Biosynthesis of 3-Acetyldeoxynivalenol and Sambucinol
IDENTIFICATION OF THE TWO OXYGENATION STEPS AFTER TRICHODIENE*

Lolita O. ZamirDagger §, Anastasia Nikolakakis§, Liren Huang§, Patrick St-Pierre§, Françoise SauriolDagger , Salvatore Sparaceparallel , and Orval Mamer**

From the § Centre de Recherche en Microbiologie Appliquée, Université du Québec, Institut Armand-Frappier, Laval, Québec H7N 4Z3, Canada, the Dagger  Department of Chemistry, McGill University, Montreal, Québec H3Z 2K6, Canada, the parallel  Plant Science Department, McGill University, McDonald Campus, Ste-Anne de Bellevue, Québec H9X 3V9, Canada, and the ** Biomedical Mass Spectrometry Unit, McGill University, Montreal, Québec H3A 1A3, Canada

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The first two oxygenation steps post-trichodiene in the biosyntheses of the trichothecenes 3-acetyldeoxynivalenol and sambucinol were investigated. The plausible intermediates 2-hydroxytrichodiene (2alpha - and 2beta -) and 12,13-epoxytrichodiene and the dioxygenated compounds 12,13-epoxy-9,10-trichoene-2-ol (2alpha - and 2beta -) were prepared specifically labeled with stable isotopes. They were then fed separately and/or together to Fusarium culmorum cultures, and the derived trichothecenes were isolated, purified, and analyzed. The stable isotopes enable easy localization of the labels in the products by 2H NMR, 13C NMR, and mass spectrometry. We found that 2alpha -hydroxytrichodiene is the first oxygenated step in the biosynthesis of both 3-acetyldeoxynivalenol and sambucinol. The stereoisomer 2beta -hydroxytrichodiene and 12,13-epoxytrichodiene are not biosynthetic intermediates and have not been isolated as metabolites. We also demonstrated that the dioxygenated 12,13-epoxy-9,10-trichoene-2alpha -ol is a biosynthetic precursor to trichothecenes as had been suggested in a preliminary work. Its stereoisomer was not found in the pathway. A further confirmation of our results was the isolation of both oxygenated trichodiene derivatives 2alpha -hydroxytrichodiene and 12,13-epoxy-9,10-trichoene-2alpha -ol as natural metabolites in F. culmorum cultures.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Trichothecenes are toxic secondary metabolites produced by fungi, in particular by Fusarium spp. They infect mostly wheat, grains, and corn and therefore affect human and animal health (1-4). Fusarium culmorum (HLX-1503) produces two major trichothecene metabolites (5): 3-acetyldeoxynivalenol (3-ADN)1 and sambucinol (SOL) (Fig. 1). These mycotoxins have been known for a long time (6); however, there is not yet an efficient detoxification procedure. Since trichodiene (TDN; 1, Fig. 1), the biosynthetic precursor to trichothecenes, is not toxic, knowledge of its first oxidized metabolite will enable the design of a potent inhibitor to trichothecenes. We therefore decided to focus on the identification of the oxygenation steps after the hydrocarbon trichodiene. The first oxygenated trichodiene derivatives isolated from F. culmorum were 9,10-trichoene-12,13-diol (2d) and 12,13-epoxy-9,10-trichoene-2alpha -ol (3a) (Fig. 1) (8). These compounds had been detected by the kinetic pulse-labeling method (7) and could accumulate when the inhibitor ancymidol was used (8). In addition, they had been isolated with a radiolabel after feeding (3RS)-[2-14C]mevalonate to F. culmorum cultures and isolating and purifying the derived radiolabeled 2d and 3a. These compounds were separately fed to F. culmorum cultures, and the derived trichothecenes 3-ADN and SOL were recovered and analyzed. Metabolite 3a was found to be incorporated in both 3-ADN and SOL, whereas the trichothecenes obtained after the feeding of 2d were unlabeled. This preliminary result suggests that 3a might be a biosynthetic precursor to 3-ADN and SOL, whereas 2d is a dead end metabolite (8). This result is not extremely rigorous, since no degradation could be done on the radiolabeled trichothecenes obtained from the feeding of compound 3a to prove the labeled site. Indeed, there is a possibility that the metabolite 3a was degraded and then resynthesized into the trichothecenes. Since we wanted to determine with certainty the sequence of oxygenation post-trichodiene, our first goal was to determine unambiguously if 3a is the dioxygenated metabolite and then what is the first oxygenated precursor: 2a, 2b, or 2c. The isolated 2d was shown to be a dead end product and probably derives from opening of the epoxide 2c (8).

In this paper, we have rigorously proven, for the first time, the two oxygenation steps of trichodiene and their sequence. A biosynthetic scheme for all of the oxidations leading to 3-ADN and SOL is proposed.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Instrumentation-- Analytical high performance liquid chromatography (HPLC) was performed on the same instrument used previously (9) or on a Perkin-Elmer Binary LC pump 250 coupled to a Waters 990 photodiode array detector or on a Waters 600 pump coupled to a Waters 996 photodiode array detector. Preparative and semipreparative HPLC were performed on a Waters Delta Prep 3000 instrument coupled to a Lambda-Max model 481 LC spectrophotometric detector. All ultraviolet detectors were set at 204 nm. Infrared spectra were measured in chloroform with a Perkin-Elmer model 683 infrared spectrophotometer. Flash chromatography was performed on silica gel 60, 230-400 mesh (EM Science). Thin layer chromatography was conducted on Silica Gel 60 F254-precoated TLC plates, 0.25 mm (EM Science). High performance TLC was run with LHP-KF, 0.2-mm plates (Whatman). Radiolabeled compounds were analyzed with a Bioscan Imaging Scanner System 200 (10). A Tracor Analytic Delta 300 instrument was used for liquid scintillation counting. F. culmorum slant cultures were homogenized with a Polytron homogenizer (10).

Strain and Cultivation Conditions-- An F. culmorum strain (HLX-1503) was grown as described previously (7). Seed cultures were grown for either 2 or 3 days. Production cultures (25 ml of production medium in a 125-ml Erlenmeyer flask) were incubated for 1, 2, or 3 days.

NMR and Mass Spectrometric Measurements-- All of the NMR spectra were obtained at room temperature with a UNITY-500 spectrometer operating at 499.843 MHz for 1H, 125.697 MHz for 13C, and 76.735 MHz for 2H. The NMR spectra were obtained on 1-2 mg (natural product) and 5-10 mg (synthetic compounds) dissolved in 0.3-0.4 ml of deuterochloroform. The nuclear Overhauser effect two-dimensional spectroscopy (NOESY) experiment (hypercomplex phase mode) was obtained using a mixing time of 0.3 s and a relaxation delay of 1 s. For the compounds studied, when the diagonal signals were phased negative, the cross-peaks resulting from dipolar relaxation (nuclear Overhauser effect (NOE)) were phased positive. Fast atom bombardment (FAB) mass spectra were obtained with a V G ZAB-HS double-focusing instrument using a xenon beam having 8 kV energy at 1 mA equivalent current. The ratio between enriched and nonenriched metabolites was obtained by comparing, in the proton NMR, the integration of the doublet derived from the coupling of 13C-H and the singlet corresponding to 12C-H. The extent of 13C (or 2H) obtained from labeled feedings was determined by correction of the appropriate protonated or sodiated quasimolecular ion clusters present in a computer average of 10 scans in low resolution FAB. The intensity of each ion of the cluster was reduced by the calculated contribution at that mass by natural heavy isotope substitution in all lighter ions of the cluster. The remaining intensities then represent the relative abundances of each labeled species. The percentage of isotope labeling estimated by NMR and by mass spectrometry (MS) agreed reasonably well.

High Performance Liquid Chromatography-- Analytical, semipreparative, and preparative HPLC was performed with the same columns as previously recorded (9). HPLC gradient A consisted of a linear gradient lasting 50 min with an initial concentration of 15% methanol, 85% water and a final concentration of 75% methanol, 25% water, which was held for 30 min and then increased linearly for 10 min to 100% methanol. The concentration was held for 20 min at 100% methanol before equilibrating to 15% methanol, 85% water. HPLC gradient B consisted of a linear gradient lasting 50 min with an initial concentration of 40% methanol, 60% water and a final concentration of 100% methanol, which was then held for 20 min. The metabolites isolated from the feedings were purified on analytical HPLC following isocratic conditions at a flow rate of 1 ml/min as follows: for 3-ADN, 35% methanol, 65% water; for SOL, 50% methanol, 50% water; for diacyl-SOL, 62% methanol, 38% water.

