Biotransformation of the diterpene ribenone by Mucor plumbeus

Article abstract of DOI:10.1016/S0040-4020(00)01052-8

The microbiological transformation of the diterpene ribenone (3-oxo-ent-13-epi-manoyl oxide) by the fungus Mucor plumbeus has been studied. Epoxidation of the vinyl group constitutes the main reaction and there exists a preference for hydroxylation at C-1(α), C-6(α or β) and C-12(β) and, to a lesser degree, at C-7(α) and C-11(β). Other observed reactions were the reduction of the 3-oxo group to a 3β-alcohol and the formation of metabolites with a 1,2-double bond. In this work the stereochemistry at C-14 of the diterpenes excoecarins A, B and C has been revised on the basis of X-ray data.

Full text of DOI:10.1016/S0040-4020(00)01052-8

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 761±770
Biotransformation of the diterpene ribenone by Mucor plumbeus
a,p
Braulio M. Fraga, Melchor G. Hernandez, Pedro Gonzalez, Matõas Lopez and Sergio Suarez
a
b
b
b
Â
Â
Â
Â
Â
a
Â
Instituto de Productos Naturales y Agrobiologõa, CSIC, P.O. Box 195, 38206-La Laguna, Tenerife, Canary Islands, Spain
b
Â
Instituto Universitario de Bio-Organica, Universidad de La Laguna, Tenerife, Spain
Received 19 June 2000; revised 18 October 2000; accepted 2 November 2000
AbstractÐThe microbiological transformation of the diterpene ribenone (3-oxo-ent-13-epi-manoyl oxide) by the fungus Mucor plumbeus
has been studied. Epoxidation of the vinyl group constitutes the main reaction and there exists a preference for hydroxylation at C-1(a),
C-6(a or b) and C-12(b) and, to a lesser degree, at C-7(a) and C-11(b). Other observed reactions were the reduction of the 3-oxo group to a
3b-alcohol and the formation of metabolites with a 1,2-double bond. In this work the stereochemistry at C-14 of the diterpenes excoecarins
A, B and C has been revised on the basis of X-ray data. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
molecular formula C20H30O3. Its 1H NMR spectrum showed,
in comparison with that of the substrate, the presence of two
new vinylic hydrogens at d 5.88 and 7.13 (Jˆ10.2 Hz). The
chemical shifts of these protons indicated that this new
double bond introduced into the molecule must be conju-
gated with the 3-oxo group. Another signal observed in the
spectrum of 5 was a proton geminal to a new hydroxyl group
at d 4.19 as a broad singlet, which could be situated at C-7
(a) or C-12 (b). 13C NMR data of this compound and of its
acetate 5a (Table 1) permitted the alcoholic function to be
assigned to C-12 (b). Thus the structure of this substance
was determined as 12b-hydroxy-3-oxo-1-en-ent-13-epi-
manoyl oxide (5). This compound 5 must be formed from
7, which was also isolated from this biotransformation.
The biotransformation of diterpenoids with fungi has been
one of the goals of our studies in recent years. In the micro-
biological transformation of manoyl oxide derivatives we
have used two microorganisms: Gibberella fujikuroi, a
fungus possessing a high substrate speci®city, and Mucor
plumbeus, a fungus with a low speci®city. We have studied
the incubation of manoyl oxide derivatives, such as jhanol
and jhanidiol, with both fungi.1,2 In addition we have also
examined the biotransformation of compounds of the
enantio series, such as ribenone (1),3 ribenol (2) and 19-
hydroxy-ent-13-epi-manoyl oxide4 with G. fujikuroi. In
this work we describe the results of the biotransformation
of ribenone with M. plumbeus. The purpose was to obtain
substances with a functionality similar to that of the bio-
active diterpene forskolin5 (3), but with a skeleton of the
enantio series. The fungus M. plumbeus has been used
previously for microbiological transformations, for example
with sesquiterpenes possessing the aromadendrane,6
cedrane,7 guaiane8 and patchoulane9 skeletons, and with
diterpenes of the labdane type.10±12 In this last case, the
main reaction observed was the hydroxylation of ring A.
Table 1. 13C NMR data of compounds 2a, 5, 5a, 7, 10 and 12±14
Carbon
2a
5
5a
7
10
12
13
14
1
2
3
37.3 157.1 156.8 77.4 37.6 40.9 40.3 37.8
23.8 125.9 126.1 45.1 32.5 34.2 33.9 33.7
80.8 205.0 205.4 215.2 218.5 216.3 215.5 216.8
4
5
6
7
37.4 44.6
55.3 53.4
16.0 20.2
42.9 41.7
44.7
53.3
20.2
41.6
47.1 47.0 49.3 48.9 47.0
50.9 58.8 56.8 56.8 52.3
20.5 68.3 69.5 69.4 27.5
41.3 51.9 50.7 50.3 79.8
8
75.7 76.4a 75.3a 75.8 74.9 74.9 75.1 78.7
2. Results and Discussion
9
58.1 44.1
36.4 38.7
19.4 23.7
34.7 68.9
44.8
38.6
21.7
71.1
49.2 47.9 58.6 49.7 55.4
41.9 36.2 36.7 36.1 36.5
25.6 23.9 16.5 23.8 15.9
69.0 68.7 34.8 68.8 34.5
10
11
12
13
14
15
16
17
18
19
20
The substrate used was ribenone (1), which had been
isolated from Sideritis canariensis.13 Its biotransformation
by M. plumbeus led to the isolation of the metabolites 4±18
and 21±28. The substances 4, 6, 8, 9, 11 and 28 had been
obtained in the incubation of 1 with G. fujikuroi,3 and were
identi®ed by direct comparison. Compound 5 possesses the
73.5 76.2a 76.2a 76.0 76.2 73.4 75.9 73.5
147.5 146.7 146.2 146.7 146.6 147.5 147.0 146.8
109.6 110.9 111.2 110.8 110.9 109.5 110.4 110.1
32.6 26.7
23.6 24.3
27.9 27.6
16.3 21.1
15.9 18.9
27.0
22.7
27.6
21.2
18.8
26.9 26.9 32.8 27.0 32.5
23.5 24.2 24.5 24.5 17.9
27.4 31.7 24.9 25.1 26.6
20.0 19.2 23.5 23.4 20.9
11.0 16.8 16.2 16.4 15.6
Keywords: microbial reactions; X-ray crystal structures; epoxidation;
terpenes and terpenoids.
* Corresponding author. Tel: 134-922-251728; fax: 134-922-260135;
e-mail: bmfraga@ipna.csic.es
a These values can be interchanged.
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)01052-8

762
B. M. Fraga et al. / Tetrahedron 57 (2001) 761±770
Scheme 1.
The structure of 7 was determined as 1a,12b-dihydroxy-3-
oxo-ent-13-epi-manoyl oxide on the basis of the following
considerations: The high resolution MS did not show the
molecular ion, but gave a fragment at m/z 318.2208
Another metabolite obtained from this incubation was
6a-hydroxy-ribenone (12). In its high resolution MS the
molecular ion appeared at m/z 320.2360 indicating that the
molecular formula of this substance was C20H32O3. The new
oxygen introduced into the molecule during the fermen-
tation must be a part of a secondary hydroxyl group, because
in its 1H NMR spectrum there appears a proton geminal to a
new alcoholic group at d 4.50 as a triplet (Jˆ3 Hz). Double
irradiation experiments showed the existence of a ±CH±
CHOH±CH2± group, indicating the presence of an equa-
torial hydrogen geminal to a hydroxyl at C-6(a) or
C-11(a). The ®rst position was assigned on the basis of
the 13C NMR spectrum (Table 1). Therefore, its structure
must be 6a-hydroxy-3-oxo-ent-13-epi-manoyl oxide (12).
