Electrophilic Properties of Patulin. Adduct Structures and Reaction

Chem. Res. Toxicol. 2000, 13, 363-372

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Electrophilic Properties of Patulin. Adduct Structures and Reaction Pathways with 4-Bromothiophenol and Other Model Nucleophiles Ralph Fliege and Manfred Metzler* Institute of Food Chemistry, University of Karlsruhe, P.O. Box 6980, 76128 Karlsruhe, Germany Received August 3, 1999

The mycotoxin patulin (PAT) is believed to exert its cytotoxic and chromosome-damaging effects by forming covalent adducts with essential cellular thiols. Since the chemical structures of such adducts are unknown to date, we have studied the reaction of PAT and its O-acetylated derivative with the monofunctional thiol model compound 4-bromothiophenol (BTP), which was chosen due to analytical advantages. By means of analytical and preparative highperformance liquid chromatography, 16 adducts of PAT and 3 adducts of acetyl-PAT were isolated and their chemical structures elucidated by 1H and 13C NMR, IR, and UV spectroscopy. Time course studies and analysis of daughter product formation from isolated intermediate adducts led to a detailed scheme for the reaction of PAT with BTP. The structures of adducts of PAT formed with other model nucleophiles, e.g., the aliphatic thiol 2-mercaptoethanol and the aromatic amine 4-bromoaniline, were also elucidated and found to corroborate the reaction scheme. In addition, one further reaction pathway was observed with 2-mercaptoethanol, which appears to be independent from those found for BTP. Our study with model nucleophiles provides insights into the electrophilic reactivity of PAT and proved to be useful for the structure elucidation of PAT adducts with biological nucleophiles of toxicological relevance, as will be reported by Fliege and Metzler [(2000) Chem. Res. Toxicol. 13, 373-381].

Introduction Patulin (PAT,1 Figure 1), a toxic secondary metabolite of various widespread fungi of the genus Penicillium, Aspergillus, and Byssochlamys, occasionally contaminates foodstuff and livestock feed (reviewed in refs 1 and 2). In addition to its high acute toxicity, in vivo and in vitro studies have shown impairment of mitosis and induction of structural and numerical chromosome aberrations by this mycotoxin (3-7). One author found sarcomigenic effects in rats when patulin was injected subcutaneously (8). Although the mechanisms of the cytotoxic and chromosome-damaging effects of PAT have not yet been elucidated, it is widely believed that the long-known reactivity of PAT toward thiol functions is of critical importance. This reaction may lead to the spontaneous covalent modification and subsequent biological inactivation of various cellular thiol-containing macromolecules, as well as to glutathione depletion (7, 9-18). In addition, reactivity of PAT toward the lysine side chains and R-amino groups of a protein (19) as well as direct effects at the DNA level (20, 21) have been reported. To date, no experimental studies on the identification of the chemical structures of adducts formed from the reaction of PAT with nucleophiles have been published, * To whom correspondence should be addressed: Institute of Food Chemistry, University of Karlsruhe, P.O. Box 6980, 76128 Karlsruhe, Germany. Phone: +49-721-608-2132. Fax: +49-721-608-7255. Email: [email protected]. 1 Abbreviations: AcPAT, O-acetylpatulin; BA, 4-bromoaniline; BTP, 4-bromothiophenol, 4-bromothiophenyl moiety; EA, ethanolamine, 2-aminoethanol; ME, 2-mercaptoethanol; MeOH, methanol; PAT, patulin, 4-hydroxy-4H-furo[3,2c]pyran-2(6H)-one.

Figure 1. Chemical structure of PAT.

although Michael-analogous addition reactions were proposed by various authors on the basis of structural considerations (10, 11, 22-24). Knowledge of the electrophilic reactions of PAT should be useful for further research on the mechanism of PAT toxicity, but may also help to establish procedures for biomonitoring and to answer the question of the “disappearance” of PAT in some foodstuffs during storage (13, 25, 26). To further characterize the thiol reactivity of PAT on a chemical basis, we have investigated the reactions of this mycotoxin and its hemiacetal ring-blocked O-acetyl derivative (AcPAT) with the monofunctional thiol model compound 4-bromothiophenol (BTP). Products formed under various incubation conditions were analyzed using analytical high-performance liquid chromatography (HPLC). Adducts were isolated by preparative HPLC and their structures determined by spectroscopic measurements. Knowledge of the chemical structures, the time course of the reactions, and the formation of daughter products from isolated intermediate adducts led to a detailed reaction pathway scheme. BTP combines high reactivity as a soft nucleophile with the analytical advantages of good reversed-phase HPLC features and facilitated interpretation of NMR, UV, IR, and MS data of the adducts that were formed. As an

10.1021/tx9901478 CCC: $19.00 © 2000 American Chemical Society Published on Web 04/15/2000

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aromatic thiol, BTP confers intense and uniform UV absorption (λmax ≈ 260 nm) to all reaction products. Furthermore, it reduces their polarity compared to that of PAT, thereby facilitating HPLC analysis and solidphase extraction from reaction mixtures or preparative HPLC eluates. The BTP residue does not interfere with signals characteristic for the PAT backbone in IR, 1H NMR, and 13C NMR spectra, in particular, the carboxyl, hydroxyl, and carbonyl regions in the IR spectra. In NMR spectra, only two kinds of proton signals (as a typical AA′XX′ spin system) and four carbon signals are added to the aromatic region. In MS spectra, the typical 79/81 isotopic pattern of bromine gives rise to characteristic ions for fragments containing the BTP moiety. A disadvantage, on the other hand, is the low water solubility of BTP. Therefore, incubations using millimolar concentrations had to be carried out in aqueous phosphate buffer containing equal amounts of methanol or tetrahydrofurane.

