. Mechanistic Studies Involving WOOH Decomposition to FMK. To elucidate mechanistic aspects involving WOOH conversion to FMK, two different strategies were employed. First, the origin of the carbonyl oxygen atoms was investigated through isotopic labeling experiments; second, the presence of an excited carbonyl derived from a previously formed dioxetane was evaluated based on chemiluminescence studies. Decomposition of Labeled WOOH to FMK. The first experiment involved incubating isolated trans and cis 18Olabeled hydroperoxides at pH 8.5 for up to 3 h. Aliquots were taken at 30 min intervals, and the solutions containing the FMK formed were analyzed by HPLC/MS. Representative spectra of the resulting FMK are depicted in Figure 7A,B, respectively, for 30 min incubations of trans- and cis-W18O18OH. As previously shown in Figure 6, unlabeled FMK displays a

molecular ion at m/z 237. On the other hand, if the carbonyl and amide oxygen atoms derive from labeled hydroperoxides, the mass spectrum must present a molecular ion at m/z at 241, showing an increase of 4 amu (two labeled oxygen atoms) as compared with the unlabeled molecule. In contrast, the FMK obtained upon incubating W18O18OH at pH 8.5 showed only molecular ions at m/z 237 and 239, leading us to infer that no labeled oxygen atom was incorporated in the FMK molecule or that, at best, only one oxygen atom was derived from the W18O18OH. However, several authors have reported that compounds containing oxygen functional groups may present a high rate of oxygen exchange in water (32, 33), and a study involving the structurally related N1-acetyl-N2-formyl-5-methoxykynurenine also displayed this exchangeability (34). Thus, it is important to ascertain whether the FMK in our system was formed with two 18O atoms, and whether these molecules were subsequently exchanged with water, or if the solvent participated in the formation mechanism. To this end, an unlabeled purified FMK was incubated with H218O. As expected, immediately after the dissolution of FMK, it is just possible to see the molecular ion at m/z 237 (Figure 8A). However, after 2 and 4 h of

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Figure 6. Characterization of N-formylkynurenine. (A) Mass spectrum, (B) UV-vis spectrum, and (C) 1H NMR chemical shifts.

Figure 7. HPLC/MS analyses of FMK formed during the incubation of isolated trans (A) and cis (B) buffer) for 30 min.

18

O-labeled hydroperoxides at pH 8.5 (tris

Figure 8. HPLC/MS analyses obtained after incubation of unlabeled purified FMK with H218O. (A) Control reaction, obtained immediately after the FMK dissolution in H218O. (B) Mass spectrum obtained after 2 h of incubation. (C) Mass spectrum obtained after 4 h of incubation.

incubation with H218O (Figure 8B,C, respectively), a molecular ion appears at m/z 239, confirming that in this system at least one oxygen atom can be exchanged with the solvent molecules. In an attempt to prevent exchanges occurring between FMK and water molecules, isolated trans- and cis-W18O18OH were dissolved in dried ethanol and heated at 70 °C. After the end points (0, 5, and 30 min), labeled (one or two oxygen atoms, m/z 239 and 241) and unlabeled (m/z 237) FMK contents were quantified by HPLC/MS/MS using the MRM mode and W as an internal standard. Interestingly, unlike FMK’s stability in water, the dissolution of FMK in ethanol, along with the increase of the temperature, causes a solvolysis of the amide group, leading to kynurenine (Kn). Thus, it was impossible to see a linear increase in the overall FMK content, since at any specific moment, the detected FMK is a balance between the produced and the consumed molecules. In contrast, the Kn quantification showed a linear profile up to 30 min of reaction (Figure 9).

Despite the instability of FMK in ethanol, it was still important to look for the two oxygen-labeled FMK (m/z 241), since an increase at this m/z during the course of the reaction is unequivocal evidence of the involvement of the two oxygen atoms of the hydroperoxides in the decomposition mechanism. Therefore, immediately after W18O18OH dissolution (0 h), it was already possible to see labeled and unlabeled FMK content (Figure 10). This FMK content derived from the decomposition of the hydroperoxides during the purification process. However, an analysis of Figure 10A (trans isomer decomposition) and B (cis hydroperoxide conversion) confirms that the content of the two labeled FMK molecules (m/z 241) increased in both WOOH incubations. Chemiluminescent Reactions. Additional supporting evidence of isotopic labeling experiments was obtained with chemiluminescence studies. It was previously demonstrated herein that basification and heating favor the decomposition of



Figure 9. Quantification of the decomposition products generated by incubation of 425 µM trans and 295 µM cis 18O-labeled hydroperoxides at 70 °C in dried ethanol for 30 min. The content of decomposition products was quantified by HPLC/MS/MS in the MRM mode using 100 µM W as an internal standard. The symbol ( corresponds to the FMK content, and the symbol 9 corresponds to Kn content. The results were expressed as the ratio of the decomposition product (FMK or Kn) to the internal standard (W). The transitions used in MRM mode were 237 to 220 for unlabeled FMK; 239 to 222 and 241 to 224, respectively, for FMK labeled with one or two oxygen atoms; 211 to 194 for Kn; and 205 to 188 for W, the internal standard. The total amount of FMK was calculated considering the values of the three MRM transitions.