This product was identical with one obtained in the bio-
transformation of ribenone (1) by Curvularia lunata.14
1
(C20H30O3), formed by loss of a water molecule. Its H
NMR spectrum showed the signal of the 1b-hydrogen as a
double doublet at d 3.98 (Jˆ7.8 and 5.0 Hz). The relatively
low value of the greater of these coupling constants
(7.8 Hz), compared with that observed in a normal chair
(10±12 Hz), indicated that the corresponding geminal
alcohol must be located at C-1 (a) in ring A, which is a
deformed chair. Moreover, the chemical shift of the
11a-hydrogen, d 2.27, is characteristic of compounds with
a 1a-hydroxyl group. The second alcohol, with the geminal
hydrogen resonating at d 4.11 (t, Jˆ3.3 Hz), was placed at
C-12(b) considering 2D NMR data, which also permitted us
to unambiguously assign the 13C NMR spectrum of this
compound (Table 1).
Compound 14, an isomer of 12, showed the resonance of the

B. M. Fraga et al. / Tetrahedron 57 (2001) 761±770
763
Table 2. 13C NMR data of compounds 14a±18, 21, 23 and 24 (n.o. not
observed)
and H-14, respectively, and by the disappearance of the
corresponding vinylic hydrogens. Therefore, the structure
of this compound was determined as 3-oxo-1-en-14S,15-
epoxy-ent-13-epi-manoyl oxide (15). On the other hand,
the assignment of the position of the alcoholic groups at
1a in 18, 6a in 21 and 7a in 25 was made on the basis of
the same reasons given above for 7, 13 and 14, respectively,
and consequently we will not repeat them. The stereo-
chemistry at C-14 in all these metabolites was given con-
sidering the reasons described below
Carbon
14a
15
16
17
18
21
23
24
1
2
37.6 157.3
33.5 125.9
39.3
33.9
40.1
33.9
77.8 40.5
45.1 34.0
37.7
33.7
37.4
33.4
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
216.6
46.8
51.9
26.2
80.6
n.o.
55.9
36.1
15.9
34.2
73.3
147.2
109.6
32.4
18.5
26.8
20.7
15.4
n.o. 216.9 217.5 214.6
n.o. 217.1 216.9
44.7
53.3
20.2
41.9
75.0
51.6
39.1
16.7
30.8
71.7
57.5
47.3
29.1
24.2
27.7
21.3
18.7
47.6
55.1
20.8
42.6
74.8
61.5
37.6
65.0
40.7
71.8
58.1
45.4
28.1
25.8
26.6
21.1
15.7
47.6
54.9
20.9
42.4
75.8
61.9
37.9
65.7
41.3
72.8
58.8
45.7
28.4
23.8
26.9
20.9
15.9
47.1 48.8
50.9 56.7
20.5 69.2
41.4 50.3
74.4 73.8
57.0 57.5
42.3 36.6
18.5 16.6
31.0 30.9
71.3 71.2
57.4 57.4
47.3 47.4
29.0 29.2
23.4 24.6
27.4 25.0
20.0 23.5
10.8 16.2
47.2
54.6
20.9
41.7
74.7
48.9
35.9
25.4
66.8
75.1
58.5
46.2
23.1
22.4
26.7
20.9
15.4
47.2
54.6
20.9
41.9
74.6
49.6
36.1
24.9
66.0
74.1
57.6
46.3
24.4
22.4
26.7
21.0
14.9
The structures 12b-hydroxy-3-oxo-14S,15-epoxy-ent-13-
epi-manoyl oxide (23) and 12b-hydroxy-3-oxo-14R,15-
epoxy-ent-13-epi-manoyl oxide (24) were assigned to
another two isomers isolated from this feeding on the
basis of the following considerations: The molecular
formula C20H32O4 was determined by high resolution MS.
Their 13C NMR data (Table 2) permitted the assignment of
the alcohol, which had been introduced in the incubation at
1
C-12 in both metabolites. In their H NMR spectra, both
showed the presence of the hydrogens of a 14,15-epoxide
and of the proton geminal to the hydroxyl group. This last
signal appears in 23 and 24 at d 3.71 (br d, Jˆ4.4 Hz) and
3.89 (br d, Jˆ4.0 Hz), respectively. These coupling
constants indicated a b-axial stereochemistry for the alcohol
at C-12 for both metabolites, and these in consequence must
be epimeric at C-14. Moreover, a nOe effect was observed
between H-12 and the H-16 methyl in the NOESY spec-
trum. These 1H NMR data and molecular mechanics
methods indicated that the lowest energy conformation for
ring C was a chair in both substances. The structures of these
metabolites were chemically con®rmed by epoxidation of 9
with m-chloroperbenzoic acid, which led to the compounds
23 and 24 and the lactones 29 and 30. The last two
substances were formed from 23 and 24, respectively, by
a Baeyer±Villiger reaction.
proton geminal to the alcoholic group as a double doublet at
d 3.58 with coupling constants of 11.6 and 4.3 Hz. These
couplings are characteristic of an axial hydrogen geminal to
an alcoholic group at C-7(a) or C-12(b). The ®rst position
was chosen considering its 13C NMR spectrum and a 2D
NMR study (COSY, NOESY, HMQC and HMBC) of the
acetate 14a (Table 1). Thus, the structure of 7a-hydroxy-
3-oxo-ent-13-epi-manoyl oxide (14) was given to this
metabolite (Scheme 1).
Other compounds isolated from this incubation were the
pair of isomers 10 and 13, which have the molecular
formula C20H32O4. Both are formed during feeding by the
introduction of two new hydroxyl groups into the substrate
1. In both substances the position of the alcoholic groups
were assigned at C-12 and C-6 by a study of their 13C NMR
data (Table 1). The stereochemistry at C-12 for the hydroxyl
group was determined as b-axial in both metabolites,
because its geminal hydrogen appears in the NMR spectrum
of 10 and 13 at d 4.13 (t, Jˆ3.7 Hz) and 4.15 (t, Jˆ3.5 Hz),
respectively. The stereochemistry of the second alcohol
group at the C-6 position of 10 and 13 was determined as
b-equatorial and a-axial, respectively, considering that the
6(a)-H and the 6(b)-H resonate at d 3.86 (td, Jˆ11.6 and
4.4 Hz) and 4.52 (t, Jˆ3 Hz), respectively. Therefore, the
structures of these compounds were determined as 6b,12b-
dihydroxy-3-oxo-ent-13-epi-manoyl oxide (10) and
6a,12b-dihydroxy-3-oxo-ent-13-epi-manoyl oxide (13).
The stereochemistry at C-14 in the epoxides isolated from
this feeding was assigned on the basis of the following
considerations: Konishi et al.15 have isolated epoxy-deriva-
tives of this type from Excoecaria agallocha. In their study,
the stereochemistry of excoecarin A (14R,15-epoxy-ribe-
none), excoecarin B (14S,15-epoxy-ribenone) and exco-
ecarin C (14R,15-epoxy-ribenol) was assigned combining
molecular mechanics calculations and the observation of a
long-range coupling in the 14S derivative, between one of
the H-15 and the H-16 methyls, and a nOe effect between
the same hydrogens in the 14R-derivative. However, we
1
could not detect this long-range coupling in the H NMR
spectra of our epoxy-derivatives. Thus, since we considered
that the stereochemistry of these compounds was not clear
we prepared the 14R- and 14S-stereoisomers of 14,15-
epoxy-ribenol acetate, by epoxidation of ribenol acetate
(2a), and then the structure of 32a was determined by
single-crystal X-ray diffraction. Fig. 1 shows a computer-
generated perspective drawing16 of the ®nal X-ray model of
compound 32a, which possesses a 14S-stereochemistry. The
bond lengths and the bond angles are within the usual
ranges. The two cyclohexane and the tetrahydropyrane
rings are in a chair conformation. The hydrolysis of 32a
led to a substance that was spectroscopically identical
with excoecarin C, to which the 14R stereochemistry had
been assigned.15 Thus, in consequence, excocaerin C must
A series of epoxy derivatives were also formed in this
fermentation by epoxidation of the 14,15-double bond.