Experimental Procedures Chemicals. PAT was a natural product of Penicillium expansum isolated and purified following the procedure reported previously (27). The identity was checked by 1H and 13C NMR, IR, and UV spectra. The substance was >99% pure according to HPLC/DAD and GC/MS of the acetyl and trimethylsilyl derivatives obtained with acetic anhydride and N,O-bis(trimethylsilyl)acetamide, respectively. AcPAT was prepared by acetylation of PAT with an acetic anhydride/sodium acetate mixture according to the procedure of Birkinshaw et al. (28). The crude product was washed with water, purified by flash chromatography of a diethyl ether solution on acidic alumina (Alumina Woelm A-Super I, Woelm Pharma, Eschwege, Germany), and recrystallized from an ethanol/water mixture (1:1 v/v). Its identity was checked by 1H and 13C NMR, IR, and UV spectroscopy. The product was >99% pure according to GC/MS and HPLC/DAD analysis but tended to slowly hydrolyze to PAT in alkaline or acidic aqueous solution; the highest stability was observed at pH 6. BTP (98% pure) and 2-ethanolamine (EA, >98% pure) hydrochloride were purchased from Lancaster (Mu¨lheim/Main, Germany); 2-mercaptoethanol (ME, g99% pure) was from Fluka (Deisenhofen, Germany), and 4-bromoaniline (BA, 97% pure) was from Aldrich (Deisenhofen, Germany). All solvents and reagents for the preparation of buffers and HPLC eluents were of the highest commercial grade. High-Performance Liquid Chromatography. Two HPLC systems were used for analytical and preparative HPLC. For the analytical system, we employed Waters (Milford, MA) model 510 and model 6000A pumps, a Waters model 680 automated gradient controller, an LDC/Milton Roy (Ivyland, PA) spectromonitor III UV absorbance detector, and a Rheodyne (Cotati, CA) type 7125 injector equipped with a 100 µL sample loop. Data acquisition was carried out using a Berthold (Wildbad, Germany) LB 506 C-1 HPLC monitor operated with Berthold HPLC software version 1.53. The analytical columns (250 mm × 4.6 mm) were filled with Nucleosil-120 5-C18 and Polygosil-60 5-C18 (Macherey-Nagel, Du¨ren, Germany). The preparative system consisted of two Rainin (Woburn, MA) model SD300 pumps, a Rainin Dynamax UV-1 UV absorbance detector, a Rheodyne 7725i injector equipped with a 2 mL sample loop, and Rainin Dynamax Method Manager version 1.4 software for gradient programming and data acquisition. Columns were filled with Nucleosil-100 7-C18 (250 mm × 21 mm) (Macherey-Nagel) and filled with Eurospher 100-C18, 5 µm (250 mm × 8 mm) (Knauer, Berlin, Germany). Incubations and Separation of Adducts by HPLC. (1) BTP Adducts. Most of the incubations for analytical purposes and some of the preparative incubations were carried out at 20 °C in 50 mM phosphate buffer (pH 7.0) prepared using a 1:1

Fliege and Metzler (v/v) mixture of methanol and water. Small volumes of reactants freshly dissolved in methanol were added to a final concentration of 0.5-5 mM. Prolonged incubations (1-2 weeks) occasionally gave rise to small amounts of a white precipitate which probably contained polymeric products as it was not soluble in common solvents. In some studies aimed at the isolation of alcohol-labile adducts, methanol was substituted by tetrahydrofurane in the incubation buffer. Most of the incubations for preparative purposes were carried out in pure methanol, so that higher concentrations of adducts could be formed without precipitation. Sodium hydroxide was added as a catalyst at onetenth the molar concentration of BTP to form the thiolate ion. Aliquots of the incubation mixtures were analyzed by analytical HPLC with a solvent gradient made of acidified methanol/water mixtures. The following solvents and the HPLC conditions were used for the analytical separation of all adducts on Nucleosil120 5-C18: solvent A, 70% methanol plus 1 mL/L formic acid; solvent B, 100% methanol plus 1 mL/L formic acid; gradient, isocratic at 100% solvent A for 15 min and then a linear change to 90% solvent B within 30 min. The flow rate was set to 1 mL/min and the UV absorbance detector to 260 nm. For the isolation of adducts, aliquots of incubations in pure methanol were directly subjected to preparative HPLC operated with gradients of acidified methanol and water mixtures optimized for the respective separation problems. Incubations carried out in methanolic, aqueous buffer were first acidified with formic acid to pH 3, partly evaporated at 40 °C, and extracted with dichloromethane. The solvent of the extract was then changed to methanol, acetone, or tetrahydrofurane prior to preparative HPLC. As an alternative, methanol-conditioned RP-8 silica for solid-phase extraction (SPE, LiChroprep RP-8, 25-40 µm, Merck, Darmstadt, Germany) was added to the incubation mixture and adsorption of the PAT adducts forced by dilution with water while stirring. The SPE material was then filtered, the adducts were desorbed with methanol, and the extract was subjected to preparative HPLC. In the case of stable adducts, the methanol component of the HPLC eluates was evaporated at 40 °C and the aqueous phase extracted with dichloromethane. For the isolation of unstable intermediate products, the HPLC eluate was collected over methanolconditioned RP-8 or RP-2 SPE material and instantly diluted with an excess of ice-cold water, which favors adsorption. The SPE material was vacuum-filtered immediately, packed into a cartridge, and eluted with dichloromethane. Solvent evaporation was achieved under a stream of nitrogen or using a nonheated evaporation centrifuge. HPLC peaks 4 and A were contaminated with coeluting BTP disulfide which could be removed using normal-phase column chromatography on silica (MN Silica Gel 60, 0.063-0.2 mm, Macherey-Nagel) with chloroform prior to preparative HPLC. The dominating product of the incubation of acetyl-PAT with BTP (AcPAT/BTP 3) was obtained by spontaneous precipitation and purified by recrystallization from methanol. The purity of products isolated by preparative HPLC was always checked by analytical HPLC. (2) BA Adducts. Incubations were carried out as described for BTP, but the temperature was raised to 60 °C to reduce the reaction time. For preparative adduct isolation, the methanol content of the incubation mixture was removed by evaporation, the adducts were extracted with ethyl acetate, and the solvent was changed to methanol prior to HPLC. (3) ME Adducts. As ME readily dissolves in water, no cosolvent was needed in the incubation buffer. Phosphate buffer solutions (1 M, pH 7.4) allowed higher PAT and ME concentrations (up to 50 mM PAT and 250 mM ME) so that the entire incubation mixture could be subjected to preparative HPLC after pH adjustment to pH 3 with formic acid. Product Characterization. The chemical structures of the isolated products were elucidated using 1H and 13C NMR, IR, and UV spectroscopy. The instrumentation comprised Bruker (Rheinstetten, Germany) AC 250, AM 400, and DRX 500 spectrometers for NMR measurements, a Bruker IFS 88 instru-

