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Structure of the Aflatoxin B1 Dialdehyde Adduct Formed from Reaction with Methylamine F. Peter Guengerich,*,†,‡ Markus Voehler,‡,§ Kevin M. Williams,†,‡ Zhengwu Deng,‡,§ and Thomas M. Harris‡,§ Departments of Biochemistry and Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37232-0146 Received January 29, 2002
Amine conjugates of activated aflatoxin (AF) B1 are formed in various systems, e.g., buffer amines and with protein lysine groups. Structures have been published in the literature, but the evidence is indirect in that (i) halogenated AFB1 was usually used as the precursor and (ii) the assignment of the structure of the five-membered ring formed by cyclization is based on NMR chemical shifts. To better define these adducts and distinguish among several possibilities, we synthesized AFB1 dialdehyde and reacted this with the surrogate methylamine at neutral pH, to simplify the system. The isolated product had the expected molecular ion (mass spectrometry) and showed pH-dependent UV spectra similar to those published for a lysine conjugate. Nuclear Overhauser enhanced spectroscopy (two-dimensional NMR, 800 MHz) of the sample (2H2O) showed proximity of the N-CH3 protons only with a singlet at δ 4.10, assigned to the methylene of the added five-membered ring, but not to a δ 6.53 singlet assigned as the vinylic proton of that ring. All protons in the coumarin-furanone portion of the system were correlated to each other but not to those in the added five-membered ring. These experiments establish the structure as 2,3-dihydro-2-oxo-4-(1,2,3,4-tetrahydro-7-hydroxy-9methoxy-3,4-dioxocyclopenta[c][1]benzopyran-6-yl)-1H-pyrrole-1-methane. The similarity of the reaction to that occurring in the reaction of AFB1 dialdehyde with lysine and the agreement of the UV spectra suggest that this structure is applicable for the lysine analogue. The NMR results support the possible structure B of Sabbioni et al. [Sabbioni, G., Skipper, P. L., Buchi, G., and Tannenbaum, S. R. (1987) Carcinogenesis 8, 819-824] and the proposed structure 8 of Sabbioni [Sabbioni, G. (1990) Chem.-Biol. Interact. 75, 1-15] but not alternative proposals. Kinetic and mechanistic considerations of the reaction of lysine with AFB1 dialdehyde are presented in the previous article in this issue [Guengerich, F. P., Arneson, K. O., Williams, K. M., Deng, Z., and Harris, T. M. (2002) Chem. Res. Toxicol. 15, 780-793].
Introduction The mycotoxin aflatoxin (AF)1 B1 is a potent hepatocarcinogen in experimental animals, and considerable epidemiological evidence has been presented that this mycotoxin is a human carcinogen (1-4). Other AFs, particularly AFG1, are also produced by molds and are carcinogenic (5, 6). The available evidence indicates that AFB1 is activated to a genotoxic form by oxidation to the exo-8,9-epoxide via the action of P450 (7, 8). Only the exo-epoxide reacts with DNA (8). However, both the exo- and endo-epoxides can be formed by different P450s and have a number of fates, including conjugation with GSH (9-12). Hydrolysis of AFB1 epoxides yields AFB1 8,9-dihydrodiol, which is in equilibrium with the ring-opened dialdehyde form (13, 14). * To whom correspondence should be addressed. Phone: (615) 322-2261. Fax: (615) 322-3141. E-mail: guengerich@ toxicology.mc.vanderbilt.edu. † Department of Biochemistry. ‡ Center in Molecular Toxicology. § Department of Chemistry. 1 Abbreviations: AF, aflatoxin; MeNH , methylamine; CHES, 2-(N2 cyclohexylamino)ethanesulfonate; MOPS, 3-(N-morpholino)propanesulfonate; Tris, tris(hydroxymethyl)aminomethane; ESI, electrospray ionization; NOESY, nuclear Overhauser effect spectroscopy (twodimensional NMR).
