Chem. Res. Toxicol. 1994, 7, 185-190
Selective Formation of Oxindole- and Formylkynurenine-Type Products from Tryptophan and Its Peptides Treated with a Superoxide-Generating System in the Presence of Iron(II1)-EDTA: A Possible Involvement with Iron-Oxygen Complex Koichi Itakura, Koji Uchida, and Shunro KawakishP Laboratory of Food and Biodynamics, Department of Applied Biological Sciences, Nagoya University, Nagoya 464-01, Japan Received October 8,1993"
The oxygenation of tryptophan and its peptides by the superoxide-generating system hypoxanthine/xanthine oxidase in the presence of iron(II1) and ethylenediaminetetraacetic acid (EDTA) has been investigated. The reaction of a tryptophan derivative, N-(tertbutoxycarbony1)-L-tryptophan,with hypoxanthine/xanthine oxidase/Fe(III)-EDTA mainly resulted in the oxygenation of the pyrrole ring of the indole nucleus. 2- [(tert-Butoxycarbony1)amino] -3-(3-oxindoly1)propionic acid and N- (tert-butoxycarbony1)-N'-formylkynureninewere identified as the major products. Similar oxindole- and formylkynurenine-type products were also obtained from the N-(tert-butoxycarbonyl) derivative of the tryptophan-containing peptides Ile-Trp, Trp-Leu, Gly-Trp-Leu, and Ala-Trp-Ile. In all cases, however, hydroxylation products of the benzene ring of the indole nucleus were scarcely detected, leading to the assumption that free hydroxyl radical did not play a role in the tryptophan oxidation of this system. Of interest with H202/horseradish was the fact that the reaction of N-(tert-butoxycarbony1)-L-tryptophan peroxidase mainly afforded the same oxindole- and formylkynurenine-type products as those obtained in the hypoxanthine/xanthine oxidaselFe(II1)-EDTA system. Taken together, ironoxygen complex-type active species may play a role in the tryptophan oxygenation in a superoxidegenerating system in the presence of iron-EDTA.
Introduction The one-electron reduction of dioxygen by enzymatic and nonenzymatic reactions universallyoccurs in biological systems to form superoxide anion (02-1. 0 2 - is one of the active oxygen species and plays important roles in various diseases caused by oxygen toxicity (1,2). Although it has been reported that 0 2 - is very toxic to living cells (3,4),the reactivity of 02-itself is not as vigorous in aqueous media as those of other active oxygen species such as singlet oxygen (l02), hydroxyl radical ('OH), and hydrogen peroxide (H2Oz). This is because, in aqueous media, 0 2 - is unstable and rapidly dismutates to H202 and 0 2 (5). However, 0 2 - can act as a suitable reducing agent for transition metal ions such as iron and copper in aqueous media. It has been reported that 0 2 - from stimulated human neutrophils causes the reduction and release of iron from transferrin (6). This iron release may be an important aspect of ischemia-reperfusion injury because the burst production of 0 2 - and H202 takes place during reperfusion (7). Furthermore, Chevion et al. (8) have recently reported that iron is mobilized following myocardial ischemia. Iron released presumably chelates to cellular constituents such as citrate, ATP, GTP, and other phosphate esters. In the presence of such chelatable iron, 0 2 - can cause serious damage to proteins. For example, Girotti et al. (9) have reported that isolated erythrocyte membranes exposed to xanthine oxidase (XO) plus xanthine and ferric ion undergo protein cross-linking.
* To whom correspondence should be addressed. 0
Abstract published in Aduance ACS Abstracts, February 1, 1994.
Stadtman et al. (10)have reported that glutamine synthetase is inactivated by hypoxanthine (Hyp)/XO/FeCls. Among the amino acid residues of proteins, tryptophan is well-known to be a preferred target of active oxygen I 0 2 (13,14), *OH(15-17),and species such as 0 2 - (11,12), H202 (18). With respect to the superoxide-generating system in the presence of iron, McCord et al. (19) have reported that a decrease in tryptophan absorbance was observed in xanthine/XO/iron-EDTA; however, the oxidation product of tryptophan residues in proteins remained to be identified. In the present study, we found that tryptophan and its peptides were selectively oxidized at the pyrrole ring of the indole nucleus when they were treated with a superoxide-generating system in the presence of iron(III), resulting in the generation of oxindole- and formylkynurenine-type products. We selected EDTA as a model of physiological chelators. EDTA has been most widely used as a chelator of iron. Iron-EDTA reacts rapidly with 0 2 and H202 (20).As far as we know, this is the first paper which addresses the chemistry of selective tryptophan oxygenation at the pyrrole ring of the indole nucleus by a superoxide-generating system.
