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J. Phys. Chem. B 1999, 103, 8606-8611
OH Radical Induced One-Electron Oxidation of Serotonin and Tryptamine P. G. Hela,† N. R. Anipindi,† K. I. Priyadarsini,‡ and P. O’Neill*,§ School of Chemistry, Andhra UniVersity, Visakhapatnam-530003, India, Radiation Chemistry and Chemical Dynamics DiVision, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India, and MRC Radiation & Genome Stability Unit, Harwell, Didcot, Oxfordshire OX11 0RD, U.K. ReceiVed: April 19, 1999; In Final Form: August 16, 1999
The interactions of the one-electron oxidants, N3• and Br2•- radicals, with serotonin, tryptamine and their analogues 5-methoxytryptamine, N-methyltryptamine and N(1)-methyltryptophan were studied using the technique of pulse radiolysis with spectrophotometric detection. One-electron oxidation of serotonin results in the formation of an indoloxyl radical with a pKa value ,3. With tryptamine, N-methyltryptamine and 5-methoxytryptamine, their one-electron oxidation gives indolyl radicals with pKa values of 4.2, 4.3 and 4.8, respectively. The reactions of OH radicals (•OH) with serotonin and tryptamine lead to the formation of the respective •OH-adducts, which decay by acid catalyzed water elimination to give almost quantitatively the corresponding indoloxyl and indolyl radicals, respectively. The first-order rate constants determined for water elimination are pH dependent, suggesting that the dehydration reaction is acid and base catalyzed. The •OHadduct of serotonin reacts with oxygen in competition with the dehydration reaction to yield a peroxyl radical adduct, which is tentatively suggested to eliminate HO2•. On the basis of the above findings, the mechanisms for the •OH -induced formation of the indoloxyl and indolyl radicals from serotonin and tryptamine respectively are proposed.
Introduction 5-Hydroxytryptamine or serotonin is a well-known neurotransmitter produced by the metabolism of the aromatic amino acid, tryptophan through the intermediate, 5-hydroxytryptophan.1,2 In the central nervous system, oxidizing free radicals such as hydroxyl radicals (•OH) are produced during the biochemical processes. Reactions of oxidizing free radicals with serotonin, its precursors, and its metabolites are implicated in many neuronal disorders. Electrochemical oxidation of several substituted indoleamines has been studied by Dryhurst et al.3-5 The precursors formed under various biologically relevant conditions have been studied.6-17 However, there are only a few reports on the actual mechanism of oxidation of indoleamines.18-25 Pulse radiolysis offers a convenient technique to study short-lived radicals generated by one-electron oxidation of substituted indoles. Using pulse radiolytically generated azide radicals as the free radical oxidant, one-electron oxidation of several hydroxy- and methoxy-substituted indoles produces indoloxyl radicals and indolyl radicals, respectively.18-25 Oneelectron oxidation of hydroxylated indoles results in deprotonation of the radical cation at the hydroxy-group, as also shown with serotonin.19,26 Photo-oxidation27 and one-electron oxidation28 produce, by one-electron transfer and deprotonation at N(1),29 the indolyl radicals of tryptophan and tryptamine with pKa values of 4.3-4.527,30,31 and 4.2-4.3,28 respectively. The spectral, redox, and acid-base properties of the radicals produced on one-electron oxidation of a series of substituted indoles were reported.19,33 The interaction of the •OH with a series of methyl substituted indoles yields hydroxyl radical * Corresponding author. E-mail:
[email protected]. † Andhra University. ‡ Bhabha Atomic Research Centre. § MRC Radiation & Genome Stability Unit.
adducts, 44-68% of which dehydrate to produce the corresponding indolyl radicals.32 In the present study, the one-electron oxidation of serotonin, tryptamine, 5-methoxytryptamine, N-methyltryptamine, and N(1)-methyltryptophan has been studied to help in the elucidation of the reaction mechanisms of hydroxyl radicals with tryptamine and serotonin under various pH conditions.