Among the macromolecular adducts formed from AFB1 are the protein adducts (15-17). These are probably not of direct relevance regarding genotoxicity but may be important in the acute toxicity of AFB1, the original observation that led to the discovery of AFB1 (18). Evidence has been presented that a Lys adduct is the principal one formed from AFB1 in vivo (19). Chemical studies with 8,9-dibromo-8,9-dihydro-AFB1, a putative surrogate for AFB1 8,9-epoxide, have been done to facilitate the assignment of the structure of the Lys adduct. Sabbioni et al. (19) showed chromatographic and UV and MS spectral identity of the major Lys adduct isolated from albumin of AFB1-treated rats with an adduct prepared from the chemical reaction of 8,9dibromo-8,9-dihydro-AFB1 with (N2-acetyl)Lys (subsequently N-deacetylated). Three possibilities were presented for the adduct, with varying structures for the five-membered ring derived from the Lys nitrogen (Scheme 1). Structure 1a was preferred, primarily on the basis of the NMR proton chemical shifts and, to a lesser extent, the UV spectra (19). Subsequently Sabbioni (20) reinterpreted the structure assignment in favor of 2a (Scheme 1) based on the 13C NMR chemical shifts. The same structure of the five-membered ring (2a) was preferred in the assignment of the analogous AFG1-N2-acetylLys adduct (21).
10.1021/tx0200055 CCC: $22.00 © 2002 American Chemical Society Published on Web 05/10/2002
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Scheme 1. Possible Structures of Amine Adducts (19)
Many studies have been done involving measurement of AF-Lys adducts in experimental animals and humans, to assess AF exposure, biological activation, and the appropriateness of cancer chemopreventive agents (2225). However, the exact structure of the principal Lys adduct is still not unambiguous, in that the assignment is based only on expected NMR chemical shifts and UV wavelength maxima. To resolve the issue, we used methylamine (MeNH2) as a model of Lys to prepare a simplified primary amine adduct with synthetic AFB1 dialdehyde. This adduct has spectra expected on the basis of the corresponding Lys adduct and is characterized as structure 2b and cannot be 1b or 3b (Scheme 1). The HPLC/MS results argue against the formation of other species with the same molecular formula, indicating that this appears to be the preferred product. The results are important in that the chemical mechanism of formation of the MeNH2 and Lys adducts should involve carbinolamine formation instead of an Amadori rearrangement (19, 20, 26). The kinetics and mechanism of formation of the Lys adduct are discussed in the previous paper in this issue (26).
Experimental Procedures Caution: AFB1 exo-8,9-epoxide is a potent mutagen and probable direct-acting carcinogen and should only be handled with gloves and other appropriate precautions. AFB1 and AFB1 dialdehyde should be treated with the same care. Glassware and unused samples should be treated with commercial bleach (HOCl). All work with AFB1 was done with institutional approval. Chemicals. AFB1 exo-8,9-epoxide was synthesized from AFB1 (Sigma Chemical Co., St. Louis, MO) by oxidation with dimethyldioxirane (27), recrystallized from CHCl3/(CH3)2CO (27, 28), and stored desiccated at -20 °C. Aliquots were dissolved in CH3CN and then added to 4 vol of 20 mM sodium 2-(Ncyclohexylamino)ethanesulfonate (CHES) buffer (pH 10.0) to form AFB1 dialdehyde. Dilutions were made into 50 mM potassium phosphate buffer (pH 5.1) to estimate concentrations, using 360 ) 21 800 M-1 cm-1 (29). Ehrlich’s reagent was a solution of 5 g of 4-dimethylaminobenzaldehyde, 20 mL of concentrated HCl, and 80 mL of 95% C2H5OH (30). HPLC. Preparative chromatography of the AFB1 dialdehyde: MeNH2 conjugate was done using a Spectra-Physics 8700 pumping system (ThermoSeparations, Piscataway, NJ) and a Beckman Ultrasphere semipreparative octadecylsilane (C18) HPLC column (5 µM, 10 × 250 mm, Phenomenex, Torrance, CA). A gradient of CH3CN increasing from 13 to 27% (v/v) in