Synthesis of Compound (Fig. 2)-- A solution of compound 4 (11, 25) (0.74 g; 2.1 mmol) in 6.3 ml of dichloromethane, at 25 °C, was treated with selenium dioxide (0.060 g; 0.54 mmol) and a 90% solution of tert-butyl hydroperoxide (0.92 ml; 8.3 mmol) for 4 h. The solution was filtered through a short dry silica gel column, eluted with ethyl acetate, and then concentrated. The residue was chromatographed on silica gel using hexane/ethyl acetate (65:35) as the eluting solvent to yield 0.65 g (84%) of the desired alcohol as a pale yellow oil. IR (CHCl3) nu max 3590, 3450 (br), 3010, 2970, 2880, 2050, 1970 (br), 1650 cm-1. The numbering system used in the figures and for the carbons in the NMR derives from trichothecenes, since this makes it easier to compare data for all of the compounds. 1H NMR delta  (ppm) 5.16 (1H, d, J = 1.9 Hz, H-13a), 5.10 (1H, br, H-11), 4.91 (1H, d, J = 1.9 Hz, H-13b), 4.32 (1H, br.q, J = 8.3 Hz, H-2), 3.65 (3H, s, OMe), 3.22 (1H, br.q, J approx  2.4 Hz, H-8), 2.50 (1H, d, J = 6.3 Hz, H-10), 2.04 (1H, dt, J = 6.8, 11.2 Hz, H-3a), 1.87 (1H, br, H-7a), 1.63 (1H, br, H-4a), 1.46 (1H, d, J = 8.8 Hz, OH-2), 1.44 (1H, dd, J = 3.0, 15.1 Hz, H-7b), 1.29 (1H, o.m, H-3b), 1.24 (1H, o.m, H-4b), 1.01 (3H, s, H-14), 0.99 (3H, s, H-15); 13C NMR delta  (ppm) 106.0 (C-13), 76.8 (C-2), 66.0 (C-11), 60.2 (C-10), 54.0 (OMe), 53.0 (C-8), 38.0 (C-7), 32.6 (C-4), 32.5 (C-3), 28.3 (C-14), 27.2 (C-15).

Synthesis of Compound (Fig. 2)-- A solution of the iron diene complex 5 (0.65 g; 1.7 mmol) in 2.5 ml of ethanol was added to a solution of CuCl2 (1.77 g; 13.2 mmol) in 17.6 ml of ethanol. After stirring at 25 °C for 18 h, the reaction mixture was poured into 50 ml of brine, extracted with ethyl acetate, dried (MgSO4), filtered, and concentrated in vacuo. The residue was chromatographed on silica gel using hexane/ethyl acetate (50:50) to yield 0.35 g (91%) of the desired enone 6 as a pale yellow oil. Compound 6 (0.35 g; 1.6 mmol) dissolved in 16 ml of toluene was treated with vanadyl acetylacetonate (0.002 g; 0.008 mmol) and then with a 90% solution of tert-butyl hydroperoxide (0.70 ml; 6.3 mmol). After 5.5 h at 25 °C, the toluene was evaporated, and the residue was chromatographed on silica gel using hexane/ethyl acetate (25:75) to yield 0.36 g (96%) of the desired epoxide as white crystals, melting point 65-67 °C. IR (CHCl3) nu max 3520 (br), 3010, 2980, 2880, 1675 cm-1; 1H NMR delta  (ppm) 6.78 (1H, dd, J = 10.5, 2.2 Hz, H-11), 5.94 (1H, dd, J = 10.5, 1.2 Hz, H-10), 3.91 (1H, dd, J = 11.0, 9.8, 6.6 Hz, H-2), 3.28 (1H, d, J = 3.9 Hz, H-13a), 2.88 (1H, d, J = 3.9 Hz, H-13b), 2.56 (1H, ddd, J = 17.6, 14.7, 5.9 Hz, H-8a), 2.42 (1H, dddd, J = 17.3, 4.9, 2.5, 1.0 Hz, H-8b), 2.12 (1H, dt, J = 12.0, 7.8, 6.6 Hz, H-3a), 2.04 (1H, d, J = 9.5 Hz, OH-2), 2.02 (1H, dt-br., J = 14.1, 14.1, 5.1 Hz, H-7a), 1.83 (1H, ddt, J = 13.9, 5.9, 2.4, 2.4 Hz, H-7b), 1.68 (1H, m, H-4a), 1.50 (1H, om, H-3b), 1.50 (1H, om, H-4b), 1.17 (3H, s, H-15), 1.01 (3H, s, H-14); 13C NMR delta  (ppm) 198.6 (C-9), 155.6 (C-11), 128.4 (C-10), 72.3 (C-2), 66.8 (C-12), 48.7 (C-13), 45.4 (C-5), 40.8 (C-6), 33.9 (C-8), 31.3 (C-4), 31.0 (C-3), 30.2 (C-7), 19.4 (C-15), 18.7 (C-14).

Synthesis of Compound (Fig. 2)-- Enone 7 (0.41 g; 1.7 mmol) dissolved in 8.5 ml of dichloromethane was treated with t-butyldimethylsilyl chloride (0.36 g; 2.4 mmol) and 4-dimethylaminopyridine (0.28 g; 2.3 mmol) at 25 °C for 18 h. The solution was treated further with t-butyldimethylsilyl chloride (0.20 g; 1.3 mmol) and 4-dimethylaminopyridine (0.16 g; 1.3 mol) at 25 °C for 3.5 h. The reaction was quenched with a 15% solution of ethyl acetate in hexane, filtered through dry silica gel, and concentrated in vacuo. The residue was chromatographed on silica gel using hexane/ethyl acetate (80:20) to yield 0.43 g (71%) of the desired silyl compound 8 as white crystals, melting point 52-54 °C. To a suspension of Ph3P13CH3I (12) (1.55 g; 3.82 mmol) in 5 ml of diethyl ether, a 0.8 M solution of potassium tert-butoxide in tert-butanol was added (4.80 ml; 3.84 mmol), and the white suspension was heated at 55 °C for 30 min. The resulting pale yellow suspension was cooled to room temperature and was treated with enone 8 (0.43 g; 1.2 mmol) dissolved in 2 ml of diethyl ether. The suspension was stirred at 55 °C for 45 min. Upon cooling, the mixture was poured into 50 ml of water and was extracted with ether, dried (MgSO4), filtered, and concentrated in vacuo. Chromatography on silica gel using hexane/ethyl acetate (90:10) yielded 0.40 g (93%) of the desired diene 9 as a colorless oil. IR (CHCl3) nu max 3010, 2960, 2890, 2860, 1630, 1580, 1255 cm-1; 1H NMR delta  (ppm) 6.06 (1H, dd, J = 10.2, 3.9 Hz, H-11), 5.65 (1H, d, J = 10.0 Hz, H-10), 4.78 (1H, d, JC-H = 156.3 Hz, H-16a), 4.76 (1H, d, JC-H = 156.5 Hz, H-16b), 3.93 (1H, dd, J = 10.3, 6.3 Hz, H-2), 3.14 (1H, d, J = 4.9 Hz, H-13a), 2.64 (1H, d, J = 4.7 Hz, H-13b), 2.44 (1H, br.m, H-8a), 2.31 (1H, dq, J = 15.4, 4.1(q) Hz, H-8b), 1.84 (1H, m, H-3a), 1.67 (1H, mo, H-4a), 1.65 (1H, mo, H-3b), 1.57 (1H, mo, H-7a), 1.53 (1H, mo, H-7b), 1.40 (1H, m, H-4b), 0.99 (3H, s, H-14), 0.99 (3H, s, H-15), 0.87 (9H, s, t-Bu), 0.045, 0.036 (6H, SiMe2); 13C NMR delta  (ppm) 136.5 (d, J = 7.3 Hz, C-11), 128.2 (s, C-10), 105.1 (s, C-13), 110.5 (s + d, JC-9,C-16 = 71.4 Hz, C-16 enriched), 74.0 (s, C-2), 33.1 (s, C-3), 31.5 (s, C-4), 30.6 (d, J = 2.0 Hz, C-8), 26.9 (d, J = 2.0 Hz, C-7), 25.9 (s, t-Bu), 21.6 (s, C-14), 19.2 (s, C-15), -4.5, -4.7 (s, SiMe2).