Products of the Reaction of Patulin with Model Nucleophiles

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Figure 3. Chemical structure of the initial product of the reaction of PAT with BTP (peak J in Figure 2).

Figure 2. Time course of the reaction of PAT with BTP (molar ratio of 1:2) in methanolic, aqueous (1:1, v/v) phosphate buffer (pH 7.0) analyzed by HPLC. (Nucleosil-120 5-C18 column, 250 mm × 4.6 mm; eluent A, 70% methanol and 1 mL/L formic acid; eluent B, 100% methanol and 1 mL/L formic acid; gradient, isocratic 100% A for 15 min, followed by a linear change to 90% B within 30 min at a flow rate of 1 mL/min). The UV absorbance detector wavelength was 260 nm. The assignment of the peak numbers is identical to the PAT/BTP series adduct code in the Supporting Information. ment for Fourier transform IR spectra, and a Kontron (Zu¨rich, Switzerland) UVIKON 860 apparatus for UV spectra. IR spectra of liquid test substances were measured as a film on potassium bromide, whereas solids and small liquid sample amounts were mixed with potassium bromide powder and the spectra recorded in the diffuse reflection (DRIFT) mode. NMR solvents were CDCl3 (99.8 at. % D, Acros Organics), acetone-d6 (99.5 at. % D, Fluka, Neu-Ulm, Germany), and methanol-d4 (99.5 at. % D, Fluka). The chemical shifts are expressed in parts per million with respect to the tetramethylsilane (TMS) signal set to 0 ppm as an internal standard. UV spectra were recorded in the doublebeam mode in methanol versus methanol (BTP and BA adducts) or in water versus water (ME adducts). In some cases, further NMR experiments (1H double resonance, 1H-1H COSY, NOE difference spectra, NOESY) and mass spectra recorded on a Finnigan (San Jose, CA) GCQ GC/ MS system were useful. In the case of AcPAT adducts, the structural relationship within the series could be ascertained by transesterification of the thioester to the respective methyl ester. The thio and methyl esters were also hydrolyzed to the corresponding free acid, using 1:1 mixtures of methanol or tetrahydrofurane and 10% aqueous sulfuric acid at 80 °C for 1 h. The methyl ester proved to be suitable for GC/MS analysis.

Results Products of the Reaction of PAT with BTP. In the course of the reaction of PAT with BTP in 50 mM phosphate buffer (pH 7.0) containing 50% methanol, at least 16 products were formed, as was shown by HPLC analysis of the incubation mixture. The quantities of the individual products depended strongly on the ratio of the reactants and on the incubation time. For example, when BTP was incubated with PAT at a molar ratio of 2:1, all products could be observed in the HPLC profile in a timedependent manner (Figure 2). Within a few seconds of

Figure 4. Lactone structure type of adducts formed during the reaction of PAT with BTP.

incubation, the initial product peak J was formed, which was unstable and gave rise to the numerous other products depicted in Figure 2. After a prolonged incubation time, the predominating peaks were F, 3, C, and 4 under these conditions with an excess of BTP. When the ratio of BTP to PAT was changed from 2:1 to 1:2, the formation of peaks K, L, 2, and G was found to predominate even after an extended time of incubation (data not shown). The 16 major reaction products (peaks J, D, 1, 2, E, 3, 4, A, B, L, K, G, F, H, C, and Fa in Figure 2) were isolated by preparative HPLC and their chemical structures elucidated by means of 1H and 13C NMR, IR, and UV spectroscopy. In some cases, further NMR experiments (1H double resonance,1H-1H COSY, NOE difference spectra, and NOESY) and mass spectra proved to be useful. These extensive spectroscopic data are provided as Supporting Information. In addition to spectroscopic analysis, the isolated adducts were incubated in a phosphate buffer/methanol mixture in the absence or presence of BTP to study the formation of daughter products, which were detected by analytical HPLC. This information was useful in confirming the chemical structures derived from the spectroscopic studies and in clarifying the reaction pathways of PAT with BTP. The chemical structure of the initial reaction product, peak J, is depicted in Figure 3. IR and 1H NMR data indicated the simultaneous presence of the tautomeric hemiacetal and aldehyde form. In acetone-d6, the equilibrium was approximately 60% hemiacetal and 40% aldehyde according to the integral ratio of the H-4 NMR signal. Incubation of isolated peak J with BTP gave rise to the complete pattern of products as obtained from the reaction of PAT with BTP. According to their chemical structures, the 15 products other than peak J can be divided into three major groups, viz. lactones (Figure 4), thioenolether ketones (Figure 5), and ketohexanoic acid derivatives (Figure 6). The lactone and thioenolether ketone adducts carry at least two BTP residues, located at C-4 and C-6 of PAT. All thioenolether ketones, i.e., peaks 1, 2, C, 4, Fa, F, H, and G (Figure 5),