Experimental Procedures Hypoxanthine, iron(II1) chloride, EDTA, dimethyl sulfoxide, and mannitol were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Superoxide dismutase (from bovine erythrocytes, EC 1.15.1.1), catalase (from bovine liver, EC 1.11.1.6), and horseradish peroxidase (type VI, EC 1.11.1.7) were purchased from Sigma Chemical Co. (St. Louis, MO).
0893-228~/94/2707-0185$04.50/0 0 1994 American Chemical Society
Itakura et al.
186 Chem. Res. Toxicol., Vol. 7,No. 2, 1994 H B~-X-N+
o
8: Y=OH
8: X d e I O : Y=Leu 12: X=Gly, Y r b u 14: X=Ala, Ydle
H
o
B ~ C - X - N ~ Y
7: Y=OH 9: X=lle 11: Y=Leu 13: X=Gly, Y=Leu 15: X=Ala, Y=lle
Figure 1. Structures of oxidation products derived from N-tBoc-L-tryptophan (1) and tryptophan-containing peptides (25). Xanthine oxidase (from cow milk, EC 1.1.3.22) was obtained from Boehringer Mannheim Corp. (Indianapolis,IN). All other chemicals were of the highest grade commercially available and were used without further purification. N-E-Boc-isoleucyltryptophanwas prepared from N-t-Boc-Lisoleucine and L-tryptophan benzyl ester hydrochlorideby using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride according to the method of Sheehan et al. (21),followed by catalytic hydrogenationwith palladium on activated carbon, and N-t-Boc-tryptophylleucinewas prepared from N-t-Boc-L-tryptophan and L-leucine bemyl ester p-toluenesulfonate salt by the same method. N-t-Boc-glycyltryptophylleucinewas prepared from N-t-Boc-glycine N-hydroxysuccininiideester and L-tryptophyl-L-leucineaccordingto the method of Anderson et al. (221, and N-t-Boc-alanyltryptophylisoleucine was prepared from N-tBoc-L-alanine N-hydrosysuccinimideester and L-tryptophyl-Lisoleucine by the same method. N-t-Boc-L-isoleucine,N-t-Boc-L-tryptophan,N-t-Boc-glycine, and N-hydrosysuccinimide were purchased from Wako Pure Chemical Industries Ltd. L-Tryptophan benzyl ester hydrochloride, leucine benzyl ester p-toluenesulfonate salt, N-t-BocL-alanine N-hydrosysuccinimide ester, and L-tryptophyl-Lleucine were obtained from Sigma Chemical Co. L-TryptophylL-isoleucine was purchased from Funakoshi (Tokyo, Japan). l-Ethyl-3-[3-(dimethylamino)propyllcar~iimide hydrochloride was obtained from Dojin Kagaku Kenkyujo (Kumamoto,Japan). Ultraviolet spectra were recorded on a Hitachi 200-0110 spectrometer. Fast atom bombardment mass spectra were obtained with a JEOL JMS-DX 705 mass spectrometer with glycerol as a matrix. 'H nuclear magnetic resonance spectra were obtained with a JEOL EX-270 spectrometer with tetramethylsilane as an internal standard. High-performance liquid chromatographywas performed with TOYO SODA high-speedliquid chromatograph (HLC-803D)equipped with a TOYO SODA UV8000. Areas of the chromatographicpeaks of each material were calculatedby use of a Shimadm Chromatopac C-R6A integrator. Amino acid analysis was done with a JEOL JLC-300 amino acid analyzer. Hydrolysates were dissolved in citrate buffer (pH 2.2) and then submitted for amino acid analyses. Thin-layer chromatography was carried out on Merck silica gel F m precoated plates. Silica gel BW-300 for column chromatography was purchased from Fuji Davison (Nagoya, Japan). Analyses of the Oxidation Products of N-t-Boc-L-tryptophan (1) and Tryptophan-Containing Peptides, Boc-IleTrp (2), Boc-Trp-Leu (3), Boc-Gly-Trp-Leu (4), and BocAla-Trp-Ile (5), by the Hyp/XO/Fe(III)-EDTA System. Each reaction mixture (total volume 1.05 mL) contained a substrate (2.5 mM), Hyp (2.5 mM), XO (0.1 unit/mL), FeCls (0.25mM), and EDTA (0.26 mM) in 0.1 M phosphate buffer (pH 7.4). After 0.5, 1,2, and 4 h, 80 pL of the reaction mixture was directlysubjected to HPLC analysis. HPLC analysiswas carried out on a DevelceilODs-7column (8X 250") (NomuraChemical Co., Ltd.), at a flow rate of 2.5 mL/min (monitored by 254 nm). Oxidation products are summarized in Figure 1. The concentrations of oxidation products were determined by comparison