Guengerich et al. 20 mM NH4HCO3 (pH 4.5) was applied to the column at a flow rate of 4.0 mL min-1 and the effluent was monitored at 300 nm. The major AFB1 dialdehyde:MeNH2 adduct was eluted at 1617 min and collected for further analysis (concentrated using lyophilization, dissolution in H2O, and repeated lyophilization to remove NH4HCO3). A similar gradient was utilized in coupled HPLC/MS, with a smaller HPLC column and reduced flow. UV and Fluorescence Spectroscopy. UV spectra were recorded using a Cary 14/OLIS spectrophotometer (On-Line Instrument Systems, Bogart, GA). Fluorescence spectra were recorded with a Varian SF-330 spectrofluorimeter (Varian, Walnut Creek, CA). All spectra were recorded at room temperature in aqueous buffers. Mass Spectrometry. Samples were separated on a Zorbax Rx octadecylsilane column (C18, 2.1 × 150 mm, Mac-Modd, Chadds Ford, PA) and introduced into a Finnigan TSQ-7000 triple quadrapole mass spectrometer (Finnigan, San Jose, CA) equipped with a standard API-I electrospray ionization (ESI) source. Further general details are presented in the previous paper in this issue (26). NMR Spectroscopy. Freshly prepared AFB1 dialdehyde: MeNH2 conjugate was dissolved in 0.5 mL of D2O, pH 6.5. 1H NMR spectra were recorded at 800.10 MHz on a Bruker instrument (Bruker Instruments, Billerica, MA). The temperature was controlled at 25 ( 0.5 °C. Chemical shifts were referenced to the water resonance at δ 4.76 ppm at 25 °C. 1-D proton and 2-D nuclear Overhauser correlated (NMR) spectroscopy (NOESY) spectra were recorded with water presaturation. Parameters for the 1-D proton spectrum were as followed: 64 scans, 8012 Hz spectral width, 16 384 complex points. A phasesensitive NOESY spectrum was recorded with 64 scans per increment, using TPPI phase cycling, a mixing time of 500 ms, 2048 complex data points in the acquisition dimension, and 512 points in the indirect dimension. A relaxation delay of 1.5 s was used for this experiment. The data were processed using Bruker XWINNMR software on an Octane workstation (Silicon Graphics, Mountain View, CA). The data were one times zero-filled in the acquisition dimension. On the NOESY, linear prediction was applied in the indirect dimension to give a matrix of 2048 × 1024 real points. A squared sine-bell apodization function with a 72° phase shift was used in both dimensions.
Results Preparation of AFB1 Dialdehyde:MeNH2 Adduct. A trial reaction of AFB1 dialdehyde with MeNH2 indicated an apparently two-part reaction (Figure 1) with general similarity to that seen with N2-acetylLys (26). Reactions were generally done with ∼3 mM AFB1 dialdehyde and 0.5 to 1.0 M MeNH2, with 25 mM sodium 3-(N-morpholino)propanesulfonate (MOPS), at pH 7.2 and room temperature for 8-16 h under Ar. A precipitate formed during the first 1 h of the reaction; this material reacted with Ehrlich’s reagent to yield a purple color with λmax 530 nm and indicative of pyrrole formation (30) [some Ehrlich-positive material was also seen in AFB1 dialdehyde reactions with N2-acetylLys (26)]. HPLC of the reaction (Figure 2) yielded several peaks, the major of which (A300) yielded a UV spectrum similar to that expected for the Lys adduct (19, 21) (broad λmax ∼300-320 nm at pH 4.5, λmax 400 nm at higher pH). HPLC/ESI-MS of the mixture yielded a major peak with the expected MH+ 342 ion corresponding to this peak (Figure 3). The spectrum of this peak yielded MH+ at m/z 342, with all of the other peaks at m/z 300-750 accounted for as dimers or conjugates of MH+ with ions or solvent (results not shown). The identities of other peaks are unknown; the mass spectra showed higher
Aflatoxin B1 Methylamine Adduct
Figure 1. UV spectral changes during reaction of AFB1 dialdehyde with MeNH2 at pH 7.2. In this reaction, AFB1 dialdehyde (from pH 10 stock solution) was added (to 59 µM) to a 1.0 M solution of MeNH2 in 25 mM sodium MOPS (adjusted to pH 7.2). The spectra labeled 1, 2, 3, and 4 were recorded at reaction times of 0, 3, 11, and 20 min, respectively. Most of the remaining traces, with the shifts associated with the arrows first in the direction of a and then b, were collected over the course of another 7.5 h, with the final trace recorded at 21 h.
Figure 2. HPLC of preparative reaction of AFB1 dialdehyde with MeNH2. AFB1 dialdehyde (∼3 mM) was reacted with 1.0 M MeNH2 in 25 mM sodium MOPS (pH 7.2) at 23 °C for 20 h (in amber glass, under Ar) and an aliquot was injected onto the preparative HPLC system described in the text. The peak eluted at tR 17 min was collected for the subsequent spectral analyses.
