 |
INTRODUCTION |
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-2
-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.
 |
EXPERIMENTAL PROCEDURES |
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 5 (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)
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
(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
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
(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 7 (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)
max 3520 (br), 3010, 2980, 2880, 1675 cm
1;
1H NMR
(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
(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 9 (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)
max 3010, 2960, 2890, 2860, 1630, 1580, 1255 cm
1; 1H NMR
(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
(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 (2
-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-2
-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
(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
(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-2
-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,
2
-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:
2
-acetoxy-12,13-epoxy-[16-13C]9,10-trichoene. IR
(CHCl3)
max 3005, 2970, 1730, 1440, 1370, 1245, 970 cm
1; 1H NMR
(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
(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
2
-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-2
-ol).
1H NMR
(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.
2
-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 2
-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: 2
-hydroxy-[16-13C]trichodiene. IR
(CHCl3)
max 3600, 3450, 3080, 2960, 1645 cm
1; 1H NMR
(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
(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
9
,10
-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)
(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
(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)
max 3090(w), 3030(w), 2960(s), 2860(s), 1670(s), 1650(m)
cm
1; 1H NMR
(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)
max 3090(w), 3060(w), 2960(s), 2860(s), 1650(w),
1625(w), 1580(w) cm
1; 1H NMR
(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
(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)
max
3090(w), 3005(w), 2960(s), 1645(w) cm
1; 1H
NMR
(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
(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)--
2
-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:
2
-hydroxy-[16-13C]trichodiene. IR (CHCl3)
max 3600(m), 3450(br), 3080(w), 2960(s), 1645(w)
cm
1; 1H NMR
(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
(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-2
-ol 3b and
Purification and Characterization of the Derived 3-ADN and
SOL--
The 12,13-epoxy-[16-13C]9,10-trichoene-2
-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-2
-ol 3a
and Purification and Characterization of the Derived 3-ADN, SOL,
pre-SOL, and 11
-2
,13
-Apotrichodiol--
The
12,13-epoxy-[16-13C]9,10-trichoene-2
-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
11
-2
,13
-apotrichodiol, which has previously been isolated from
cultures of F. culmorum (14). The 13C NMR of
11
-2
,13
-apotrichodiacetate shows incorporation of 13C at 23.4 ppm corresponding to C-16. The 1H
NMR spectra of the diacetylated compound
(11
-2
,13
-apotrichodiacetate) is as follows:
(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
(ppm) 2.01, 1.97 (6H, s, OAc); 13C NMR
(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 2
-Hydroxy-[16-13C]trichodiene
2a and/or [15-2H]12,13-Epoxytrichodiene
2c and Purification and Characterization of the Derived
3-ADN and
SOL--
-2
-Hydroxy-[16-13C]trichodiene
2a, 12 mg in total dissolved in acetone, was equally
distributed to six 125-ml Erlenmeyer flasks. A mixture of
2
-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 2
-Hydroxy-[16-13C]trichodiene
2b and Purification and Characterization of the Derived
3-ADN and SOL--
The 2
-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
2
-Hydroxytrichodiene 2a and
12,13-Epoxy-9,10-trichoene-2
-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-2
-ol (3a, tR = 52.5 min) and 2
-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 2
-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 |
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-2
-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-2
-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 (2
- and
2
-) 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
-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:
2 -hydroxytrichodiene 2a;
2 -hydroxytrichodiene 2b;
12,13-epoxytrichodiene 2c;
12,13-epoxy-9,10-trichoene-2 -ol 3a;
12,13-epoxy-9,10-trichoene-2 -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
2 -hydroxy-[16-13C]trichodiene
2a;
12,13-epoxy-[16-13C]9,10-trichoene-2 -ol
3a; and
12,13-epoxy-[16-13C]9,10-trichoene-2 -ol
3b. TBDMS, t-butyldimethylsilyloxy.
|
|

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Fig. 3.
A, synthesis of
[15-2H]12,13-epoxytrichodiene 2c;
B, synthesis of 2 -hydroxy-[16-13C]
trichodiene 2b. OTBDMS,
t-butyldimethylsilyloxy.
|
|
Syntheses of
12,13-Epoxy-[16-13C]9,10-trichoene-2
-ol
3b,
12,13-Epoxy-[16-13C]9,10-trichoene-2
-ol
3a, 2
-Hydroxy-[16-13C]trichodiene
2a, 2
-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-
)-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
-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
-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-2
-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
-OH on C-2.
In order to synthesize compound 3a containing the C-2
hydroxy with an
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-2
-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
-OAc on C-2.
After hydrolysis, compound 3a has therefore an
-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 ( 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
11 -2 ,13 -apotrichodiacetate with a large enriched
13C-16; the natural abundance of 13C-4 is shown
to emphasize the incorporation at C-16.
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When acetate 12 was treated with WCl6 and
n-butyllithium (19), the olefin 13 was produced,
and upon hydrolysis, 2
-hydroxy-[16-13C]trichodiene
2a was isolated.
2
-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
-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-2
-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-2
-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 2
-stereoisomer is the precursor, we also synthesized
12,13-epoxy-[16-13C]9,10-trichoene-2
-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,
11
-2
,13
-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
11
-2
,13
-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 11
-2
,13
-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-2
-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 2
, leading to 2
-hydroxytrichodiene,
followed by the epoxidation at C-12-C-13 to give
12,13-epoxy-9,10-trichoene-2
-ol, which has been successfully
incorporated to both 3-ADN and SOL (see above). The feeding of
2
-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,
2
-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 2
-hydroxy is absolutely required, we also synthesized the
stereoisomer: 2
-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 2
-hydroxytrichodiene. In addition, we succeeded in
finding 2
-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 2
-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
2
-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
2
-[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 ( 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.
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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-2
-ol 3a
to F. culmorum cultures, which leads to 90% of the
13C being incorporated into the apotrichothecene
11
-[16-13C]2
,13
-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.