of HPLC peak areas with those of the authentic 6 and 7. The product yields (%) were calculated on the basis of the consumed substrates. Common Procedure for the Isolation of Oxidation Products of 1-6 by the Hyp/XO/Fe(III)-EDTA Syetem. Each reaction mixture (total volume 50 mL) contained a substrate (2.5 mM), Hyp (2.5 mM), XO (0.1 unit/mL), FeCh (0.25 mM), and EDTA (0.26 mM) in 0.1 M phosphate buffer (pH 7.4). After incubation at room temperature under air for 4 h, the reaction mixture was directly applied on a column of Amberlite XAD-2 The (Organ0 Co., Ltd.) equilibrated with 1% CHSCO~H-H~O. products were eluted successively by 100 mL of 99:l H&-CHgCOzH, 2079:l CHSOH-H~OCH~CO~H, 4059~1CHaOH-HaOCHgC02H, 60391 CHgOH-H2OCH&02H, 80:191 CHgOHHzMH~COZH and , 991 CHSOH-CHSCOZH.HPLC purification was carried out on a Develosil ODs-7 column (8 X 250 mm), at a flow rate of 2.5 mL/min (monitored by 254 nm). Isolation of 6 and 7. The fractions eluted with 80% CHsOH-H20 and 80% C&OH-H20 were combined and concentrated to dryness. The residue was purified by HPLC, eluting with 5050:l CHSOH-H~O-CH~CO~H. The HPLC fractions eluted at 12.1and 16.8min were collected and concentrated to provide the diastereomersof 6. 6 was dissolved in CHsOH, and diazomet.be in ether was added. The resulting solution was concentrated to dryness, and the residue was purifiedby silicagel TLC, developing with 1:l ethyl acetate-hexane. Elution with ethyl acetate provided the methyl ester of 6. The HPLC fraction eluted at 14.0 min was collected and concentrated to provide the crude 7. Since 7 gradually decomposed during the HPLC purification, it was converted into a methyl ester to purify it under neutral conditions. The residue was dissolved in CHgOH, and dimomethane in ether was added. The resulting solution was concentrated to dryness, and the residue was purified by silica gel TLC, developing with 2 5 ethyl acetatehesane. Elution with ethyl acetate provided the crude methyl ester of 7. The methyl ester of 7 was further purified by silica gel TLC, developing with 2:98 CHSOH-CH2C12. Elution with ethyl acetate provided the methyl ester of 7 in a pure form. The methyl ester of 6: 'HNMR (CDCb) 6 1.57 (8, Boc), 2.20-2.51 (m, CHZCHNHBW), 3.48-3.61 (m, 3-H), 3.71 (8, COzCHs), 4.42-4.59 (m, CH~CHNHBOC), 4.61-4.78 (m, CH&HNHBoc), 5.43 (d, NHBoc, J = 8.1Hz), 5.65 (d, NHBoc, J = 8.1 Hz), 6.81-7.55 (m, aromatic protons and NH of osindole); UV (CHsOH),A, 250 and 280 nm; ELMS mlz 334 (M+),278,275,261,247,234,201,174,158,146, 132, 130, and 117. The methyl ester of 7 'H-NMR (CDCh) 6 1.45 (9H, s, Boc), 3.77 (3H, s, CO&Hg), 3.62-3.81 (2H, m, CH25.53 (lH, d, CHNHBoc), 4.64-4.76 (lH, m, CH~CHNHBOC), NHBoc, J = 8.0 Hz),7.19 (lH, t, J = 8.0 Hz), 7.60 (lH, t, J = 8.0 Hz), 7.92 (lH, d, J = 8.4 Hz), 8.49 (lH, 8, CHO), 8.77 (lH, 230, d, J 8.4 Hz), 11.40 (lH, 8, NHCHO); UV (CHgOH) 259, 266(sh), and 320 nm; ELMS mlz 350 (M+), 294,291, 277, 266, 235, 218, 205, 191, 174, 162, 148, 146, 133, and 120. Synthesis of the Methyl Ester of 6 (23,24). A solution of tert-butyl hypochlorite (180 pL, 1.60 mmol) in carbon tetrachloride (2.0 mL) was added dropwise over 35 min to a wellstirred solution of N-t-Boc-L-tryptophanmethyl ester (501 mg, 1.57 mmol) in methylene chloride (30 mL) containing triethylamine (880 rL, 6.35 "01). The resulting solution was stirred at -20 "C for 2 h and then allowed to warm up to room temperature. f i r standing overnight,it was washed twice with cold water, dried over anhydrous NaaO,, and concentrated to dryness. The residue was purified by silica gel column, eluting withmethylenechloride toaffordmethyl l-(tert-butoxycarbonyl)2,3-dihydropyrrolo[2,341indole-2-carboxylate(111mg, 22.3 % ). Methyl 1-(tert-buto~~bonyl)-2,3-dihydropyrrolo[2,3-~lindole2-carboxylate (78.0 mg, 0.25 "01) was treated with 20% CHaC02H-H20 (18.0 mL) at room temperature for 50 min. The product was purified by silica gel column, eluting with 4 5 ethyl acetate-hexane to afford the methyl ester of 6 (32.7 mg, 39.7 5%). Isolation of 8 and 9. The fractions eluted with 60% CHaOH-H20 and 80%CHsOH-H@ were combined and concentrated