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Scheme 1. Proposed Mechanism for the Thiol-Catalyzed Z/E-Isomerization Process Observed for the Thioenolether Ketonesa

a

A similar thiol-catalyzed isomerization has also been described for the mycotoxin and PAT precursor ascladiol (29).

Figure 5. Thioenolether ketone structure type of adducts formed during the reaction of PAT with BTP.

Figure 6. Ketohexanoic acid derivative structure type of adducts formed during the reaction of PAT with BTP.

Figure 7. E/Z-isomeric structure assignment of peaks 1 and 2 of the reaction of PAT with BTP as determined by NOE difference spectra experiments.

contain the structural element of a thioenolether R,βunsaturated ketone, involving carbon atoms 4, 3, and 7 of PAT. This structure acts as a powerful chromophor, causing a symmetrical absorption band with a maximum at 325-330 nm for the Z-isomer or 310-315 nm for the E-isomer in the UV spectrum. The configurations of peaks 1 and 2 were determined to be E and Z, respectively, by NOE difference spectra experiments (Figure 7), and analogous configurations are assumed for the daughter products of peak 1, viz. C and Fa (E-configuration), and of peak 2, viz. 4 and F (Z-configuration). As peaks 2, 4, and F predominated over isomeric forms 1,

C, and Fa, respectively, after short periods of incubation (Figure 2), it was assumed that the Z-isomers are formed first. When the lactones peak A and B were incubated in an aqueous buffer/methanol mixture, the corresponding thioenolether ketones peak 4 and C were formed in a nonreversible reaction. Likewise, peaks D and E transformed into peaks 2 and 1. Conversion of the isolated peak 2 into 1, peak 4 into C, and peak F into Fa was observed in the presence of BTP. A possible mechanism for the thiol-catalyzed Z- to E-isomerization is proposed in Scheme 1. The methoxy group of peak F and Fa most likely originates from methanol as it depends on the presence of this solvent in the buffer. The ketohexanoic acid derivatives peak 3, L, and K (Figure 6) appear to be independent of the lactones and thioenolether ketones, as no mutual transformation could be detected. The most obvious common feature of the ketohexanoic acid derivatives is the loss of hemiacetal C-4 of the PAT backbone. As in the case of peaks F and Fa, the methoxy group probably originates from the solvent methanol, although these products could not be observed in incubations performed in pure methanol. Scheme 2 could be established for the reaction of PAT with BTP in methanol-containing aqueous phosphate buffer (pH 7.0) at 20 °C from the chemical structures of the adducts, the time course of the reaction, and the formation of daughter products from isolated intermediate adducts. The initial step for all observed products is a Michael-like addition of the thiol group of BTP to C-6 of the R,β,γ,δ-unsaturated lactone-carbonyl system (step 1, Scheme 2), combined with a simultaneous shift of the C-6dC-7 double bond to the C-3dC-7 position. This reaction appears to destabilize the hemiacetal ring, which opens to the aldehyde form. The subsequent reaction of either the aldehyde or the hemiacetal form of the initial adduct determines whether lactones and thioenolether ketones are formed on one hand or ketohexanoic acid derivatives on the other hand (Scheme 2). With an excess of BTP, reactions are favored starting from the aldehyde form, which undergoes a nucleophilic 1,2-addition reaction at the carbonyl C-4 with another molecule of BTP (step 2). In the postulated thiohemiacetal intermediate, the original lactone ring is transesterified to a new lactone, followed by tautomerization of the emerging enol to its keto form (step 3). The resulting structure is characteristic for the entire group of lactone-type adducts (Figure 4). Only two of the four possible diastereomers, viz. peaks D and E, appear to be formed, possibly due to steric reasons, although other diastereomers may be generated in amounts too small to detect by HPLC. The dominating pathway following step 3 is the opening of the lactone ring (step 4), leading to the major product peak 2 and its E-isomer (peak 1). In a rather slow reaction, these thioenolether ketones and their lactone



Scheme 2. Proposed Mechanism of the Reaction of PAT with BTP in Methanolic, Aqueous (1:1 v/v) Phosphate Buffer at pH 7.0a

a Bold and dotted arrows: pathways preferred for an excess and lack of BTP, respectively. Shaded compounds have been isolated and characterized.

