Biotransformation of ent-13-epi-manoyl oxides difunctionalized at C-3 and C-12 by filamentous fungi

Article abstract of DOI:10.1016/j.phytochem.2003.09.017

Biotransformation of ent-3β,12α-dihydroxy-13-epi-manoyl oxide with Fusarium moniliforme gave the regioselective oxidation of the hydroxyl group at C-3 and the ent-7β-hydroxylation. The action of Gliocladium roseum in the 3,12-diketoderivative originated monohydroxylations at C-1 and C-7, both by the ent-β face, while Rhizopus nigricans produced hydroxylation at C-7 or C-18, epoxidation of the double bond, reduction of the keto group at C-3, and combined actions as biohydroxylation at C-2/epoxidation of the double bond and hydroxylation at C-7/reduction of the keto group at C-3. In the ent-3-hydroxy-12-keto epimers, G. roseum originated monohydroxylations at C-1 and C-7 and R. nigricans originated the oxidation at C-3 as a major transformation, epoxidation of double bond and hydroxylation at C-2. Finally, in the ent-3β-hydroxy epimer R. nigricans also originated minor hydroxylations at C-1, C-6, C-7 and C-20 and F. moniliforme produced an hydroxylation at C-7 and a dihydroxylation at C-7/C-11.

Full text of DOI:10.1016/j.phytochem.2003.09.017

Phytochemistry 65 (2004) 107–115
www.elsevier.com/locate/phytochem
Biotransformation of ent-13-epi-manoyl oxides difunctionalized
at C-3 and C-12 by filamentous fungi
Andres Garcıa-Granadosa,*, Antonia Fernandeza, Marıa C. Gutierreza,
Antonio Martıneza, Raquel Quirosa, Francisco Rivasa, Jose M. Ariasb
a
´
Departamento de Quımica Organica, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain
´
Departamento de Microbiologıa, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain
b
´
Received 28 July 2003; received in revised form 2 September 2003
Abstract
Biotransformation of ent-3b,12a-dihydroxy-13-epi-manoyl oxide with Fusarium moniliforme gave the regioselective oxidation of
the hydroxyl group at C-3 and the ent-7b-hydroxylation. The action of Gliocladium roseum in the 3,12-diketoderivative originated
monohydroxylations at C-1 and C-7, both by the ent-b face, while Rhizopus nigricans produced hydroxylation at C-7 or C-18,
epoxidation of the double bond, reduction of the keto group at C-3, and combined actions as biohydroxylation at C-2/epoxidation
of the double bond and hydroxylation at C-7/reduction of the keto group at C-3. In the ent-3-hydroxy-12-keto epimers, G. roseum
originated monohydroxylations at C-1 and C-7 and R. nigricans originated the oxidation at C-3 as a major transformation, epox-
idation of double bond and hydroxylation at C-2. Finally, in the ent-3b-hydroxy epimer R. nigricans also originated minor hydroxy-
lations at C-1, C-6, C-7 and C-20 and F. moniliforme produced an hydroxylation at C-7 and a dihydroxylation at C-7/C-11.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Filamentous fungi; Biotransformation; Diterpenoids; ent-Manoyl oxides; Biohydroxylation
1. Introduction
demand of the end products. Problems with substrate
acceptance, undesired side reactions, selectivity and
prediction of the hydroxylation position all hamper the
general synthetic utility of the biohydroxylation. Bio-
transformations are typically carried out using either
whole cells or isolated enzymes. Although in recent
years some enzymes responsible for fungal hydroxyl-
ation have been isolated, whole-cell fermentation is the
technique most often employed in fungal hydroxylation
(Lehman et al., 2001; Urlacher and Schmid, 2002; Van
Beilen et al., 2003). A great difficulty for the biohydrox-
ylation of a certain substrate is to find the appropriate
microorganism, so that, traditionally, one of the most
widely used techniques is the screening with different
fungal strains. In this context, the microbial transforma-
tion of ent-manoyl oxides—labdane-type diterpenoids—
by filamentous fungi constitutes one of the aims of the
present study. These biotransformation processes have
been used to introduce hydroxyl groups, at positions dif-
ficult to achieve by chemical means, on the substrates.
The objective was to produce new bioactive, highly oxy-
genated ent-manoyl oxide analogues of ent-forskolin. In
Biotransformation is today considered to be a routine
economically and ecologically competitive technology
by the synthetic organic chemists, in search of new pro-
duction routes for fine chemical, pharmaceutical and
agrochemical compounds (Huisman et al., 2002; Lau-
men et al., 2002; Patel, 2001, 2002; Rasor et al., 2001;
Straathof et al., 2002). The selectivity and mildness of
the biotransformations can make these processes super-
ior to similar, chemical-based methods (Davis et al.,
2001; Loughlin, 2000; Roberts, 2000; Thomas et al.,
2002; Zaks, 2001). From the different transformations
catalysed by enzymatic systems, the selective hydroxy-
lation of non-activated carbon atoms is particularly
interesting, because this transformation is difficult to
achieve by classical chemical methods. These biohy-
droxylations have been developed mainly in the fields of
the steroids and terpenoids because of the industrial
*Corresponding author. Tel./Fax: +34-958-243364.
E-mail address: agarcia@ugr.es (A. Garcia-Granados).
0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2003.09.017

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108
previous papers, we reported the incubation of several
ent-manoyl oxides, with functions at C-3 or at C-3 and
C-12, with the filamentous fungi Curvularia lunata,
Cunninghamella elegans, Fusarium moniliforme, Rhizo-
pus nigricans and Gliocladium roseum, yielding, in some
cases, products with biological activity (Garcıa-Grana-
dos et al., 1990, 1994, 1995a, 1999). New ent-manoyl
oxides also were obtained by biotransformation of some
ent-13-epi-manoyl oxides by the fungus Gibberella fuji-
kuroi (Fraga et al., 1989, 1999, 2001, 2003). In the pre-
sent work, we used the fungi Fusarium moniliforme
CECT 2152, a synonym of Fusarium verticilliodes (EAN
337), Rhizopus nigricans CECT 2672, a synonym of
Rhizopus stolonifer (ATCC 10404) and Gliocladium
roseum CECT 2733, the anamorphic form of Nectria
ochroleuca (ATCC 10523), to complete the earlier bio-
transformation studies.
Metabolites 9 and 10 had the molecular formula
C20H30O4, which indicated the presence of an additional
oxygen atom. In their H NMR spectra, three double
1
doublet signals of geminal protons to a 14,15-epoxy
group were observed (ꢀ 3.29, 2.72 and 2.67 for metabo-
lite 9 and ꢀ 3.10, 2.75 and 2.63 for metabolite 10). These
epoxide groups were confirmed by their 13C NMR data.
Both metabolites (9 and 10) were epimers at C-14 with a
structure of ent-3,12-dioxo-8a,13;14,15-diepoxylabdane.