The least polar of these compounds was 15. Its MS showed
the molecular ion at m/z 318.2198 corresponding to a molec-
ular formula C20H30O3. The presence of the a,b-unsaturated
ketone on ring A was determined considering the same
arguments given above for 4, and by the absorbances at
1670 cm21 and 227 nm observed in the IR and UV spectra,
respectively. The epoxidation of the 14,15-double bond of
1
the substrate during the incubation was indicated in the H
NMR spectrum of 15 by the appearance of three double
doublets at d 2.82, 2.86 and 2.95, due to the two H-15

764
B. M. Fraga et al. / Tetrahedron 57 (2001) 761±770
Figure 1. ORTEP drawing of 32a (displacement ellipsoids are drawn at 40% probability level).
be a 14S derivative (32) and not 14R. Moreover, the stereo-
chemistry at this carbon in excoecarins A and B must also be
revised to 14S (19) and 14R (20), respectively, because in
the original work these compounds had been related to
excoecarin C (32) (Scheme 2).
position to be assigned to this alcohol in both metabolites.
Their 1H NMR spectra showed a proton geminal to a
hydroxyl group with similar chemical shifts and coupling
constants. Thus, this last signal appears in 16 and 17 as a
double triplet at d 4.13 (Jˆ9.1 and 5.4 Hz) and as a broad
multiplet at d 4.29, respectively. In this latter case, double
resonance experiments on 17 allowed observation of the
couplings of this carbinol with the protons of the C-12
methylene at d 1.34 and 1.85 (Jˆ10 and 5.0 Hz) and with
that of the C-9 methine at d 1.36 (Jˆ10.2 Hz). These facts
indicated a b-equatorial stereochemistry for the alcohol in
both substances. Moreover, a nOe effect was observed
between H-11 and the methyl groups at C-8 and C-10 in
the NOESY spectra of both compounds. These resonance
studies indicated that the low energy conformation of ring C
was a chair, which was con®rmed by molecular mechanics
calculations. Thus, these two metabolites must be stereo-
isomers at C-14, with the structures 11b-hydroxy-3-oxo-
14S,15-epoxy-ent-13-epi-manoyl oxide (16) and 11b-
We have noted NMR differences between the 14R- and 14S-
epoxy derivatives, concretely between the two epoxy-
acetates 31a and 32a, the pair of epimeric compounds 16
and 17, in 23 and 24, and also in those reported for exco-
ecarins A and B, that now permitted the stereochemistry at
C-14 in the epoxides of this type isolated from this feeding
to be resolved using only NMR data. These differences,
taking for example 23 and 24, are: (a) the NOESY spectrum
of the 14S-derivative showed a nOe effect between H-15
and H-17, whilst in the 14R derivative this effect was not
observed. (b) In the epoxide 24, which possesses a 14R
stereochemistry, the H-17 methyl resonates at a lower
®eld (d 1.42) in comparison with that of the unepoxidated
compound 9 (d 1.23)3, whilst in 23, with a 14S stereo-
chemistry, it is practically not affected (d 1.27). (c) The
deshielding of the H-17 methyl in the 14R derivative 24 is
accompanied by another deshielding of H-14 (d 3.11) and
by the shielding of one of the H-15 (d 2.56) in this
compound, in comparison with the respective hydrogens
in the 14S-derivative 23 (d 2.81 and 3.03, respectively).
These differences in the resonances of H-14, H-15 and
H-17 between the 14R- and 14S-stereoisomers were also
observed in all the 14,15-epoxides described in this work
and permitted them to be assigned the C-14 stereochemistry.
In consequence, this is a facile and direct procedure for the
assignment of the sterochemistry at C-14 in 14,15-epoxy-
13-epi-manoyl oxides.
hydroxy-3-oxo-14R,15-epoxy-ent-13-epi-manoyl
oxide
(17). The stereochemistry at C-14 was determined in the
same way as described above for 23 and 24.
Compound 22 and the acetate 26a, which have been identi-
®ed considering their 1H NMR data, were present as traces,
contaminating fractions of 16 and of the acetate 25a, respec-
tively. Finally, a diol 27 was isolated in the form of its
diacetate 27a by acetylation and chromatography of the
fractions containing it. Its mass spectrum does not show
the molecular ion, but at m/z 407.2418 (C23H35O6) a frag-
ment ion was observed, which is formed by loss of a methyl
group. Thus, the molecular formula of the corresponding
diol must be C20H34O4. The presence of the 14,15 epoxide
1
was observed in the H NMR spectrum of 27a as three
Another two stereoisomers with structures 16 and 17 and
molecular formula C20H32O4 were isolated from this feed-
ing. The molecular ion was not observed in the MS of either,
a fragment appearing at m/z 321, which is formed by loss of
a methyl group. 13C NMR data (Table 2) permitted the C-11
double doublets at d 2.81 (Jˆ4.6 and 3.6 Hz), 2.85
(Jˆ4.6 and 3.0 Hz) and 2.97 (1H, dd, Jˆ3.6 and 3.0 Hz),
due to H-15 and H-14, respectively. In this spectrum,
resonances of protons geminal to two acetoxyl groups at
4.67 (t, Jˆ3.0 Hz) and 4.79 (t, Jˆ4.1 Hz) were also

B. M. Fraga et al. / Tetrahedron 57 (2001) 761±770
765
Scheme 2.
observed, which, considering its molecular formula, indi-
cated that the 3-oxo group of ribenone (1) had been reduced
to an hydroxyl group during the biotransformation, and that
a new hydroxyl group had also been introduced into the
molecule. 13C NMR data (Table 3) and 2D NMR studies
of the diacetate 27a (COSY, NOESY, HMQC and HMBC)
con®rmed the presence of a C-3 acetoxyl and permitted the
C-12 position to be assigned to the second acetate group. On
the other hand, considering the coupling constants of the
geminal hydrogen, an axial stereochemistry was assigned
to both acetoxyl groups. The chemical shifts of H-14 (d
2.97) and the two H-15 (d 2.81 and 2.85) indicated the
14S stereochemistry of the epoxy group, which was
con®rmed by the nOe effect observed between the two
H-15 and H-17 (see above). Therefore, the structure
3b,12b-dihydroxy-14S,15-epoxy-ent-13-epi-manoyl oxide
(27) was assigned to the corresponding diol. An analogous
bioreduction of the 3-oxo group of ribenone (1) to the 3b-
alcohol has been observed in its biotransformation with both
C. lunata14 and G. fujikuroi,3 whilst the borohydride reduc-
tion of the same group afforded the a-alcohol.3
3. Conclusions
In addition to the structure revision of excoecarins A±C and
to the 1H NMR method described for the assignment of the
stereochemistry at C-14 in this type of diterpene, several

766
B. M. Fraga et al. / Tetrahedron 57 (2001) 761±770
Table 3. 13C NMR spectroscopic data of compounds 25, 25a, 27a and 29±
32a
and HRMS in a Micromass Autospec spectrometer. Confor-
mations of minimum energy were determined by compu-
tational methods employing the Hyperchem program from
Hypercube. Dry column chromatographies were made on Si
gel Merck 0.02±0.063 mm. The fungal strain was Mucor
plumbeus CMI 116688 and was a gift from Dr J. R. Hanson,
School of Chemistry, Physics and Environmental Sciences,
University of Sussex, UK.
