Plant Catechols and Their S-Glutathionyl Conjugates as

Oct 23, 2008 - With a view to elucidating the structural requisites for effective antinitrosating properties in plant polyphenolics and their metaboli...
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Chem. Res. Toxicol. 2008, 21, 2407–2413

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Plant Catechols and Their S-Glutathionyl Conjugates as Antinitrosating Agents: Expedient Synthesis and Remarkable Potency of 5-S-Glutathionylpiceatannol Maria De Lucia, Lucia Panzella, Alessandro Pezzella, Alessandra Napolitano,* and Marco d’Ischia Department of Organic Chemistry and Biochemistry, UniVersity of Naples “Federico II”, Via Cinthia 4, I-80126 Naples, Italy ReceiVed July 31, 2008

With a view to elucidating the structural requisites for effective antinitrosating properties in plant polyphenolics and their metabolites, we have undertaken a comparative investigation of the nitrite scavenging effects of representative catechol derivatives of dietary relevance in the 2,3-diaminonaphthalene (DAN) nitrosation and tyrosine nitration assays. Compounds tested included caffeic acid (1), chlorogenic acid (2), piceatannol (3), hydroxytyrosol (4), and the corresponding S-glutathionyl conjugates 5-8, which were prepared using either tyrosinase (5 and 6) or a novel, o-iodoxybenzoic acid (IBX)-based oxygenation/ conjugation methodology (7b and 8). In the DAN nitrosation assay at pH 4.0, the rank order of inhibitory activities was found to be 5-S-glutathionylpiceatannol (7b) > 3 > 1 > 2 > 2-S-glutathionylcaffeic acid (5) > 2-S-glutathionylchlorogenic acid (6) > 4 ≈ 5-S-glutathionylhydroxytyrosol (8). Quite unexpectedly, in the tyrosine nitration assay in 0.5 M HCl, 2 was the most efficient inhibitor followed by 1 > 4 > 3 > 7b ≈ 5 > 8 > 6. Under the assay conditions, the glutathionyl conjugates were usually consumed at faster rates than the parent catechols (decomposition rates: 3 > 1 > 4 > 2). The 2,2-diphenyl-1picrylhydrazyl radical (DPPH) assay indicated that the most effective hydrogen donors were 4 > 7b > 1 ≈ 3. Overall, these results indicated that catechol compounds and their glutathionyl conjugates may exhibit profoundly different inhibitory properties depending on the specific conditions of the assay, including especially pH, and that their antinitrosating properties do not correlate tout-court with their hydrogen donor capacity. The glutathionyl-piceatannol conjugate 7b was found to be one of the most potent inhibitors in the physiologically relevant DAN assay and may provide a new structural lead for the design of effective antinitrosating agents based on dietary polyphenolic compounds. Introduction The existence of a correlative association between excessive nitrite intake and the onset of various forms of cancer in the gastrointestinal tract is a subject of continuing interest (1). Nitrite sources of dietary origin are multiple and include vegetables (e.g., spinaches, beets, radishes, celery, and cabbages), cured meats (bacon, fermented sausage, hot dogs, ham, and smoked meat), and polluted drinking waters (2). In all of these sources, nitrite may be found associated with nitrate, which can be reduced to nitrite by certain microorganisms present in the oral cavity, accounting for significant nitrite formation in the saliva and in the gastrointestinal tract, thus contributing to the overall nitrite load (3). In addition, nitrite is the main physiological product of nitric oxide metabolism, being generated in locally high amounts at sites of chronic inflammation. In the acidic environment of the stomach (pH 2.5-4.0 during digestion), nitrite is in equilibrium with nitrous acid (pKa ) 3.2) (4), which is unstable and decomposes to a range of reactive nitrogen species (RNS),1 according to equations reported in Figure 1. The pathological consequences of RNS formation are rooted in the ability of these species to cause a series of structural and * To whom correspondence should be addressed. Tel: +39081674133. Fax: +39081674393. E-mail: [email protected]. 1 Abbreviations: RNS, reactive nitrogen species; IBX, o-iodoxybenzoic acid; HR, high resolution; ESI+, electrospray ionization in positive ion mode; DAN, 2,3-diaminonaphthalene; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical.