Synthesis of Compound 3b (Fig. 2)-- A three-necked 100-ml flask equipped with a KOH drying tube was charged with 3.6 ml of tetrahydrofuran. Upon cooling in a dry ice/acetone bath to -78 °C, 17 ml of ammonia gas were condensed into the flask. Sodium (0.17 g; 7.4 mmol) was added, and after 15 min the diene 9 (0.40 g; 1.1 mmol) dissolved in 2 ml of tetrahydrofuran was added dropwise. After stirring at -78 °C for 60 min, the reaction was quenched with excess ethanol. Upon standing at room temperature, the ammonia was allowed to evaporate, and the solution was diluted with water, extracted with ether, dried (MgSO4), filtered, and concentrated. Chromatography on silica gel using hexane/ethyl acetate (97.5:2.5) yielded 0.24 g (60%) of the desired trichoene 10 (2beta -t-butyldimethylsilyloxy-12,13-epoxy-[16-13C]9,10-trichoene) as a colorless oil. A solution of compound 10 (0.13 g; 0.37 mmol) in 2 ml of tetrahydrofuran at 25 °C was treated with a 1 M solution of tetrabutylammonium fluoride (0.95 ml; 0.95 mmol) for 15 min. The reaction was quenched with 2.5 ml of water, and the solution was extracted with ethyl acetate, dried (MgSO4), filtered, and evaporated. The residue was chromatographed on silica gel using hexane/ethyl acetate (90:10) and then (80:20) to yield 0.073 g (83%) of the desired alcohol 3b as a colorless oil: 12,13-epoxy-[16-13C]9,10-trichoene-2beta -ol. Compound 3b was unstable in chloroform, thereby decomposing during NMR spectroscopy. Subsequently, it was acetylated (3 mg with 30 µl of deuterated acetic anhydride and 15 µl of pyridine) and purified by analytical HPLC, using 74% methanol, 26% water for 30 min and then increasing the concentration of methanol linearly to 100% over a 40-min period (tR = 55.5 min) at 1 ml/min. After evaporation, a colorless oil, Ac-3b, was obtained. 1H NMR delta  (ppm) 5.26 (1H, br.t, J = 5.4 Hz, H-10), 5.07 (1H, dd, J = 10.7, 6.8 Hz, H-2), 3.21 (1H, d, J = 4.4 Hz, H-13a), 2.75 (1H, d, J = 4.4 Hz, H-13b), 2.05 (1H, m, H-3a), 1.82 (1H, om, H-4a), 2.05, 1.67 (2H, m, H-11), 1.67 (1H, om, H-3b), 1.62 (1H, br.d, JC-H = 125.0 Hz, H-16), 1.46 (1H, br.m, H-8a), 1.46 (1H, m, H-4b), 1.40 (1H, q, H-8b), 1.24 (2H, om, H-7), 0.96 (3H, s, H-14), 0.87 (3H, s, H-15); 13C NMR delta  (ppm) 132.2 (C-9), 119.6 (C-10), 73.5 (C-2), 65.4 (C-12), 48.0 (C-13), 46.2 (C-5), 36.5 (C-6), 33.1 (C-11), 31.1 (C-4), 28.2 (C-7), 27.1 (C-8), 26.8 (C-3), 23.0 (C-16 enriched), 19.0 (C-14), 18.2 (C-15).

Synthesis of Compound 12 (Fig. 2)-- A solution of 12,13-epoxy-[16-13C]9,10-trichoene-2beta -ol 3b (0.073 g; 0.31 mmol) in 1.9 ml of pyridine was treated with para-toluenesulfonyl chloride (0.46 g; 2.4 mmol) at 25 °C. After 1 h, the solution was poured into ice water, extracted with ethyl acetate, dried (MgSO4), filtered, and evaporated. Chromatography on silica gel using hexane/ethyl acetate (85:15) yielded 0.106 g (88%) of the desired sulfonate, 2beta -p-toluenesulfonyloxy-12,13-epoxy-[16-13C]9,10-trichoene 11. A solution of 11 (0.064 g; 0.16 mmol) in 1.5 ml of dimethylformamide was added to a suspension of cesium acetate (0.063 g; 0.33 mmol) in 1.5 ml of dimethylformamide. After stirring at 65 °C for 24 h, the solution was cooled, diluted with water, and extracted with methylene chloride/ether (1:3). The extracts were dried (MgSO4), filtered, and concentrated. Chromatography on silica gel using hexane/ethyl acetate (90:10) yielded 0.032 g (75%) of compound 12 as a colorless oil: 2alpha -acetoxy-12,13-epoxy-[16-13C]9,10-trichoene. IR (CHCl3) nu max 3005, 2970, 1730, 1440, 1370, 1245, 970 cm-1; 1H NMR delta  (ppm) 5.27 (1H, br.m, H-10), 4.75 (1H, dd, J = 5.4, 2.2 Hz, H-2), 3.30 (1H, d, J = 4.6 Hz, H-13a), 2.98 (1H, d, J = 4.4 Hz, H-13b), 2.17 (1H, td, J = 12.2, 12.2, 7.1 Hz, H-4a), 2.14 (1H, om, H-11a), 2.05 (1H, dddd, J = 13.1, 12.2 (H-3, H-4a), 7.1 (H-3, H-4b), 5.4 (H-2, H-3) Hz, H-3a), 2.02 (3H, s, OAc), 1.95 (1H, br.m, H-8a), 1.84 (1H, br.dm, J = 17.1 Hz, H-8b), 1.70 (1H, mo, J = 7.0, 2.4, 1.2 Hz, H-4b), 1.69 (1H, om, H-11b), 1.66 (1H, ddt, J = 13.9, 7.1, 2.2, 2.2 Hz, H-3b), 1.64 (3H, br.d, JC-H = 124.7 Hz, H-16), 1.44 (1H, m, H-7a), 1.42 (1H, m, H-7b), 1.01 (3H, s, H-14), 0.88 (3H, s, H-15); 13C NMR delta  (ppm) 170.1 (s, OAc), 133.1 (s, C-9), 119.7 (s, C-10), 81.9 (s, C-2), 69.1 (s, C-12), 50.3 (s, C-13), 34.4 (s, C-4), 33.3 (d, J = 3.7 Hz, C-11), 28.3 (s, C-3), 28.2 (s, C-7), 27.4 (d, J = 3.7 Hz, C-8), 23.3 (s, C-16 enriched), 19.4 (s, C-15), 18.7 (s, C-14).

Synthesis of Compound 3a (Fig. 2)-- A solution of 2alpha -acetoxy-12,13-epoxy-[16-13C]9,10-trichoene 12 (0.087 mg; 0.311 mmol) in 4 ml of methanol was treated with 4 ml of 0.1 N NaOH for 3 h at 25 °C. The methanol was evaporated, and the aqueous portion was saturated with NaCl and extracted with ethyl acetate. The organic layer was dried (MgSO4), filtered, and concentrated in vacuo. The residue (74 mg) was further purified by analytical HPLC on HPLC gradient A at 1 ml/min. The peak at tR = 83.5 min was isolated, and after evaporation a colorless oil was obtained, 3a: 12,13-epoxy-[16-13C]9,10-trichoene-2alpha -ol). 1H NMR delta  (ppm) 5.27 (1H, br.s, H-10), 3.85 (1H, br.s, H-2), 3.31 (1H, d, J = 4.4 Hz, H-13a), 3.04 (1H, d, J = 4.2 Hz, H-13b), 2.16 (1H, om, H-11a), 2.00 (1H, m, H-3a), 1.68 (1H, om, H-11b), 1.64 (3H, d, JC-H = 125.2 Hz, H-16), 1.56 (1H, m, H-3b), 1.43 (1H, m, H-7b), 1.04 (3H, s, H-14), 0.88 (3H, s, H-15).