Carbon
25
25a
27a
29
30
31a
32
32a
1
2
37.6
33.6
37.5 32.5
33.9 22.6a
37.1
31.8
37.9 37.2 37.4 37.1
31.9 23.6 27.2 23.5
3
215.7 215.6 77.8
174.4 174.6 80.8 78.8 80.7
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
47.0
52.3
27.6
79.8
78.0
54.6
36.4
16.5
31.7
71.8
58.6
45.7
28.5
16.6
26.7
20.9
15.4
47.0 36.0
52.0 49.5b
26.4 19.3
80.6 42.2
76.0. 75.1
55.3 50.2b
36.2 36.6
16.6 22.5a
31.5 68.8
71.7 72.9
58.8 57.1
45.9 46.4
28.6 24.3
17.3 22.5
26.8 27.7
20.9 21.4
15.3 14.9
85.5
53.2
25.3
41.4
74.1
50.2
38.8
24.4
66.1
73.9
57.4
46.2
22.3
23.6
31.4
26.3
16.6
85.6 37.8 38.8 37.8
52.8 55.4 55.2 55.3
25.8 19.5 19.6 19.5
41.2 42.8 42.8 42.7
74.7 75.0 74.8 74.7
49.8 57.7 57.2 57.1
38.5 36.5 36.6 36.5
24.7 16.7 16.5 16.5
66.8 32.0 30.9 30.9
74.6 71.7 71.3 71.3
58.5 58.9 57.6 57.6
46.3 45.9 47.5 47.4
21.6 28.0 29.2 29.1
23.1 22.7 23.9 24.0
31.1 28.6 28.0 28.0
26.7 16.3 15.6 16.4
17.4 15.8 15.2 15.7
4.2. Incubation procedure and isolation of products
The fungus M. plumbeus was grown in shake cultures at
258C in conical ¯asks (250 mL), each containing 50 mL
of a sterile medium comprising (per L) glucose (80 g),
NH4NO3 (0.48 g), KH2PO4 (5 g), MgSO4 (1 g) and trace
elements solution containing (per 100 mL) Co(NO3)2
(0.01 g), CuSO4 (0.015 g), ZnSO4 (0.16 g), MnSO4
(0.01 g) and (NH4)6Mo7O24 (0.01 g). The substrate
(260 mg) dissolved in EtOH (5.2 mL) was equally distri-
buted among the 26 conical ¯asks after one day of growth.
After a further six days, the fermentation was harvested. The
mycelium was ®ltered, and the culture ®ltrate was extracted
with EtOAc. The extract was dried over Na2SO4, the solvent
evaporated, and the residue chromatographed on a Si gel
column using a petroleum ether±EtOAc gradient, giving:
starting material (1) (5 mg), 3-oxo-1-en-ent-13-epi-manoyl
oxide (4) (3 mg), 3-oxo-1-en-14S,15-epoxy-ent-13-epi-
manoyl oxide (15) (2 mg), 6a-hydroxy-3-oxo-ent-13-epi-
manoyl oxide (12) (0.8 mg), 7a-hydroxy-3-oxo-ent-13-
epi-manoyl oxide (14) (1.7 mg), 1a-hydroxy-3-oxo-ent-
13-epi-manoyl oxide (6) (20 mg), 6a-hydroxy-3-oxo-
14S,15-epoxy-ent-13-epi-manoyl oxide (21) (1.7 mg),
11b-hydroxy-3-oxo-ent-13-epi-manoyl oxide (11) (5 mg),
6b-hydroxy-3-oxo-ent-13-epi-manoyl oxide (8) (1.3 mg),
12b-hydroxy-3-oxo-1-en-ent-13-epi-manoyl oxide (5)
(1.2 mg), 12b-hydroxy-3-oxo-ent-13-epi-manoyl oxide (9)
a These values can be interchanged.
b These values can be interchanged.
consequences may be deduced from the results obtained in
this biotransformation of ribenone (1) by M. plumbeus: (1)
The epoxidation of the vinyl group constitutes the main
reaction and there exists a preference for hydroxylation at
C-1(a) (6, 7 and 18), C-6(a or b) (8, 10, 12, 13, 21 and 22),
and C-12(b) (5, 7, 9, 10, 13, 23, 24, 27 and 28) and, to a
lesser degree, at C-7(a) (14, 25 and 26) and C-11(b) (11, 16
and 17). (2) The epoxidations produced in this biotrans-
formation are not stereospeci®c. This lack of stereospeci®-
city has also been observed in the chemical epoxidation of
this type of compounds or in its occurrence in plants, for
example in excoecarins A and B, 19 and 20, previously
cited.15 In all these cases the main stereoisomer obtained
was 14S. (3) The hydroxylations produced with this fungus
are not very regioselective, permitting the functionalization
of all the methylene groups of ribenone, except C-2, which,
on the other hand, is the most chemically accessible. (4) The
metabolites with a 1,2-double bond (5 and 15) are probably
formed by dehydration of 1a-hydroxy-derivatives. (5)
Another observed reaction was the reduction of the 3-oxo
group to a 3b-alcohol (27 and 28), which is also produced in
the biotransformation of 1 by G. fujikuroi. The chemical
reduction of the 3-oxo group formed the 3a-alcohol.
(16 mg),
1a-hydroxy-3-oxo-14S,15-epoxy-ent-13-epi-
manoyl oxide (18) (4.2 mg), 11b-hydroxy-3-oxo-14S,15-
epoxy-ent-13-epi-manoyl oxide (16) (1.4 mg), 6b-hy-
droxy-3-oxo-14S,15-ent-13-epi-manoyl oxide (22) (traces),
11b-hydroxy-3-oxo-14R,15-epoxy-ent-13-epi-manoyl oxide
(17) (1.8 mg), 7a-hydroxy-3-oxo-14R,15-epoxy-ent-13-
epi-manoyl oxide (25) (0.6 mg), 7a-hydroxy-3-oxo-
14S,15-epoxy-ent-13-epi-manoyl oxide (26) (traces),
6b,12b-dihydroxy-3-oxo-ent-13-epi-manoyl oxide (10)
(1 mg), 6a,12b-dihydroxy-3-oxo-ent-13-epi-manoyl oxide
(13) (1.3 mg), 12b-hydroxy-3-oxo-14S,15-epoxy-ent-13-
epi-manoyl oxide (23) (3.2 mg), 12b-hydroxy-3-oxo-
14R,15-epoxy-ent-13-epi-manoyl oxide (24) (2.9 mg),
3b,12b-dihydroxy-ent-13-epi-manoyl oxide (28) (13 mg),
3b,12b-dihydroxy-14S,15-epoxy-ent-13-epi-manoyl oxide
(27) and 1a,12b-dihydroxy-3-oxo-ent-13-epi-manoyl
oxide (7) (16 mg). Compound 27 was identi®ed as the
3b,12b-diacetate 27a (0.8 mg) by acetylation and chroma-
tography of the fraction containing it.
4. Experimental
4.1. General procedures
Mps were determined with a Reichert Thermovar apparatus
and are uncorrected. IR and UV spectra were recorded in a
Perkin±Elmer 1600 FT and a Varian Cary 1E spectrophoto-
meter, respectively. 1H NMR spectra were recorded in
CDCl3 solutions at 200.13 and 500.13 MHz, with a Bruker
AC-200 or a Bruker AMX2-500 spectrometer, respectively.