The configuration at C-14 for metabolites 9 and 10 was
established by chemical correlation with the epoxy deriv-
atives 16 and 17, achieved by epoxidation of metabolite
2 with MCPBA. These compounds (16 and 17) had been
previously obtained in the biotransformation of ribe-
none by Mucor plumbeus (Fraga et al., 2001), and their
stereochemistry unequivocally determined, ‘‘14S’’ being
for the epoxy derivative 16 and ‘‘14R’’ for 17. Oxidation
of 16 gave compound 9 ((13S,14S)-ent-3,12-dioxo-
8a,13;14,15-diepoxylabdane) while identical oxidation of
17 gave compound 10 ((13S,14R)-ent-3,12-dioxo-
8a,13;14,15-diepoxylabdane). Metabolite 11 was ent-3a-
hydroxy-12-oxo-13-epi-manoyl oxide, and was pre-
viously obtained from the incubation of 5 with baker’s
yeast (Garcıa-Granados et al., 1999).
2. Results and discussion
ent-3b,12a-Dihydroxy-13-epi-manoyl oxide (varodiol,
1) was isolated from Sideritis varoi (Algarra et al.,
1983). Biotransformation of substrate 1 with Fusarium
moniliforme gave metabolites 2 (16%) and 3 (35%), and
substrate 1 (37%). Metabolite 2, the result of the
regioselective oxidation of the hydroxyl group at C-3 in
substrate 1, presented spectroscopic data identical to
those of ent-12a-hydroxy-3-oxo-13-epi-manoyl oxide
obtained in the biotransformation of ribenone 4 with
Gibberella fujikuroi (Fraga et al., 1999). Metabolite 3
was identified as ent-3b,7b,12a-trihydroxy-13-epi-man-
oyl oxide, previously obtained in the biotransformation
of ribenone 4 with F. moniliforme (Garcıa-Granados et
al., 1995a).
Oxidation of diol 1 gave ent-3,12-dioxo-13-epi-man-
oyl oxide (varodione, 5), which was incubated with
Gliocladium roseum to give 6 (7%) and 7 (19%). Meta-
bolite 6 had a molecular formula C20H30O4, which
indicated the presence of an additional hydroxyl group
in the molecule. Its 1H NMR spectrum revealed a signal
at ꢀ 3.72 (dd, J=10.5, 4.4 Hz) due to an axial geminal
proton to a hydroxyl group. The position of the new
function was established by oxidation to give 8 (Garcıa-
Granados et al., 1995a), and therefore, the structure of 6
was determined as ent-7b-hydroxy-3,12-dioxo-13-epi-
manoyl oxide. The second metabolite (7), with a struc-
ture of ent-1b-hydroxy-3,12-dioxo-13-epi-manoyl oxide,
has been previously isolated in the biotransformation of
diketone 5 with F. moniliforme (Garcıa-Granados et al.,
1995a).
Compounds 12 and 13 had the same molecular for-
mula (C20H30O5) and very similar spectroscopic data.
Their 1H NMR spectra showed three double-doublet
signals, analogous to those observed for metabolites 9
and 10, which correspond to the geminal protons to the
epoxy group on C-14 and C-15. In addition, a deshield-
ing signal at 4.52 ppm (1H, dd, J=12.5, 6.6 Hz) due to
a geminal proton to an equatorial hydroxyl group at
C-2 was noted. The a- and b-effects detected in their
13C NMR spectra (Table 1) confirmed this position. Thus,
metabolite 12 was (13S,14S)-ent-2a-hydroxy-3,12-
dioxo-8a,13;14,15-diepoxylabdane and 13 (13S,14R)-
ent-2a-hydroxy-3,12-dioxo-8a,13;14,15-diepoxylabdane.
The molecular formula of 14 indicated the presence of
an additional hydroxyl group in the molecule. Compar-
1
isons of the H NMR data of this metabolite (14) with
those of substrate 5 revealed the existence of two sig-
nals, forming an AB system (ꢀ 3.71 and 3.39, J=11.3
Hz), and the absence of one of the methyl signals. The
a-effect on C-18, the b-effect on C-4 and the g-effect on
C-19 positioned the new hydroxyl group at C-18, which
was confirmed by comparison of the 13C NMR data
with those of triol 18—obtained by reduction of meta-
bolite 14—and those previously published for triol 19
(Arias et al., 1988). These compounds showed similar
chemical shifts values for C-3, C-4, C-5, C-18 and C-19,
which was in accordance with a structure of ent-
3b,12b,18-trihydroxy-13-epi-manoyl oxide for 18, and
consequently metabolite 14 was identified as ent-18-
hydroxy-3,12-dioxo-13-epi-manoyl oxide.
Biotransformation of varodione 5 with Rhizopus
nigricans originated metabolites 6 (4%), 9 (5%), 10
(6%), 11 (3%), 12 (13%), 13 (14%), 14 (10% ) and a
polar mixture of compounds, from which, after acetyl-
ation, diacetate 15 (2%) was obtained.
1
Diacetate 15 presented in its H NMR spectrum two
signals of geminal hydrogens to an acetoxyl group at ꢀ

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109
Table 1
13C NMR chemical shifts
spectrum of compound 22 showed hydroxyl, acetoxyl
and carbonyl group bands. Comparison of the 1H
NMR spectrum of this compound with that of 20
revealed the chemical acetylation only on the hydroxyl
group at C-3, and the presence of a new signal at ꢀ 4.49,
due to an axial geminal proton to a hydroxyl group.
The position of this alcohol was determined by analys-
ing the 1H NMR chemical shifts of H-17, H-19 and
H-20 in both compounds. The signals of these protons
for 22 were more deshielded (1.45, 1.27 and 1.18 ppm,
respectively) than those for compound 20 (1.17, 0.86 or
0.85 and 0.78 ppm, respectively), due to the 1,3-diaxial
interactions produced by a hydroxyl group located at
C-6, as in other ent-6b-hydroxyderivatives (Garcıa-
Granados et al., 1990, 1995b). Thus, 22 was identified as
ent-3a-acetoxy-6b-hydroxy-12-oxo-13-epi-manoyl oxide.
Treatment of varodiol (1) with CCL (lipase of Can-
dida cylindracea) originated the regioselective acetyl-
ation of the hydroxyl group at C-3 to give ent-3b-
acetoxy-12a-hydroxy-13-epi-manoyl oxide (23) (71%).
Oxidation of this monoacetate (23) yielded compound
24, which was then hydrolysed to give ent-3b-hydroxy-
12-oxo-13-epi-manoyl oxide (25) (Garcıa-Granados et
al., 1999). The biotransformation of this substrate (25)
by R. nigricans over a period of 6 days produced meta-
bolites 9 (6%), 10 (5%), 12 (11%), 13 (9%), 26 (6%), 27
(1%), 28 (9%), together with a more polar mixture of
metabolites which was acetylated to give compounds 29
(1%), 30 (8%) and 31 (1%).