Synthesis of Compound 2a (Fig. 2)-- Tungsten hexachloride (0.30 g; 0.756 mmol) was added to 2.0 ml of tetrahydrofuran at -78 °C. A dark brown suspension was obtained. n-Butyllithium (1.4 ml; 1.6 M solution in hexanes; 2.24 mmol) was added to the above suspension at -78 °C. The mixture was stirred for 15 min at this temperature and then allowed to warm to room temperature. 2alpha -Acetoxy-12,13-epoxy-[16-13C]9,10-trichoene 12 (0.053 g; 0.19 mmol) dissolved in 0.8 ml of tetrahydrofuran was added dropwise to the mixture at 25 °C. After stirring at 25 °C for 30 min, the solution was heated for 30 min at 50 °C. Upon cooling to room temperature, the mixture was evaporated in vacuo. The black residue was chromatographed on silica gel using hexane/ethyl acetate (90:10). This yielded 37 mg (74%) of the desired compound 2alpha -acetate-[16-13C]trichodiene 13 as a colorless oil. Compound 13 (0.030 g; 0.114 mmol) dissolved in 1.5 ml of methanol, was treated with 10 N NaOH to pH 12. Hydrolysis was completed after 5.5 h at 25 °C. The mixture was neutralized with 5% aqueous HCl, diluted with brine, and extracted with ethyl acetate. The organic layer was dried (MgSO4), filtered, and evaporated. The residue was chromatographed on silica gel using hexane/ethyl acetate (80:20). This yielded 25 mg (99%) of the desired compound 2a as a colorless oil: 2alpha -hydroxy-[16-13C]trichodiene. IR (CHCl3) nu max 3600, 3450, 3080, 2960, 1645 cm-1; 1H NMR delta  (ppm) 5.30 (1H, br.m, J = 1.2 Hz, H-10), 5.26 (1H, d, J = 0.7 Hz, H-13a), 5.07 (1H, s, H-13b), 4.41 (1H, dd, J = 2.2, 4.9 Hz, H-2), 2.25-1.78 (5H, m, H-4a, H-8a/b, H-11a/b), 1.76-1.59 (3H, m, H-3a/b, H-7a), 1.64 (3H, br.d, JC-H = 125.0 Hz, H-16), 1.47-1.32 (2H, m, H-4b, H-7b), 1.07 (3H, s, H-14), 0.92 (3H, s, H-15); 13C NMR delta  (ppm) 161.8 (s, C-12), 132.5 (s, C-9), 120.3 (d, JC-10,C-16 = 3.7 Hz, C-10), 108.2 (s, C-13), 78.5 (s, C-2), 33.5 (d, JC-7,C-16 = 4.6 Hz, C-7), 28.7 (d, JC-8,C-16 = 2.7 Hz, C-8), 27.8 (d, JC-11,C-16 = 3.7 Hz, C-11), 23.3 (s, C-16 enriched), 20.8 (s, C-14), 18.5 (s, C-15).

Synthesis of Compound 2c (Fig. 3)-- A solution of 9beta ,10alpha -dibromo-[15-2H]12,13-epoxytrichodiene 15 (13, 14) (0.025 g; 0.066 mmol) in 5 ml of absolute ethanol was treated with pyridine (0.41 ml; 5.07 mmol) and freshly activated zinc (0.125 g; 1.91 mmol). The solution was heated at 79 °C for 2 h. Upon cooling to room temperature, the solution was filtered and diluted with ethyl acetate. The organic layer was washed with a saturated solution of NaHCO3 and brine to neutrality. The ethyl acetate was dried (MgSO4), filtered, and evaporated to dryness. The residue was chromatographed on silica gel using hexane/ethyl acetate (95:5) to yield 10 mg (69%) of the desired deuterated 12,13-epoxytrichodiene 2c as a colorless oil. 1H NMR (2H6-acetone) delta  (ppm) 5.27 (1H, br.s, H-10), 3.19 (0.4H, d, J = 4.4 Hz, H-13a), 3.16 (0.6H, d, J = 4.9 Hz, H-13a), 2.73 (0.6H, d, J = 4.4 Hz, H-13b), 2.70 (0.4H, d, J = 4.4 Hz, H-13b), 1.62 (3H, br.s, H-16), 0.89 (1.2H, s, H-14), 0.87 (1.8H, s, H-14), 0.86 (1.2H, br.m, H-15), 0.82 (0.8H, t, JH,D = 2.0 Hz, H-15); 2H NMR delta  (ppm) 0.87 (0.6H, s, 2H-15), 0.84 (0.4H, s, 2H-15).

Synthesis of Compound 16 (Fig. 3)-- Compound 16 was prepared from compound 6 employing the same procedure as for the preparation of compound 8 from compound 7. Compound 16 was isolated as white crystals, melting point 66-68 °C. IR (CHCl3) nu max 3090(w), 3030(w), 2960(s), 2860(s), 1670(s), 1650(m) cm-1; 1H NMR delta  (ppm) 6.94 (1H, dd, J = 10.6, 2.0 Hz, H-11), 5.93 (1H, d, J = 10.6 Hz, H-10), 5.21 (1H, d, J = 2.6 Hz, H-13a), 5.01 (1H, d, J = 2.6 Hz, H-13b), 4.25 (1H, m, H-2), 2.46-1.36 (8H, m, H-3, H-4, H-7, H-8), 1.17 (3H, s, H-15), 1.15 (3H, s, H-14), 0.93 (9H, s, t-Bu), 0.11, 0.08 (6H, s, SiMe2).

Synthesis of Compound 17 (Fig. 3)-- Compound 17 (a colorless oil) was prepared from compound 16 following the procedure for the preparation of compound 9 from compound 8. IR (CHCl3) nu max 3090(w), 3060(w), 2960(s), 2860(s), 1650(w), 1625(w), 1580(w) cm-1; 1H NMR delta  (ppm) 6.08 (1H, dd, J = 10.6, 4.0 Hz, H-11), 5.81 (1H, d, J = 10.6 Hz, H-10), 5.13 (1H, d, J = 2.0 Hz, H-13a), 4.96 (1H, d, J = 2.0 Hz, H-13b), 4.66 (1H, d, JC-H = 156.4 Hz, H-16a), 4.64 (1H, d, JC-H = 156.4 Hz, H-16b), 4.23 (1H, m, H-2), 2.34-1.25 (8H, m, H-3, H-4, H-7, H-8), 1.12 (3H, s, H-14), 1.02 (3H, s, H-15), 0.93 (9H, s, t-Bu), 0.11, 0.08 (6H, s, SiMe2); 13C NMR delta  (ppm) 153.6, 142.9, 136.5, 128.3, 110.0 (C-16 enriched), 106.7, 47.7, 40.3, 32.2, 31.6, 30.1, 27.1, 26.0, 25.1, 22.7, 20.7, 14.1, -4.8.

Synthesis of Compound 18 (Fig. 3)-- Compound 18 (a colorless oil) was prepared from compound 17 similarly to the preparation of compound 10 from compound 9. IR (CHCl3) nu max 3090(w), 3005(w), 2960(s), 1645(w) cm-1; 1H NMR delta  (ppm) 5.29 (1H, br, H-10), 5.12 (1H, d, J = 1.3 Hz, H-13a), 4.87 (1H, d, J = 1.3 Hz, H-13b), 4.22 (1H, m, H-2), 1.64 (3H, br.d, JC-H = 125.4 Hz, H-16), 2.08-1.16 (10H, m, H-3, H-4, H-7, H-8, H-11), 1.09 (3H, s, H-14), 0.93 (9H, s, t-Bu), 0.83 (3H, s, H-15), 0.11, 0.08 (6H, s, SiMe2); 13C NMR delta  (ppm) 159.9, 132.4, 120.3, 106.7, 48.7, 36.5, 32.7, 32.1, 31.6, 30.7, 27.9, 26.0, 24.6, 23.3 (s, C-16 highly enriched), 22.7, 18.4, 17.7, -4.8.