13C NMR spectra were run in CDCl3 at 50.32 MHz, with a
Bruker AC-200. Chemical shifts are given in ppm (d). Mass
spectra were taken at 70 eV (probe) in a Shimadzu Q2000,
4.2.1. 12b-Hydroxy-3-oxo-1-en-ent-13-epi-manoyl oxide
(5). Colourless gum; H NMR (500 MHz) d: 1.00 (3H, s,
1
H-20), 1.09 (3H, s, H-19), 1.16 (3H, s, H-18), 1.22 (3H, s,
H-16), 1.33 (3H, s, H-17), 4.19 (1H, br s, H-12), 5.01 (1H, d,
Jˆ18.1 Hz, H-15), 5.03 (1H, d, Jˆ11.5 Hz, H-15), 5.88
(1H, d, Jˆ10.2 Hz, H-1), 6.10 (1H, dd, Jˆ18.1, 11.5 Hz,
H-14), 7.13 (1H, d, Jˆ10.2 Hz, H-2); EIMS m/z (rel. int.):

B. M. Fraga et al. / Tetrahedron 57 (2001) 761±770
767
318 [M]1 (1), 303 (4), 300 (10), 285 (4), 248 (66), 233 (4),
205 (68); [M]1 at m/z 318.2190, C20H30O3 requires
318.2194.
H-11), 2.31 (1H, m, H-2), 2.84 (1H, dd, Jˆ14.3, 6.4 Hz,
H-2), 4.15 (1H, t, Jˆ3.5 Hz, H-12), 4.52 (1H, t, Jˆ6.0,
3.0 Hz, H-6), 4.98 (1H, d, Jˆ17.5 Hz, H-15), 5.01 (1H, d,
Jˆ11.2 Hz, H-15), 6.09 (1H, dd, Jˆ17.5 Hz, H-15), 5.01
(1H, d, Jˆ11.2 Hz, H-15), 6.09 (1H, dd, Jˆ17.5, 11.2 Hz,
H-14; EIMS m/z (rel. int.): 336 [M]1 (1), 321 (2), 318 (6),
303 (3), 300 (3), 293 (14), 285 (4), 248 (7), 223 (81), 205
(88); [M]1 at m/z 336.2299, C20H32O4 requires 336.2300.
4.2.2. Acetate (5a). IR (®lm) nmax 2930, 1740, 1670, 1460,
1380, 1240, 1080 cm21
; UV (EtOH) lmax 226 nm
(eˆ4.99£103), H NMR (500 MHz) d: 0.97 (3H, s, H-20),
1.08 (3H, s, H-19), 1.13 (3H, s, H-18), 1.17 (3H, s, H-16),
1.33 (3H, s, H-17), 2.14 (3H, s), 5.05 (1H, d, Jˆ11.4 Hz,
H-15), 5.11 (1H, d, Jˆ18.2 Hz, H-15), 5.44 (1H, t,
Jˆ3.0 Hz, H-12), 5.88 (1H, d, Jˆ10.1 Hz, H-1), 65.06
(1H, dd, Jˆ18.2, 11.4 Hz, H-14), 6.98 (1H, d, Jˆ10.1 Hz,
H-2); EIMS m/z (rel. int.): 360 [M]1 (1), 345 (3), 333 (1),
300 (2), 290 (3), 285 (5), 248 (40), 230 (14), 215 (8), 204
(100); [M]1 at m/z 360.2283, C22H32O4 requires 360.2300.
1
4.2.7. 7a-Hydroxy-3-oxo-ent-13-epi-manoyl oxide (14).
Colourless gum; 1H NMR (500 MHz) d: 0.84 (3H, s,
H-20), 1.03 (3H, s, H-19), 1.11 (3H, s, H-18), 1.13 (3H, s,
H-16), 1.23 (3H, s, H-17), 1.48 (1H, m, H-6), 1.77 (1H, ddd,
Jˆ13.0, 4.3, 3.1 Hz, H-6), 3.58 (1H, dd, Jˆ11.6, 4.3 Hz,
H-7), 4.96 (1H, d, Jˆ11.4 Hz, H-15), 4.99 (1H, d,
Jˆ18.2 Hz, H-15), 5.97 (1H, dd, Jˆ18.2, 11.4 Hz, H-14);
EIMS m/z (rel. int.): 320 [M]1 (2), 305 (80), 302 (1) 287
(28), 275 (5), 269 (10), 250 (6), 235 (9), 217 (8); [M]1 at m/z
336.2310, C20H32O3 requires 336.2300.
4.2.3. 1a,12b-Dihydroxy-3-oxo-ent-13-epi-manoyl oxide
(7). Colourless oil; IR (®lm) nmax 3420, 2980, 2940, 2870,
1700, 1460, 1390, 1290, 1240, 1090 cm21 1H NMR
;
(500 MHz) d: 0.80 (3H,s, H-20), 1.03 (3H, s, H-19), 1.07
(3H, s, H-18), 1.20 (3H, s, H-16), 1.28 (3H, s, H-17), 1.50
and 1.81 (each 1H, m, H-7), 1.68 (1H, m, H-6), 1.89 (1H, dd,
Jˆ13.0, 2.4 Hz, H-9), 2.02 (1H, td, Jˆ13.8, 2.8 Hz, H-11),
2.27 (1H, ddd, Jˆ13.8, 4.0, 2.6 Hz, H-11), 2.36 (1H, dd,
Jˆ14.9, 5.0 Hz, H-2), 2.96 (1H, dd, Jˆ14.9, 7.8 Hz, H-2),
3.98 (1H, dd, 7.8, 5.0 Hz, H-1), 4.11 (1H, t, Jˆ3.3 Hz,
H-12), 4.97 (1H, d, Jˆ18.2, H-15), 4.99 (1H, d, Jˆ
11.4 Hz, H-15), 6.07 (1H, dd, Jˆ18.2, 11.4 Hz, H-14);
EIMS m/z (rel. int.): 318 [M2H2O]1 (15), 300 (66), 285
(16), 257 (7), 248 (21), 235 (7), 223 (10), 205 (33);
[M2H2O]1 at m/z 318.2208, C20H30O3 requires 318.2194.
1
4.2.8. Acetate (14a). H NMR (500 MHz) d: 0.83 (3H, s,
H-20), 1.01 (3H, s, H-18), 1.08 (3H, s, H-19), 1.10 (3H, s,
H-16), 1.28 (3H, s, H-17), 1.45 (2H, complex signal, H-1
and H-6), 1.77 (1H, dt, Jˆ12.0, 4.5 Hz, H-6), 1.84 (1H, m,
H-1), 2.09 (3H, s), 2.50 (1H, dd, Jˆ8.6, 6.1 Hz, H-2), 4.77
(1H, dd, Jˆ12.0, 4.5 Hz, H-7), 4.93 (1H, d, Jˆ11.0 Hz,
H-15), 4.96 (1H, d, Jˆ18.0 Hz, H-15), 5.96 (1H, dd,
Jˆ18.0, 11.0 Hz, H-14); EIMS m/z (rel. int.): 362 [M]1
(3), 347 (100), 305 (14), 287 (93), 277 (5), 269 (52), 253
(18), 232 (27), 217 (33), 203 (23),; [M]1 at m/z 362.2468,
C22H34O4 requires 362.2457.