C 9
10
12 13 14 15 18 20 21
26
30
1
2
37.4 37.3 47.5 47.5 37.0 32.2 37.4 32.5 77.0 37.0 31.7
33.7a 33.7a 68.8 68.9 33.8 22.5 26.9 22.6 37.2 27.0 23.5
3
4
5
6
7
8
9
216.2 216.4 215.5 215.5 217.4 77.2 77.6 77.8 74.7 78.7 79.9
47.4 47.4 47.6 47.6 36.3 42.0 36.7 37.4 38.8 37.6
—
54.7 54.7 57.0 57.1 53.7 47.5 49.7 49.9 47.5 52.9 55.5
20.8 20.8 20.2 20.2 20.1 25.0 19.6 19.3 19.4 19.6 19.1
41.4 41.4 41.5 41.7 41.2 80.3 42.1 42.0 42.0 42.0 42.2
74.9 74.8 74.8 74.8 75.1
52.3 54.4 52.5 54.7 48.1 53.2 57.7 54.5 54.1 54.9a 52.2
—
77.3 75.5 75.7 75.2 75.2
10 36.5 36.5 37.1 37.2 52.5 36.7 36.6 36.7 42.5 36.7 39.9
11 33.3a 35.5a 33.3 35.4 34.9 33.1 25.3 33.7 36.1 33.3 36.2
12 211.4 208.4 211.0 207.9 210.7 210.9 76.2 211.2 213.0 212.1 210.1
13 78.6 81.0 78.5 81.0 82.1 81.6 75.6 81.9 81.4 78.5 82.6
14 54.9 57.1 54.7 57.0 142.2 141.8 140.3 142.5 142.2 55.0a 142.7
15 43.1 45.5 43.0 45.5 113.4 113.3 117.3 113.2 113.2 43.2 113.1
16 24.9 24.0 24.8 24.0 28.6 29.1 28.0 28.6 28.7 24.7 28.7
17 21.8b 21.8b 22.3 22.4 22.1 16.7 25.4 22.3 22.1a 22.1 22.3
18 26.5 26.6 24.9 24.9 66.7 27.8 71.5 27.9 28.0 28.2 28.9
19 21.4b 21.2b 21.5 21.4 16.8 21.6 11.2 21.6 21.9a 15.5 17.0
20 14.2 14.6 15.3 15.5 14.5 14.5 16.4 14.5 10.3 14.7 63.2
a,b
Values bearing the same superscript may be interchanged.
4.96 (dd, J=11.8, 4.8 Hz) and at ꢀ 4.67 (t, J=2.9 Hz),
due to axial and equatorial protons, respectively. The
comparison of the 13C NMR spectrum of 15 with that
of 20, obtained by acetylation of metabolite 11, revealed
that the microorganism had reduced the keto group at
C-3 to give an axial hydroxyl group, as in metabolite 11,
and had introduced a hydroxyl group at C-7, which
were then acetylated. Consequently, compound 15 was
ent-3a,7b-diacetoxy-12-oxo-13-epi-manoyl oxide.
The regioselective and stereoselective reduction of the
carbonyl group at C-3 of varodione 5 with baker’s yeast
gave ketol 11 as the main metabolite (Garcıa-Granados
et al., 1999). Biotransformation of this ketol (11) with
R. nigricans for 3 days yielded metabolite 5 (7%) (varo-
dione), and the previously isolated metabolites in the
above biotransformation of varodione (5) with R. nigri-
cans, 9 (11%), 10 (9%), 12 (11%) and 13 (10%).
The incubation of the same ketol (11) with G. roseum
for 13 days produced 21 (32%) and a mixture of more
polar metabolites, which was acetylated, yielding diace-
tate 15 (4%), previously obtained in the biotransforma-
tion of varodione (5) with R. nigricans, and compound
22 (3%). The molecular formula of metabolite 21
(C20H32O4) suggested the presence of an additional
1
The H NMR spectrum of metabolite 26 revealed the
presence of three double-doublets signals characteristic
of a 14,15-epoxide group. The stereochemistry at C-14
of this function was determined by comparison with the
spectral data of 10, being ‘‘R’’ and resulting in a struc-
ture of (13S,14R)-ent-3b-hydroxy-12-oxo-8a,13;14,15-
1
diepoxylabdane. The H NMR spectrum of metabolite
27 showed a signal at ꢀ 3.91 that corresponded to an
axial geminal proton to a hydroxyl group, coupled with
two axial protons (J=11.0, 11.0 Hz) and an equatorial one
(J=3.9 Hz). In this compound the equatorial hydroxy-
lation can be located only at C-6. Thus, 27 was
identified as ent-3b,6a,-dihydroxy-12-oxo-13-epi-man-
oyl oxide. Metabolite 28 presented spectroscopic data
identical to those of ent-3b,7b-dihydroxy-12-oxo-13-epi-
manoyl oxide, previously obtained in the bio-
transformation of compound 24 with Nectria ochroleuca
(Garcıa-Granados et al., 1999). Compound 29 was the
diacetate of 28. In the 1H NMR spectrum of compound
30, only four signals of methyl groups and an AB sys-
tem signal with doublets centred at ꢀ 4.58 and 4.07
(J=12.4 Hz) were detected, indicating that one of
methyl groups of substrate (25) was functionalized by
the microorganism. This oxygenated functional group
was positioned at C-20 by comparison of the 13C NMR
spectra of diacetate 30 and monoacetate 24, and con-
firmed by other bibliographical data (Arias et al., 1988;
1
hydroxyl group in the molecule. Its H NMR spectrum
confirmed this proposal with the presence of a new sig-
nal due to a geminal axial proton to a hydroxyl group at
ꢀ 3.88 (dd, J=11.4, 4.7 Hz). The b-effect on C-2
(Áꢀ=+12.1), the g-effect on C-20 (Áꢀ=À4.2), and the
ꢀ-effect on C-11 (Áꢀ=+2.5), positioned this hydroxyl
group at C-1, establishing its structure as ent-1b,3a-
dihydroxy-12-oxo-13-epi-manoyl oxide (21). The IR

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A. Garcia-Granados et al. / Phytochemistry 65 (2004) 107–115
110
Garcıa-Granados et al., 1997). Thus, compound 30 was
identified as ent-3b,20-diacetoxy-12-oxo-13-epi-manoyl
oxide. Compound 31 had in its H NMR spectrum a
signal at ꢀ 4.72 (dd, J=11.5, 4.7 Hz) originated by a
geminal proton to an acetoxyl group that could be
located at C-1 or C-7. The first position was confirmed
by acetylation of an ent-1b,3b-dihydroxy derivative
(32), isolated in the biotransformation of compound 24
by N. ochroleuca (Garcıa-Granados et al., 1999), which
proved to be identical with 31. Therefore, this compound
had the structure of ent-1b,3b-diacetoxy-12-oxo-13-epi-
manoyl oxide (31).
1
The biotransformation of 25 with F. moniliforme gave
unaltered substrate 25 (60%), and the metabolites 28
(8%), isolated in the biotransformation of ketol 25 with
R. nigricans, and 33 (7%). This metabolite (33) had a
molecular formula of C20H32O5, indicating that two new
oxygen atoms were introduced in the biotransformation
process. In its 1H NMR spectrum, as in that of 28,
appeared two signals of the equatorial geminal protons

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A. Garcia-Granados et al. / Phytochemistry 65 (2004) 107–115
111
to the hydroxyl groups at C-3 and C-7, together with a
new signal at 4.59 ppm (d, J=12.4 Hz) originated by an
axial geminal proton to a hydroxyl group at C-11. This
ent-11a-hydroxylation was also observed in the bio-
transformation of varodione (5) with F. moniliforme
(Garcıa-Granados et al., 1995a). Hence, the structure of
this compound was determined as ent-3b,7b,11a-trihy-
droxy-12-oxo-13-epi-manoyl oxide (33).
