Biotransformation of 4β-hydroxyeudesmane-1,6-dione by Gliocladium roseum and Exserohilum halodes

Article abstract of DOI:10.1016/S0031-9422(01)00340-5

Biotransformation of sesquiterpene 4β-hydroxyeudesmane-1,6-dione by the filamentous fungi Gliocladium roseum and Exserohilum halodes was achieved. With Exserohilum halodes, only one metabolite was obtained, as a result of the regio- and stereo-selective reduction of the keto group at C-1, which is difficult to achieve by chemical means. Five metabolites were produced with Gliocladium roseum, three of which, the 7α-hydroxylated, the 7α, 11- and the 1α,8α-dihydroxylated derivatives, have not previously been reported. The hydroxylation at C-11 is the main action of this microorganism. These 11-hydroxylated compounds can be chemically transformed into 6β.12-eudesmanolides.

Full text of DOI:10.1016/S0031-9422(01)00340-5

Phytochemistry 58 (2001) 891–895
www.elsevier.com/locate/phytochem
Biotransformation of 4b-hydroxyeudesmane-1,6-dione by
Gliocladium roseum and Exserohilum halodes
Andres Garcıa-Granadosa,*, Marıa C. Gutierreza, Francisco Rivasa, Jose M. Ariasb
a
´
Departamento de Quımica Organica, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain
´
Departamento de Microbiologıa, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain
b
´
Received 8 June 2001; received in revised form 24 July 2001
Abstract
Biotransformation of sesquiterpene 4b-hydroxyeudesmane-1,6-dione by the filamentous fungi Gliocladium roseum and Exsero-
hilum halodes was achieved. With Exserohilum halodes, only one metabolite was obtained, as a result of the regio- and stereo-
selective reduction of the keto group at C-1, which is difficult to achieve by chemical means. Five metabolites were produced with
Gliocladium roseum, three of which, the 7a-hydroxylated, the 7a,11- and the 1a,8a-dihydroxylated derivatives, have not previously
been reported. The hydroxylation at C-11 is the main action of this microorganism. These 11-hydroxylated compounds can be
chemically transformed into 6b,12-eudesmanolides. # 2001 Published by Elsevier Science Ltd.
Keywords: Biotransformation; Sesquiterpene; Eudesmane; Hydroxylation; Gliocladium roseum; Exserohilum halodes
1. Introduction
In previous papers (Garcıa-Granados et al., 1991,
1993) we reported the incubations of several 4b-hydro-
xyeudesmane compounds by the filamentous fungi Cur-
vularia lunata (ATCC 12017) and Rhizopus nigricans
(ATCC 10404). The action of these microorganisms was
directed mainly to the isopropyl moiety, Curvularia
lunata to C-12 and Rhizopus nigricans to C-11. In both
cases, the best results were obtained on the 1,6-diketo
derivative. Consequently, we have chosen this substrate
to carry out several biotransformations with the
microorganisms Exserohilum halodes and Gliocladium
roseum.
Gliocladium roseum (CECT 2733), the anamorphic
form of Nectria ochroleuca (IMI 40022), is a filamentous
fungus that had been used previously to hydroxylate the
sesquiterpene patchoulol (Becher et al., 1978) as well as
to biotransform a derivative of the diterpene varodiol in
order to produce an ent-ambrox1 derivative (Garcıa-
Granados et al., 1999). Exserohilum halodes (CECT
2716) is a synonym of Exserohilum rostratum, the ana-
morphic form of Setosphaeria rostrata (IMI 76563).
Eudesmane sesquiterpenes are common in nature
(Fischer et al., 1979; Fraga, 2000) and they possess sig-
nificant biological properties (Rodrıguezet al., 1976;
Picman, 1986; Robles et al., 1995; Marles et al., 1995;
Fraga, 1997; Schmidt, 1999). Several biotransforma-
tions of eudesmane compounds have been accomplished
in the last decade (Amate et al., 1991; Atta-ur-Rahman
et al., 1994; Shimizu et al., 1994; Garcıa et al., 1995; El-
Sharkawy et al., 1996; Miyazawa et al., 1997; Garcıa-
Granados et al., 1998, 2000a,b).