Figure 1. Equilibria showing acid-promoted conversion of nitrite ions to nitrous acid and its subsequent decomposition.

functional modifications of biomolecules. The possible toxic events associated with excessive nitrous acid accumulation in the stomach include (i) DNA base deamination by diazotization of primary amino groups, for example, conversion of adenine and guanine to hypoxanthine and xanthine, respectively, and mutagenesis (5); (ii) reaction of nitrosonium ion or N2O3 with secondary amines leading to the formation of carcinogenic nitrosamines (6); (iii) nitration of biomolecules, including chiefly tyrosine residues in proteins but also unsaturated fatty acids and nucleic acids (7); and (iv) methemoglobin formation, with a consequent decrease in oxygen availability and tissue asphyxia, a condition that may pose serious health risks for infants (8). Inhibition and scavenging of RNS produced by nitrite decomposition in the stomach may therefore be critical to protect the gastrointestinal tract from mutagenesis and other damaging chemical interactions and to decrease the risk of cancer. Evidence accumulated during the past two decades supports a protective role of polyphenolic compounds of plant origin as efficient scavengers of nitrosating species, for example, by

10.1021/tx800283d CCC: $40.75  2008 American Chemical Society Published on Web 10/23/2008

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Figure 2. Main products formed by reaction of 1-4 with nitrite at acidic pH.

lowering the impact of nitrosation reactions on DNA base deamination and carcinogenic N-nitrosamine formation in the stomach following elevated nitrite intake (9-11). Caffeic acid (1) and its esters, for example, chlorogenic acid (2), which is widely found in vegetables, fruits, cereals, coffee, and honeybee propolis, rank among the most efficient inhibitors of nitrosation reactions (12-16), but new data suggest that piceatannol (3), a resveratrol-related stilbene found in red wine polyphenolics and in Vaccinium berries, may be even more efficient in preventing nitrosamine formation (17). Other phenolic scavengers of nitrosating species include epigallocatechingallate from green tea (10, 18), quercetin (19), resveratrol (20), and hydroxytyrosol (4) and related phenols from extra virgin olive oil (21), although the list can be further extended. Chemical investigations of the reaction products of these polyphenolic compounds with acidic nitrite indicated unexpectedly different nitrosation/nitration patterns of reactivity depending not only on the structural features of the polyphenolic scavenger but also on the specific reaction conditions (11). This is well apparent by comparing the reaction behavior of catechol derivatives. For example, 1 undergoes mainly nitrosation at the propenyl sector, whereas its ester 2 is nitrated at the catechol moiety (14, 16). To add to this complex scenario, the catechol stilbene 3, despite structural analogies with 1 and its esters, suffers mainly nitration at the double bond (17). The structures of the catechol compounds previously investigated and their reaction products with acidic nitrite are shown in Figure 2. At a more general level, marked differences in the nature of reaction products with acidic nitrite are also observed with other polyphenolic compounds, which appear to depend on the structural characteristics of the reactive sites (18, 20). Although the chemistry of these interactions suggests differential inhibitory effects on the various nitrite-derived RNS via a broad range of mechanisms, very little is presently known as to the structural features imparting specific RNS scavenging abilities under different experimental conditions. An interesting related issue is the existence of any relationships between the RNS scavenging properties, with the underlying reactivity patterns, and the antioxidant/hydrogen donor properties of these phenols. In this paper, we report the results of an investigation aimed at assessing the relative RNS scavenging abilities of some representative catechol compounds of plant origin, namely, 1-4. For each phenolic compound examined, the effects were

compared with those of the corresponding S-glutathionyl conjugate because of the biological relevance of these derivatives, for example, to phase II metabolic transformations (22-29). These latter experiments were also prompted by recent reports showing that sulfur substituents can affect the antioxidant properties of monophenols (30) and green tea catechins (31), whereby it seemed of interest to extend the investigation to the RNS scavenging properties of catechols.