Chem. Res. Toxicol., Vol. 7, No. 2, 1994 187
Superoxide-Mediated Oxygenation of Tryptophan Scheme 1 0
H
Boc-x-i y - Y
Boc-X-N &Y
9: 11: 13: 15:
16: 17: 10: 19:
X=iie Y=Leu X=Gly, Yoleu X=Ala, Y=ile
o
X=iie Y=Leu X=Giy, Y=bu X=Aia, Y=iie
I
0
to dryness. The residue was purified by HPLC, eluting with 5050:l CH~OH-H~O-CH~COZH. The HPLC fraction eluted at 18.9 min was collected and concentrated to provide 9. As shown in Scheme 1,9 was gradually hydrolyzed to form a kynureninetype compound (16) during purification; the identification of 9 was made from the UV, FAB-MS, and amino acid analysis of 16. 16 was separated by silica gel TLC, developing with 5:94:1 CH3OH-CH2C12-CH3C02H and eluting with 20:791 CH30H-CH2C12-CH3C02H4 The HPLC fraction eluted at 22.6 min was collected and concentrated to dryness. The residue was purified by silica gel TLC, developing with 5:941 CHSOH-CH~CI~-CH~C02H. Elution with 20:79:1CH30H-CH2C12-CH3C02H afforded 249 and 280 nm; the diastereomers of 8. 8: UV (CH30H) ,A, FAB-MS (+HCl) mlz 456 (M Na)+, 434 (M H)+, 378, and 334; amino acid analysis of the acid hydrolysate of 8 (6 N HCI, 105 OC, 24 h) revealed the presence of isoleucine. 1 6 UV (CH3OH) ,A, 227,257, and 363 nm; FAB-MS (+HCl) mlz 444 (M Na)+, 422 (M H)+, and 322; amino acid analysis of the acid hydrolysate of 16 (6 N HC1,105 OC, 24 h) revealed the presence of isoleucine and kynurenine. Isolation of 10 and 11. The fraction eluted with 80% CH3OH-H20 was concentrated. The residue was purified by HPLC, eluting with 55:45:1 CH30H-H20-CH&02H. The HPLC fractions eluted at 24.7,25.7,and 33.2 min were collected. To purify the products simultaneously, the three fractions were combined and concentrated. The residue was purified by silica gel TLC, developingwith 5:941 CHsOH-CH2C12-CH3C02H. Elution with 10:89:1 CH3OH-ethyl acetate-CHsCOzH provided 11 and the diastereomers of 10. As shown in Scheme 1, 11 was gradually hydrolyzed to form 17 during purification; the identification of 11 was made from the UV, FAB-MS, and amino acid analysis of 17. 17 was separated by HPLC, eluting with 55:45:1 CH30H250 and 279 nm; FABH20-CH3C02H. 10: UV (CH3OH) A,, MS m/z 456 (M + Na)+,434 (M + H)+,378, and 334; amino acid analysis of the acid hydrolysate of 10 (6 N HC1, 105 "C,24 h) revealed the presence of leucine. 17: UV (CH3OH) ,A, 228,256, and 363 nm; FAB-MS mlz 444 (M + Na)+,422 (M + H)+, and 322; amino acid analysis of the acid hydrolysate of 17 (6 N HCl, 105 "C, 24 h) revealed the presence of leucine and kynurenine. Isolation of 12 and 13. The fractions eluted with 60% CH3OH-HzO and 80 % CH30H-H20 were combined and concentrated to dryness. The residue was purified by HPLC, eluting with 55:45:1 CHsOH-H20-CHsC02H. The HPLC fractions eluted at 16.5, 17.2, and 21.2 min were collected and combined. The combined solution was concentrated to dryness, and the residue was purified by silica gel TLC, developing with 5:94:1 CHsOHCH2CI2-CHsC02H. Elution with 15:84:1CH3OH-ethyl acetateCHaCOzH provided 13 and the diastereomers of 12. As shown in Scheme 1, 13 was gradually hydrolyzed to form 18; the identification of 13 was made from the UV, FAB-MS, and amino acid analysis of 18. 18 was separated by HPLC, eluting with 45:55:1 CH~OH-H~O-CHSCO~H. 12: UV (CHsOH) ,A, 249 and 280 nm; FAB-MS mlz 513 (M + Na)+, 491 (M + H)+, and 435; amino acid analysis of the acid hydrolysate of 12 (6 N HCI, 105 "C, 24 h) revealed the presence of glycine and leucine. 18: UV 227.5,257, and 365 nm; FAB-MS mlz 501 (M + (CHsOH) A,, Na)+, 479 (M + H)+, and 423; amino acid analysis of the acid hydrolysate of 18 (6 N HC1,105 "C, 24 h) revealed the presence of glycine, leucine, and kynurenine.