Synthesis of Compound 2b (Fig. 3)-- 2beta -t-Butyldimethylsilyloxy-[16-13C]trichodiene 18 (56 mg; 0.17 mmol), in a 1 M solution of tetrabutylammonium fluoride (0.50 ml; 0.50 mmol) was treated with acetic acid (29 µl; 0.50 mmol) at room temperature for 36 h. The reaction was quenched with 0.5 ml of water, extracted with ether, dried on MgSO4, filtered, and evaporated to dryness. Flash chromatography on silica gel using hexane/ethyl acetate (90:10) yielded 23 mg (62%) of the title compound as a colorless oil: 2beta -hydroxy-[16-13C]trichodiene. IR (CHCl3) nu max 3600(m), 3450(br), 3080(w), 2960(s), 1645(w) cm-1; 1H NMR delta  (ppm) 5.28 (1H, br.t, J = 4.3 Hz, H-10), 5.15 (1H, d, J = 2.6 Hz, H-13a), 4.93 (1H, d, J = 2.6 Hz, H-13b), 4.25 (1H, br.s, H-2), 2.18-1.70 (5H, m), 1.70-1.46 (3H, m), 1.46-1.23 (2H, m), 1.63 (3H, d, J = 126.0 Hz, H-16), 1.10 (3H, s, H-14), 0.84 (3H, s, H-15); 13C NMR delta  (ppm) 161.2 (s, C-12), 132.4 (d, J = 44.0 Hz, C-9), 120.1 (d, J = 2.7 Hz, C-10), 106.5 (s, C-13), 76.9 (s, C-2), 49.5 (s, C-5), 36.6 (s, C-6), 32.7 (br, C-7), 30.6 (s, C-4), 27.9 (d, J = 2.7 Hz, C-8), 27.7 (d, J = 3.7 Hz, C-11), 27.2 (s, C-3), 24.4 (s, C-14), 23.2 (s, C-16 enriched), 17.5 (s, C-15).

Feeding of 12,13-Epoxy-[16-13C]9,10-trichoene-2beta -ol 3b and Purification and Characterization of the Derived 3-ADN and SOL-- The 12,13-epoxy-[16-13C]9,10-trichoene-2beta -ol 3b, 45 mg in total, was dissolved in methanol and was equally distributed among nine sterile 125-ml Erlenmeyer flasks. The methanol was allowed to evaporate from the stoppered flasks overnight. To each of the nine flasks was added 0.1 ml of a 5% Brij 35 solution (15) and one 48-h production culture (previously prepared from a 3-day-old seed culture). Three controls were prepared by adding 0.1 ml of a 5% Brij 35 solution to each of three 48-h production cultures. The cultures were incubated for 7 days at 25 °C and 220 rpm. After 7 days, the three controls and the nine samples were filtered separately. The filtrates were saturated with NaCl and extracted with ethyl acetate. The organic extracts were dried over MgSO4, filtered, and concentrated in vacuo. The sample crude extract was fractionated by preparative HPLC using gradient A at 18 ml/min. Fraction 1 and fraction 3, corresponding to 3-ADN (tR = 36 min) and SOL (tR = 48 min), respectively, were further purified by HPLC. The pure 3-ADN (tR = 37 min) that was isolated contained no 13C incorporation. SOL was acetylated by incubation at 25 °C for 18 h with 90 µl of deuterated acetic anhydride (Ac2O(D6)) and 60 µl of pyridine, purified by HPLC, and the pure diacyl-SOL (tR = 49 min) also contained no 13C incorporation; there was no enriched carbon visible.

Feeding of 12,13-Epoxy-[16-13C]9,10-trichoene-2alpha -ol 3a and Purification and Characterization of the Derived 3-ADN, SOL, pre-SOL, and 11alpha -2beta ,13beta -Apotrichodiol-- The 12,13-epoxy-[16-13C]9,10-trichoene-2alpha -ol, 38 mg in total, was dissolved in ether and equally distributed among eight sterile 125-ml Erlenmeyer flasks. The ether was allowed to evaporate from the stoppered flasks overnight. To each of the eight flasks was added 0.5 ml of a 5% Brij 35 solution and one 24-h production culture (previously prepared from a 2-day-old seed culture). Three controls were prepared by adding 0.5 ml of a 5% Brij 35 solution to each of three 24-h production cultures. The cultures were incubated for 5 days at 25 °C and 220 rpm. After 5 days, the three controls and the eight samples were filtered separately. The filtrates were saturated with NaCl and extracted with ethyl acetate. The organic extracts were dried over MgSO4, filtered, and concentrated in vacuo. This crude extract was fractionated by analytical HPLC using gradient A at 1 ml/min. Fraction 1, corresponding to 3-ADN (tR = 38-43 min) was further purified by HPLC (tR = 39 min). After evaporation, the structure of 3-ADN was confirmed by high resolution mass spectrometry. FAB-MS: (M + Na)+: 361.1264; C17H22O7Na+ requires 361.1263. The 13C NMR spectra of the 3-ADN show that the site of incorporation of 13C is C-16 at 15.3 ppm. The percentage of 13C incorporation calculated by low resolution mass spectrometry was 44.1%. From NMR we obtained the ratio of 32:68 between 13C-enriched and 12C-nonenriched metabolites by comparing the integration peaks of the doublet derived from the coupling of 13C-H-16 and the singlet 12C-H-16 at 1.92 ppm (JC-H = 129.0 Hz). Fraction 3, corresponding to SOL (tR = 55 min) was isolated and evaporated. FAB-MS: (M + H)+: 267.1597; C15H22O4H+ requires 267.1596. The 13C NMR spectra of SOL show incorporation of 13C at 23.0 ppm, corresponding to C-16. The percentage of 13C incorporation calculated by low resolution mass spectrometry was 49.9%. From NMR we obtained the ratio of 57:43 between 13C-enriched and 12C-nonenriched metabolites by comparing the integration peaks of the doublet derived from the coupling of 13C-H-16 and the singlet 12C-H-16 at 1.76 ppm (JC-H = 126.5 Hz). Fraction 5, corresponding to pre-SOL (tR = 62-64 min) was further purified by analytical HPLC using 55% methanol, 45% water at 1 ml/min (tR = 64 min). After evaporation, the structure was confirmed by high resolution mass spectrometry. FAB-MS: (M + Na)+: 273.1466; C15H22O3Na+ requires 273.1467. The 13C NMR spectra of pre-SOL show incorporation of 13C at 23.0 ppm, corresponding to C-16. The percentage of 13C incorporation calculated by low resolution mass spectrometry was 51.7%. From NMR, we obtained the ratio of 44:56 between 13C-enriched and 12C-nonenriched metabolites by comparing the integration peaks of the doublet derived from the coupling of 13C-H-16 and the singlet 12C-H-16 at 1.84 ppm (JC-H = 126.0 Hz). Fraction 6 (tR = 64-69 min), from HPLC gradient A, was acetylated with 145 µl of deuterated acetic anhydride and 135 µl of pyridine at 25 °C overnight. The acetic anhydride and pyridine were evaporated under a stream of nitrogen, and the acetylated fraction was purified by analytical HPLC using 65% methanol, 35% water at 1 ml/min (tR = 56 min). NMR characterization identified this compound as 11alpha -2beta ,13beta -apotrichodiol, which has previously been isolated from cultures of F. culmorum (14). The 13C NMR of 11alpha -2beta ,13beta -apotrichodiacetate shows incorporation of 13C at 23.4 ppm corresponding to C-16. The 1H NMR spectra of the diacetylated compound (11alpha -2beta ,13beta -apotrichodiacetate) is as follows: delta  (ppm) 5.55 (1H, br.m, H-10), 5.16 (1H, t, J2,3 = 5.1 Hz, H-2), 4.38 (1H, d, JA,B = 11.7 Hz, H-13A), 4.12 (1H, d, JA,B = 11.7 Hz, H-13B), 3.77 (1H, br.d, J10,11 = 5.3 Hz, H-11), 1.94 (1H, m, H-3A), 1.71 (3H, d, J = 126.0 Hz, H-16), 1.64 (1H, om, H-3B), 1.04 (3H, s, H-14), 0.82 (3H, s, H-15); 2H NMR delta  (ppm) 2.01, 1.97 (6H, s, OAc); 13C NMR delta  (ppm) 118.4 (d, JC-10,C-16 = 1.8 Hz, C-10), 81.6 (s, C-2), 79.0 (s, C-11), 66.0 (s, C-13), 34.4 (s, C-4), 30.2 (s, C-3), 23.4 (s, C-16 enriched), 17.9 (s, C-14), 15.1 (s, C-15). From this 1H NMR, we obtained a ratio of 90:10 between 13C-enriched and 12C-nonenriched metabolites by comparing the integration peaks of the doublet derived from the coupling of 13C-H-16 and the singlet 12C-H-16 at 1.71 ppm (JC-H = 126.0 Hz).