Experimental Section Chemicals. Serotonin (creatinine sulfate complex), 5-methoxytryptamine, N-methyltryptamine, tryptamine base, and N(1)methyl-DL-tryptophan were obtained from Sigma Aldrich. All the chemicals were used as supplied. The solutions were prepared in phosphate buffer (5 mmol dm-3) using water purified by a Milli-Q system (Millipore). AR grade HClO4 or NaOH were used to adjust the pH of the solutions to acidic or alkaline conditions, respectively. Solutions were purged for >20 min with nitrous oxide (BOC special gases, zero grade), oxygen
10.1021/jp991260+ CCC: $18.00 © 1999 American Chemical Society Published on Web 09/23/1999
OH Radical Induced Oxidation Serotonin and Tryptamine
J. Phys. Chem. B, Vol. 103, No. 40, 1999 8607
(BOC), or mixtures of N2O/O2 prior to irradiation. A concentration of 0.1 mmol dm-3 indoleamines was used for determination of optical absorption spectra, whereas concentrations of 5-50 µmol dm-3 were used for kinetic and pKa measurements. For determination of the pKa values of the radicals obtained from one-electron oxidation of indoleamines, Br2•-was employed since it does not have a pKa in the pH range 3.0-10.0. Pulse Radiolysis. Pulse radiolysis measurements were carried out with a 4.3 MeV Mullard Linear Accelerator, SL46 at the Radiation and Genome Stability Unit, Harwell, U.K. as described previously.34 The doses/pulse used for determination of the optical absorption spectra were 10.0-12.0 Gy/pulse. The kinetic and radical pKa measurements were made using a dose/ pulse of 1.5-2.0 Gy, to minimize the competitive radicalradical interactions which would lead to loss of dibromide radical anion/N3• or the indole radicals. The photomultiplier signals were stored using a Datalab transient digitizer (model DL 905) interfaced with a PC 386/20 (Tandon) which utilizes a customized version of ASYST Software (Technologies Inc.) to process the data obtained. Air-saturated, aqueous solutions of 10 mmol dm-3 KSCN were used as dosimeter.35 The value for G(SCN)2•- of 2.6 × 104 m2 J-1 determined at 475 nm was used. The G value is the radiation chemical yield in µmol/J and , the extinction coefficient, expressed as mol-1 dm3 cm-1. Radiolysis of aqueous solutions produces three primary radicals, eaq- (G ) 0.28), •OH (G ) 0.28), and H atoms (G ) 0.06). The solutions were saturated with nitrous oxide to convert hydrated electrons into •OH (eq 1).
N2O + eaq- + H2O f •OH + OH- + N2
(1)
The one-electron oxidants N3• and Br2•- were generated as shown in the following reactions:
OH• + N3- f OH- + N3•
(2)
OH• + Br- f OH- + Br•
(3)
•
-
Br + Br f Br2
•-
(4)
Results One-Electron Oxidation of Substituted Indoles. The reactions of N3• and Br2•- with serotonin, tryptamine, and their analogues 5-methoxytryptamine, N-methyltryptamine, and N(1)methyltryptophan were studied in the pH range 3.0-10.0, to provide benchmark spectra to assist in the characterization of the species produced from the reaction of the •OH with serotonin and tryptamine. The optical absorption spectra of the transients obtained on pulse radiolysis of nitrous oxide saturated aqueous solutions containing 0.1 mmol dm-3 5-methoxytryptamine, N-methyltryptamine, N(1)-methyltryptophan, or tryptamine and 0.1 mol dm-3 NaN3 or KBr at pH 7.0 and 3.0 are shown in Figure 1 and Figure 1S of the Supporting Information, respectively. The optical absorption spectra at pH 7 are assigned to the formation of indolyl radicals of these substituted indoles. The resulting indolyl radicals exhibit pronounced absorbance in the 300600 nm range as already reported for methylindoles,29,31 methoxyindoles,18 tryptophan,29 and dimethoxyindoles.18 The optical absorption spectra of the one-electron oxidized species from tryptamine and N-methyltryptamine are similar. With the exception of N(1)-methyltryptophan, the optical absorption spectra, produced on one-electron oxidation of these indoles, are significantly different at pH 3, consistent with formation of
Figure 1. Difference absorption spectrum of the transient obtained 20 µs after pulse radiolysis of nitrous oxide saturated, aqueous solutions containing 0.1 mmol dm-3 indoleamines and 0.1 mol dm-3 NaN3 at pH 7.0: 5-methoxytryptamine (b), tryptamine (2), N(1)-methyltryptophan (3) and N-methyltryptamine (]). The absorbed dose was 1012 Gy.