masses, and these products may be associated with further reactions of pyrroles. UV and Fluorescence Spectra. UV spectra of the purified AFB1 dialdehyde:MeNH2 conjugate (two subsequent HPLC runs, with intermittent lyophilization) are shown at varying pH values in Figure 4. The spectra can be compared to those presented for AFB1- and AFG1derived trihydroxymethane (Tris) and Lys conjugates (17, 19, 21) and an AFB1 dialdehyde:Lys conjugate (26). The spectra are very similar to the AFB1-Lys spectra presented by Sabbioni et al. (19) and almost identical to the
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Figure 3. HPLC/MS of preparative reaction of AFB1 dialdehyde with MeNH2. An aliquot of the preparation described under Figure 2 was submitted to HPLC/MS using a modified gradient (NH4HCO2, pH 4.5, increasing CH3CN) and monitored for (A) the parent ion (MH+ 342) and (B) A340. The peak eluting at tR 31.4 min was analyzed and yielded MH+ 342.1, with all other major peaks with m/z 300-750 corresponding to dimers or ion adducts.
Figure 4. UV spectra of the isolated AFB1 dialdehyde:MeNH2 adduct as a function of pH. The concentration of the adduct was ∼4.1 µM [estimated using 399 ) 25 400 M-1 cm-1 at pH 7 (19)] in 50 mM potassium phosphate buffers with the indicated pH values. Compare with Figure 1 of ref 19 and Figure 1 of ref 21.
AFB1 dialdehyde:N2-acetylLys conjugate we characterized (26). The pH titration, which was reversible, shows more detail than the Lys conjugate spectra presented elsewhere (19, 21, 26) (in which some of the lower UV changes may be due to the Lys carboxyl). The UV titration indicates more than two species, as shown by the presence of at least two sets of isosbestic points. The earlier 8,9-dibromo-8,9-dihydro-AFB1:Lys conjugate of Sabbioni et al. (19) also shows this phenomenon upon inspection. Thus, the pH-dependent equilibrium involves more than two species. Fluorescence spectra were also recorded at various pH values (Figure 5). With excitation at either 370 or 400
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Guengerich et al. Table 1. 1H NMR Shifts (800 MHz) for the AFB1 Dialdehyde:MeNH2 Adduct
Figure 5. Fluorescence spectra of AFB1 dialdehyde:MeNH2 adduct. The concentration of the adduct was 1.6 µM in 20 mM potassium phosphate buffers with the indicated pH values for the emission spectra (excitation at 400 nm). The bottom trace was recorded with only buffer. The inset shows plots of the fluorescence emission at 475 nm (400 nm excitation) as a function of pH [Femis (0)], as well as the intensity of the excitation band (at 400 nm, with 480 nm emission) [Fexcit (b)] as a function of pH.
Figure 6. NOESY spectrum of AFB1 dialdehyde:MeNH2 adduct at 800 MHz. See Experimental Procedures for details and Table 1 and Scheme 2 for assignments and connectivity patterns.
nm, the λmax for emission was ∼475 nm (results with 370 nm excitation similar but not presented; spectra were weaker than with 400 nm excitation). When the wavelength emission was set at 480 nm and the emission wavelength was scanned, a peak at 400 nm was seen at pH values of >4, with a weaker broad band from 310 to 345 nm and resembling the neutral pH UV spectra (Figure 4) (results not shown). Plots of fluorescence versus pH are presented in the inset of Figure 5 and show the involvement of more than two species, as judged by the behavior at pH >6. NMR. NMR spectra were recorded in a slightly acidic D2O solution. The conjugate was not completely stable in the aqueous solution but other experiments in Me2SO indicated even less stability, in confirmation of a previous
assignment
chemical shift δ (ppm)
CH2 (a) CH2 (b) -OCH3 -NCH3 CH2 (c) CH (d) CH (e)
2.40 2.99 3.63 2.99 4.10 5.74 6.53
Scheme 2. Structure of MeNH2 Adduct with 1H Chemical Shifts and Connectivity
report (19). The sample was not stable in D2O indefinitely, but only limited degradation was observed in the course of a 48 h sample collection. The 2-D NOESY (including the 1-D spectrum) spectrum is shown in Figure 6, and the assignments are compiled in Table 1 and Scheme 3. The NCH3 group overlaps the pentenone ring methylene protons designated as b. The a, c, d, e and OCH3 protons are clearly distinguished and show expected chemical shifts (Table 1). The connectivity pattern (Figure 6, Scheme 2) clearly links a to b to OCH3 to d. The NCH3 protons are linked only to the c protons and not to e. Peak e has the most downfield chemical shift and must be the vinylic proton; it is unlinked to any other protons in the system and must be assigned an isolated position. The pattern of connectivity (Scheme 2) is interrupted by the exchange of the phenolic proton with deuterium, yielding the two sets of connectivity. The small, unassigned peaks including δ 4.1 and the peaks near the OCH3 are attributed to residual solvents and to degradation products of the sample. The set at δ 3.6-3.9 increased in intensity with further time. The NOESY connectivity (Scheme 2) is consistent with structure 2b (Scheme 1) but not with 1b or 3b.