+
+
+
+
1
10 20 30 Retention Time (min)
0
10 20 30 Retention Time (min)
Figure 2. HPLC profiles of (a) N-t-Boc-L-tryptophan (1) and (b)N-t-Boc-isoleucyltryptophan(2) treated with hypoxanthine/ xanthine oxidase / Fe(II1)-EDTA after 4 h. The reaction and HPLC analysiswere carried out as described under Experimental Procedures. Elution was with 5 0 5 0 1 CH30H-H204H&OaH. Isolation of 14 and 15. The fractions eluted with 60% CH3OH-H20 and 80%CHSOH-H~Owere combined and concentrated to dryness. The residue was purified by HPLC, eluting with 5545:l CH~OH-HZO-CH~CO~H. The HPLC fractions eluted at 19.4 and 25.1 min were collected and combined. The combined solution was concentrated, and the residue was purified by silica gel TLC, developing with 5:941 CH30H-CHzCl&HsCOzH. Elution with 1089:l CH3OH-ethyl acetate-CHsCO2H provided 15 and the diastereomers of 14. As shown in Scheme 1, 15 was gradually hydrolyzed to form 19 during purification; the identification of 15 was made from the UV, FAB-MS, and amino acid analysis of 19. 19 was separated by HPLC, eluting with 175 22-54 CH30H-H20-CH&02H. 14: UV (CHaOH) A, 250 and 280 nm; FAB-MS mlz 505 (M + H)+, 449, and 405; amino acid analysis of the acid hydrolysate of 14 (6 N HCl, 105 "C, 24 h) revealed the presence of alanine and isoleucine. 1 9 UV (CH3OH) ,A, 228,257, and 365 nm; FAB-MS mlz 493 (M + H)+,437, and 393; amino acid analysis of the acid hydrolysate of 19 (6 N HCl, 105 "C, 24 h) revealed the presence of alanine, isoleucine, and kynurenine. Reaction of N-t-Boc-L-tryptophan (1) with HzOz/Horseradishperoxidase. The reactionmixture (totalvolume 1.0 mL) contained 1 (2.5mM), HzO2 (2.5 mM), and horseradish peroxidase (2.0 pM) in 0.1 M phosphate buffer (pH 7.4). After incubation at room temperature under air for 4 h, 80 pL of the reaction mixture was directly subjected to HPLC analysis. Analysis was carried out on a Develosil ODS-7 column (8 X 250 mm), eluting with 5050:l CH30H-H20-CH&02H at a flow rate of 2.5 mL/ min (monitored by 254 nm). When the reaction was carried out under oxygen or argon, oxygen or argon gas was bubbled into the solution of 1 for 10 min before the reaction was started by the addition of Hz02 and horseradish peroxidase in a flask with a serum rubber stopper.