indolyl radicals, which undergo prototropic equilibrium at N(1). From the pH dependence of the optical absorbance, the pKa values for the indolyl radicals of tryptamine, N-methyltryptamine, and 5-methoxytryptamine were determined to be 4.2, 4.3, and 4.8, respectively. The value with tryptamine is consistent with reported values.33 At pH 7, the nature of the spectra does not change in the presence of oxygen, inferring that the one-electron oxidized species of these indoles do not interact with oxygen at these early times. With N(1)-methyltryptophan, methyl substitution at N(1) prevents deprotonation of the radical cation formed, consistent with the optical absorption spectrum of the one-electron oxidized species of N(1)-methyltryptophan being independent of pH (3-10). The optical absorption spectrum (Figure 2S of the Supporting Information) of the one-electron oxidized species of serotonin is also independent of pH (3-10). However, in this case, the spectrum, which has a λmax at 420 nm, is attributed to formation of the indoloxyl radical, reflecting deprotonation at the C(5) hydroxy group of serotonin. This assignment is supported by the differences seen when comparing this spectrum with that for the one-electron oxidized radical of 5-methoxytryptamine. The effect of oxygen on the one-electron oxidized transient species of serotonin was assessed by pulse irradiation of N2O/ O2 (4:1 v/v) saturated aqueous solutions containing 0.1 mmol dm-3 serotonin and 0.1 mol dm-3 NaN3 at pH 7.0. The nature of the spectrum did not change, inferring that the one-electron oxidized species from serotonin does not interact with oxygen. Hydroxyl Radical Interactions with Serotonin and Tryptamine. The hydroxyl radical reactions were studied with serotonin and tryptamine only. The time-resolved optical absorption spectra of the intermediates produced by •OH interactions with serotonin were measured by pulse irradiation
8608 J. Phys. Chem. B, Vol. 103, No. 40, 1999
Figure 2. Time-resolved transient optical absorption spectra obtained 30 (0) and 140 µs (O) after pulse irradiation of a nitrous oxide saturated, aqueous solution containing 0.1 mmol dm-3 serotonin at pH 7.0 at a dose/pulse of 11.4 Gy. Inset: effect of pH on the rate of formation of the one-electron oxidized radical from serotonin-•OH adduct at 420 nm determined using a dose of 2.4 Gy/pulse.