Experimental Procedures Preparation of Piceatannol (trans-3,3′,4,5′-Tetrahydroxystilbene, 3). A solution of resveratrol (150 mg, 0.66 mmol) in methanol (9 mL) was treated with o-iodoxybenzoic acid (IBX) (450 mg, 1.6 mmol) under vigorous stirring at -78 °C. Aliquots of the reaction mixture were periodically withdrawn and analyzed by HPLC, using binary gradient elution conditions as follows: 3% trifluoroacetic acid, solvent A; acetonitrile, solvent B; from 2 to 20% B, 0-40 min; from 20 to 55% B, 40-55 min; 55% B, 55-65 min; and flow rate, 1.0 mL/min. After 70 min, the reaction mixture was reduced with a solution of Na2S2O4 (300 mg) in water (26 mL), acidified to pH 3 with 3 M HCl, and extracted with chloroform (3 × 10 mL) and then with ethyl acetate (3 × 10 mL). The combined ethyl acetate layers were dried over sodium sulfate and taken to dryness to give 3 (93 mg, 58% yield, >98% purity). Preparation of 2-S-Glutathionylcaffeic Acid (5) and of 2-S-Glutathionylchlorogenic Acid (6). 2-S-Glutathionylcaffeic acid (5) and 2-S-glutathionylchlorogenic acid (6) were prepared by a general procedure reported in literature (23). Briefly, to a solution of 1 or 2 (100 mg) in methanol, 0.1 M sodium phosphate buffer (pH 7.4) was added up to a 2 mM final concentration followed by GSH (8 mM) and tyrosinase (50 U/mL) in the case of 1 and by GSH (10 mM) and tyrosinase (100 U/mL) for 2. The reaction mixture was taken under vigorous stirring at room temperature and periodically analyzed by HPLC (0.5% acetic acid-methanol 75:25 v/v; flow rate, 0.8 mL/min). After 1 h, the reaction mixture was acidified to pH 3 with 3 M HCl and then fractionated by preparative HPLC (0.5% acetic acid-methanol 80:20 v/v; flow rate, 30 mL/min) to give 5 (32) (125 mg, 47% yield, > 98% purity) or 6 (23) (169 mg, 90% yield, > 98% purity). Preparation of trans-2,5,6-Tri-S-glutathionylpiceatannol (7a). To a solution of 3 (100 mg, 0.4 mmol) in methanol, 400