(Toyota et al., 1988), natural ptychantins (Hashimoto et
al., 1994; Wu et al., 2001), etc., but with a skeleton of
the enantio series.
4. Experimental
Melting points were determined using a Kofler
(Reichter) apparatus and are uncorr. Measurements of
1
NMR spectra (300.13 MHz, H, and 75.47 MHz, 13C)
3. Conclusions
were made in CDCl3 in a Bruker AM-300 spectrometer.
Assignments of 13C chemical shifts (Table 1) were made
with the aid of distortionless enhancement by polariz-
ation transfer (DEPT) using a flip angle of 135ꢀ. IR
spectra were recorder on a Nicolet 20SX FT-IR spec-
trometer. High-resolution mass spectra were made by
LSIMS (FAB) ionization mode in a MICROMASS
AUTOSPEC-Q spectrometer (EBE geometry). Optical
rotations were measured on a Perkin-Elmer 240 polari-
a). The regioselective oxidation of varodiol 1 with
F. moniliforme gave better yields for compound 2 (16%)
than did biohydroxylation at C-12 of ribenone (4) by
G. fujikuroi (6%) (Fraga et al., 1999). The ent-7b-
hydroxylation (35%) of varodiol (1) with F. mon-
iliforme was improved with regard to the correspond-
ing biohydroxylation of ribenol (34) with the same
microorganism (7%), indicating that a second oxyge-
nated function at C-12 seems to accelerate the action of
the microorganism.
ꢀ
meter at 25 C. Silica gel Scharlau 60 (40–60 mm) was
used for flash chromatography. CH2Cl2 with increasing
amounts of Me2CO was used as eluent. Analytical
plates (silica gel, Merck 60 G) rendered visible by
spraying with H2SO4–HOAc–H2O, followed by heating
at 120 ꢀC. Varodiol (1) used as starting material in these
biotransformations was isolated from Sideritis varoi
(Algarra et al., 1983).
b). The biotransformation of varodione (5) by
G. roseum was focussed only on two monohydroxylations
at C-1 (19%), while F. moniliforme produced only 9%
(Garcıa-Granados et al., 1995a), and at C-7 (7%), both
by the ent-b face. Whereas R. nigricans showed lower
selectivity with biohydroxylations at C-2, C-7 and C-18,
reduction of the carbonyl group at C-3, and epoxida-
tions of the double bond. G. roseum and F. moniliforme
have in common the non-functionalization of the dou-
ble bond of the molecule, as was also observed with R.
nigricans, and in previous biotransformations with
Curvularia lunata (Garcıa-Granados et al., 1994) and
Cunninghamella elegans (Garcıa-Granados et al., 1995a).
c). In the biotransformation processes by R. nigricans
of the epimer ketols 11 and 25, the main action of the
microorganism was the oxidation at C-3 and epoxid-
ation of the double bond of the molecule, with or without
hydroxylation at C-2. In addition, with substrate 25,
R. nigricans also originated minor hydroxylations at
C-1, C-6, C-7 and C-20. The configuration at C-3 of
ketols 11 and 25 (Garcıa-Granados et al., 1999) does
not appear to influence the action of G. roseum, produ-
cing monohydroxylations at C-1 and C-7 in both sub-
strates. The action of F. moniliforme on ketol 25 was
focused at C-7, producing ent-7b-hydroxyl and ent-
7b,11a-dihydroxyl derivatives.
4.1. Organism, media and culture conditions
Rhizopus nigricans CECT 2672 (ATCC 10404), Glio-
cladium roseum CECT 2733 (ATCC 10523) and Fusar-
ium moniliforme CECT 2152 (EAN 337) were obtained
from the Coleccion Espanola de Cultivos Tipo, Depar-
tamento de Microbiologıa, Universidad de Valencia,
Spain. Medium YEPGA containing 1% yeast extract
1% peptone, 2% glucose, 2% agar, at pH 5 was used
for storage of microorganisms. In all transformations
experiments a medium containing 0.1% peptone, 0.1%
yeast ext., 0.1% beef ext. and 0.5% glucose at pH 5.7 in
H2O was used. Erlenmeyer flasks (250 ml) containing 80
ml of medium were inoculated with a dense suspension
of the correspondiꢀng microorganism. Incubations were
maintained at 28 C with gyratory shaking (150 rpm)
for 6 days after which the substrates (5–10%) in EtOH
were added.
4.2. Recovery and purification of metabolites
d). The hydroxylations at non-activated positions of
the carbocyclic system of this type of compounds are
difficult to achieve by classical chemical means, this
being tenable by microbial transformation, selecting the
microorganism and the functional groups of the diter-
pene molecule. Some of the isolated metabolites have
similar structures to several remarkable compounds,
such as forskolin (Bhat et al., 1977), hamachilobenes
Cultures were filtered and pooled, and cells were
washed thoroughly with water and the liquid was
saturated with NaCl and extracted twice with CH2Cl2.
Both extracts were pooled, dried with dry Na2SO4,
and evaporated at 40 ꢀC in vacuum. The mixt. of
compounds obtained was chromatographed on silica
gel column.

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112
4.3. Biotransformation of 1 by F. moniliforme
starting material 5 (110 mg, 31%); ent-7b-hydroxy-3,12-
dioxo-13-epi-manoyl oxide (6, 14 mg, 4%); (13S,14S)-
ent-3,12-dioxo-8a,13;14,15-diepoxylabdane (9, 19 mg,
5%): syrup; [ꢁ]D À34ꢀ (CHCl3; c 0.5); IR ꢂmax (CHCl3),
Substrate 1 (150 mg) was dissolved in EtOH (2 ml),
distributed among 2 Erlenmeyer-flask cultures (F. mon-
iliforme) and incubated for 6 days, after which the cul-
tures were processed as indicated above to obtain
starting material 1 (56 mg, 37%), ent-12a-hydroxy-3-
oxo-13-epi-manoyl oxide (2, 24 mg, 16%) (Fraga et al.,
1999), and ent-3b,7b,12a-trihydroxy-13-epi-manoyl
oxide (3, 55 mg, 35%) (Garcıa-Granados et al., 1995a).
1
1709, 1098 and 1078 cmÀ1; H NMR (CDCl3), ꢀ 3.29
(1H, dd, J=4.0, 2.8 Hz, H-14), 2.72 (1H, dd, J=5.5, 2.8
Hz) and 2.67 (1H, dd, J=5.5, 4.0 Hz ) (2H-15), 2.52
(1H, dd, J=19.1, 6.5 Hz, Heq-11), 2.37 (1H, dd, J=19.1,
12.7 Hz, Hax-11), 2.00 (1H, dd, J=12.7, 6.5 Hz, H-9),
1.27 and 1.16 (3H each, s, 3H-16 and 3H-17), 1.11, 1.04
and 0.94 (3H each, s, 3H-18, 3H-19 and 3H-20); HR-
LSIMS m/z: 357.2055 [M+Na]+ (calcd for
C20H30O4Na, 357.2042); (13S,14R)-ent-3,12-dioxo-
8a,13;14,15-diepoxylabdane (10, 21 mg, 6%): syrup; IR
4.4. Oxidation of 1
Jones’s reagent was added dropwise to a stirrꢀed soln.
of varodiol (1, 500 mg) in Me2CO (20 ml) at 0 C until
an orange-brown colours persisted (15 min), following
the oxidation by TLC. MeOH was them added, the
reaction mixture was diluted with H2O and extracted
with CH2Cl2. The organic layer was washed with aq.