of nitrous oxide saturated, aqueous solutions containing 0.1 mmol dm-3 serotonin at a dose per pulse of 11.4 Gy. The optical absorption spectra at pH 7, determined 30 µs and 140 µs after the pulse, are shown in Figure 2. The optical absorption spectrum at 30 µs changes with time, and at 140 µs an optical absorption spectrum was observed with a maximum centered at 420 nm. This latter spectrum is similar to that of the indoloxyl radical of serotonin as shown in Figure 2S of the Supporting Information. It is therefore inferred that some of the •OH adducts of serotonin initially formed are converted into the indoloxyl radical of serotonin. The pH dependence for the rate of formation of the indoloxyl radical from serotonin, determined at 420 nm, is shown in the inset of Figure 2. The first-order rate constants for formation of the indoloxyl radical decrease with increase in pH from 3.0 to 7.0. At pH > 7.0, the rate constant for formation of the indoloxyl radical increases again on increasing the pH. These changes are consistent with an acid/ base-catalyzed reaction. From comparison of the yield of the indoloxyl radical produced from the •OH interaction with serotonin at pH 3.0 with that determined on its reaction with the Br2- radical, >90% of the •OH adducts of serotonin are converted by acid catalysis into the indoloxyl radical. The time-resolved optical absorption spectra for the reaction of the •OH with tryptamine measured 35 and 150 µs after pulse irradiation of nitrous oxide saturated, aqueous solution containing 0.1 mmol dm-3 tryptamine at pH 7.0 are shown in Figure 3. The initially formed species, which absorbs at 90% of the •OH adducts of tryptamine are converted by acid and base catalysis into indolyl radicals. To investigate whether oxygen reacts with the •OH adducts of tryptamine and serotonin, pulse radiolysis of oxygenated solutions containing 0.1 mmol dm-3 serotonin or tryptamine at pH 7.0 was carried out using a dose per pulse of 9-10 Gy. With serotonin as shown in Figure 4a, the intensity of the absorbance at 420 nm (normalized for dose/pulse) is less and the peak is shifted to around 460 nm. In O2 saturated solution, the hydrated electrons react with O2 to give O2•-. The solutions containing serotonin were also saturated with N2O/O2 mixtures (N2O (5.7-17.3 mmol dm-3), O2 (0.96-0.32 mmol dm-3)) to minimize the formation of O2•- directly from the hydrated electrons. The optical absorption spectrum of the serotonin species formed is similar to that shown in Figure 4a, indicating that the species observed is not produced in reactions involving O2•-. The rate constant for formation of the serotonin radical was determined to be (8 ( 1) × 104 s-1 and is essentially independent of the concentration of O2 (0.32-0.96 mmol dm-3). This rate constant is significantly larger than that for formation of the indoloxyl radical, shown in the inset of Figure 2. The rate constant for interaction of O2 with the •OH adducts of serotonin could not be determined accurately but was estimated
OH Radical Induced Oxidation Serotonin and Tryptamine
Figure 4. Difference optical absorption spectrum of the transient formed, 200 µs after the pulse, in the pulse radiolysis of oxygen saturated aqueous solutions containing 0.1 mmol dm-3 serotonin/ tryptamine at pH 7.0, using a dose per pulse of 9.5-10 Gy: (a) serotonin (9); (b) tryptamine (b).
to be ∼109 dm3 mol-1 s-1 from the small optical absorbance changes at 360 nm. With tryptamine, the •OH adduct interacts with O2 in competition with the dehydration reaction of the •OH adducts, since the radical observed (Figure 4b) has the same spectrum as that formed from dehydration of the •OH adducts of tryptamine (Figure 2). The yield of the tryptamine radical determined at 500 nm is 50-60% of that determined in the absence of O2 (N2O saturated), after normalization for the different yields of •OH. Since the rate of dehydration of the •OH adducts of tryptamine at pH 7 (Figure 3, inset) is 105 s-1, it was not possible to monitor accurately the dependence for the rate of loss of the •OH adducts on the concentration of O2. Discussion In the present study, N3• and Br2•- are suggested to oxidize the indoleamine analogues by one-electron transfer giving their corresponding one-electron oxidized species, as shown in Scheme 1. Depending on the substituent, the radical cations of the indoleamine analogues formed may deprotonate at N(1) or at the hydroxy group (where RdOH). With serotonin, it is suggested that one-electron oxidation of serotonin results in deprotonation at the hydroxy group to produce an indoloxyl radical with a pKa , 3. This assignment is also consistent with the formation of an indoloxyl radical, since its optical absorption spectrum is similar to that previously assigned to the indoloxyl radical of 5-hydroxyindole.21 This deprotonation process is consistent with the low pKa values known for phenoxyl radical.36 With 5-methoxytryptamine, where deprotonation of the radical cation at the C(5) position is blocked by the methyl group, it is suggested that an indolyl radical is formed with a pKa of 4.8. The radical cation deprotonates at N(1) of the indole ring, as shown in Scheme 1. The pKa value determined for the indolyl