Catechols and GSH Conjugates as HNO2 ScaVengers

mL of 0.1 M sodium phosphate buffer (pH 7.4) was added, followed by 4 mol equiv of GSH and tyrosinase (50 U/mL), and the mixture was taken under vigorous stirring. Aliquots of the reaction mixture were periodically withdrawn and analyzed by HPLC (3% trifluoroacetic acid, solvent A; acetonitrile, solvent B; from 2 to 30% B, 0-15 min; from 30 to 60% B, 15-45 min; flow rate, 1 mL/min, eluant A). After 2 h, the reaction mixture was treated with Na2S2O5, acidified to pH 3 with 3 M HCl, and washed with ethyl acetate. The aqueous phase was fractionated by preparative HPLC (0.5% trifluoracetic acid-acetonitrile 90:10 v/v; flow rate, 40 mL/min) to give 7a (251 mg, 53% yield, > 98% purity). Compound 7a. UV: λmax (H2O) 282, 304 nm. For 1H and 13C NMR spectra and positional assignment, see the Supporting Information. High resolution (HR) electrospray ionization in positive ion mode (ESI+)/MS: m/z 1160.2862; calcd for C44H58N9O22S3, 1160.2858 ([M + H]+). Preparation of trans-5-S-Glutathionylpiceatannol (7b). A solution of resveratrol (100 mg, 0.4 mmol) in methanol (6 mL) was treated with IBX (300 mg, 1.0 mmol) under vigorous stirring at -78 °C. After 70 min, a solution of GSH (540 mg, 1.8 mmol) in 0.1 M sodium phosphate buffer (pH 7.4) (6 mL) was added. Aliquots of the reaction mixture were periodically withdrawn and analyzed by HPLC (eluant A). After 50 min, the reaction mixture was treated with Na2S2O5 and acidified to pH 3 with 3 M HCl and washed with ethyl acetate. The water phase was fractionated by preparative HPLC (0.5% acetic acid-acetonitrile 82:18 v/v; flow rate, 45 mL/min) to give 7b (130 mg, 43% yield, >98% purity). Compound 7b. UV: λmax (H2O) 255, 311, 328 nm. For 1H and 13C NMR spectra and positional assignment, see the Supporting Information. HR ESI+/ MS: m/z 550.1490; calcd for C24H28N3O10S, 550.1495 ([M + H]+). Preparation of trans-5-S-Glutathionylhydroxytyrosol (8). The reaction was run on 100 mg of tyrosol as described above for the preparation of 7b but at -25 °C and with 1.5 mol equiv of IBX. Aliquots of the reaction mixture were periodically withdrawn and analyzed by HPLC (0.2% trifluoroacetic acid, solvent A; acetonitrile, solvent B; from 0 to 5% B, 0-5 min; from 5 to 40% B, 5-40 min; from 40 to 70% B, 40-55 min; flow rate, 0.7 mL/min). The reaction mixture was worked up as above, and the water phase was fractionated by preparative HPLC (0.2% trifluoracetic acid-acetonitrile 85:15 v/v; flow rate, 30 mL/min) to give 8 (27) (110 mg, 66% yield, > 98% purity). Reaction of Catechols with Nitrite Ions. To a solution of the appropriate catechol 1-4 (1.2 × 10-5 mol) in methanol, 30 mL of 0.05 M sodium acetate buffer (pH 4.0) or 0.5 M HCl was added followed by NaNO2 (1.2 × 10-5 mol) under stirring. Aliquots of the reaction mixture were periodically withdrawn and analyzed by HPLC (1% acetic acid-acetonitrile 85:15 v/v; flow rate, 0.7 mL/min). When required, the reaction was carried out under an argon atmosphere. In other experiments, (i) catechols 1-4 were reacted all together with nitrite ions under the above conditions or (ii) each catechol (1-4) was reacted with nitrite ions at pH 4.0 in the presence of equimolar amounts of the corresponding glutathionyl conjugate (5-8). 2,3-Diaminonaphthalene (DAN) Assay (12). Compounds 1-8 were incubated separately at 0-1 mM concentration in 50 mM sodium acetate buffer (pH 4.0, 200 µL) in the presence of DAN (0.2 mM) and sodium nitrite (20 mM). After 30 min, 50 mM sodium phosphate buffer (pH 7.4, 1.8 mL) was added to stop the reaction. Naphtho[2,3-d]triazole was quantified by

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Figure 3. Structural formulas of the synthetized glutathionyl conjugates 5-8.

measuring the fluorescence of each sample using an excitation wavelength of 375 nm and an emission wavelength of 450 nm. Inhibition of Tyrosine Nitration (10). Compounds 1-8 were incubated separately at 25-150 µM concentration in 0.5 M HCl at 37 °C in the presence of tyrosine (400 µM) and nitrite (400 µM). After 4 h and 30 min, the mixture was put on ice to stop the reaction, and 3-nitrotyrosine formation was quantified by HPLC analysis (1% acetic acid/acetonitrile 85:15 v/v; flow rate, 0.7 mL/min) at 275 nm (calibration ranges for 3-nitrotyrosine of 15-50 µM). 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) Assay (33). To 3.0 mL of a freshly prepared 0.2 mM solution of DPPH in methanol, compounds 1-8 (50 µM) were added separately. The reaction was monitored spectrophotometrically at 25 °C over 0-300 s.

Results Catechol Preparation and Assays. Compounds 1-4 were selected because of their dietary importance and/or structural features, which were suitable for comparative studies of the effects of side chains and the sulfur substituent on the scavenging properties of the catechol system. Whereas 1 and 2 were commercially available, compounds 3 and 4 were easily obtained from the corresponding monophenols, that is, resveratrol and tyrosol, by an expedient procedure involving oxidation with IBX at low temperature (-78 °C) followed by a reductive treatment (34, 35).The method was simple, cost-effective, and gave the desired catechols in pure form and in sufficient yields for chemical studies. Glutathionyl conjugates of 1 and 2, namely, 5 and 6, were obtained in good yields by tyrosinase-catalyzed oxidation of 1 and 2 in the presence of GSH (Figure 3). Attempts to extend the procedure to the synthesis of the glutathionyl adduct of 3 met with failure, due to unexpected difficulties in arresting the reaction at the monoadduct stage, and the triadduct 7a was usually the prevalent species, with only little 7b. Accordingly, an alternate procedure was developed, involving oxidation of resveratrol with IBX to give the corresponding o-quinone, which was then reacted with GSH. By this method, the desired monoadduct 7b was obtained in pure form in 43% isolated yield (Figure 4). A similar IBX-based procedure was conveniently employed to prepare the GSH adduct of 4, 8, from tyrosol. The advantage