4.2.4. 6b,12b-Dihydroxy-3-oxo-ent-13-epi-manoyl oxide
(10). Colourless prisms, mp 88±918C (benzene±EtOAc); 1H
NMR (500 MHz) d: 0.65 (3H, s, H-20), 1.21 (3H, s, H-16),
1.28 (3H, s, H-17), 1.34 (3H, s, H-19), 1.57 (3H, s, H-18),
1.68 (1H, dd, Jˆ11.4, 3.7 Hz, H-11), 1.79 (1H, d, Jˆ3.7 Hz,
H-11), 2.11 (1H, dd, Jˆ11.6, 4.4 Hz, H-7), 2.31 (1H, m,
H-2), 2.74 (1H, m, H-2), 3.86 (1H, td, 11.6, 4.4 Hz, H-6),
4.13 (1H, t, Jˆ3.7 Hz, H-12), 4.97 (1H, d, Jˆ18.2, H-15),
5.01 (1H, d, Jˆ11.6 Hz, H-15), 6.07 (1H, dd, Jˆ18.2,
11.6 Hz, H-14); EIMS m/z (rel. int.): 336 [M]1 (3), 318
(2), 301 (1), 266 (13), 248 (10), 223 (77), 205 (66); [M]1
at m/z 336.2300, C20H32O4 requires 336.2300.
4.2.9. 3-Oxo-1-en-14S,15-epoxy-ent-13-epi-manoyl oxide
(15). Colourless gum; IR (®lm) nmax 2930, 1735, 1670,
1460, 1380, 1260, 1110, 1080 cm21; UV (EtOH) lmax
1
227 nm (eˆ6.44£103) H NMR (500 MHz) d: 1.01 (3H,
s, H-20), 1.09 (3H, s, H-19), 1.17 (3H, s, H-18), 1.24 (3H,
s, H-16), 1.36 (3H, s, H-17), 2.82 (1H, dd, Jˆ4.6, 2.9 Hz,
H-15), 2.86 (1H, dd, Jˆ4.6, 3.9 Hz, H-15), 2.95 (1H, dd,
Jˆ3.9, 2.9 Hz, H-14), 5.88 (1H, d, Jˆ10.2 Hz, H-1), 7.07
(1H, d, Jˆ10.2 Hz, H-2); EIMS m/z (rel. int.): 318 [M]1
(1), 303 (23), 275 (100), 257 (50), 217 (31), 199(35); [M]1
at m/z 318.2198, C20H30O3 requires 318.2194.
4.2.10. 11b-Hydroxy-3-oxo-14S,15-epoxy-ent-13-epi-manoyl
oxide (16). 1H NMR (500 MHz) d: 1.01 (3H, s, H-20), 1.03
(3H, s, H-19), 1.09 (3H, s, H-18), 1.23 (3H, s, H-17), 1.31
(3H, s, H-16), 1.46 (2H, m, H-9 and H-12b), 1.78 (1H, m,
H-1), 2.21 (1H, dd, Jˆ14.6, 5.4 Hz, H-12a), 2.24 (1H, m,
H-1), 2.42 (1H, ddd, Jˆ15.8, 7.3, 3.9 Hz, H-2a), 2.51 (1H,
ddd, Jˆ15.8, 10.4, 7.4 Hz, H-2b), 2.72 (1H, dd, Jˆ5.0,
4.1 Hz, H-15), 2.81(2H, m, H-14 and H-15), 4.13 (1H, dt,
Jˆ9.1, 5.4 Hz, H-11); EIMS m/z (rel. int.): 321 [M2CH3]1
(6), 303 (3), 293 (100), 275 (68), 257 (37), 235 (15), 217
(12), 207 (10), 201 (6), 199 (10); [M2CH3]1 at m/z
321.2064, C19H29O4 requires 321.2065.
4.2.5. 6a-Hydroxy-3-oxo-ent-13-epi-manoyl oxide (12).
1H NMR (500 MHz) d: 1.13 (3H, s, H-18), 1.16 (3H, s,
H-16), 1.33 (3H, s, H-20), 1.40 (3H, s, H-19), 1.57 (3H, s,
H-17), 1.35 (1H, d, Jˆ3.3 Hz, H-5), 1.66 (1H, dd, Jˆ15.1,
3.3, H-7), 1.95 (1H, ddd, Jˆ6.2, 2.9 Hz, H-1), 2.85 (1H, td,
Jˆ14.6, 6.2 Hz, H-2), 4.50 (1H, t, Jˆ3 Hz, H-6), 4.95 (1H,
d, Jˆ11.0 Hz, H-15), 4.99 (1H, d, Jˆ17.8 Hz, H-15), 6.04
(1H, dd, Jˆ17.8, 11.0 Hz, H-14); EIMS m/z (rel. int.): 320
[M]1 (1), 305 (100), 287 (18), 269 (24), 250 (6), 235 (1),
232 (2), 217 (100), 199 (10); [M]1 at m/z 320.2360,
C20H32O3 requires 320.2351.
4.2.6. 6a,12b-Dihydroxy-3-oxo-ent-13-epi-manoyl oxide
(13). Colourless gum; H NMR (500 MHz) d: 1.17 (3H, s,
4.2.11. 11b-Hydroxy-3-oxo-14R,15-epoxy-ent-13-epi-manoyl
oxide (17). Colourless gum; H NMR (500 MHz) d: 1.04
1
1
H-18), 1.19 (3H, s, H-16), 1.35 (3H, s, H-20), 1.41 (3H, s,
H-19), 1.60 (3H, s, H-17), 1.37 (1H, d, Jˆ3.0 Hz, H-5), 1.71
(1H, dd, Jˆ14.0, 2.9 Hz, H-7), 1.91 (1H, m, H-1), 2.00 (1H,
dd, Jˆ14.0, 6.0 Hz, H-7), 2.03 (1H, dd, Jˆ13.8, 3.5 Hz,
(3H, s, H-20), 1.06 (3H, s, H-19), 1.12 (3H, s, H-18), 1.24
(3H, s, H-16), 1.42 (3H, s, H-17), 1.35 (2H, m, H-9 and
H-12b), 1.74 (1H, m, H-1), 1.85 (1H, dd, 13.2, 5.0 Hz,
H-12a), 2.46 (1H, m, H-2), 2.48 (2H, m, H-1 and H-2),

768
B. M. Fraga et al. / Tetrahedron 57 (2001) 761±770
2.56 (1H, dd, Jˆ4.4, 2.9 Hz, H-15), 2.83 (1H, t, Jˆ4.4 Hz,
H-15), 3.04 (1H, dd, Jˆ4.4, 2.9 Hz, H-14), 4.29 (1H, br m,
W1/2ˆ30 Hz, H-11); EIMS m/z (rel. int.): 336 [M]1 (1), 321
(7), 303 (9), 293 (100), 275 (51), 257 (20), 235 (14), 217
(11), 201 (5); [M]1 at m/z 336.2298, C20H32O4 requires
336.2300.
(1H, t, Jˆ4.4 Hz, H-15), 3.11 (1H, dd, Jˆ4.4, 2.9 Hz,
H-14), 3.89 (1H, br d, Jˆ4.0 Hz, H-12); EIMS m/z (rel.
int.): 336 [M]1 (1), 321 (6), 318 (2), 303 (1), 293 (47),
275 (10), 257 (5), 249 (18), 235 (2), 217 (2), 206 (100),
191 (51); [M]1 at m/z 336.2295, C20H32O4 requires
336.2300.
4.2.12. 1a-Hydroxy-3-oxo-14S,15-epoxy-ent-13-epi-manoyl
oxide (18). White needles, mp 134±1358C (petroleum
ether±EtOAc), [a]Dˆ252 (c 0.21, CHCl3); 1H NMR
(500 MHz) d: 0.81 (3H, s, H-20), 1.04 (3H, s, H-19), 1.09
(3H, s, H-18), 1.22 (3H, s, H-16), 1.32 (3H, s, H-17), 1.36
(1H, dt, Jˆ12.5, 5.7 Hz, H-12), 1.44 (1H, dd, Jˆ12.2,
2.4 Hz, H-9), 1.47 (3H, br m, H-5, H-6 and H-7), 1.49
(1H, m, H-11a), 1.61 (1H, m, H-12), 1.69 (1H, m, H-6),
1.83 (1H, m, H-7), 2.06 (1H, m; H-11b), 2.35 (1H, dd,
Jˆ14.8, 4.9 Hz, H-2b), 2.81 (2H, m, H-15), 2.92 (1H, t,
Jˆ3 Hz, H-14), 2.97 (1H, dd, Jˆ14.8, 7.8 Hz, H-2a), 3.94
(1H, dd, Jˆ7.8, 4.9 Hz, H-1); EIMS m/z (rel. int.): 321
[M2CH3]1 (1), 303 (2), 293 (56), 275 (100), 257 (34),
217 811), 201 (7), 199 (10); [M2CH3]1 at m/z 321.2063,
C19H29O4 requires 321.2065.