precursors eliminate the C-5 hydroxy group (steps 5 and 7), giving rise to a new R,β-unsaturated carbonyl system. The latter reacts preferentially with BTP (steps 6 and 8) but to a small extent also with the solvent methanol in another Michael-analogous 1,4-addition. Spontaneous opening of the lactone ring can also be observed at this stage of a tertiary adduct (step 9). If the amount of BTP is limited, the ketohexanoic acid derivatives are the preferred adducts that are formed. This reaction pathway presumably starts from the hemiacetal tautomer of the initial product (peak J) with the methanolysis of the lactone ring (step 10). The hypotheti-

cal solvolysis product is proposed to undergo opening of the hemiacetal ring (step 11), followed by quick elimination of C-4 of the PAT backbone as formic acid (step 12). The resulting product peak K is an R,β-unsaturated ketone that adds BTP or methanol (step 13), in analogy to the reactions described above for the lactone and thioenolether ketone-type adducts of PAT. Products of the Reaction of Acetyl-PAT with BTP. When the products of the reaction of acetyl-PAT with BTP in 50 mM aqueous phosphate buffer (pH 7.0) containing 50% methanol were analyzed by HPLC, a much simpler pattern was observed than for the reaction

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Scheme 3. Proposed Reaction of AcPAT and BTP (molar ratio of 1:2) in Methanolic, Aqueous (1:1 v/v) Phosphate Buffer at pH 7.0a

a

Shaded adducts have been isolated and characterized.

of PAT. One single major and two minor peaks were observed (data not shown). Whereas the major product (designated AcPAT/BTP 3) spontaneously crystallized, the minor products (AcPAT/BTP 1 and 2) were isolated by preparative HPLC. The chemical structures of all three products were derived from their 1H NMR, IR, and UV spectra, and, in part, 13C NMR spectra. Product AcPAT/BTP 2 was suitable for GC/MS and proved to be identical to a derivative of AcPAT/BTP 3 obtained from an acid-catalyzed transesterification experiment with methanol. On the basis of the adduct structures and on the basis of the insights gained from the reaction of PAT with BTP (see above), the pathways depicted in Scheme 3 are proposed for the reaction of acetyl-PAT with BTP. The three adducts belong to the same structural type, containing a dihydropyran-4-one ring and thus termed the dihydropyranones. The hypothetical primary adduct for the reaction of acetyl-PAT with BTP (step 1, Scheme 3) is unable to tautomerize to an aldehyde because of its acetal structure. Instead, opening of the lactone ring occurs, preferentially by thio-transesterification with BTP (step 2) but also by methanolysis (step 3) or hydrolysis (step 4). The resulting intermediates could not be observed in the HPLC profile, which may be explained by their rapid tautomerization to the keto forms, followed by elimination of acetic acid (step 5). Apparently, these R,β-unsaturated ketones do not readily add another nucleophile, possibly because of deactivation by the ring oxygen. The major differences between the adducts of acetyl-PAT and PAT with BTP are the lack of tautomerization of the primary adduct and the easy elimination of acetic acid from subsequent adducts of acetyl-PAT. Adducts Formed with Other Model Nucleophiles. Adduct structures corresponding to those of the BTP adducts could also be observed when PAT was reacted with other model nucleophiles with low molecular weights,

Figure 8. Time course of the reaction of PAT with ME (molar ratio of 1:5) in aqueous phosphate buffer at pH 7.4 and 20 °C analyzed by HPLC (Polygosil-60 5-C18 column, 250 mm × 4.6 mm; eluent A, 6% methanol and 5 mL/L formic acid; eluent B, 35% methanol and 5 mL/L formic acid; gradient, linear change from 100% A to 100% B within 60 min at a flow rate of 1 mL/ min). The UV absorbance detector wavelength was 300 nm. The peak number assignment is identical to the PAT/ME series adduct code in the Supporting Information.

viz. the aliphatic thiol 2-mercaptoethanol (ME), the aliphatic amine ethanolamine (EA), and the aromatic amine 4-bromoaniline (BA). A summary of typical product structures found in incubations of PAT with single nucleophiles or in mixed incubations containing both thiol and amine is given in Tables 1-4. The HPLC profile of the reaction products of PAT with ME in aqueous phosphate buffer (pH 7.4) is depicted in Figure 8 together with the designation of the adduct peaks as used in Tables 1-4. The reaction appeared to follow identical pathways as described above for BTP, but two additional product types could be observed with this aliphatic thiol. (i) Peak 4 represents a dihydropyranonetype adduct (Table 4), which was only observed in the reaction of BTP with acetyl-PAT but not with PAT (see above). It is presumably formed as depicted for the reaction of acetyl-PAT and BTP in Scheme 3 with the difference that water instead of acetic acid is eliminated in the last step. (ii) Peaks 1 and 3 represent adducts of a completely new class, i.e., thioenolether ketones with thiol substitution at position C-2 of the PAT backbone (Table 3). They are probably formed by an initial thiol addition at C-2 (Scheme 4), followed by subsequent reactions analogous to those postulated for the C-6-thiolsubstituted thioenolether ketones of BTP (see above). An alternative pathway could start with the hydrolysis of the lactone ring and tautomerization of the enol, followed by thiol addition to C-2 of the resulting C-2-, C-3-, and C-7-unsaturated carbonyl system (not shown). However, the first route is more plausible because PAT does not hydrolyze readily at pH 7.4. In contrast to BTP, the ME moiety does not exhibit a marked UV absorption at wavelengths above 220 nm. PAT adducts can therefore only be detected by the absorption of chromophores located in the former PAT



Table 1. PAT Adducts of the Ketohexanoic Acid Derivative Type Formed with the Aliphatic Thiol 2-Mercaptoethanol (ME), the Aliphatic Amine Ethanolamine (EA), the Aromatic Amine 4-Bromoaniline (BA), and Mixtures of Thiols and Amines

nucleophiles in the incubation mixture ME (H2O) ME/EA (H2O) BTP/EA (H2O/MeOH) ME/BA (H2O/MeOH) BA (H2O/MeOH)