NaHCO3, dried over Na2SO4 and evaporated to dry-
ness. Chromatography on a silica gel column yielded
varodione (5, 450 mg, 91%) (Garcıa-Granados et al.,
1994).
nmax (CHCl3), 1710, 1115 and 1093 cmÀ1 1H NMR
;
(CDCl3), ꢀ 3.10 (1H, dd, J=4.3, 2.8 Hz, H-14), 2.75
(1H, dd, J=4.8, 4.3 Hz) and 2.63 (1H, dd, J=4.8, 2.8
Hz) (2H-15), 1.53 and 1.30 (3H each, s, 3H-16 and
3H-17), 1.11, 1.05 and 0.94 (3H each, s, 3H-18, 3H-19
and 3H-20); HR-LSIMS m/z: 357.2040 [M+Na]+
(calcd for C20H30O4Na, 357.2042); ent-3a-hydroxy-12-
oxo-13-epi-manoyl oxide (11, 10 mg, 3%) (Garcıa-
Granados et al., 1999); (13S,14S)-ent-2a-hydroxy-3,12-
dioxo-8a,13;14,15-diepoxylabdane (12, 52 mg, 13%):
syrup; IR ꢂmax (CHCl3), 3456, 1705, 1105 and 1090
4.5. Biotransformation of 5 by G. roseum
1
cmÀ1; H NMR (CDCl3), ꢀ 4.52 (1H, dd, J=12.5, 6.6
Substrate 5 (90 mg) was dissolved in EtOH (2 ml),
distributed among
Hz, H-2), (1H, dd, J=4.0, 2.8 Hz, H-14), 2.70 (1H, dd,
J=5.5, 2.8 Hz) and 2.66 (1H, dd, J=5.5, 4.0 Hz)
(2H-15), 2.55 (1H, dd, J=19.1, 7.1 Hz, Heq-11), 2.40
(1H, dd, J=19.1, 12.6 Hz, Hax-11), 2.00 (1H, dd,
J=12.6, 7.1 Hz, H-9), 1.24, 1.18, 1.16, 1.14 and 1.11
(3H each, s, 3H-16, 3H-17, 3H-18, 3H-19 and 3H-20);
HR-LSIMS m/z: 351.2169 [M+H]+ (calc. for
C20H31O5, 351.2172); (13S,14R)-ent-2a-hydroxy-3,12-
dioxo-8a,13;14,15-diepoxylabdane (13, 56 mg, 14%):
syrup; IR ꢂmax (CHCl3), 3466, 1712, 1110 and 1096
2
Erlenmeyer-flask cultures
(G. roseum) and incubated for 7 days, after which the
cultures were processed as indicated above to obtain
starting material 5 (50 mg, 56%); ent-7b-hydroxy-3,12-
dioxo-13-epi-manoyl oxide (6, 7 mg, 7%): syrup; [ꢁ]D
À28ꢀ (CHCl3; c 0.5); IR ꢂmax (CHCl3), 3466, 3080, 1705,
1
1085, 1000 and 925 cmÀ1; H NMR (CDCl3), ꢀ 6.09
(1H, dd, J =13.1, 6.3 Hz, H-14), 5.12 (1H, dd, J=13.1,
0.8 Hz) and 5.07 (1H, dd, J=6.3, 0.8 Hz) (2H-15), 3.72
(1H, dd, J=10.5, 4.4 Hz, H-7), 1.32, 1.24 (3H each, s,
3H-16 and 3H-17), 1.13, 1.05 and 0.91 (3H each, s,
3H-18, 3H-19 and 3H-20); HR-LSIMS m/z: 335.2219
[M+H]+ (calc. for C20H31O4, 335.2222); and ent-1b-
hydroxy-3,12-dioxo-13-epi-manoyl oxide (7, 18 mg,
19%) (Garcıa-Granados et al., 1995a).
1
cmÀ1; H NMR (CDCl3), ꢀ 4.52 (1H, dd, J=12.5, 6.6
Hz, H-2), 3.09 (1H, dd, J=4.3, 2.8 Hz, H-14), 2.76 (1H,
dd, J=4.7, 4.3 Hz) and 2.63 (1H, dd, J=4.7, 2.8 Hz)
(2H-15), 2.50 (1H, dd, J=16.9, 13.6 Hz, Hax-11), 2.40
(1H, dd, J=16.9, 5.0 Hz, Heq-11), 1.77 (1H, dd, J=13.6,
J2=5.0 Hz, H-9), 1.53 and 1.27 (3H each, s, 3H-16 and
3H-17), 1.18, 1.16 and 1.11 (3H each, s, 3H-18, 3H-19
and 3H-20); HR-LSIMS m/z: 351.2176 [M+H]+ (calc.
for C20H31O5, 351.2172); ent-18-hydroxy-3,12-dioxo-13-
epi-manoyl oxide (14, 35 mg, 10%): syrup; [ꢁ]D À87ꢀ
(CHCl3, c 1); IR ꢂmax (CHCl3), 3460, 3080, 1707, 1090,
4.6. Oxidation of 6
Metabolite 6 (7 mg) was dissolved in Me2CO (2 ml)
and oxidized with Jones’ reagent to obtain ent-3,7,12-
trioxo-13-epi-manoyl oxide (8, 5 mg, 72%) (Garcıa-
Granados et al., 1995a).
1
1000 and 925 cmÀ1; H NMR (CDCl3), ꢀ 6.10 (1H, dd,
J=17.5, 10.8 Hz, H-14), 5.15 (1H, dd, J=17.5, 1.0 Hz)
and 5.06 (1H, dd, J=10.8, 1.0 Hz) (2H-15), 3.71 (1H, d,
J=11.3 Hz) and 3.39 (1H, d, J=11.3 Hz) (2H-18), 2.61
(1H, ddd, J=16.5, 13.0, 6.9 Hz, H-2), 2.51 (1H, dd,
J=17.8, 6.2 Hz, Heq-11), 2.39 (1H, dd, J=17.8, 12.9 Hz,
Hax-11), 2.35 (1H, ddd, J=16.5, 5.8, 2.6 Hz, H-3), 2.00
(1H, dd, J=12.9, 6.1 Hz, H-9); 1.31 and 1.24 (3H each,
s, 3H-16 and 3H-17), 1.00 and 0.98 (3H each, s, 3H-19
4.7. Biotransformation of 5 by R. nigricans
Substrate 5 (350 mg) was dissolved in EtOH (10 ml),
distributed among 10 Erlenmeyer-flask cultures
(R. nigricans) and incubated for 6 days, after which the
cultures were processed as indicated above to obtain

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113
and 3H-20); HR-LSIMS m/z: 357.2043 [M+Na]+
(calcd for C20H32O4Na, 357.2042); together with a mixture
which was acetylated to give ent-3a,7b-diacetoxy-12-
oxo-13-epi-manoyl oxide (15, 10 mg, 2% of overall
yield): syrup; [ꢁ]D À2ꢀ (CHCl3, c 0.5); IR ꢂmax (CHCl3),
12), 1.34 and 1.24 (3H each, s, 3H-16 and 3H-17), 0.84 and
0.81 (3H each, s, 3H-19 and 3H-20); HR-LSIMS m/z:
361.2351 [M+Na]+ (calc. for C20H32O4Na, 361.2355).