Results Oxidation of N - ( t e r t - B u t o x y c a r b o n y l ) ( B o c ) - T r p with Hyp/XO/Fe(III)-EDTA. When Boc-Trp (1) was treated with H y p (2.5 mM), XO (0.1 unit/mL), and Fe(111)-EDTA (0.25 mM) for 4 hat room temperature, 19.7 % of the starting material was consumed. Figure 2a demonstrates the H P L C profile of the reaction mixture after 4 h of incubation. Three major peaks (A-C) were detected and isolated by reverse-phase HPLC. When the A and C peaks were analyzed by HPLC again, both of them again provided a pair of peaks, A and C, with t h e same relative intensity as shown in Figure 2a. This reversible interconversion indicates that they are isomers. T h e structural identification of these isomers was performed bylH-NMR, UV, and mass spectrometries. The UV spectrum of the
188 Chem. Res. Toxicol., Vol. 7, No. 2, 1994
Table 1. Reaction of N-t-Boc-L-tryptophan and Tryptophan-Containing Peptides with Hypoxanthine/ Xanthine Oxidase/Fe(III)-EDTA* consumed oxindole- formylkynureninesubstrate type product type product substrate (%)* (%)C (%)C 7 (9.9) Boc-Trp (1) 19.7 6 (13.2) 9 (8.1) Boc-Ile-Trp (2) 19.3 8 (14.0) 20.5 10 (10.2) 11 (10.5) Boc-Trp-Leu (3) 13 (10.1) Boc-Gly-Trp-Leu (4) 20.1 12 (11.9) 20.2 14 (12.6) 15 (7.8) Boc-Ala-Trp-Ile (5) a Each of the reaction mixtures (1.05 mL) containing a substrate (2.5 mM), hypoxanthine (2.5 mM), xanthine oxidase (0.1 unit/mL), FeCla (0.25 mM), and EDTA (0.26 mM) in 0.1 M phosphate buffer (pH 7.4) was incubated at room temperature under air for 4 h. The consumed substrateswere determined by HPLC. The product yields (%) were calculated on the basis of the consumed substrates.
mixture of the isomers exhibited absorption maxima at 249 and 280 nm which are characteristic of the oxindole nucleus. The FAB-MS spectrum of the mixture provided a quasimolecular ion peak at mlz 321 (M + H)+,indicating the incorporation of one oxygen atom into 1. On the basis of this information, A and C were confirmed to be the diastereomers of 2-[(tert-butoxycarbonyl)aminol-3-(3oxindoly1)propionicacid (6) as shown in Figure 1. The 1H-NMR spectrum of the mixture was identical to the authentic sample independently prepared. The UV spectrum of the methyl ester of B exhibited absorption maxima at 261,268 (sh), and 322 nm, which are characteristic of formylkynurenine-type compounds. The EI-MS spectrum of the methyl ester of B gave a parent ion peak at mlz 350 (M+). From these spectral data, B was confirmed to be N-t-Boc-N'-formylkynurenine(7) as shown in Figure 1. The 'H-NMR spectrum of the methyl ester of B also supported this structure. The yields of 6 and 7 based on the consumption of 1 were 13.2% and 9.9%, respectively (Table 1). Thus, we found that the reaction of a Trp derivative with a Hyp/XO/Fe(III)-EDTA system was highly specific on the pyrrole ring of the indole nucleus, resulting in the generation of oxindole- and formylkynurenine-type products. Oxidation of Tryptophan-ContainingPeptideswith Hyp/XO/Fe(III)-EDTA. When Boc-Ile-Trp (2) was treated with Hyp/XO/Fe(III)-EDTA for 4 h, 19.3% of the starting material was consumed. Figure 2b shows the HPLC profile of the reaction mixture after 4 h of incubation. Two major peaks (Dand E) were detected and isolated by HPLC. When isolated E was analyzed further by silica gel TLC, two major spots were detected. They turned out to be intraconvertible isomers because both of them showed an original pair of two spots when each of them was again analyzed by silicagel TLC. From UV and FAB-MS spectra of a mixture of the two spots, they were confirmed to be the diastereomers of an oxindole-type compound (8) as illustrated in Figure 1. Amino acid analysis of the hydrolysate revealed the presence of isoleucine. On the other hand, D was confirmed to be a formylkynurenine-type compound (9) from the UV and FAB-MS spectra as illustrated in Figure 1. The spectral data were obtained using the deformylated product (16) (Scheme 1) since 9 was gradually deformylated during purification. Amino acid analysis of the acid hydrolysate of 16 revealed the presence of isoleucine and kynurenine, directly supporting the structures illustrated in Figure 1.
Itakura et al. Table 2. Effects of Inhibitors on the Oxidation of N-t-Boc-L-tryptophan (1) by Hypoxanthine/Xanthine Oxidase/Fe( 111)-EDTA* addition % inhibition none 0.0 SOD (500 units/mL) 80.7 59.4 catalase (500 units/mL) SOD (500 units/ 97.0 mL) + catalase (500 units/mL) DTPAb,l(0.25mM) 91.4 a Each of the reaction mixtures (1.05 mL) containing 1 (2.5 mM), hypoxanthine (2.5 mM), xanthine oxidase (0.1 unit/mL), FeCh (0.25 mM), and EDTA (0.26 mM) in 0.1 M phosphate buffer (pH 7.4) was incubated at room temperature under air for 4 h. The inhibitions were calculated on the basis of the residual substrates determined by HPLC. b In the absence of FeC13 and EDTA.