2. Results and discussion
4b-Hydroxyeudesmane-1,6-dione (1) was obtained
from a natural compound (2) isolated from Sideritis
varoi subsp. cuatrecasasii (Garcıa-Granados et al.,
1985), a synonym of Sideritis leucantha Cav. subsp.
meridionalis (Font Quer) O. Socorro (Socorro, 2001).
Deacetylation of 2 gave the trihydroxyl derivative 3,
which was oxidized to give the 1,6-diketo derivative
1.
The incubation of 1 with Exserohilum halodes for 13
days produced only a limited quantity (15%) of meta-
* Corresponding author. Tel./Fax: +34-958-243364.
E-mail address: agarcia@ugr.es (A. Garcıa-Granados).
0031-9422/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.
PII: S0031-9422(01)00340-5

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892
Fig. 1. Bioconversion of sesquiterpene 4b-hydroxyeudesmane-1,6-dione by Gliocladium roseum and Exserohilum halodes.
bolite 4. This metabolite (4) was the result of the regio-
data of this metabolite (7) with those of metabolites 5
and 6. Hence, metabolite 7 was 4b,7a,11-trihydrox-
yeudesmane-1,6-dione.
and stereoselective reduction of the keto group at C-1 of
substrate 1 from the b face giving a (1S)-hydroxyl deri-
vative, which, due to the steric hindrance, is difficult to
achieve by chemical means. The S configuration at C-1
could easily be deduced from the signal in the 1H NMR
spectrum (ꢀ 3.52, 1H, dd, J1,2b=J1,2a=3.0 Hz), with
coupling constants corresponding to an equatorial pro-
ton. This metabolite previously appeared in the incuba-
tion of substrate 1 with the microorganism Curvularia
lunata (Garcıa-Granados et al., 1991).
The main metabolite isolated (8) showed spectral data
that, by comparison with those of metabolites 4 and 6,
indicated that Gliocladium roseum, besides the intro-
duction of a hydroxyl group at C-11, also had reduced
the carbonyl group at C-1 of the molecule, producing an
S-alcohol. This metabolite (8) also resulted from the
incubation of substrate 1 with Rhizopus nigricans (Gar-
cıa-Granados et al., 1993).
Substrate 1 was more efficiently biotransformed by
Gliocladium roseum. Incubation of this compound for 5
days yielded the metabolites 5 (2%), 6 (27%), 7 (3%), 8
(58%) and 9 (5%).
Metabolite 5 had a molecular formula of C15H24O4,
which indicated the presence of an additional hydroxyl
group in the molecule. The 13C NMR spectrum revealed
b-effects on C-8 and C-11 and g-effects on C-5, C-9, C-
12 and C-13, positioning the new hydroxyl group at C-
7. Consequently, metabolite 5 was 4b,7a-dihydroxy-
eudesmane-1,6-dione.
The last metabolite isolated (9) had the same mole-
1
cular formula (C15H26O4) as metabolite 8. Its H NMR
spectrum revealed two signals of geminal protons to
hydroxyl groups at ꢀ 3.55 (1H, dd, J1,2b=J1,2a=2.9 Hz),
characteristic of a 1a-hydroxyl group, and at ꢀ 4.04 (1H,
ddd, J8,9a=10.8, J8,7=10.1, J8,9b=5.0 Hz) due to an
axial proton. The position of the new function was
determined by the b-effects found on the adjacent car-
bons (C-7 and C-9). Therefore, metabolite 9 was
1a,4b,8a-trihydroxyeudesman-6-one.
Metabolite 6 had the same molecular formula as the
above metabolite (5), and its spectral data suggested a
new hydroxyl group at C-11. This metabolite (6) was
previously obtained in the incubation of substrate 1
with Rhizopus nigricans (Garcıa-Granados et al., 1993).