4.12. Acetylation of 11
1
3085, 1736, 1243 and 926 cmÀ1; H NMR (CDCl3), ꢀ
6.15 (1H, dd, J=17.4, 10.8 Hz, H-14), 5.16 (1H, dd,
J=17.4, 1.4 Hz) and 5.04 (1H, dd, J=10.8, 1.4 Hz) (2H-
15), 4.96 (1H, dd, J=11.8, 4.8 Hz, H-7), 4.67 (1H, t,
J=2.9 Hz, H-3), 2.10 and 2.08 (3H each, s, AcO group),
1.29 and 1.25 (3H each, s, 3H-16 and 3H-17), 1.19, 0.89
and 0.83 (3H each, s, 3H-18, 3H-19 and 3H-20); HR-
LSIMS m/z: 421.2588 [M+H]+ (calcd for C24H37O6,
421.2590).
Metabolite 11 (10 mg) was dissolved in pyridine (3 ml)
and Ac2O (1.5 ml). The reaction was maintained for 72
h at room temp., and extracted in the usual way. The
solvent was evapd. to give a mixt. of compounds which
was chromatographed on silica gel to give ent-3a-acet-
oxy-12-oxo-13-epi-manoyl oxide (20, 8 mg, 71%): white
solid, mp: 143–145 ꢀC; IR ꢂmax (KBr), 3088, 1732, 1716,
1639, 1245, 1087, 991 and 924 cmÀ1; 1H NMR (CDCl3), ꢀ
6.09 (1H, dd, J=17.6, 10.8, H-14), 5.12 (1H, dd, J=17.6,
1.1 Hz) and 5.07 (1H, dd, J=10.8, 1.1 Hz) (2H-15); 4.64
(1H, t, J=2.8 Hz, H-3), 2.45 (1H, dd, J=17.8, 5.9 Hz,
Heq-11), 2.32 (1H, dd, J=17.8, 13.1 Hz, Hax-11), 2.05
(3H, s, AcO group), 1.31 and 1.17 (3H each, s, 3H-16 and
3H-17), 0.86 and 0.85 (3H each, s, 3H-18 and 3H-19),
0.78 (3H, s, 3H-20); HR-LSIMS m/z: 385.2354 [M
+Na]+ (calc. for C22H34O4Na, 385.2355).
4.8. Epoxidation of 2
Metabolite 2 (20 mg) was dissolved in CHCl3 (3 ml)
ꢀ
and MCPBA (15 mg) was added. After 24 h at 0 C the
mixt. was diluted with CHCl3 (20 ml) and washed with
aq. FeSO4, NaHCO3 and H2O. The organic layer was
dried with dry Na2SO4, concentrated in vacuum and
chromatographed on a silica gel column to give
(13S,14S)-ent-12a-hydroxy-3-oxo-8a,13;14,15-diepoxy-
labdane (16, 5 mg, 24%) and (13S,14R)-ent-12a-
hydroxy-3-oxo-8a,13;14,15-diepoxylabdane (17, 4 mg,
19%) (Fraga, 2001).
4.13. Semisynthesis of ketol 11 and biotransformation
by R. nigricans
Ketol 11 was previously obtained in the bio-
transformation of varodione 5 with baker’s yeast (Gar-
cıa-Granados et al., 1999). Ketol 11 (100 mg) was
dissolved in EtOH (2 ml), distributed among 2 Erlen-
meyer-flask cultures (R. nigricans) and incubated for 3
days, after which the cultures were processed as indi-
cated above to give starting material 11 (30 mg, 40%);
varodione (5, 7 mg, 7%); 9 (12 mg, 11%); 10 (10 mg,
9%); 12 (12 mg, 11%); and 13 (11 mg, 10%).
4.9. Oxidation of diepoxylabdane 16
Diepoxylabdane 16 (5 mg) was dissolved in Me2CO
(1 ml) and oxidized with Jones’ reagent to give
(13S,14S)-ent-3,12-dioxo-8a,13;14,15-diepoxylabdane (9,
4 mg, 80%).
4.10. Oxidation of diepoxylabdane 17
4.14. Biotransformation of 11 by G. roseum
Diepoxylabdane 17 (4 mg) was dissolved in Me2CO
(1 ml) and oxidized with Jones’ reagent to give
(13S,14R)-ent-3,12-dioxo-8a,13;14,15-diepoxylabdane
(10, 3 mg, 75%).
Substrate 11 (100 mg) was dissolved in EtOH (3 ml),
2
distributed among
Erlenmeyer-flask cultures
(G. roseum) and incubated for 13 days, after which the
cultures were processed as indicated above to obtain
starting material (11, 58 mg, 58%) and ent-1b,3a-dihy-
droxy-12-oxo-13-epi-manoyl oxide (21, 34 mg, 32%):
4.11. Reduction of 14
ꢀ
Metabolite 14 (10 mg) was dissolved in EtOH (5 ml)
and NaBH4 (5 mg) was added. The mixture was main-
tained for 2 h at room temp. after which the reaction
mixture was treated with HCl (10%) and extracted with
CH2Cl2 in the usual way to give ent-3b,12b,18-trihydroxy-
13-epi-manoyl oxide (18, 8 mg, 80%): syrup; [ꢁ]D À45ꢀ
(CHCl3, c 0.5); IR ꢂmax (CHCl3), 3446, 3081, 1050, 990
and 915 cmÀ1; 1H NMR (CDCl3), ꢀ 6.28 (1H, dd, J=17.8,
11.2 Hz, H-14), 5.42 (1H, dd, J=17.8, 1.2 Hz) and 5.24
(1H, dd, J=11.2, 1.2 Hz) (2H-15), 3.70 (1H, d, J=11.3
Hz) and 3.40 (1H, d, J=10.3 Hz) (2H-18), 3.64 (1H, dd,
J=10.7, 5.3 Hz, H-3), 3.49 (1H, dd, J=11.5, 4.6 Hz, H-
white solid, mp: 138–140 C; [ꢁ]D À73ꢀ (CHCl3, c 0.5);
IR ꢂmax (KBr), 3433, 3091, 1704, 1640, 1091, 996 and
1
925 cmÀ1; H NMR (CDCl3), ꢀ 6.13 (1H, dd, J=17.4,
10.8 Hz, H-14), 5.18 (1H, dd, J=17.4, 1.3 Hz) and 5.03
(1H, dd, J=10.8, 1.3 Hz) (2H-15), 3.88 (1H, dd,
J=11.4, 4.7 Hz, H-1), 3.50 (1H, t, J=2.3 Hz, H-3), 3.12
(1H, dd, J=19.2, 6.1 Hz, Heq-11), 2.58 (1H, dd, J=19.2,
6.1 Hz, Hax-11), 2.18 (1H, dd, J=12.9, 6.1 Hz, H-9),
1.30 and 1.13 (3H each, s, 3H-16 and 3H-17), 0.94, 0.83
and 0.82 (3H each, s, 3H-18, 3H-19 and 3H-20);
HR-LSIMS m/z: 359.2197 [M+Na]+ (calcd for
C20H32O4Na, 359.2198); together with a mixture of

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A. Garcia-Granados et al. / Phytochemistry 65 (2004) 107–115
114
polar metabolites which was acetylated to give 15 (5 mg,
4% of overall yield) and ent-3a-acetoxy-6b-hydroxy-12-
oxo-13-epi-manoyl oxide (22, 4 mg, 3% of overall
yield): syrup; IR ꢂmax (CHCl3), 3500, 3090, 1728, 1716,
1245, 990 and 925 cmÀ1; 1H NMR (CDCl3), ꢀ 6.14 (1H,
dd, J=17.6, 10.9 Hz, H-14), 5.16 (1H, dd, J=17.6, 1.1
Hz) and 5.06 (1H, dd, J=10.9, 1.1 Hz) (2H-15), 4.66
(1H, t, J=2.8 Hz, H-3), 4.49 (1H, m, W1/2=8.3 Hz, H-
6), 2.07 (3H, s, AcO group), 1.45 (3H, s, 3H-17), 1.34
(3H, s, 3H-16), 1.27 (3H, s, 3H-19), 1.18 (3H, s, 3H-20),
and 0.97 (3H, s, 3H-18); HR-LSIMS m/z: 401.2305
[M+Na]+ (calcd for C22H34O5Na, 401.2304).