0
20
40
60
80
100
Mannltol (mM)
Figure 3. Effects of concentration of mannitol on the oxidation of N-t-Boc-L-tryptophan (1)by hypoxanthine/xanthine oxidase/ Fe(II1)-EDTA. 1 (2.5 mM) waa treated with hypoxanthine (2.5 mM), xanthine oxidase (0.1 unit/mL), FeC13 (0.25 mM), and EDTA (0.26 mM) in the presence of mannitol (0-100mM) in 0.1 M phosphate buffer (pH 7.4, 1.05 mL) at room temperature for 4 h.
Those oxindole- and formylkynurenine-type products were also formed in other tryptophan-containing peptides, i.e., Boc-Trp-Leu (3), Boc-Gly-Trp-Leu (4), and Boc-AlaTrp-Ile (51, exposed to the Hyp/XO/Fe(III)-EDTA system. Table 1 summarizes the consumption of peptides and the yield of products based on the peptides consumed. These results demonstrate that oxindole-and formylkynureninetype products were generally formed in tryptophan and its peptides exposed to the superoxide-generating system in the presence of Fe(II1)-EDTA. Effect of Inhibitors. In order to get insight into the active species which participated in the formation of the oxindole- and formylkynurenine-typecompounds, effects of several inhibitors were examined using 1as the substrate (Table 2). Superoxide dismutase (SOD) and catalase significantly suppressed the oxidation of 1. Furthermore, when both SOD and catalase were added to the reaction mixture, the oxidation was completely suppressed. The oxidation of 1 did not proceed in the absence of Fe(II1)EDTA. These results suggest that 0 2 - , HzO2, and Fe(II1) are essential for the oxidation of 1. Mannitol, which is an .OH scavenger, showed a poor inhibitory effect (Figure 31, indicating that .OH may not take part in the oxidation of the tryptophan derivative. Oxidation of Boc-Trpwith HzOz/Horseradish Peroxidase. As already demonstrated, the participation of *OHin the formation of oxindole- and formylkynureninetype products was precluded. Alternatively, an ironoxygen complex is a possible candidate for the oxidation of tryptophan and its peptides with the Hyp/XO/Fe(III)EDTA system. 1
Abbreviation: DTPA,diethylenetriaminepentaaceticacid.
Chem. Res. Toxicol., Vol. 7, No. 2, 1994 189
Superoxide-Mediated Oxygenation of Tryptophan BOC-TQ
20;
/I
E
C
H,O,
I
I
I
I
0
10
20
30
Retention Time (min) Figure 4. HPLC profile of N-t-Boc-L-tryptophan (1) treated with HaOz/horseradish peroxidase. The reaction and HPLC
analysis were carried out as described under Experimental Procedures. (a): Ar
(b): 02 Boc-Trp
I
I
I
0
10
20
L
30
0
10
20
30
Retention Time (min)
Figure 5. HPLC profiles of N-t-Boc-L-tryptophan(1) treated with HzOz/horseradishperoxidase under (a)argon or (b)oxygen. The reaction and HPLC analysis were carried out as described
under Experimental Procedures.
Hence, the product distribution in Hyp/XO/Fe(III)EDTA was compared with that in HpO~lhorseradish peroxidase, in which the high-valent iron-oxygen complexes, known as compound I and compound 11, are generated at the active site of this enzyme (25). The HPLC pattern in HpOdhorseradish peroxidase (Figure 4) was almost identical to that in Hyp/XO/Fe(III)-EDTA (Figure 2a), suggesting that a similar active species was generated in both systems. When Boc-Trp was treated with HpOz/horseradish peroxidase under an argon atmosphere, the formation of 6 and 7 was suppressed completely (Figure 5a). In contrast, the reaction was dramatically enhanced under an oxygen atmosphere (Figure 5b), suggesting that molecular oxygen was incorporated into the pyrrole ring of the indole nucleus.