4.2.17. 7a-Hydroxy-3-oxo-14R,15-epoxy-ent-13-epi-manoyl
oxide (25). 1H NMR (500 MHz) d: 0.90 (3H, s, H-20), 1.05
(3H, s, H-19), 1.11 (6H, s, H-16 and H-18), 1.44 (3H, s,
H-17), 1.52 and 1.79 (each 1H, m, H-6), 2.51 (3H, m, H-15
and H-2), 2.82 (1H, t, Jˆ4.0 Hz, H-15), 3.09 (1H, t,
Jˆ4.0 Hz, H-14), 3.63 (1H, dd, Jˆ11.5, 4.3 Hz, H-11);
EIMS m/z (rel. int.): 336 [M]1 (1), 321 (2), 293 (100),
275 (32), 257 (15), 217 (8), 199 (7); [M]1 at m/z
336.2308, C20H32O4 requires 336.2300.
1
4.2.18. Acetate (25a). H NMR (500 MHz) d: 0.87 (3H, s,
H-20), 1.03 (3H, s, H-19), 1.07 (3H, s, H-16), 1.11 (3H, s,
H-18), 1.49 (3H, s, H-17), 1.47 and 1.84 (each 1H, m, H-1),
1.74 (1H, dd, Jˆ12.6, 3.0 Hz, H-5), 1.78 (2H, m, H-6), 2.48
(3H, m, 2H-15 and H-2), 2.81 (1H, t, Jˆ4.0 Hz, H-15), 3.07
(1H, t, Jˆ4.0 Hz, H-14), 4.85 (1H, dd, Jˆ11.7, 4.6 Hz,
H-7), EIMS m/z (rel. int.): 378 [M]1 (1), 335 (78), 275
(100), 257 (83), 239 (6), 217 (21), 199 (14); [M]1 at m/z
378.2408, C22H34O5 requires 378.2406.
4.2.13. 6a-Hydroxy-3-oxo-14S,15-ent-13-epi-manoyl oxide
(21). Colourless gum; H NMR (500 MHz) d: 1.15 (3H, s,
1
H-18), 1.19 (3H, s, H-16), 1.28 (1H, d, Jˆ3.0 Hz, H-5), 1.33
(3H, s, H-20), 1.39 (3H, s, H-19), 1.59 (3H, s, H-17), 1.68
(1H, dd, Jˆ14.0, 3.0 Hz, H-7), 1.89 (1H, ddd, Jˆ14.0, 6.1,
2.8 Hz, H-2), 2.00 (1H, dd, Jˆ14, 3.0 Hz, H-7), 2.25 (1H,
ddd, 14, 4.5, 2.8 Hz, H-1), 2.81 (3H, m, 2H-15 and H-2),
2.92 (1H, t, Jˆ3.2 Hz, H-14), 4.49 (1H, t, Jˆ3.0 Hz, H-6);
EIMS m/z (rel. int.): 336 [M]1 (1), 321 (22), 303 (8), 293
(100), 275 (40), 257 (64), 215 (15), 199 (18); [M]1 at m/z
336.2310, C20H32O4 requires 336.2300).
4.2.19. 7a-Acetoxy-14S,15-epoxy-ent-13-epi-manoil oxide
(26a). This compound was observed contaminating 24a, 1H
NMR (500 MHz) d: 0.86 (3H, s, H-20), 1.02 (3H, s, H-19),
1.11 (3H, s, H-18), 1.18 (3H, s, H-16), 1.35 (3H, s, H-17),
2.77 (1H, dd, Jˆ4.9, 2.8 Hz, H-15), 2.81 (1H, t, Jˆ4.9 Hz,
H-15), 2.87 (1H, t, Jˆ2.8 Hz, H-14), 4.85 (1H, dd, Jˆ 11.7,
4.6 Hz, H-7).
4.2.14. 6b-Hydroxy-3-oxo-14S,15-ent-13-epi-manoyl oxide
(22). This compound was observed contaminating a fraction
of 15, 1H NMR (500 MHz) d: 0.67 (3H, s, H-20), 1.23 (3H,
s, H-16), 1.26 (3H, s, H-19), 1.29 (3H, s, H-17), 1.32 (3H, s,
H-18), 2.10 (1H, dd, Jˆ11.5, 3.9 Hz, H-7a), 2.75 (1H, dd,
Jˆ4.8, 3.0 Hz, H-15), 2.80 (1H, t, Jˆ4.0 Hz, H-14), 2.89
(1H, t, Jˆ3.0 Hz, H-14), 3.82 (1H, br m, H-6).
4.2.20. 3b,12b-Diacetoxy-14S,15-epoxy-ent-13-epi-manoyl
oxide (27a). White prisms, mp 184±1868C (petroleum
ether±EtOAc), [a]Dˆ238 (c 0.04, CHCl3); 1H NMR
(500 MHz) d: 0.79 (3H, s, H-20), 0.87 (3H, s, H-18), 0.89
(3H, s, H-19), 1.12 and 1.35 (each 1H, m, H-1), 1.20 (3H, s,
H-16), 1.31 (3H, s, H-17), 1.51 (1H, dd, Jˆ11.8, 2.4 Hz,
H-5), 1.64 (1H, m, H-2), 1.67 (1H, dd, Jˆ13.0, 4.1 Hz,
H-11), 1.73 (1H, dd, Jˆ12.5, 3.0 Hz, H-9), 1.79 (1H, dd,
Jˆ13.0, 4.1 Hz, H-11), 1.84 and 1.87 (each 1H, m, H-7),
1.90 (1H, m, H-2), 2.09 and 2.11 (each 3H, s), 2.81 (1H, dd,
Jˆ4.6, 3.6 Hz, H-15), 2.85 (1H, dd, Jˆ4.6, 3.0 Hz, H-15),
2.97 (1H, dd, Jˆ3.6, 3.0 Hz, H-14), 4.67 (1H, t, Jˆ3.0 Hz,
H-3), 4.67 (1H, t, Jˆ3.0 Hz, H-3), 4.79 (1H, t, Jˆ4.1 Hz,
H-12); EIMS m/z (rel. int.): 407 [M2CH3]1 (9), 379 (16),
347 (6), 319 (64), 302 (5), 259 (46), 250 (20), 241 (52), 201
(10); [M2CH3]1 at m/z 407.2418, C23H35O6 requires
407.2433.