(1H, dd, J=11.6, 4.8 Hz, H-3), 3.91 (1H, ddd, J=11.0,
11.0, 3.9 Hz, H-6), 1.33, 1.31, 1.22, 0.97 and 0.83 (3H
each, s, 3H-16, 3H-17, 3H-18, 3H-19 and 3H-20); HR-
LSIMS m/z: 359.2190 [M+Na]+ (calcd for
C20H32O4Na, 359.2198); ent-3b,7b-dihydroxy-12-oxo-
13-epi-manoyl oxide (28, 19 mg, 9%) (Garcıa-Granados
et al., 1999); together with a mixture which was acety-
lated to give ent-3b,7b-diacetoxy-12-oxo-13-epi-manoyl
oxide (29, 5 mg, 1% of overall yield): syrup; [ꢁ]D À1.2ꢀ
(CHCl3, c 0.3); IR ꢂmax (CHCl3), 3060, 1736, 1242 and
1
926 cmÀ1; H NMR (CDCl3), ꢀ 6.14 (1H, dd, J=17.4,
10.8 Hz, H-14), 5.16 (1H, dd, J=17.4, 1.4 Hz) and 5.04
(1H, dd, J=10.8, 1.4 Hz) (2H-15), 4.50 (1H, dd,
J=11.7, 4.7 Hz, H-3), 4.90 (1H, dd, J=11.6, 4.9 Hz,
H-7), 2.10 and 2.04 (3H each, s, AcO group), 1.27, 1.17,
0.90, 0.85 and 0.84 (3H each, s, 3H-16, 3H-17, 3H-18,
3H-19 and 3H-20); HR-LSIMS m/z: 421.2596 [M+H]+
(calc. for C24H37O6, 421.2590); ent-3b,20-diacetoxy-12-
oxo-13-epi-manoyl oxide (30, 21 mg, 8% of overall
yield): syrup; [ꢁ]DÀ13.1ꢀ (CHCl3, c 0.5); IR ꢂmax
4.15. Semisynthesis of ent-3ꢃ-hydroxy-12-oxo-13-epi-
manoyl oxide (25)
ent-3b-Hydroxy-12-oxo-13-epi-manoyl oxide (25) was
obtained from monoacetate 23, which had been
obtained in the regioselective acylation of varodiol (1)
with Candida cylindracea lipase and vinyl acetate.
Monoacetate 23 (500 mg) was dissolved in acetone (20
ml) and oxidized with Jones’ reagent as indicate for
varodiol (1) to give ent-3b-acetoxy-12-oxo-13-epi-man-
oyl oxide (24, 460 mg, 93%). Product 24 (300 mg) was
dissolved in MeOH/H2O (70%) (10 ml) containing
KOH (5%) (0.5 g). The mixture reaction was main-
tained for 12 h at room temp. The reaction mixture was
diluted with H2O, extracted in the usual way and chro-
matographed on a silica gel column to yield ketol 25
(240 mg, 90%) (Garcıa-Granados et al., 1999).
(CHCl3), 3082, 1738, 1240 and 924 cmÀ1 1H NMR
;
(CDCl3), ꢀ 6.07 (1H, dd, J=17.6, 10.9 Hz, H-14); 5.07
(1H, d, J=17.6 Hz) and 5.02 (1H, d, J=10.9 Hz)
(2H-15), 2.81 (1H, dd, J=17.4, 14.1 Hz, Hax-11), 2.61
(1H, dd, J=17.4, 4.5 Hz, Heq-11), 4.58 and 4.07 (2H,
AB system, J=12.4 Hz, 2H-20), 4.50 (1H, dd, J=11.8,
4.7 Hz, H-3), 1.33, 1.31, 0.91 and 0.88 (3H-16, 3H-17,
3H-18 and 3H-19); HR-LSIMS m/z: 421.2595 [M+H]+
(calc. for C24H37O6, 421.2590); and ent-1b,3b-diacetoxy-
12-oxo-13-epi-manoyl oxide (31, 3 mg, 1% of overall
yield): syrup; [ꢁ]D À2.95ꢀ (CHCl3, c 0.5); IR ꢂmax
4.16. Biotransformation of 25 with R. nigricans
(CHCl3), 3087, 1740, 1713, 1246 and 925 cmÀ1 1H
;
NMR (CDCl3), ꢀ 6.19 (1H, dd, J=17.6, 10.8 Hz, H-14),
5.26 (1H, dd, J=17.6, 1.7 Hz) and 5.08 (1H, dd,
J=10.8, 1.7 Hz) (2H-15), 4.72 (1H, dd, J=11.5, 4.7 Hz,
H-1), 4.62 (1H, dd, J=12.3, 4.6 Hz, H-3), 2.03 and 2.03
(3H each, s, AcO group), 1.29, 1.08, 0.97, 0.89 and 0.86
(3H each, s, 3H-16, 3H-17, 3H-18, 3H-19 and 3H-20);
HR-LSIMS m/z: 421.2590 [M+H]+ (calcd for
C24H37O6, 421.2590).