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Figure 4. Preparation of monoadduct 7b from resveratrol through IBXmediated oxidation to the o-quinone of 3 followed by nucleophilic addition of GSH.

of this procedure lies not only in the control exerted over the reaction course, preventing repeated coupling with GSH, but also in the possibility of using as substrates the easily accessible monophenols in the place of the more expensive catechols. It is noted that whereas 1 and its ester 2 give the 2-S adduct, catechols 3 and 4 give the corresponding 5-S adducts. The different regiochemistry of the conjugation process does not depend on the oxidation procedure but reflects the intrinsic positional reactivity of the quinones, as discussed recently (23, 25). To assess the scavenging properties of 1-4 and glutathionyl conjugates 5-8 on nitrite-derived RNS, two different assays were used, namely, the DAN nitrosation assay and the tyrosine nitration assay. The DAN assay involves determination of fluorescent naphtho[2,3-d]triazole generated by N-nitrosation of DAN in 50 mM acetate buffer at pH 4.0 in the presence of excess nitrite (12). The assay allows selective monitoring of the levels of nitrosating species, such as NO+ or N2O3, that are produced by decomposition of nitrite in acidic media and that specifically account for fluorescence development. The tyrosine nitration assay is based on the HPLC quantitation of the 3-nitrotyrosine produced by reaction of tyrosine (400 µM) and equimolar nitrite in 0.5 M HCl at 37 °C (10). The reaction is complete after about 4.5 h and leads to 3-nitrotyrosine in an acid-dependent reaction, due to the generation in the strongly acidic medium of efficient nitrating species. These assays were well-suited for the comparative purposes of the present study because of their different experimental conditions and predictive significance, allowing us to distinguish selective effects of potential inhibitors in the nitrosation and nitration processes. Effects of Catechols and Their Glutathionyl Conjugates on DAN Nitrosation at pH 4.0. The relative effects of the various catechols and their glutathionyl conjugates on DAN nitrosation were compared using 0.2 mM DAN, 20 mM NaNO2, and varying concentrations of the catechols (12). Relative fluorescence values determined in the presence and in the absence of the inhibitor are shown in Figure 5. A noticeable finding is that adduct 7b proved more effective than 3 in inhibiting fluorophore development over the whole concentration range examined. From fluorescence measurements, the ratio of the kinetic constants k7b/k3 for the reactions of 7b and 3 with nitrite could be calculated as 1.7 ( 0.5 using the equation below:

f ⁄ F ) 1 - kIn[In] ⁄ kDAN[DAN]

(1)

where f and F are the fluorescence intensities determined in the

Figure 5. Inhibition of N-nitrosation of DAN by 1 (9), 2 (2), 3 ([), 4 (×), 5 (0), 6 (4), 7b (]), and 8 (O) measured as fluorescence emission at 450 nm of naphtho[2,3-d]triazole. Relative fluorescence represents the ratio of fluorescence values measured in the presence and in the absence of the inhibitor. Shown are the mean ( SD values for two separate experiments.