Figure 10. N-Formylkynurenine generated by incubation of trans (425 µM) and cis (295 µM) 18O-labeled hydroperoxides at 70 °C in dried ethanol for 30 min. The FMK content was quantified by HPLC/MS/MS in the MRM mode using W (100 µM) as an internal standard. The results were expressed as the ratio of the FMK to the internal standard (W). The transitions used in MRM mode were 237 to 220 for unlabeled FMK; 239 to 222 and 241 to 224, respectively, for FMK labeled with one or two oxygen atoms; and 205 to 188 for the internal standard (W).

WOOH into FMK. We therefore decided to test if the conversion is chemiluminescent under these conditions. To this end, a mixture containing both WOOH was placed in a cuvette in front of the photomultiplier and stabilized at 37 °C. After 1 min, the photomultiplier shutter was opened, revealing that the solution was already slightly luminescent under these conditions (Figure 11A, trace 1). After 2 min, a NaOH solution was injected into the cuvette. Immediately after the NaOH injection, an increase in light emission was observed. A control reaction replacing the NaOH solution with water did not present the flash of light emission (Figure 11A, trace 2). Indeed, analyzing Figure 11B, one can see that heating the WOOH solution also led to a chemiluminescence emission. Comparison of FMK Fluorescence and WOOH Decomposition Chemiluminescence Spectra. It is well-known that dioxetane decomposition produces excited carbonyl compounds that emit light as they decay (35, 36). Assuming that the light emission is caused by an excited FMK generated by a dioxetane cleavage, the fluorescence spectrum of FMK should match the spectrum obtained with the WOOH decomposition. To address this issue, a FMK fluorescence spectrum was recorded using 320 nm as the excitation wavelength. In parallel, chemiluminescence spectra for each isolated WOOH were obtained using cutoff filters. The overall results are depicted in Figure 12. The emission spectrum of FMK shows a broad peak with the maximum at 436 nm. In addition, the thermal decomposition of trans- and cis-WOOH demonstrate the same maximum near 436 nm, along with another band at around 510 nm.

Discussion W oxidation has long been a matter of concern. The first photoproducts isolated and characterized were the two diastereoisomeric 3a-hydroxypyrroloindoles and FMK (19). Subsequently, other studies performed under mild conditions successfully identified a mixture of trans- and cis-3a-hydroperoxypyrroloindoles as the primary W photoproducts (17, 18). The analytical data obtained herein and in previous studies are consistent with a type ene reaction between 1O2 and W. This mechanism has been widely accepted to substitute indole derivatives, although the exact nature of the process, that is, whether it is a concerted or stepwise reaction involving shortlived species such as a perepoxide or a zwitterion, is still a matter of controversy (37–39). The reaction is postulated to give rise to an unstable hydroperoxide, which is rapidly converted to the isolable tricyclic hydroperoxide by intramolecular addition of the ethylamino side chain to the azomethine unsaturation (18). In the current study, we were able to isolate and fully characterize each distinct WOOH isomer as a tricyclic hydroperoxide. Thus, HPLC/MS/MS and isotopic labeling analyses showed two separated product peaks that contained two additional oxygen atoms when compared with the W molecule. Moreover, these products were completely reduced, giving rise to the corresponding alcohols. Proposed structures were confirmed by 1H and 13C NMR analyses. C8a and H8a chemical shifts support the proposed cyclic structures, since in the case of open forms, these atoms would belong to an olefinic system and should show a more pronounced downfield shift. Stereochemical assignments for trans- and cis-WOOH were estab-

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Figure 11. Chemiluminescence generated by WOOH decomposition. (A) Light emission upon basification of a mixture of hydroperoxides (3 mM). The reactions were carried out at 37 °C. After 1 min, the photomultiplier shutter was opened, and after 2 min, NaOH (final concentration, 20 mM; trace 1) was injected into the cuvette. The control reaction was done by replacing the NaOH solution with water (trace 2). (B) Light emission upon heating a mixture of 3 mM hydroperoxides for 16 min at 70 °C. The photomultiplier shutter was opened after 1 min.