The molecular formula (C15H24O5) of the third meta-
bolite (7) was in agreement with the presence of two new
hydroxyl groups. These functional groups were posi-
tioned at C-7 and C-11, by comparison of the spectral
3. Conclusions
Several conclusions can be drawn from the above
biotransformation results. On the one hand, bioconver-
sion of substrate (1) by Exserohilum halodes is more
limited, reducing only the carbonyl group at C-1, produ-
cing an S-alcohol, as is usual in enzymatic reductions (Sih
and Rosazza, 1976). On the other hand, Gliocladium

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A. Garcıa-Granados et al. / Phytochemistry 58 (2001) 891–895
893
roseum achieved the complete bioconversion of sub-
strate (1), producing mainly 11-hydroxyl derivatives
(88%). These 11-hydroxylated compounds can be che-
mically transformed into 12-hydroxyl derivatives, pre-
cursors of 6b,12-eudesmanolides (Garcıa-Granados et
al., 1993). The main action achieved on the substrate (1)
by Gliocladium roseum is similar to the carried out by
Rhizopus nigricans (Garcıa-Granados et al., 1993),
although the first one, also produced 7a- and 8a-
hydroxylated derivatives. All the microorganisms tested
on the substrate (1) have reduced only the carbonyl
group at C-1, while the one situated at C-6 remained
unaltered (Garcıa-Granados et al., 1991, 1993).
mixture was extracted with CH2Cl2, dried over Na2SO4
and evaporated to dryness. Chromatography on a silica
gel column yielded 1b,4b,6b-trihydroxyeudesmane (3,
952 mg, 92%) (Garcıa-Granados et al., 1991).
4.3. Oxidation of 3
Jones’ reagent was added dropwise to a stirred solu-
tion of 1b,4b,6b-trihydroxyeudesmane (3, 905 mg) in
acetone at room temp. until an orange-brown colour
persisted (30 min), following the oxidation by TLC.
Methanol was then added and the reaction mixture was
diluted with water and extracted with CH2Cl2. The
organic layer was dried over anhydrous Na2SO4 and
evaporated to dryness. Chromatography on a silica gel
column yielded 4b-hydroxyeudesmane-1,6-dione (1, 815
mg, 91%) (Garcıa-Granados et al., 1991). Syrup;
[ꢂ]D=+67ꢀ (CHCl3, c 1); IR ꢃmax (CHCl3): 3527, 1712
In summary Exserohilum halodes produced only one
known compound of difficult access by chemical means
while Gliocladium roseum originated five compounds,
three of which, have not previously been reported.
1
and 1705 cmÀ1, H NMR (CDCl3): ꢀ 0.87 and 0.91 (3H
4. Experimental
each, d, J=6.6 Hz, 3H-12 and 3H-13), 1.24 and 1.25
(3H each, s, 3H-14 and 3H-15), 2.58 (1H, s, H-5) and
3.07 (1H, ddd, J2b,2a=J2b,3a=14.2, J2b,3b=6.0 Hz, H-
2b).
1
Measurements of NMR spectra (300.13 MHz H and
75.47 MHz 13C) were made in CDCl3 (which also pro-
vided the lock signal) in a Bruker AM-300 spectrometer.
The assignments of 13C chemical shifts (Table 1) were
made with the aid of distortionless enhancement by
polarization transfer (DEPT) using a flip angle of 135ꢀ.
IR spectra were recorded on a Nicolet 20SX FT–IR
spectrometer. High-resolution mass spectra were made
by LSIMS (FAB) ionization mode in a MICROMASS
AUTOSPEC-Q spectrometer (EBE geometry). Melting
points (uncorr.) were determined using a Kofler (Reich-
ter) apparatus. Optical rotations were measured on a
Perkin-Elmer 241 polarimeter at 25ꢀ. Silica gel Scharlau
60 (40–60 mm) was used for flash chromatography.