R1

R2

R3

adduct codea

SCH2CH2OH SCH2CH2OH S-Ph-Br SCH2CH2OH OH NH-Ph-Br NH-Ph-Br

SCH2CH2OH SCH2CH2OH S-Ph-Br SCH2CH2OH NH-Ph-Br NH-Ph-Br OH

OH NHCH2CH2OH NHCH2CH2OH NH-Ph-Br OCH3 OCH3 OCH3

PAT/ME 6 PAT/ME/EA 1 PAT/BTP/EA 1b (PAT/BTP/EA 2b) PAT/ME/BA 1 PAT/BA 1 PAT/BA 2 PAT/BA 3

a For spectroscopic data, see the Supporting Information. b In solution, this derivative appears to slowly form an equilibrium with its ring-closed aminohemiacetal form:

Table 2. PAT Adducts of the C-6-Thiol-Substituted Thioenolether Ketone Type Formed with the Aliphatic Thiol 2-Mercaptoethanol (ME)

nucleophiles in the incubation mixture ME (H2O)

a

R1

R2

R3

adduct codea

OH OH SCH2CH2OH

SCH2CH2OH SCH2CH2OH SCH2CH2OH

SCH2CH2OH SCH2CH2OH SCH2CH2OH

PAT/ME 2 (Z-isomer) PAT/ME 5 (E-isomer) PAT/ME 7 (E-isomer)

For spectroscopic data, see the Supporting Information. Table 3. PAT Adducts of the C-2-Thiol-Substituted Thioenolether Ketone Type Formed with the Aliphatic Thiol 2-Mercaptoethanol (ME)

nucleophiles in the incubation mixture ME (H2O) a

R1

R2

R3

adduct codea

OH OH

SCH2CH2OH SCH2CH2OH

SCH2CH2OH SCH2CH2OH

PAT/ME 3 (Z*-isomer) PAT/ME 1 (E*-isomer)

For spectroscopic data, see the Supporting Information.

moiety, which in some cases (e.g., peak 6) have only very low absorption coefficients. This fact could also account for the failure to find lactone-type adducts and an “initial adduct”. On the other hand, the UV spectra of the ME adducts represent the pure spectra of the adduct chromophores, without superposition of thiol chromophores as in the case of the BTP adducts. All adduct types exhibit typical band shapes in their UV spectra (Figure 9) from which they can be recognized. C-6- and C-2-thiolsubstituted thioenolether ketones have identically shaped UV bands but different maxima of absorbance. Their Zand E-isomers, like the corresponding C-6-substituted BTP adducts, differ in their UV absorbance maxima and in 1H NMR spectra with respect to the chemical shift of

Table 4. PAT Adduct of the Dihydropyranone Type Formed with the Aliphatic Thiol 2-Mercaptoethanol (ME)

nucleophiles in the incubation mixture

R

adduct codea

ME (H2O)

SCH2CH2OH

PAT/ME 4

a

For spectroscopic data, see the Supporting Information.

the 4-H proton. As for the respective adducts with BTP, an assignment of the E- and Z-conformation was made

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Scheme 4. Proposed Reaction Sequence Leading to C-2-Thiol-Substituted Thioenolether Ketone Adducts of PAT and MEa

a The initial step involves a Michael-like 1,4-addition of the thiol group to the cryptic R,β-unsaturated hemiacetal aldehyde. The subsequent reactions are assumed to be similar to those postulated for the C-6-thiol-substituted thioenolether ketones formed with BTP (see Scheme 2).

Figure 9. UV absorption spectra and molar absorption coefficients for the major ME adducts of PAT, measured in water and water. Three types of spectra can be distinguished: type I, thioenolether ketones; type II, dihydropyranones; and type III, ketohexanoic acid derivatives.

from NOE experiments. For the sake of direct comparability, a uniform E/Z-nomenclature was used, indicating the relative positions of C-4-thiol and C-7, despite the interchange of CIP priorities of C-7 and C-2 between C-6and C-2-thiol-substituted thioenolether ketones. This non-IUPAC nomenclature is marked by an asterisk. NOESY spectra indicated the spatial neighborhood of H-4 and H-2 in peak 3 but not in peak 1, thus suggesting Z*-geometry for peak 3 and E*-geometry for peak 1. NOESY of peak 2 indicated the spatial neighborhood of H-4 and particularly one of the two H-2 protons, suggesting Z-geometry for peak 2 and leaving E-geometry for its isomerization product (peak 5). This conformational assignment is in agreement with the observation already made for the BTP adducts that Z(*)-isomers of thioenolether ketones exhibit UV absorbance maxima at higher wavelengths and H-4 proton NMR signals at higher field than the respective E(*)-isomers. As with the BTP adducts, Z(*)-isomers are formed first and transformed to the energetically favored E(*)-isomers later in a thiol-catalyzed isomerization process. This conversion appears to be quantitative with ME adducts, as no

Z-isomer of peak 7 could be detected in the incubation mixtures. Isolated peak 7 did not isomerize to the Z-form when incubated with an excess of the thiol. Co-incubation of PAT with a 2-fold molar amount of thiol (BTP or ME) and an up to 100-fold excess of amine (EA) gave rise to several mixed thiol-amine adducts (Table 1, PAT/BTP/EA 1 and 2, and PAT/ME/EA 1) in addition to the dominating “thiol-only” adducts. Even at the markedly higher amine concentrations, no “amineonly” adducts could be observed. All mixed adducts were of the ketohexanoic acid derivative type (Table 1), and the EA always formed an amide bond. Adduct formation of the aromatic amine BA with PAT (molar ratio of 5:1) in the absence of thiol was markedly slower than with the thiols BTP or ME and led to three major products (Table 1, PAT/BA 1-3) after a prolonged period of time or elevated temperature. The three adducts proved to be amino analogues of the ketohexanoic acid derivative type, containing 1 or 2 equiv of amine instead of thiol at C-5 and/or C-6, and 1 equiv of the cosolvent methanol as methyl ester (Table 1). In addition to the characterized adducts, a large amount of products with a dark-brownish color was formed which could not be analyzed by HPLC.