Figure 12. Comparison of FMK fluorescence spectrum and chemiluminescence spectra generated by the decomposition of each isolated WOOH. FMK fluorescence spectrum was recorded using 320 nm as the excitation wavelength. Chemiluminescence spectra were obtained for each isolated WOOH using cutoff filters.

lished indirectly from that obtained by comparison with the previously identified WOH and with the literature (18). The most notable difference in the 1H NMR spectra of the two WOOH isomers is the fact that C3 methylene hydrogens of the trans isomer appear as two-proton doublets (2.89 and 2.52 ppm). These results indicate that these two protons are magnetically nonequivalent, being the AB part of an ABX system where X is the H2 proton. This phenomenon was observed in similar compounds by Zang et al. (38) (Table 2). In contrast, C3 methylene hydrogens of the cis isomer present only one doublet (2.62 ppm), showing in this case these two protons are

magnetically equivalent. Interestingly, in the case of WOH, C3 methylene hydrogens of the cis isomer clearly appear as twoproton doublets (2.93 and 2.59 ppm), while in the trans isomer these hydrogens have close chemical shifts, resulting in a multiplet. Nonequivalence of these two protons could also be noted in the C2multiplicity (a double-doublet). Besides, the J2,3′ scalar coupling constant for the cis-WOH present a high magnitude (close to 12 Hz), which is indicative of a trans diaxial orientation for the concerned protons, suggesting that the carboxylic group adopts a preferential equatorial conformation (Table 1). Apparently, this is not the case of the carboxylic group


of the trans-WOOH. Although this hydroperoxide also has two protons magnetically nonequivalent, the two vicinal J2,3′ and J2,3′′ scalar coupling constants are close to 8 Hz. Another contrast observed between WOOH and WOH is a difference of about 10 ppm at C3a chemical shifts, due to a more electronegative hydroperoxyl group. Aiming to obtain mechanistic information about 1O2-mediated oxidation of W, the reactivity of the resulting hydroperoxides was investigated. The WOOH decayed only slightly when the solutions were heated to 37 °C. Indeed, Cu(II) ions did not exert a significant effect toward the hydroperoxides. This apparent stability upon Cu2+ oxidation could be due to the formation of peroxyl radicals, which may abstract a proton from a suitable donor, regenerating the hydroperoxides. In contrast, Fe(II) proved to be a good one-electron reducing agent to the WOOH, leading to the corresponding alcohols. In this case, an organic Fenton reaction is expected, giving rise to related alcoxyl radicals, Fe(III) and OH-. Moreover, the hydroperoxides were shown to decompose upon heating or increased levels of pH, leading to the formation of FMK. Although the exact mechanism for this transformation has been studied exhaustively by many researchers, the detailed chemistry is still not well understood. Thus, as already shown in Scheme 1, there are three main pathways for the conversion of hydroperoxides into FMK. As previously proposed, the rearrangement of the tricyclic hydroperoxides could be explained by assuming either the hydrated indolenine (2) (26, 27) or the eight-membered hydroxyketone (5) (28) as a possible intermediate. The first experiment undertaken with a mechanistic purpose was the decomposition of each isolated W18O18OH in tris buffer (pH 8.5) (Figure 7). The data obtained tend toward the hydrated indolenine pathway, since the resulting FMK shows a lack of 18 O-labeled incorporation (m/z 237) or the incorporation of only one oxygen atom (m/z 239). On the basis of these data, it could be suggested that a water molecule participates in the mechanism of W18O18OH decomposition to FMK. However, it has been shown that oxygen functional groups may present a high rate of oxygen exchange in water (32–34). Thus, one cannot exclude the possibility of the two 18O-labeled FMK formation, followed by an oxygen exchange of the carbonyl molecules with water. In fact, an experiment carried out with an unlabeled purified FMK dissolved in H218O proved the exchangeability of the FMK carbonyl oxygen atoms (Figure 8). In light of these results, another strategy was employed as follows: isolated trans- and cis-W18O18OH were dissolved in dried ethanol. Immediately thereafter, they were heated, and the FMK content thus formed (labeled and unlabeled) was quantified (Figure 10). The results indicate that two 18O-labeled FMK were formed during the decomposition of trans- and cis-W18O18OH. This is unequivocal evidence of the involvement of the two oxygen atoms of the hydroperoxides in the FMK formation mechanism. In addition, the WOOH decomposition proved to be chemiluminescent. The chemiluminescence produced by substituted indole derivatives and their hydroperoxides in the presence of alkali has been studied since the 1960s (40–44). A basecatalyzed decomposition of a dioxetane is the postulated mechanism. Furthermore, luminescence has also been demonstrated in reactions involving the peroxidase-catalyzed aerobic oxidation of indole acetic acid, a plant hormone. Indole-3aldehyde is a major product expected from a hypothetical R-peroxylactone intermediate (45–47). Recent researches have also shown that oxidation of melatonin (N-acetyl-5-methoxytryptamine), an important hormone structurally related to W, is luminescent (48–50). Moreover, Alarcoń et al. (51) reported