CH2Cl2 or CHCl3 containing increasing amounts of
Me2CO were used as eluents. Analytical plates (silica
gel, Merck 60 G) were rendered visible by spraying with
H2SO4–AcOH, followed by heating to 120ꢀ. The iden-
tity of compound 2 was confirmed by direct comparison
with an authentic sample (IR, MS, NMR, etc.).
4.4. Organism, media and culture conditions
Gliocladium roseum CECT 2733 and Exserohilum
halodes CECT 2716 were obtained from the Coleccion
Espanola de Cultivos Tipo, Departamento de Micro-
biologıa, Facultad de Ciencias, Universidad de Valen-
cia, Spain, and was kept in YEPGA medium containing
yeast extract (1%), peptone (1%), glucose (2%) and
agar (2%) in H2O at pH 5. In all transformation
experiments a medium of peptone (0.1%), yeast extract
(0.1%), beef extract (0.1%) and glucose (0.5%) in H2O
at pH 5.7 was used. Erlenmeyer flasks (250 ml) con-
taining 80 ml of medium were inoculated with a dense
suspension of the corresponding microorganism. The
cultures were incubated by shaking (150 rpm) at 28ꢀ for
6 days, after which, substrate 1 (5–10%) in EtOH was
added.
4.1. Isolation of 6ꢁ-acetoxy-1ꢁ,4ꢁ-dihydroxyeudesmane
(2)
4.5. Biotransformation of substrate 1 with Exserohilum
halodes
6b-Acetoxy-1b,4b-dihydroxyeudesmane was isolated
from Sideritis varoi subsp. cuatrecasasii (Garcıa-Grana-
dos et al., 1985), a synonym of Sideritis leucantha Cav.
subsp. meridionalis (Font Quer) O. Socorro (Socorro,
2001).
Substrate 1 (124 mg) was dissolved in EtOH (2 ml),
distributed between 2 Erlenmeyer flask cultures and
incubated for 13 days, after which the cultures were fil-
tered and pooled; the cells were washed thoroughly with
water and the liquid was satd. with NaCl and extracted
twice with CH2Cl2. Both extracts were pooled, dried
with anhydrous Na2SO4, and evaporated at 40ꢀ in
vacuum to give a mixture of compounds (102 mg). This
mixture was chromatographed on a silica gel column to
obtain 83 mg of starting material 1 and 19 mg (15%) of
1a,4b-dihydroxyeudesman-1-one (4) (Garcıa-Granados
4.2. Deacetylation of 2
6b-Acetoxy-1b,4b-dihydroxyeudesmane (2, 1.20 g)
was dissolved in MeOH/H2O (70%) (70 ml) containing
KOH (5%) (3.5 g) and refluxed for 1 h. The reaction

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A. Garcıa-Granados et al. / Phytochemistry 58 (2001) 891–895
894
et al., 1991). Syrup; [ꢂ]D=+21 (CHCl3, c 1); IR ꢃmax
(CHCl3): 3444 and 1690 cmÀ1 1H NMR (CDCl3): ꢀ
Table 1
13C NMR chemical shifts for compounds 1, 4, 5, 6, 7, 8 and 9
;
0.85 and 0.88 (3H each, d, J=6.6 Hz, 3H-12 and 3H-
13), 0.98 (3H, s, 3H-14), 1.18 (3H, s, 3H-15), 2.64 (1H, s,
H-5) and 3.52 (1H, dd, J1,2b=J1,2a=3.0 Hz, H-1).