Discussion Amines such as Tris and Lys react with AFB1 dialdehyde to form adducts (17, 19). Characterization of the exact structure has not been trivial. Although there is general agreement on the structure of the coumarin/ furanone ring system, at least three generally reasonable structures can be proposed for the associated five-
Aflatoxin B1 Methylamine Adduct
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Scheme 3. Possible Mechanism of Formation of Adducta
a
See also ref 20.
membered ring containing the amine nitrogen (Scheme 1). Sabbioni et al. (19) argued in favor of structure 1 (Scheme 1) on the basis of 1H NMR chemical shifts; later Sabbioni (20) preferred structure 2 on the basis of 13C NMR chemical shifts. 1 and 2 both have more conjugated systems and might be expected to differ from 3 in their UV and NMR spectral properties. The products used in the previous characterizations (19-21) were generated from 8,9-dibromo-8,9-dihydro-AFB1, although an AFB1 conjugate with identical chromatographic properties was reported to also be generated from a reaction of Lys with AFB1 8,9-epoxide (20). The NMR spectra are useful in the assignment of the structure but shifts alone may not be unambiguous in distinguishing among the possibilities (Scheme 1). A simplified amine adduct was prepared from the reaction of MeNH2 and chemically characterized AFB1 dialdehyde (14, 31) at pH 7.2. The purified product showed UV and fluorescence spectra very similar to those reported for the Lys and N2-acetylLys adducts (19, 21) and recorded in our work with the N2-acetylLys adducts (26). The 1H NMR NOESY spectra are consistent only with structure 2b of Scheme 1 (Figure 5, Scheme 2). Thus, the structure of the major Lys adduct must be 2a, in agreement with the latter papers of Sabbioni on the subject (20, 21). The assignment of the structure as 2 has mechanistic implications for the process of formation of this adduct. In particular, a mechanism involving an Amadori rearrangement (following Schiff base formation) was postulated to be involved in the formation of 1b (19). However, with the assignment of the adduct as 2, the Amadori mechanism is probably untenable because the product is an amide (equivlent of 2-hydroxylpyrrole), not a pyrrolin-3-one (equivalent of a 3-hydroxypyrrole) (Scheme 1). A more likely mechanism involves carbinolamine formation followed by dehydration and rearrangement, in the manner proposed in Scheme 3 (see also ref 20). These mechanistic differences are relevant to kinetic considerations discussed in the previous paper in this issue (26). A MeNH2 adduct has been characterized but the possibility of reaction of AFB1 dialdehyde to form other amine adducts can be considered, particularly in proteins in which Lys groups might conceivably influence the
formation of other products due to unique environments and steric constraints. The reaction of AFB1 dialdehyde with MeNH2 is not quantitative, as seen in the HPLC trace of Figure 2. However, HPLC/MS analysis indicated the presence of only trace (and unidentified) peaks showing the expected m/z 342 ion (i.e., MH+ for adduct). This result would argue that a simple amine does not form simple conjugates other than that characterized here (as 2b), which is argued to be analogous to the Lys adduct. However, as pointed out earlier, the reaction of AFB1 dialdehyde with MeNH2 yielded insoluble material giving a positive reaction with Ehrlich’s reagent (30). The indicated pyrrole(s) could be the product of the facile degradation of the penultimate compound in Scheme 3 or other related species. Ehrlich’s positive material could also be identified in the reaction of AFB1 dialdehyde with N2-acetylLys (26). The extent of pyrrole formation from AFB1 dialdehyde in proteins is not known. However, recoveries of the Lys-AFB1 adduct from albumin have been reported to be less than quantitative due to instability (19, 21). Bifurcation between pyrroles and lactams has been studied with other bis-carbonyl electrophiles and protein Lys groups in work with 2,5-hexanedione (32, 33) and levuglandins (34). The structure and mechanism of formation of Lys adducts are considered in the kinetic analyses presented in the previous article in this issue (26).
Acknowledgment. This work was supported in part by U.S. Public Health Service (USPHS) Grants R35 CA44353, R01 ES10546, and P30 ES00267. K.M.W. was supported in part by USPHS Training Program Grant T32 ES07028.
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