low level chemiluminescence after the Rose Bengal irradiation of free W and dipeptides containing W. On the basis of previous studies, these authors suggested a mechanism through a dioxetane intermediate, leading to a FMK derivative. However, despite extensive work on the luminescence of indole compounds, there are still many gaps in our knowledge of the nature of intermediates and in the steps involved in the mechanism, although a dioxetane pathway is widely accepted. In addition, the majority of studies have been carried out with W derivatives, since these compounds are more soluble in organic solvents and, hence, easier to work with. Indeed, the free amino group of W has been argued to quench luminescence, making its detection more difficult (52). In this work, the decomposition of W-derived hydroperoxides under heating and basification was shown to be luminescent. Moreover, in our search for the emitting species, the spectra of the thermal decomposition of trans- and cis-WOOH were recorded (Figure 12). These spectra showed a maximum close to 436 nm, matching the FMK fluorescence spectrum perfectly. However, another band was also visible at around 510 nm. In a study of the peroxidase-catalyzed oxidation of indole derivatives, this shift to red luminescence was attributed to the formation of an excited dimmer (52). Another study, which showed the effect of melatonin on the Cu(II)/H2O2 system, suggested that this spectral red shift was derived from 1O2 (48). In contrast, this emission can be understood in terms of the mechanism involved in excited FMK formation. Thus, light emission at around 520 nm was demonstrated when indolyl peroxides where dissolved in ethanol (43) or dimethyl sulfoxide (40) containing potassium hydroxide. This light emission was attributed to an excited carbonyl anion formed during dioxetane decomposition. The authors also showed that the neutral amide formed by protonation of the anion loses that fluorescence. Indeed, it has been shown that intramolecular hydrogen bond formation between the N-H and the ortho carbonyl group of FMK plays an important role in the observation of an abnormally red-shifted fluorescence emission. It is postulated that the hydrogen-bonding ability could, for instance, favor excited state proton transfer between these two groups, one of which (the N-H group) becomes strongly acidic in the excited state and the other strongly basic. This was suggested as being responsible for the 510 nm fluorescence emission of FMK detected by decreasing the solvent’s polarity. The lack of such emission in the N-CH3 derivative supports this hypothesis (53, 54). Taken together, these considerations can well explain the red-shifted fluorescence obtained during WOOH conversion to FMK. In summary, we have shown that 1O2-mediated oxidation of W gives rise to a mixture of trans- and cis-hydroperoxides, which are fully characterized in this paper. Also, they were shown to be relatively stable at ambient and physiological temperatures, leading to a slow decomposition to the corresponding alcohols. Increasing the pH or heating the solutions gives rise to a luminescent decomposition of the WOOH to FMK. Mechanistic aspects of WOOH decomposition were identified by different techniques, 18O-isotopic labeling studies coupled with HPLC/mass spectrometry analyses, and light emission measurements. The results are in agreement and are all consistent with a dioxetane pathway (Scheme 1, bold arrows). The relevance of this study is underscored by various pieces of evidence linking W-derived oxidation products with covalent aggregation in proteins. It has been proposed that the covalent attachment of the kynurenine-derived UV filter compounds may underlie the modifications of the crystalline lens and the development of cataracts (21, 22). It has also been shown that

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covalent aggregates formed in human superoxide dismutase are due to oxidative modification of W residues to kynurenine type products. It is believed that superoxide dismutase aggregation can be implicated in amyotrophic lateral sclerosis, a degenerative disease of motor neurons (23–25). In this context, this work may offer further insights into the potential involvement of W in oxidative reactions and in the development of these pathological conditions. Acknowledgment. This work was financially supported by FAPESPsFundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo, CNPqsConselho Nacional para o Desenvolvimento Cientı´fico e Tecnolo´gico, and CNPqsInstituto do Mileˆnio, Redoxoma. We are deeply indebted to Fernanda Manso Prado for her contribution to the mass spectrometry analyses. G.E.R. and M.C.B.O. are recipients of FAPESP fellowships.

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