J. Phys. Chem. B, Vol. 103, No. 40, 1999 8609 radical of 5-methoxytryptamine is less than the value of 6.1 previously reported.33 The mechanism of one-electron oxidation of tryptamine is similar to that for one-electron oxidation of 5-methoxytryptamine, as shown in Scheme 1. The low pKa value of the tryptamine radical (4.2) compared with that of the one-electron oxidized radical from 5-methoxytryptamine (4.8) is consistent with the electron donation effect of the methoxy group. The decay of the indolyl radical of tryptamine is not affected by the presence of oxygen. The absence of an effect of oxygen on the lifetime of the indoloxyl radical of serotonin is consistent with the lack of interaction of the N-centered radical of N-acetyltryptophan methyl ester with oxygen.37 Verification that the deprotonation site of the radical cation of tryptamine and 5-methoxytryptamine occurs at N(1) of the indole ring was obtained through one-electron oxidation of N(1)-methyltryptophan. The optical absorption spectra of the radical cations of tryptamine and of N(1)-methyltryptophan are similar, whereas when the pH is much greater than the pKa of the one-electron oxidized radical of tryptamine, the spectra are different, since methyl substitution at N(1) prevents deprotonation of the radical cation of N(1)-methyltryptophan. Therefore, deprotonation of the radical cation of tryptamine occurs at N(1) of the indole ring (Scheme 1). Methylation of the amino group of the chain, e.g., N-methyltryptamine, does not influence the one-electron oxidation chemistry of the indoleamines. The interaction of the •OH with serotonin and tryptamine produces •OH adducts which absorb at 360 and 410 nm, respectively. Since the •OH is electrophilic in nature, it interacts with serotonin and tryptamine by addition to the indole ring at electron-rich centers. These initial optical absorption spectra of the •OH adducts of serotonin and tryptamine are different from those assigned to their respective one-electron oxidized radicals. The •OH adducts of serotonin and tryptamine are subsequently converted into new species which have optical absorption spectra similar to those of the corresponding one-electron oxidized species of serotonin and tryptamine. With serotonin, the optical absorption spectrum centered around 420 nm is attributed to the indoloxyl radical. With tryptamine, the optical absorption spectrum centered around 520 nm is characteristic of the indolyl radical. It is proposed that the •OH adducts of serotonin and tryptamine undergo dehydration reactions and not redox interactions with the respective indoleamines, since the rate of transformation into the indoloxyl radical or indolyl radical is independent of the concentration of the respective indoleamines. From the pH dependences of the rate constants for transformation of the •OH adducts of these indoleamines, it is inferred that the dehydration reaction is acid catalyzed with tryptamine and acid and base catalyzed in the case of serotonin. As the rate of dehydration increases, the yield of indoloxyl radical increases and accounts quantitatively for the yield of •OH adducts of serotonin at pH 3. At the dose per pulse used, the dehydration reaction competes favorably with the loss of •OH adducts through radical-radical interactions. Since the decay of the •OH adduct of tryptamine at 410 nm is independent of the concentration of the substrate, it is inferred that water is eliminated from the •OH adduct of tryptamine. The reaction mechanisms for the interaction of both serotonin and tryptamine with •OH are shown in Scheme 2. From the interaction of oxygen with •OH adducts of serotonin, it is inferred that this interaction competes with dehydration of the •OH adducts of serotonin to produce a new transient species (Scheme 2). It is proposed that the •OH adducts of serotonin interact initially with O2 to produce a peroxyl radical adduct,
8610 J. Phys. Chem. B, Vol. 103, No. 40, 1999
Hela et al.