Substrate 25 (200 mg) was dissolved in EtOH (5 ml),
distributed among
5
Erlenmeyer-flask cultures
(R. nigricans) and incubated for 6 days, after which the
cultures were processed as indicated above to obtain
starting material (25, 58 mg, 29%); 9 (12 mg, 6%); 10
(10 mg, 5%); 12 (24 mg, 11%); 13 (20 mg, 9%);
(13S,14R)-ent-3b-hydroxy-12-oxo-8a,13;14,15-diepoxy-
labdane (26, 13 mg, 6%): syrup; [ꢁ]D À36ꢀ (CHCl3, c
0.5); IR ꢂmax (CHCl3), 3470, 1712, 1127, 1097, 1068 and
4.17. Acetylation of 28
1
1039 cmÀ1; H NMR (CDCl3), ꢀ 3.26 (1H, dd, J=4.0,
2.8 Hz, H-14), 3.23 (1H, dd, J=11.3, 4.8 Hz, H-3), 2.70
(1H, dd, J=5.5, 2.8 Hz) and 2.65 (1H, dd, J=5.5, 4.0
Hz) (2H-15), 2.48 (1H, dd, J=19.1, 6.8 Hz, Heq-11),
2.30 (1H, dd, J=19.1, 12.9 Hz, Hax-11), 1.91 (1H, dd
J=12.9, 6.8 Hz, H-9), 1.25 and 1.11 (3H each, s, 3H-16
and 3H-17), 1.00, 0.79 and 0.77 (3H each, s, 3H-18, 3H-
19 and 3H-20); HR-LSIMS m/z: 359.2208 [M+Na]+
(calc. for C20H32O4Na, 359.2198); ent-3b,6a-dihydroxy-
12-oxo-13-epi-manoyl oxide (27, 3 mg, 1%): syrup; [ꢁ]D
À11.3ꢀ (CHCl3, c 0.3); IR ꢂmax (CHCl3), 3436, 3414,
Metabolite 28 (10 mg) was dissolved in pyridine (0.5
ml) and Ac2O (0.25 ml). The reaction was stirred for 24
h. at room temp. The reaction mixture was processed as
indicated for acetylation of metabolite 11 yielding 29 (9
mg, 72%).
4.18. Acetylation of 32
ent-1b,3b-Dihydroxy-12-oxo-13-epi-manoyl oxide (32,
20 mg), previously isolated from the biotransformation
of product 24 with N. ochroleuca (Garcıa-Granados et
al., 1999), was dissolved in pyridine (1 ml) and Ac2O
(0.5 ml). The reaction was stirred for 24 h at room
1
3090, 1711 and 923 cmÀ1; H NMR (CDCl3), ꢀ 6.12
(1H, dd, J=17.5, 10.8 Hz, H-14), 5.18 (1H, dd, J=17.5,
1.1 Hz) and 5.06 (1H, dd, J=10.8, 1.1 Hz) (2H-15), 3.21

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115
temp., and then processed as indicated for acetylation of
11 to give 31 (18 mg, 72%).
epi-manoyl oxide by Curvularia lunata: a possible route to the synthesis
of ent-forskolin analogues. Journal of Chemical Research (S) 94–95.
Garcıa-Granados, A., Jimenez, M.B., Martınez, A., Rivas, F., Onor-
ato, M.E., Arias, J.M., 1994. Chemical-microbiological synthesis of
ent-13-epi-manoyl oxides with biological activities. Phytochemistry
37, 741–747.
4.19. Biotransformation of 25 by F. moniliforme
Substrate 25 (50 mg) was dissolved in EtOH (1.5 ml),
distributed among 1 Erlenmeyer-flask cultures (F. mon-
iliforme) and incubated for 6 days, after which the cul-
tures were processed as indicated above to obtain
starting substrate (25, 30 mg, 60%); 28 (4 mg, 8%); and
Garcıa-Granados, A., Linan, E., Martınez, A., Rivas, F., Arias, J.M.,
1995a. Preparation of polyoxygenated ent-13-epi-manoyl oxides by
chemical-microbiological semisyntheses. Phytochemistry 38, 1237–
1244.
Garcıa-Granados, A., Linan, E., Martınez, Onorato, M.E., Arias,
J.M., 1995b. New polyoxygenated ent-13-manoyl oxides obtained
by biotransformation with filamentous fungi. Journal of Natural
Products 58, 1695–1701.
ent-3b,7b,11a-trihydroxy-12-oxo-13-epi-manoyl
oxide
(33, 4 mg, 7%): syrup; [ꢁ]D À1.5ꢀ (CHCl3, c 0.5); IR ꢂmax
Garcıa-Granados, A., Gutierrez, J.J., Martınez, A., Rivas, F., Arias,
J.M., 1997. Biotransformation of ent-6a-acetoxy and ent-6-ketoma-
noyl oxides with Rhizopus nigricans and Curvularia lunata cultures.
Phytochemistry 45, 283–291.
(CHCl3), 3453, 3082, 1720 and 927 cmÀ1 1H NMR
;
(CDCl3), ꢀ 6.02 (1H, dd, J=18.0, 10.9 Hz, H-14), 5.06
(1H, d, J=10.9 Hz) and 4.87 (1H, d, J=18.0 Hz) (2H-15),
4.59 (1H, d, J=12.4 Hz, H-11), 3.60 (1H, dd, J=11.3, 3.4
Hz, H-7), 3.21 (1H, dd, J=11.4, 5.5 Hz, H-3), 1.60 (1H, d,
J=12.4, H-9), 1.39, 1.37, 1.02, 1.01 and 0.80 (3H each, s,
3H-16, 3H-17, 3H-18, 3H-19 and 3H-20); HR-LSIMS m/
z: 353.2322 [M+H]+ (calcd for C20H33O5, 353.2328).
Garcıa-Granados, A., Martınez, A., Quiros, R., Extremera, A.L.,
1999. Chemical-microbiological semisynthesis of enantio-ambrox
derivatives. Tetrahedron 55, 8567–8578.
Hashimoto, T., Horie, M., Toyota, M., Taira, Z., Takeda, R., Tori,
M., Asakawa, Y., 1994. Structures of five highly oxygenated
labdane-type diterpenoids, ptychantins A–E, closely related to for-
skolin from the liverwort Ptychanthus striatus. Tetrahedron Letters
35, 5457–5460.
Huisman, G.W., Gray, D., 2002. Towards novel processes for the fine-
chemical and pharmaceutical industries. Current Opinion in Bio-
technology 13, 352–358.
Acknowledgements
This work was financially supported by grants from
the Comision Interministerial de Ciencia y Tecnologıa
(PM98-2013) and the Conserjerıa de Educacion y Cien-
cia de la Junta de Andalucıa (FQM 0139). We thank
David Nesbitt for improving the use of English in the
manuscript.
Laumen, K., Kittelmann, M., Ghisalba, O., 2002. Chemo-enzymatic
approaches for the creation of novel chiral building blocks and
reagents for pharmaceutical applications. Journal of Molecular
Catalysis B: Enzymatic 19-20, 55–66.
Lehman, L.R., Stewart, J.D., 2001. Filamentous fungi: potentially
useful catalysts for the biohydroxylations of non-activated carbon
centers. Current Organic Chemistry 5, 439–470.
Loughlin, W.A., 2000. Biotransformations in organic synthesis. Bio-
resource Technology 74, 49–62.
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