Feeding of 2alpha -Hydroxy-[16-13C]trichodiene 2a and/or [15-2H]12,13-Epoxytrichodiene 2c and Purification and Characterization of the Derived 3-ADN and SOL-- -2alpha -Hydroxy-[16-13C]trichodiene 2a, 12 mg in total dissolved in acetone, was equally distributed to six 125-ml Erlenmeyer flasks. A mixture of 2alpha -hydroxy-[16-13C]trichodiene 2a (10 mg) and [15-2H]12,13-epoxytrichodiene 2c (10 mg) dissolved in acetone was equally distributed to each of five 125-ml Erlenmeyer flasks. The acetone was allowed to evaporate, and to each of the 11 flasks was added 0.5 ml of a 5% Brij 35 solution and one 48-h production culture (previously prepared from a 3-day-old seed culture). Two controls were prepared by adding 0.5 ml of a 5% Brij 35 solution to each of two 48-h production cultures. The cultures were incubated for 5 days at 25 °C and 220 rpm. After 5 days, the two controls and the samples from each individual feeding experiment were filtered separately. The filtrates were saturated with NaCl and extracted with ethyl acetate. The organic extracts were dried over MgSO4, filtered, and concentrated in vacuo. The crude extracts were fractionated by analytical HPLC, and 3-ADN and SOL were isolated and purified as described for the feeding of 3a. When 2a or a mixture of 2a and 2c was fed, the 3-ADN structure was confirmed by high resolution mass spectrometry. FAB-MS: (M + Na)+: 361.1264; C17H22O7Na+ requires 361.1263. The 13C NMR spectra of the 3-ADN derived from the feeding of 2a or of 2a and 2c showed incorporation at C-16 at 15.2 or 15.3 ppm, respectively. No deuterated 3-ADN was obtained from the feeding of the mixture 2a and 2c. The percentage of 13C incorporation calculated by low resolution mass spectrometry when 2a or 2a and 2c were fed was 25.9 and 28.8%, respectively. From the 1H NMR of the 3-ADN isolated from the feeding of 2a or 2a and 2c to F. culmorum, we obtained the ratio of 29:71 and 36:64, respectively, between 13C-enriched and 12C-nonenriched metabolites by comparing the integration peaks of the doublet derived from the coupling of 13C-H -16 and the singlet 12C-H -16 at 1.90 ppm (JC-H = 128.9 Hz). When 2a was fed, the purified isolated SOL (obtained as described for the feeding of 3a) was acetylated by incubation at 25 °C with 120 µl of acetic anhydride and 90 µl of pyridine. The diacyl-SOL was purified by analytical HPLC using 65% methanol, 35% water at a flow rate of 1 ml/min (tR = 25.8 min), and the structure was confirmed by high resolution mass spectrometry. FAB-MS: (M + Na)+: 373.1627; C19H26O6Na+ requires 373.1627. The 13C NMR spectra of the diacyl-SOL derived from the feeding of 2a give the exact site of incorporation at C-16 at 23.0 ppm. The percentage of 13C incorporation calculated by low resolution mass spectrometry was 34.2%. From NMR, we obtained the ratio of 39:61 between 13C-enriched and 12C-nonenriched metabolites by comparing the integration peaks of the doublet derived from the coupling of 13C-H-16 and the singlet 12C-H-16 at 1.76 ppm (JC-H = 126.2 Hz). When a mixture of 2a and 2c was fed there was not enough sambucinol to isolate.

Feeding of 2beta -Hydroxy-[16-13C]trichodiene 2b and Purification and Characterization of the Derived 3-ADN and SOL-- The 2beta -hydroxy-[16-13C]trichodiene, 50 mg in total, was dissolved in methanol and equally distributed among 10 sterile 125-ml Erlenmeyer flasks. The methanol was allowed to evaporate from the stoppered flasks overnight. To each of the 10 flasks was added 0.1 ml of a 5% Brij 35 solution and one 48-h production culture (previously prepared from a 3-day-old seed culture). Two controls were prepared by adding 0.1 ml of a 5% Brij 35 solution to each of two 48-h production cultures. The cultures were incubated for 5 days at 25 °C and 220 rpm. After 5 days, the two controls and the 10 samples were filtered separately. The filtrates were saturated with NaCl and extracted with ethyl acetate. The organic extracts were dried over MgSO4, filtered, and concentrated in vacuo. The crude extract was fractionated by semipreparative HPLC, using gradient A, at 3 ml/min. The 3-ADN that was isolated, as described for the feeding of 3a, showed no incorporation of 13C at C-16 in the 13C NMR. There was not enough SOL to isolate due to inhibition during the feeding.

Isolation, Purification, and Characterization of 2alpha -Hydroxytrichodiene 2a and 12,13-Epoxy-9,10-trichoene-2alpha -ol 3a from F. culmorum Cultures-- Three-day-old production cultures of F. culmorum were filtered through miracloth (Calbiochem) to remove the mycelia. Each liter of the filtrate (5 liters in total) was extracted with ethyl acetate (3 × 500 ml), the organic layer was washed with a saturated solution of NaCl (3 × 500 ml), dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure to give a yellow oil (1.4 g). This crude extract was dissolved in methanol and fractionated on preparative HPLC using program B at 18 ml/min. The region with a retention time between 49 and 60 min was collected. The combined fractions were evaporated to a 10-ml volume under reduced pressure and extracted with pentane (3 × 5 ml). Evaporation of the pentane was done with a stream of nitrogen, and the extract was dissolved in methanol to be repurified by fractionation on semipreparative HPLC using program B at 3 ml/min. The peaks with a retention time corresponding to standard 12,13-epoxy-9,10-trichoene-2alpha -ol (3a, tR = 52.5 min) and 2alpha -hydroxytrichodiene (2a, tR = 55.8 min) were collected. Each fraction was extracted with pentane, which was then removed under nitrogen. There was enough of metabolite 3a to be characterized by NMR, and indeed its proton NMR was identical to the standard compound synthesized in our laboratory as well as the NMR published from its accumulation in the media with ancymidol (8). In order to rigorously identify the metabolite that had the same retention time on HPLC as 2a, we acetylated it with radiolabeled acetic anhydride. The compound was dissolved in a mixture of 120 µl of pyridine and 25 µl (12.5 µCi) of [1'-14C]acetic anhydride (100 mCi/mmol). The mixture was incubated for 24 h at 25 °C, and then 145 µl of unlabeled acetic anhydride was added, and the mixture was incubated for an additional 24 h at 25 °C. Following the removal of acetic anhydride and pyridine, methanol was added, and the solution was fractionated by analytical HPLC using 80% methanol and 20% water at a flow rate of 1 ml/min. The peak observed at tR = 56 min was collected and extracted using a ChemElut tube (Extube®, Varian) with 4 × 10 ml of pentane and evaporated under a stream of nitrogen to 20 µl and spotted on a high performance TLC plate. The plate was developed with ethyl acetate/hexane (1:4) and then analyzed with the Bioscan imaging scanner system. One symmetrical radioactive peak was detected with an RF of 0.81. In order to ensure that the compound was pure and was identical to synthesized 2alpha -acetyl-trichodienol, it was purified on HPLC using three different conditions of elution at 1 ml/min, and its retention time coincided with the standard. The conditions were as follows: 80% methanol, 20% water, tR = 56 min; 85% methanol, 15% water, tR = 32 min; and HPLC program B, tR = 61 min.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Putative Post-trichodiene Oxygenated Trichothecene Precursors (Fig. 1)-- Putative post-trichodiene oxygenated precursors were synthesized with a stable isotope label (13C or 2H) at a nonlabile position and fed to F. culmorum cultures. The derived metabolites were analyzed by 13C or 2H NMR and mass spectrometry to determine if the precursor was incorporated and the site and extent of incorporation. In order to confirm the preliminary data (8) suggesting that 12,13-epoxy-9,10-trichoene-2alpha -ol (3a, Fig. 1) is a post-trichodiene biosynthetic precursor to trichothecenes, we synthesized it with a 13C label at C-16 (Fig. 2). Its stereoisomer 12,13-epoxy-[16-13C]9,10-trichoene-2beta -ol (3b, Fig. 2) was also prepared with a 13C label at C-16. We wanted to determine if both stereoisomers were precursors to trichothecenes, implying a 2-keto-12,13-epoxy-9,10-trichoene intermediacy. The syntheses of two of the three plausible monooxygenated trichodiene intermediates (2a and 2b, Figs. 2 and 3B) are closely related to that of 3a and 3b. We therefore decided to synthesize the different stereoisomers of the putative monooxygenated precursors (2a-2c, Figs. 2 and 3) as well as 3a and 3b, specifically labeled with stable isotopes (13C or 2H). The dioxygenated trichodiene intermediates 3a and 3b (Fig. 2) and the monooxygenated stereoisomers of 2-hydroxytrichodiene (2alpha - and 2beta -) were labeled with a 13C label at C-16 (2a and 2b, Figs. 2 and 3B), while 12,13-epoxytrichodiene was labeled with a deuterium at C-15 (2c, Fig. 3A). We have prepared only the beta -epoxide (2c), since all natural trichothecenes have this stereochemistry. The rationale for using a different isotope for 2a, 2b, and 2c is to enable the simultaneous feeding of two plausible monooxygenated precursors (2a and 2c; 2b and 2c) under the exact same conditions. The analysis of the derived products will therefore determine the relative incorporations. We confirmed these results by separate feedings of each compound. Below, the synthetic methods for specifically labeling 3a and 3b and 2a, 2b, and 2c will be described.