presence and in the absence of the inhibitor (In), respectively. For the reaction of DAN with nitrite, the reported rate constant of 8.6 × 109 M-1 s-1 was used (12). By contrast, triadduct 7a was devoid of activity. Moreover, and quite unexpectedly, adduct 5 was much less active than 1, indicating that the glutathionyl residue can exert opposite effects on the inhibitory properties of catechol compounds. To gain an insight into the origin of these effects, competition experiments were performed in which the rates of decay of each catechol against its glutathionyl conjugate were determined under the typical conditions of the assay but in the absence of DAN, with the compounds 0.4 mM each and nitrite ions at 0.4 mM concentration in acetate buffer at pH 4.0. HPLC analysis indicated that 7b was consumed at a faster rate than 3 and that all of the conjugates were more reactive than the corresponding catechols, with the exception of the 1/5 couple, for which no significant difference in the rates of decay was observed. In a related competition experiment, 1-4 at 0.4 mM concentration each were mixed together with 0.4 mM nitrite in acetate buffer at pH 4.0, both in the presence and in the absence of oxygen (argon flushed mixture). After 2 h of incubation, the extent of decay was 3 > 1, while 2 and 4 were little consumed, and no difference was noted under oxygen-depleted atmosphere. These data confirmed the higher reactivity toward acidic nitrite displayed by the catechol ring incorporated into a stilbene scaffold, as in 3. In subsequent experiments, the nature of the products formed by exposure of the catechols to nitrite was briefly investigated. All attempts at isolating reaction products from 7b were unsuccessful, due to their fast degradation to very polar and ill-defined materials eluding chromatography. Detectable amounts of ring nitration products were formed from 2 (14, 16) and 4 (21), whereas 1 gave as a main product a previously reported hydroxyoxime (16), as determined by HPLC analysis in comparison with authentic standards. Effects of Catechols and Their Glutathionyl Conjugates on Tyrosine Nitration in 0.5 M HCl. The effect of catechols on tyrosine nitration was assessed as reported (10), measuring 3-nitrotyrosine formation by reaction of 0.4 mM tyrosine with equimolar nitrite in 0.5 M HCl in the presence and in the absence of the inhibitor (25-150 µM). 3-Nitrotyrosine detection was carried out by HPLC with the analytical wavelength set at 275 nm, under conditions in which no interference by the catechols and reaction products was observed. Typically, 3-nitrotyrosine yield in the absence of inhibitor was 8 µM. Data in Figure 6 indicated that 2 was the most effective inhibitor of nitrotyrosine formation (>95% inhibition at 100

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Figure 6. Inhibition of 3-nitrotyrosine formation by 1 (9), 2 (2), 3 ([), 4 (×), 5 (0), 6 (4), 7b (]), and 8 (O) in the reaction mixture of tyrosine (400 µM) with nitrite ions (400 µM) in 0.5 M HCl, 37 °C, at 4 h and 30 min after the addition of nitrite. Shown are the mean ( SD values for two separate experiments.

Figure 7. Decay of compounds 1-4 (400 µM each) with nitrite ions (400 µM) in 0.5 M HCl in the presence (black bars) or in the absence (open bars) of oxygen at 2 h after the addition of nitrite. Shown are the mean ( SD values for two separate experiments: p < 0.05 for compounds 1, 2, and 4 in the presence and absence of oxygen.

µM concentration) followed by 1, 4, and 3, in that order. Conjugation with GSH caused a marked drop in the inhibitory activity in all catechols examined; in particular, adduct 6 was about 60% less active than 2. To inquire into the factors determining the different rank order of activity of the various catechols in the two assays, the decay rates of 1-4 at 0.4 mM concentration each were investigated under the specific conditions of the tyrosine nitration assay but without tyrosine, that is, with 0.4 mM nitrite in 0.5 M HCl. Figure 7 shows that after 2 h of incubation, the more reactive catechols were 3 and 1, as in the previous experiment at pH 4, and that substrates decayed with significantly slower kinetics under conditions of oxygen depletion, with the sole exception of the highly oxidizable 3. Overall, these results were suggestive of prevalent oxidative processes contributing to catechol reactions with nitrite ions under strongly acidic conditions. To support this conclusion, the products formed by reaction of the catechols with nitrite ions in 0.5 M HCl were investigated. Although HPLC analysis of the reaction mixtures from 1, 2, and 3 was little informative, spectrophotometric monitoring showed in the case of 1 the development of a chromophore similar to that attributed to the o-quinone (36). HPLC analysis of the mixture from 4 showed after 2 min the formation of a main product (Figure 8, trace a) whose chromatographic properties were superimposable to those of a synthetic sample of the o-quinone of 4 prepared by the IBX-based methodology (trace b). Accordingly, reductive treatment of the mixture showed conversion of the product back to 4 (trace c). The UV spectrum of the reaction mixture at that time showed a species with a maximum at 390 nm (see the Supporting Information) in good agreement with that of a

Figure 8. HPLC elution profiles of the reaction mixture of (a) 4 (400 µM) with nitrite ions (400 µM) in 0.5 M HCl at 2 min reaction time; (b) tyrosol with IBX in methanol at -25 °C after 1 h; and (c) 4 with nitrite ions as in trace a but after reduction treatment. Elution conditions: 3% trifluoroacetic acid, solvent A; acetonitrile, solvent B; 5% B, 5 min; from 5 to 90% B, 5-45 min; flow rate, 0.7 mL/min; and detection at 280 nm.