SCHEME 1: One-Electron Oxidation of Indoleamines by the Azide Radical/Br2•-
SCHEME 2: Reaction Mechanism for the Interaction of the •OH with (a) Serotonin and (b) Tryptamine in the Presence and Absence of O2
as shown in Scheme 2. The rate constant for the interaction of the •OH adducts with O2 is estimated to be greater than that for the interaction of O2 with the •OH adducts of anisole.38 It has previously been shown that the formation of peroxyl radical adducts from the •OH adducts of substituted benzenes is reversible, occurring with rate constants in the range of (1-7) × 104 s-1 for the release of O2.38-40 Since the interaction of O2 with the •OH adduct of serotonin produces a species (Figure 4a), which has a different optical absorption spectrum from that of the indoloxyl radical and at a rate that is independent of the concentration of O2, it is inferred that release of O2 from the peroxyl radical is not significant. It is tentatively suggested that the peroxyl radical, see Scheme 2, preferentially eliminates
HO2•. The elimination of HO2• occurs with a rate constant that is >10 times that for elimination of HO2• from the corresponding peroxyl radical of anisole.38 With tryptamine, it is suggested that the •OH adducts of tryptamine in the presence of O2 dehydrate to give the indolyl radical in competition with O2 addition, since the yield of the indolyl radical is significantly less than that in the absence of O2. It was not possible to confirm if the resulting peroxyl radicals, produced on interaction of the •OH adduct of tryptamine with O , eliminate O , due to the fast 2 2 dehydration reaction of the •OH adducts and the possible interference through the interaction of O2•- with the indolyl radical. An addition reaction of O2•- with tyrosine-derived phenoxyl radicals has previously been reported.40 As already
OH Radical Induced Oxidation Serotonin and Tryptamine discussed above, the indoloxyl radical of serotonin and the indolyl radical of tryptamine do not however interact with oxygen. In summary, the •OH adducts of serotonin and tryptamine decay by an acid catalyzed reaction to give quantitatively the respective indoloxyl and indolyl radicals. The •OH adducts of tryptamine and serotonin interact with O2 in competition with the dehydration reaction. The reactions of the indoleamine radicals, if produced in vivo, may therefore depend critically on the oxygen tension. Supporting Information Available: Optical absorption spectra of one-electron oxidized species of indoleamines (Figure 1S) and serotonin (Figure 2S). This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Novonta, R. Physiol. BohemosloV. 1980, 29, 243. (2) Wrona, M. Z.; Yang, Z.; Waskiewicz; Dryhurst, G. NeurodegenerratiVe diseases; Fiskum, G., Ed.; Plenum Press: New York, 1966; p 285. (3) Wrona, M. Z.; Dryhurst, G. J. Org. Chem. 1987, 52, 2817. (4) Wrona, M. Z.; Dryhurst, G. J. Org. Chem. 1989, 54, 2718. (5) Wrona, M. Z.; Dryhurst, G. Bioorg. Chem. 1990, 18, 219. (6) Wrona, M. Z.; Yang, Z.; McAdams, M.; O’Connor-Coates, S.; Dryhurst, G. J. Neurochem. 1995, 64, 1390. (7) Wong, K. S.; Goyal, R. N.; Wrona, M. Z.; LeRoy, B. C.; Dryhurst, G. Biochem. Pharmacol. 1993, 46 (9), 1637. (8) Wrona, M. Z.; Dryhurst, G. J. Pharm. Sci. 1988, 77 (11), 911. (9) Bjorklund, A.; Nobin, A.; Stenevi, U. Brain Res. 1973, 53, 117. (10) Bjorklund, A.; Nobin, A.; Stenevi, U. Z. Zellforsch. 1973, 145, 479. (11) Saner, A.; Pieri, L.; Moran, J.; DaPradam, M.; Pletscher, A. Brain Res. 1974, 76, 109. (12) Baumgarten, H. G.; Lachenmeyer, L. Z. Zellforsch. 1972, 135, 399. (13) Baumgarten, H. G.; Goethert, M.; Holstein, A. F.; Schlossberger, H. G. Z. Zellforsch 1972, 128, 115. (14) Baumgarten, H. G.; Bjorklund, A.; Lachenmeyer, L.; Nobin, A.; Stenevi, U. Acta Physiol. Scad. Suppl. 1971, 373, 1. (15) Baumgarten, H. G.; Evetts, K. D.; Holman, R. B.; Iversen, L. L.; Wilson, G. J. Neurochem. 1972, 19, 1587. (16) Wrona, M. Z.; Goyal, R. N.; Turk, D. J.; Blank, C. L.; Dryhurst, G. J. Neurochem. 1992, 9, 1392.
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