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Fig. 1.   Plausible bicyclic precursors post-trichodiene (1) to the trichothecenes 3-ADN and SOL: 2alpha -hydroxytrichodiene 2a; 2beta -hydroxytrichodiene 2b; 12,13-epoxytrichodiene 2c; 12,13-epoxy-9,10-trichoene-2alpha -ol 3a; 12,13-epoxy-9,10-trichoene-2beta -ol 3b. Deoxynivalenol (DON), also called vomitoxin, is the cause of vomitoxicosis affecting animals ingesting corn infected with Fusarium species.


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Fig. 2.   Syntheses of 2alpha -hydroxy-[16-13C]trichodiene 2a; 12,13-epoxy-[16-13C]9,10-trichoene-2alpha -ol 3a; and 12,13-epoxy-[16-13C]9,10-trichoene-2beta -ol 3b. TBDMS, t-butyldimethylsilyloxy.


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Fig. 3.   A, synthesis of [15-2H]12,13-epoxytrichodiene 2c; B, synthesis of 2beta -hydroxy-[16-13C] trichodiene 2b. OTBDMS, t-butyldimethylsilyloxy.

Syntheses of 12,13-Epoxy-[16-13C]9,10-trichoene-2beta -ol 3b, 12,13-Epoxy-[16-13C]9,10-trichoene-2alpha -ol 3a, 2alpha -Hydroxy-[16-13C]trichodiene 2a, 2beta -Hydroxy-[16-13C]trichodiene 2b, [15-2H]12,13-Epoxytrichodiene 2c (Figs. 2 and 3)-- This is the first time these labeled compounds were prepared. The 13C label was introduced via a Wittig reaction of a ketone with Ph3P13CH3I. No unlabeled reagent was added in order to ensure 100% incorporation of 13C. The 2H label was derived from reduction of an aldehyde group at C-15 with NaB2H4, thereby introducing one deuterium in the resulting primary alcohol in the two prochiral hydrogens, prior to reducing it to a methyl group. The reactions involved in the syntheses of 3a, 3b, and 2a are the following. The tricarbonyl [4-methoxy-1-methyl-1-(1-methyl-2-methylenecyclopentyl)-(2-5-eta )-cyclohexa-2,4-dienyl] iron (4) was prepared according to Pearson's method (11, 25) and was oxidized using selenium dioxide and a 90% solution of tert-butyl hydroperoxide (16). Compound 5 was obtained with an allylic alcohol at C-2 with a beta -orientation as confirmed by NOESY experiments. Indeed, the NOE correlations of 5 show a strong interaction between H-2 and Me-15, thereby confirming the existence of a beta -OH on C-2. Decomplexation of compound 5, using CuCl2 in ethanol (17) produced enone 6. Subsequent epoxidation of enone 6 using vanadyl acetylacetonate with a 90% solution of tert-butyl hydroperoxide (16) and silylation of the epoxide 7 with t-butyldimethylsilyl chloride produced compound 8. Wittig olefination of compound 8 with Ph3P13CH3I (12) enabled the introduction of the 13C label at C-16 (trichothecene numbering) to afford compound 9, which was subsequently reduced with sodium in liquid ammonia to give compound 10 (11, 25). Desilylation of compound 10 with tetrabutylammonium fluoride produced 12,13-epoxy-[16-13C]9,10-trichoene-2beta -ol (3b). This compound was acetylated (Ac-3b), and its NOE correlations determined by the NOESY experiment show a strong interaction between H-2 and Me-15, thereby confirming the existence of a beta -OH on C-2.

In order to synthesize compound 3a containing the C-2 hydroxy with an alpha  configuration, the allylic alcohol of compound 3b had to be inverted. Alcohol 3b was inverted via its para-toluenesulfonyloxy intermediate 1 with cesium acetate in dimethylformamide (18) to produce acetate 12, which was then hydrolyzed to obtain 12,13-epoxy-[16-13C]9,10-trichoene-2alpha -ol 3a. The NOE correlations of 12 determined by the NOESY experiment show a strong interaction between H-2 and H-13b and between Me-14 and H-3a, thereby confirming the existence of an alpha -OAc on C-2. After hydrolysis, compound 3a has therefore an alpha -OH on C-2. The proton NMR spectra of 3a show C-16 as a doublet (J = 125.2 Hz, Fig. 4A) derived by the coupling 13C-H-16 and no singlet that would arise from 12C-H-16. This constitutes a proof that we succeeded in synthesizing 3a practically 100% with 13C at position 16. 


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Fig. 4.   Feeding results of metabolite 3a to F. culmorum cultures (black-square represents 13C). A, 1H NMR region at H-16 of 3a showing the doublet resulting from H-16-13C coupling with no singlet in the middle corresponding to H-16-12C: 3a is 100% labeled with 13C at C-16. B, 13C NMR of the isolated 3-ADN with a large enriched 13C-16; the natural abundance 13C-13 is shown to emphasize the incorporation at C-16. C, 13C NMR of SOL also with a large enriched 13C-16 peak. D, 13C NMR of pre-SOL with a large enriched 13C-16; the natural abundance of 13C-13 is shown to emphasize the incorporation at C-16. E, 13C NMR of 11alpha -2beta ,13beta -apotrichodiacetate with a large enriched 13C-16; the natural abundance of 13C-4 is shown to emphasize the incorporation at C-16.

When acetate 12 was treated with WCl6 and n-butyllithium (19), the olefin 13 was produced, and upon hydrolysis, 2alpha -hydroxy-[16-13C]trichodiene 2a was isolated.

2beta -Hydroxy-[16-13C]trichodiene 2b was also prepared from enone 6 using the same procedure as for the preparation of compound 3b except that 6 was not epoxidized. The NOE correlations of 2b determined by the NOESY experiment show a strong interaction between H-2 and Me-15, thereby confirming the existence of a beta -OH on C-2.

[15-2H]12,13-Epoxytrichodiene (2c) was prepared from [15-2H]trichodiene (14) by bromination of the deuterated trichodiene 14 to give the 9,10-dibromide, which was epoxidized, using m-chloroperoxybenzoic acid, to give the 12,13-epoxy-9,10-dibromide 15 (13). Compound 15 was debrominated using zinc dust in ethanol (20) to yield the desired deuterated epoxytrichodiene 2c.

Biosynthesis of Trichothecenes: Dioxygenated Precursor Post-trichodiene (Fig. 4)-- Radiolabeling experiments suggested that 12,13-epoxy-9,10-trichoene-2alpha -ol was a precursor to trichothecenes (8). The site of incorporation could not be obtained, since no degradation was done on the derived radiolabeled trichothecenes. We therefore decided to confirm it by synthesizing this potential precursor with 13C at position 16: 12,13-epoxy-[16-13C]9,10-trichoene-2alpha -ol 3a. After the feeding, no degradations are necessary, since we can locate the label by 13C NMR spectroscopy. In order to ensure that only the 2alpha -stereoisomer is the precursor, we also synthesized 12,13-epoxy-[16-13C]9,10-trichoene-2beta -ol (3b, Figs. 1 and 2). When 3b was fed to F. culmorum cultures, the 3-ADN isolated showed no enrichment in 13C, and after 927 scans, all carbons were of equal intensity. Similarly, the isolated SOL, which was acetylated with deuterated acetic anhydride for purification reasons, also showed no enrichment in 13C.