Discussion In the study presented here, the chemical structures of the adducts formed in the reaction of the mycotoxin PAT with thiol nucleophiles were elucidated for the first time. On the basis of the adduct structures and the time course of adduct formation, schemes for the sequence of reactions are proposed (Schemes 2-4). Despite the variety of products observed even with simple thiol compounds such as BTP or ME, only a small number of reaction pathways are involved. With BTP, the initial step is a Michael-analogous 1,6-addition of the thiol group to the R,β,γ,δ-unsaturated lactone-carbonyl system (Scheme 2). With ME, a Michael-analogous 1,4addition of the thiol to the R,β-unsaturated hemiacetal carbonyl system was observed (Scheme 4) in addition to the 1,6-addition. Either addition appears to activate PAT


for various subsequent reactions, which depend on the availability of nucleophiles. Various nucleophiles such as thiol, amino, or aliphatic hydroxyl groups can participate in these subsequent reactions. Preliminary experiments showed that the reaction of PAT with aliphatic or aromatic amines is relatively slow compared with that of thiols, but co-incubation with thiol and amine compounds leads to rapid incorporation of both nucleophiles into a mixed-type adduct. In our studies, no indications have been found for the formation of Schiff base-type adducts between primary amine nucleophiles and the aldehyde moiety of patulin or patulin derivatives. It cannot be ruled out that Schiff base-type adducts contribute to the unknown brownish products observed in incubations of PAT with BA; however, they are formed as major products neither in this nor in mixed thiol-amine reactions. Since proteins contain a wide variety of nucleophilic sites, with nucleophilicity often modulated by the tertiary and quaternary structure, an even greater diversity of PAT adducts can be expected at the protein level than observed with the simple model nucleophiles used in this study. The fact that PAT can react with two or more nucleophilic sites of the same or different chemical nature implies that proteins may form complex adducts involving intra- and intermolecular cross-links in addition to monoadducts. In recent studies with the thiol-containing proteins bovine serum albumin and tubulin, and the thiol-free protein hen egg lysozyme, we have shown that PAT is in fact able to give rise to such covalent proteinprotein cross-links. The reaction of PAT with two cysteine moieties was preferred in these cross-links, but mixed thiol-amine or pure amine-amine bonds were also found (27). As PAT can react with amine nucleophiles and is activated for such reactions by initial thiol incorporation, reactions of PAT at the DNA level might also be possible. Preliminary studies in our lab have shown evidence for a low but detectable potential of the mycotoxin to generate base modification, DNA-DNA cross-links, and protein-DNA cross-links in vitro at high concentrations and long incubation times (30). The adduct structures and reaction pathways of PAT presented here for some model nucleophiles should prove useful for future specific research on the reaction products of PAT with toxicologically relevant but analytically more complicated nucleophiles, e.g., glutathione. Such studies will be reported in a subsequent paper (31). Knowledge about the chemical reactivity of PAT should also help to further characterize the mechanisms of the cytotoxicity and genetic effects of this mycotoxin on a molecular basis, and possibly facilitate the development of biomonitoring procedures for PAT based on macromolecular adducts.

Acknowledgment. We are greatly indebted to Mrs. Anja Kaiser, Mrs. Angelika Kernert, Mrs. Ingrid Rossnagel, Mrs. Annelie Kuiper, Mrs. Pia Lang, and Dr. Herbert Roettele from the spectroscopy laboratory of the Institute of Organic Chemistry, University of Karlsruhe, for their kind cooperation and expert measurement of IR and NMR spectra. This study was supported by a fellowship from the Fonds der Chemischen Industrie to R.F. and by a grant from the Deutsche Forschungsgemeinschaft (Me 574/14-1).