Discussion Selective formation of oxindole-and formylkynureninetype products from tryptophan and its peptides may be due to a unique active speciesgenerated from a superoxidegenerating system in the presence of the iron(II1)-EDTA complex. The active oxygen species participating in the oxidation of substrates has been generally accepted to be the hydroxyl radical (‘OH) produced via Fenton reaction, as shown (26): 0; + Fe(III)-EDTA-O,
+ Fe(I1)-EDTA
+ 2H’
+ Fe(I1)-EDTA
-
-
H20, + 0,
‘OH + HO-
+ Fe(II1)-EDTA
Aruoma et al. (27) have reported that the hypoxanthine/ xanthine oxidase/Fe(III)-EDTA system produced typical *OH-inducedbase products in DNA, similar to the pattern of base products produced in DNA when *OHis generated by ionizing radiation. With respect to the oxidation of tryptophan by the ‘OH-generating system, Maskos et al. (17) have reported that the y-radiation of tryptophan mainly resulted in the hydroxylation of the benzene ring of the indole nucleus, and oxindol-3-ylalanine and N’formylkynurenine were detected as minor products. Contrary to their findings, the. major products generated in the Hyp/XO/Fe(III)-EDTA system were not hydroxylation products but oxindole- and formylkynurenine-type compounds. Our results suggest that an active oxygen species other than *OHparticipates in the oxidation of tryptophan by Hyp/XO/Fe(III)-EDTA. As a matter of fact, Rushet al. (28)and Yamazaki et al. (29)have recently reported that an active oxygen species, presumably the ferryl ion (FeN=O), was detected in HpOp/Fe(II)-EDTA. It seems likely, therefore, that a similar active species participates in the oxidation of tryptophan by HypiXOl Fe(II1)-EDTA. To examine this possibility, HpOz/horseradish peroxidase was used for the oxidation of 1. Mechanistic studies of HpOdhorseradish peroxidase have established that the ferric enzyme reacts with Hp02 to give compound I, a two-electron-oxidized species in which the heme is oxidized to a ferryl porphyrin radical cation (P+*FeIV=O). And sequential electron abstraction from two substrate molecules reduces the porphyrin radical cation first to the ferryl porphyrin (PFeIV=O) species known as compound 11, and subsequently to the ferric resting state (25). The product distribution in HpOp/ horseradish peroxidase (Figure 4) was almost identical to that in Hyp/XO/Fe(III)-EDTA (Figure 2a). This result suggests that, in the Hyp/XO/Fe(III)-EDTA system, a similar iron-oxygen complex participates in the oxidation of tryptophan. As shown in Figure 5, oxidation of tryptophan was significantly suppressed when the reaction was carried out under an argon atmosphere. In contrast, the reaction was significantly enhanced by oxygen bubbling. These results suggest that, in the HZOdhorseradish peroxidase system, oxygen atoms incorporated into 6 and 7 are derived from molecular oxygen. If an iron-oxygen complex such as compound I and compound I1 observed in the HzOdhorseradish peroxidase system is also operating in the Hyp/XO/Fe(III)-EDTA system, it seems possible that oxygen atoms incorporated into 6 and 7 obtained by the reaction of 1with Hyp/XO/Fe(III)-EDTA are also derived from molecular oxygen. With respect to this, further investigation will be needed. N’-Formylkynurenine (15,30,31) and oxindol-3-ylalanine (32-34) are well-known as oxidation products of tryptophan. N’-Formylkynurenine is one of the most important tryptophan metabolites, and its formation is catalyzed by indoleamine 2,3-dioxygenase (35).Indoleamine 2,3-dioxygenase predominantly utilizes 02-as the active species (36). Therefore, our results that N’-formylkpurenine was produced in the Hyp/XO/Fe(III)-EDTA system as a major product are of interest in view of the mechanism for the metabolic conversion of tryptophan into N’formylkynurenine. Formation of N’-formylkynurenine was also confirmed in nonenzymatic oxidations such as
190 Chem. Res. Toxicol., Vol. 7, No. 2, 1994
ozonolysis (30),radiolysis (151, and photooxidation (31). N'-Formylkynurenine has been detected in the mature human lens (37, 38). It is generally accepted that fluorescence pigments accumulate in the human lens due partly to the photooxidation of tryptophan to N'formylkynurenine. On the other hand, oxindol-3-ylalanine was found to be a significant inhibitor of tryptophan synthase and tryptophanase (39). As far as we know, oxindol-3-ylalanine has not been detected in vivo and is not a tryptophan metabolite. However, this indicates that oxindol-3-ylalanine may be a useful chemical marker for assessing tryptophan modifications in biologicalsystems. In conclusion, we found that the tryptophan residue in peptides treated with the Hyp/XO/Fe(III)-EDTA system was selectively oxidized to form oxindole- and formylkynurenine-type compounds. On the basis of the fact that the same product distribution was observed in HzOz/horseradish peroxidase as in Hyp/XO/Fe(III)EDTA, an iron-oxygen complex was proposed as a possible candidate for the formation of the oxindole- and formylkynurenine-type products. On the basis of our results, we propose the use of these oxidation products as valuable markers for oxidatively modified proteins by an iron-oxygen complex.
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