On the other hand, the 3-ADN derived from the feeding of 3a under the exact same conditions showed a peak very clearly in the 13C NMR spectra at C-16 (15.3 ppm) (Fig. 4). Also, the average percentage of 13C incorporation (ratio between 13C-enriched and nonenriched 3-ADN) as calculated by mass spectrometry and 1H NMR data is 38%. Three more compounds isolated from the feeding of 3a showed in the 13C NMR spectra a large peak at C-16 (Fig. 4): SOL, pre-SOL (a precursor to SOL (21)), and a dead end metabolite, 11alpha -2beta ,13beta -apotrichodiol, previously isolated from F. culmorum (14). This last metabolite was diacetylated after isolation to facilitate its purification (Fig. 4). The structure of this apotrichothecene was rigorously proven by independent synthesis (14) (Fig. 4). No mass spectral data are available for 11alpha -2beta ,13beta -apotrichodiol, because it decomposed prior to analysis. We have, however, the detailed 1H and 13C NMR characterization, and they are identical to the ones reported except for the enrichment in 13C. It is interesting to note that the conversion of 3a to these three metabolites seems even more efficient than to 3-ADN. Indeed, the percentage of incorporation of 3a into SOL is ~54%; into pre-SOL ~48%, and into 11alpha -2beta ,13beta -apotrichodiol ~90% (experimental). This very large incorporation of 3a into this apotrichothecene will be discussed below.

We also succeeded in isolating 12,13-epoxy-9,10-trichoene-2alpha -ol from a large amount of crude extract prepared from F. culmorum. There was enough metabolite to characterize by NMR spectroscopy, and the data were identical to those of the unlabeled synthesized compound.

Biosynthesis of Trichothecenes: Sequence of the Oxygenations Post-trichodiene (Figs. 5 and 6)-- The results shown in Fig. 5 prove conclusively for the first time that the sequence of the two oxygenation steps of the hydrocarbon trichodiene are as follows: first hydroxylation at position 2alpha , leading to 2alpha -hydroxytrichodiene, followed by the epoxidation at C-12-C-13 to give 12,13-epoxy-9,10-trichoene-2alpha -ol, which has been successfully incorporated to both 3-ADN and SOL (see above). The feeding of 2alpha -hydroxy-[16-13C]trichodiene 2a to F. culmorum cultures gave 3-ADN and SOL (which was diacetylated in order to purify it) with a clearly enriched C-16 in the 13C NMR spectra at 15.2 and 23.0 ppm, respectively (Fig. 5). In addition, the incorporation of 2a into 3-ADN and SOL was significant as calculated by mass spectrometry and 1H NMR data (experimental): ~28% into 3-ADN and ~37% into SOL. When the two possible monooxygenated trichodiene precursors, 2alpha -hydroxy-[16-13C]trichodiene 2a and [15-2H]12,13-epoxytrichodiene 2c, were simultaneously fed to F. culmorum cultures, the production of 3-ADN and SOL was inhibited to the extent that no SOL could be isolated for characterization. The only trichothecene that was produced, 3-ADN, was highly enriched with 13C at position 16 (Fig. 5). There was no deuterated 3-ADN produced. In order to ensure that the 2alpha -hydroxy is absolutely required, we also synthesized the stereoisomer: 2beta -hydroxy-[16-13C]trichodiene 2b and fed it to F. culmorum cultures. It seems to inhibit considerably the production of trichothecenes. SOL could not be isolated, and the 3-ADN obtained was unlabeled. We are therefore very confident that the first oxygenation step post-trichodiene is the formation of 2alpha -hydroxytrichodiene. In addition, we succeeded in finding 2alpha -hydroxytrichodiene as a metabolite by radioactive dilution. A large amount of crude extract was prepared from production cultures of F. culmorum. After successive purifications on HPLC, a very small peak was found with the same retention time as unlabeled 2alpha -hydroxytrichodiene. The amount was too small to isolate and characterize by NMR. We therefore decided to acetylate that minute quantity with [1-14C]acetic anhydride and obtained a single peak on HPLC that had all of the radioactivity transferred to the new peak at a retention time identical to that of standard 2alpha -acetyltrichodienol. In one experiment, the compound was purified to constant specific activity under three different analytical HPLC conditions that gave peaks with retention times identical to that of the synthetic acetylated standard. A Bioscan tracing of 2alpha -[1'-14C]trichodienol acetate demonstrated the symmetrical distribution.


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Fig. 5.   Feeding results of metabolite 2a or of 2a and 2c to F. culmorum cultures (black-square represents 13C). a, 13C NMR of 2a and 2H NMR of 2c. b, 13C NMR of the derived 3-ADN from both feedings; no deuterated 3-ADN was detected. c, 13C NMR of the derived SOL from the feeding of 2a.


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Fig. 6.   Biosynthesis of 3-ADN and SOL. The metabolite in brackets has not been isolated in F. culmorum but has been found in other fungi (24). The thick arrows emphasize metabolic steps that have been rigorously proven. The other arrows are postulated based on the present work.

A trioxygenated derivative of trichodiene (4a, Fig. 6) has been isolated and converted to 3-ADN (22, 23). The conversion of 4a to sambucinol has never been shown, but it seems that the fungal strain utilized did not produce sambucinol, since it was not reported. We can therefore postulate that 4a is probably the more plausible trioxygenated intermediate of 3-ADN and SOL. The biosynthetic pathway of 3-ADN and SOL seem to bifurcate at an early stage (21). An attractive metabolite that would be converted to both trichothecenes could be 4a. A fourth hydroxylation could give compound 5a, which is a natural product and has been isolated from Fusarium sporotrichiodes (24). Metabolite 5a has a hydroxyl at position 3 with the correct stereochemistry and would be converted to the tricyclic metabolite isotrichodermin (Fig. 6), a proven biosynthetic precursor to 3-ADN (9, 21). The conversion of isotrichodermin to 3-ADN involves four oxidations and six metabolic conversions from 3a. 12,13-Epoxytrichothec-9-ene (Fig. 6, EPT) has been converted to pre-SOL and SOL with no connection to the isotrichodermin metabolic pathway (21). Therefore, we can see (Fig. 6) that more steps are involved from 3a to 3-ADN than from 3a to pre-SOL and SOL, which accounts for the relative incorporations. One conversion that seems particularly efficient is the feeding of 12,13-epoxy-[16-13C]9,10-trichoene-2alpha -ol 3a to F. culmorum cultures, which leads to 90% of the 13C being incorporated into the apotrichothecene 11alpha -[16-13C]2beta ,13beta -apotrichodiol (Fig. 6). One possibility could be that the SN2-type water attack on C-2, the cleavage of the ether bond, and opening of the epoxide of 12,13-epoxytrichothec-9-ene could happen simultaneously on the enzyme surface. On the other hand, the conversion of 12,13-epoxytrichothec-9-ene to pre-SOL and SOL is probably more involved. Knowledge of the sequence of the different oxygenations will be very helpful in understanding these ubiquitous enzymes.

    FOOTNOTES

* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Canadian Breast Cancer Research Initiative (to L. O. Z.) and the Medical Research Council (to O. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Centre de Recherche en Microbiologie Appliquée, Université du Québec, Institut Armand-Frappier, 531 Blvd. des Prairies, Laval, Québec H7N 4Z3, Canada. Tel.: 450-687-5010 (ext. 4260); Fax: 514-481-2797; E-mail: Lolita_Zamir{at}iaf.uquebec.ca.

    ABBREVIATIONS

The abbreviations used are: 3-ADN, 3-acetyldeoxynivalenol; SOL, sambucinol; pre-SOL, 3-deoxysambucinol; HPLC, high performance liquid chromatography; s, d, t, q and m, the usual singlet, doublet, triplet, quartet, and multiplet signals in NMR spectra; br and o, broad or overlapping lines, respectively; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect two-dimensional spectroscopy; FAB, fast atom bombardment; MS, mass spectrometry; tR, retention time.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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