Table 1. H-Atom Transfer Reactions from Catechols 1-8 (50 µM Each) to DPPH (0.2 mM)a 1 2 3 4 5 6 7b 8

DPPH inhibition (%)

k (M-1 s-1)

nkb

ntotb

63 ( 4 58 ( 3 62 ( 3 72 ( 4 38 ( 5 48 ( 3 66 ( 5 50 ( 3

509 ( 46 325 ( 60 493 ( 38 584 ( 60 191 ( 52 311 ( 43 449 ( 42 389 ( 46

2.04 1.30 1.97 2.33 0.77 1.18 1.79 1.55

2.69 2.46 2.64 3.10 1.60 2.05 2.82 2.14

a Values are means ( SD (n ) 4). b nk and ntot, moles of radicals scavenged by each mole of antioxidant at 20 or 300 s, respectively; calcd as described (33).

4-alkyl-o-quinone (37). No detectable formation of nitrosation/ nitration products was observed by HPLC analysis. In a final set of experiments, the various catechols were assessed for their hydrogen donor abilities using the DPPH assay. This assay is based on the chromophoric changes measurable at 515 nm that accompany reaction of the DPPH radical with a hydrogen donor. Data in Table 1 indicate that 4 is the most potent hydrogen donor followed by 7b and 3 ≈ 1,

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whereas the remainder of the GSH conjugates was by far less efficient than the parent catechols.

Discussion The acid-promoted decomposition of nitrite is a complex process that involves an interplay of reactions and leads to a range of RNS. Because of the pKa value of 3.2 for HNO2 (4), nitrite in the stomach may be partially protonated, whereby even slight changes in the gastric pH during digestion (pH 2.5-4.0) may affect the delicate balance of competing pathways. Depending on such factors as nitrite concentration, acidity of the medium, and oxygen availability, the final outcome may be nitrosation, nitration, and/or oxidation of crucial biomolecules in the gastric compartment causing toxic, mutagenic, and carcinogenic effects (5-8). The elucidation of scavenging properties of polyphenolic constituents of the diet against nitritederived nitrosating and nitrating species is therefore an important goal in the prospects of developing dietary cancer chemoprevention strategies aimed at controlling tumor-initiating events in the gastrointestinal tract. In this paper, we have demonstrated markedly different inhibitory effects of catechol compounds of plant origin and their GSH conjugates in two different nitrite scavenging assays. The DAN assay represents a model system of N-nitrosation processes, because under the conditions of low acidity and excess nitrite a significant formation of N2O3 can be expected (see eq 3 in Figure 1). Under these conditions, the S-glutathionyl derivative 7b was found to be one of the most effective inhibitors so far tested, even more potent than the parent 3, recently proposed as a new reference compound in the field (17). This finding is of significant interest also in the light of the opposite effect of the sulfur substituent on the inhibitory activity of 1, which was unexpectedly blunted in 5. Previous studies (16, 17) suggested that the scavenging effects of 3 and 1 may be due to free radical nitration of the double bond and to nitrosation/decarboxylation of the propenoate side chain, respectively. Thus, the potent inhibitory activity of 7b may be due to the stabilizing effect of the GSH moiety on the phenoxyl radical intermediate crucial to the inhibition mechanism. On the contrary, the same S-glutathionyl group would decrease the reactivity of the propenoate chain of 5 relative to 1, due possibly to the bulky GSH group forcing the side chain double bond out of coplanarity with the catechol ring and hindering efficient electron delocalization over the phenylpropenoate π-framework. In this line is the lack of activity of the triglutathionyl derivative of 3, 7a, whose absorption maxima are hypsochromically shifted with respect to those of the parent catechol. A different mechanistic scenario can be envisaged in the tyrosine nitration assay, in which 3 and its conjugate 7b, the most active compounds in the DAN nitrosation assay, were found to be the least efficient inhibitors. The efficient inhibitory effects of 2 on tyrosine nitration were likewise unpredicted, since 2 was a very poor inhibitor in the DAN assay and was consumed at relatively slower rate with respect to the other catechols tested. In 0.5 M HCl, the nitrite protonation equilibrium is completely shifted toward nitrous acid formation (eq 1 of Figure 1), and an increase in the pseudofirst-order rate constant for nitrite decomposition is anticipated with decreasing pH (38). It follows that nitration of tyrosine occurs probably via oxidation to a tyrosyl radical by HNO2 (E0 ) 0.996 V) (4) and subsequent radical coupling with NO2. The apparent prevalence of catechol oxidation over nitrosation/nitration processes under these conditions was also suggested by HPLC analysis of the reaction mixtures. The lack of correlation between the catechol con-