Chem. Res. Toxicol., Vol. 13, No. 5, 2000 371 Supporting Information Available: Spectroscopic data sets and NMR signal assignment for the isolated and characterized adducts. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) McKinley, E. R., and Carlton, W. W. (1991) Patulin. In Mycotoxins and phytoalexins (Sharma, R. P., and Salunkhe, D. K., Eds.) pp 191-236, CRC Press, Boca Raton, FL. (2) Scott, P. M. (1974) Patulin. In Mycotoxins (Purchase, J. F. H., Ed.) pp 383-403, Elsevier, Amsterdam. (3) Ciegler, A., Detroy, R. W., and Lillehoj, E. B. (1971) Patulin, penicillic acid, and other carcinogenic lactones. In Microbial Toxins (Ciegler, A., Kadis, S., and Ajil, S. J., Eds.) Vol. VI, pp 409-434, Academic Press, New York. (4) Thust, R., Kneist, S., and Mendel, J. (1982) Patulin, a further clastogenic mycotoxin, is negative in the SCE assay in chinese hamster V79-E cells in vitro. Mutat. Res. 10, 91-97. (5) Korte, A. (1980) Chromosomal analysis in bone-marrow cells of Chinese hamsters after treatment with mycotoxins. Mutat. Res. 78, 41-49. (6) Roll, R., Matthiaschk, G., and Korte, A. (1990) Embryotoxicity and mutagenicity of mycotoxins. J. Environ. Pathol. Toxicol. Oncol. 10, 1-7. (7) Pfeiffer, E., Gross, K., and Metzler, M. (1998) Aneuploidogenic and clastogenic potential of the mycotoxins citrinin and patulin. Carcinogenesis 19, 1313-1318. (8) Dickens, F., and Jones, H. K. (1961) Carcinogenic activity of a series of reactive lactones and related substances. Br. J. Cancer 15, 85-100. (9) Freerksen, E., and Bo¨nicke, R. (1951) Die Inaktivierung des Patulins in vivo. Z. Hyg. Infektionskrankh. 132, 274-291. (10) Geiger, W. B., and Conn, J. E. (1945) The mechanism of the antibiotic action of clavacin and penicillic acid. J. Am. Chem. Soc. 67, 112-116. (11) Hofmann, K., Mintzlaff, H.-J., Alperden, I., and Leistner, L. (1971) Untersuchungen u¨ber die Inaktivierung des Mykotoxins Patulin durch Sulfhydrylgruppen. Die Fleischwirtschaft 51, 1534-1537. (12) Lindroth, S., and Wright, A. v. (1990) Detoxification of patulin by adduct formation with cysteine. J. Environ. Pathol. Toxicol. Oncol. 10, 254-259. (13) Lieu, F. Y., and Bullerman, L. B. (1978) Binding of patulin and penicillic acid to glutathione and cysteine and toxicity of the resulting adducts. Milchwissenschaft 33, 16-20. (14) Barhoumi, R., and Burghardt, R. C. (1996) Kinetic analysis of the chronology of patulin- and gossypol-induced cytotoxicity in vitro. Fundam. Appl. Toxicol. 30, 290-297. (15) Riley, R. T., and Showker, J. L. (1991) The mechanism of patulin’s cytotoxicity and the antioxidant activity of indole tetramic acids. Toxicol. Appl. Pharmacol. 109, 108-126. (16) Burghardt, R. C., Barhoumi, R., Lewis, E. H., Bailey, R. H., Pyle, K. A., Clement, B. A., and Phillips, T. D. (1992) Patulin-induced cellular toxicity: a vital fluorescence study. Toxicol. Appl. Pharmacol. 112, 235-244. (17) Ashoor, S. H., and Chu, F. S. (1973) Inhibition of alcohol and lactic dehydrogenases by patulin and penicillic acid in vitro. Food Cosmet. Toxicol. 11, 617-624. (18) World Health Organization (1995) Evaluation of certain food additives and contaminants. WHO Technical Report Series, Vol. 859, pp 36-38, 44th Report of the Joint WHO/FAO Expert Committee on Food Additives. (19) Ashoor, S. H., and Chu, F. S. (1973) Inhibition of muscle aldolase by penicillic acid and patulin in vitro. Food Cosmet. Toxicol. 11, 995-1000. (20) Lee, K. S. (1991) Cytotoxicity of patulin and its effect on lambda DNA cleavage by restriction endonuclease. Korean J. Toxicol. 7, 157-163. (21) Lee, K. S., and Ro¨schenthaler, R. (1987) Strand scissions of DNA by patulin in the presence of reducing agents and cupric ions. J. Antibiot. 40, 692-696. (22) Ciegler, A. (1977) Patulin. In Mycotoxins in Human and Animal Health (Rodricks, J. V., Ed.) pp 609-624, Pathotox Publishers Inc., Park Forest South, IL. (23) Krivobok, S., Seigle-Murandi, F., Steiman, R., Benoit-Guyod, J.-L., and Bartoli, M.-H. (1994) Antitumoral activity of patulin and patulin-cysteine adducts. Pharmazie 49, 277-279. (24) Ciegler, A., Beckwith, A. C., and Jackson, L. K. (1976) Teratogenicity of patulin and patulin adducts formed with cysteine. Appl. Environ. Microbiol. 31, 664-667. (25) Scott, P. M., and Somers, E. (1968) Stability of patulin and penicillic acid in fruit juices and flour. J. Agric. Food Chem. 16, 483-485.

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(26) Harwig, J., Blanchfield, B. J., and Jarvis, G. (1977) Effect of water activity on disappearance of patulin and citrinin from grains. J. Food Sci. 42, 1225-1228. (27) Fliege, R., and Metzler, M. (1999) The mycotoxin patulin induces intra- and intermolecular protein crosslinks in vitro involving cysteine, lysine, and histidine side chains, and R-amino groups. Chem.-Biol. Interact. 123, 85-103. (28) Birkinshaw, J. H., Michael, S. E., Bracken, A., and Raistrick, H. (1943) Patulin in the common cold. II. Biochemistry and chemistry. Lancet 245, 625-630. (29) Sekiguchi, J., Shimamoto, T., Yamada, Y., and Gaucher, G. M. (1983) Patulin biosynthesis: enzymatic and nonenzymatic trans-

Fliege and Metzler formation of the mycotoxin (E)-ascladiol. Appl. Environ. Microbiol. 45, 1939-1942. (30) Fliege R. (1999) Zum molekularen Wirkungsmechanismus des Mykotoxins Patulin: Charakterisierung der spontanen Reaktivitaet gegenueber nucleophilen Modellsubstanzen und biologischen Makromolekuelen in vitro. Ph.D. Thesis, University of Karlsruhe, Karlsruhe, Germany. (31) Fliege, R., and Metzler, M. (1999) Electrophilic properties of patulin. N-Acetylcysteine and glutathione adducts. Chem. Res. Toxicol. 13, 373-381.

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Electrophilic Properties of Patulin. Adduct Structures and Reaction

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