Compounds
1
4
5
6
7
8
9
C-1
C-2
213.8
33.1
39.6
69.6
63.3
215.0
57.7
25.7
33.9
53.8
26.2
21.4
18.7
19.7
29.3
73.6
25.3
33.8
70.1
57.1
218.5
58.1
26.5
35.1
45.9
26.1
21.6
18.8
19.5
30.4
213.5
33.9
39.7
69.9
57.2
214.2
80.2
32.2
28.9
53.7
31.5
17.0
16.6
19.1
29.1
213.1
32.7
39.5
69.5
63.5
217.2
60.0
24.9
33.7
53.7
71.0
28.5
26.0
19.5
29.2
213.0
33.8
39.5
69.8
58.1
216.4
79.8
29.0
29.3
52.6
74.9
25.1
24.0
19.2
29.1
73.3
25.3
32.5
70.1
57.7
221.2
60.3
25.7
34.7
46.2
71.1
28.7
26.1
19.4
30.3
73.2
24.9
32.5
69.9
56.3
214.1
64.5
69.9
45.4
40.5
25.0
20.4
19.6
19.5
30.2
4.6. Biotransformation of substrate 1 with Gliocladium
roseum
C-3
C-4
C-5
C-6
670 mg of substrate 1 were dissolved in EtOH (9 ml),
distributed between 9 Erlenmeyer flask cultures and
incubated for 5 days, after which the cultures were pro-
cessed as indicated above for the biotransformation of
substrate 1 with Exserohilum halodes. The resulting
mixture (667 mg) was chromatographed on a silica gel
column to obtain 11 mg (2%) of 4b,7a-dihydroxy-
eudesmane-1,6-dione (5); syrup; [a]D=+91ꢀ (CHCl3, c
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
1
1); IR ꢃmax (CHCl3): 3514 and 1701 cmÀ1; H NMR
(CDCl3): ꢀ 0.92 and 0.94 (3H each, d, J=6.8 Hz, 3H-12
and 3H-13), 1.24 and 1.25 (3H each, s, 3H-14 and 3H-
15), 3.08 (1H, ddd, J2b,2a=J2b,3a=14,3, J2b,3b=6.1 Hz,
H-2b) and 3.54 (1H, s, H-5); HRLSIMS, m/z:
[M+Na]+ 291.1565, (C15H24O4Na, 291.1572, PPM
+2.4); 190 mg (27%) of 4b,11-dihydroxyeudesmane-
1,6-dione (6) (Garcıa-Granados et al., 1993); syrup;
[a]D=+61ꢀ (CHCl3, c 1); IR ꢃmax (CHCl3): 3527 and
m/z: [M+Na]+ 293.1727, (C15H26O4Na, 293.1729,
PPM +0.7).
Acknowledgements
1700 cmÀ1 1H NMR (CDCl3): ꢀ 1.18 and 1.21 (3H
;
each, s, 3H-12 and 3H-13), 1.23 and 1.24 (3H each, s,
3H-14 and 3H-15), 2.39 (1H, dd, J7,8b=12.2, J7,8a=7.3
Hz, H-7), 2.55 (1H, s, H-5) and 3.03 (1H, ddd,
This work was financially supported by grants from
the Comision Interministerial de Ciencia y Tecnologıa
(PM98-0213) and the Consejerıa de Educacion y Cien-
cia de la Junta de Andalucıa (FQM 0139). We thank
Laura Lopez(CECT) for her assistance with the tax-
onomy of the microorganisms used in this work and
David Nesbitt for improving the use of English in the
manuscript.
J
2b,2a=J2b,3a=14.2, J2b,3b=6.0 Hz, H-2b); 20 mg (3%)
of 4b,7a,11-trihydroxyeudesmane-1,6-dione (7); syrup;
[a]D=+89ꢀ (CHCl3, c 1); IR ꢃmax (CHCl3): 3498 and
1700 cmÀ1 1H NMR (CDCl3): ꢀ 1.22 and 1.26 (3H
;
each, s, 3H-14 and 3H-15), 1.30 and 1.44 (3H each, s,
3H-12 and 3H-13), 1.70 (1H, ddd, J3a,3b=J3a,2b=14.3,
J
J
J
3a,2a=4.5 Hz, H-3a), 2.04 (1H, ddd, J3b,3a=14.3,
3b,2b=5.9, J3b,2a=2.8 Hz, H-3b), 2.15 (1H, ddd,
2a,2b=14.3, J2a,3a=4.5, J2a,3b=2.8 Hz, H-2a), 3.07
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