De Lucia et al.

sumption and the inhibitory effects suggests that oxidizability per se is not a requisite for inhibition of tyrosine nitration at strongly acidic pH, consistent with the observation that reducing agents such as ascorbic acid (E0 ) 0.28 V) (39) are inactive in the tyrosine nitration assay (10). This can be explained considering that the nitrogenous species formed by redox interaction of nitrite-derived RNS with catechols, for example, NO, can be reoxidized in the mixture. On this basis, it can be speculated that the inhibitory effects of 2 are due to conversion to polymeric species acting as more efficient scavengers via covalent reactions. Finally, the general lack of correlation between DPPH inhibition data and nitr(os)ation inhibitory effects suggests that the RNS scavenging and catechol decay mechanisms do not depend to any significant extent on hydrogen abstraction steps.

Conclusions Catechol compounds of dietary relevance (1-4) and their glutathionyl conjugates (5-8) have been compared for their relative RNS scavenging properties in two different assays. When tested in the DAN nitrosation assay at pH 4, which is relevant for assessing inhibitory effects on toxic nitrosamine formation in the stomach, the most efficient inhibitor was the S-glutathionyl conjugate of 3, 7b, for which a novel and convenient synthetic procedure was reported. Conversely, under the strongly acidic conditions of the tyrosine nitration assay, compound 2 was the most effective inhibitor, whereas 3 and its conjugate 7b were much less active. The apparently contradictory results obtained in the two assays have been explained in the light of the markedly different reaction conditions affecting the main pathways of nitrite decomposition and the consequent competition between oxidation, nitrosation, and nitration processes. In particular, a significant formation of N2O3 can be expected under the conditions of the DAN assay, implying that an efficient inhibitory activity is exerted by those compounds with specific structural requisites for high reactivity toward nitrosating agents. In this connection, the lack of welldefined relationship between the antinitrosating properties and the antioxidant/hydrogen donor capacity of the catechols investigated is another interesting finding of this study. On the other hand, at strongly acidic pH, as in the tyrosine nitration assay, the rapid and efficient formation of nitrous acid and NO2 would favor oxidation and nitration processes over nitrosation reactions, whereby other structural factors become important for effective inhibitory properties. The actual physiologic relevance of nitrite scavenging by glutathionyl conjugates of plant catechols in the stomach remains to be addressed; however, the discovery of the piceatannol conjugate 7b as one of the most potent inhibitors of N-nitrosation reactions under physiologically relevant conditions may provide a useful structural lead for the design of novel effective antinitrosating agents for cancer chemoprevention. Acknowledgment. This work was carried out in part with the financial support of Regione Campania 2006. L.P. is thankful to “L’ORE´AL Italia Per le Donne e la Scienza” for a research fellowship. Supporting Information Available: General experimental methods; 1H NMR spectra of compounds 5, 6, and 8; NMR spectral data of 7a,b; 1H NMR, 13C NMR, 1H, 1H COSY, 1 H,13C HSQC-DEPT, and 1H,13C HMBC spectra of compounds 7a,b; and UV spectrum of the reaction mixture of 4 with nitrite ions in 0.5 M HCl. This material is available free of charge via the Internet at http://pubs.acs.org.

Catechols and GSH Conjugates as HNO2 ScaVengers

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