4.2.15. 12b-Hydroxy-3-oxo-14S,15-epoxy-ent-13-epi-manoyl
oxide (23). White needles, mp 142±1458C (petroleum
ether±CH2Cl2), [a]Dˆ246 (c 0.16, CHCl3); 1H NMR
(500 MHz) d: 0.89 (3H, s, H-20), 1.03 (3H, s, H-19), 1.10
(3H, s, H-18), 1.26 (3H, s, H-16), 1.27 (3H, s, H-17), 1.64
(1H, m, H-11), 1.87 (1H, dd, Jˆ13.0, 5.0 Hz, H-11), 2.43
(1H, m, H-2b), 2.51 (1H, m, H-2a), 2.81 (1H, dd, 4.6,
4.0 Hz, H-15), 2.86 (1H, dd, Jˆ4.6, 2.9 Hz, H-15), 3.03
(1H, dd, Jˆ4.0, 2.9 Hz, H-14), 3.71 (1H, br d, Jˆ4.4 Hz,
H-12); EIMS m/z (rel. int.): 336 [M]1 (1), 321 (5), 318 (1),
303 (1), 293 (53), 275 (12), 257 (7), 249 (21), 235 (2), 206
(100); [M]1 at m/z 336.2295, C20H32O4 requires 336.2300).
4.2.21. Epoxidation of 9. Compound 9 (15 mg) in CHCl3
(2 mL) was treated with m-chloroperbenzoic acid (11 mg) at
08C for 6 h and washed with a saturated solution of
NaHCO3. Usual work-up and chromatograpy of the residue,
eluting with petrol±EtOAc (1:1), afforded 23, 24, 29 and 30.
The ®rst two compounds were identical with those obtained
in this incubation.
4.2.16. 12b-Hydroxy-3-oxo-14R,15-epoxy-ent-13-epi-manoyl
oxide (24). Colourless needles, mp 178±1808C (petroleum
ether±CH2Cl2), [a]Dˆ242 (c 0.21, CHCl3); 1H NMR
(500 MHz) d: 0.88 (3H, s, H-20), 1.01 (3H, s, H-19), 1.08
(3H, s, H-18), 1.14 (3H, s, H-16), 1.42 (3H, s, H-17), 2.03
(1H, td, Jˆ14 and 4 Hz, H-11a), 2.44 (1H, m, H-2b), 2.52
(1H, m, H-2a), 2.56 (1H, dd, Jˆ4.4, 2.9 Hz, H-15), 2.82
4.2.22. Compound 29. 1H NMR (500 MHz) d: 1.01 (3H, s,
H-20), 1.26 (3H, s, H-16), 1.29 (3H, s, H-17), 1.41 (3H, s,

B. M. Fraga et al. / Tetrahedron 57 (2001) 761±770
769
H-19), 1.49 (3H, s, H-18), 2.59 (1H, dq, Jˆ15, 11.1, 7.1,
3.4 Hz, H-2b), 1.90 (1H, dd, Jˆ12, 3.5 Hz, H-5), 2.67 (1H,
dq, Jˆ15.5, 15, 11.1, 4.4 Hz, H-2a), 2.81 (1H, t, Jˆ4.5 Hz;
H-15), 2.85 (1H, dd, Jˆ4.5, 3.2 Hz, H-15), 3.02 (1H, t,
Jˆ3.2 Hz, H-14), 3.70 (1H, br s, H-12); EIMS m/z (rel.
int.): 352 [M]1 (2), 337 (4), 334 (4), 316 (2), 309 (24),
291 (19), 223 (36), 154 (38), 164 (100), 149 (47); [M]1 at
m/z 352.2249, C20H32O5 requires 352.2250.
2.4 Hz, H-5), 0.98 (1H, td, Jˆ12.5, 3.8 Hz, H-1), 1.19 (3H,
s, H-16), 1.26 (3H, s, H-17), 1.40 (3H, dt, Jˆ13.2, 3.6 Hz,
H-7b), 1.48 (1H, m, H-11), 1.81 (1H, dt, Jˆ12.3, 3.2 Hz,
H-7a), 2.78 (1H, dd, Jˆ4.8, 2.9 Hz, H-15), 2.80 (1H, t,
Jˆ3.8 Hz, H-15), 2.90 (1H, t; Jˆ3.1 Hz, H-14), 3.21 (1H,
Jˆ11.5, 4.0 Hz, H-3); EIMS m/z (rel. int.): 322 (0.2), 307
(6), 279 (50), 261 (61), 243 (100), 203 (25), 189 (29), 187
(30), 173 (16), 159 (20), 147 (21), 135 (77), 121 (45). [M]1
at m/z 322.2521, C20H34O3 requires 322.2508.
4.2.23. Compound 30. White needles, mp 138±1408C
(petroleum ether±EtOAc), [a]Dˆ235 (c 0.26, CHCl3); IR
(®lm) nmax 3460, 2930, 2860, 1720, 1455, 1380, 1295, 1255,
4.2.28. Crystallographic data for compound 32a. Colour-
less needles, C22H36O4, Mw 364.5, orthorhombic, space
1
1110 cm21; H NMR (500 MHz) d: 0.98 (3H, s, H-20),
Ê
Ê
group P212121, aˆ7.330(2) A, bˆ11.083(2) A, cˆ
25.311(5) A, Vˆ2056.2(8) A , Zˆ4, Dcˆ1.18 g cm23
,
Ê
Ê 3
1.11(3H, s, H-16), 1.38 (3H, s, H-17), 1.42 (3H, s, H-19),
1.46 (3H, s, H-18), 2.53 (1H, m, H-2b), 2.55 (1H, t, Jˆ
4.3, Hz, H-15), 2.83 (1H, t, Jˆ4.4 Hz, H-15), 3.10 (1H, t,
Jˆ3.0 Hz), 3.38 (1H, t, Jˆ3.3 Hz); EIMS m/z (rel. int.): 352
[M]1 (1), 337 (5), 309 (17), 291 (14), 223 (31), 164 (100),
149 (40); [M]1 at m/z 352.2200, C20H32O5 requires
352.2250.
F(000)ˆ800, m(Mo Ka)ˆ0.079 mm21
.
The intensity data of all unique re¯ections within u range
2.5±26.38 were collected at 273 K in an Enraf±Nonius
CAD4 diffractometer, using Mo Ka radiations and graphite
monochromator. Three standard re¯ections monitored every
2 h of X-ray exposure showed not signi®cative intensity
variation. A total of 1610 unique re¯ections were recorded,
of which 759 were considered as observed under Fo.4sFo.
The intensities were corrected for Lorentz and polarisation
factors, but no absorption correction was made. The struc-
ture was solved by direct methods using shelxs86,17 re®ne-
ment was performed with shelxl93,18 using full-matrix
least squares with anisotropic thermal paramethers for
non-H atoms (the C-15 was kept isotropic). The H-atoms
were placed in calculated positions and added to the re®ne-
ment as ®xed isotropic contribution. The re®nement
has converged at R1ˆ6.17% and wR2ˆ14.37% with a good-
ness of ®t on F2 of 1.007, the largest peak on the ®nal
4.2.24. Epoxidation of ribenol acetate. Compound 2a
(40 mg) was epoxidised as described above for 9 giving a
mixture of the two stereoisomers at C-14 (34.5 mg).
Chromatography of the mixture by recycled HPLC, using
as solvent n-hexane±EtOAc (8:2), afforded (32a) (9 mg)
and (31a) (2 mg).
4.2.25. 3a-Acetoxy-14R,15-epoxy-ent-13-epi-manoyl oxide
(31a). This compound was obtained slightly contaminated
by (32a), H NMR (500 MHz) d: 0.80 (3H, s, H-20), 0.83
1
(3H, s, H-19), 0.87 (3H, s, H-18), 1.01 (1H, dd, Jˆ12.1,
2.1 Hz, H-5), 1.10 (3H, s, H-16), 1.14 (1H, dd, Jˆ12.4,
2.6 Hz, H-9), 1.40 (3H, s, H-17), 1.50 (1H, dt, Jˆ13.3,
3.7 Hz, H-12), (1.82 (1H, dt, Jˆ12, 3.1 Hz, H-7), 2.04
(3H, s), 2.49 (1H, dd, Jˆ4.7, 3.1 Hz, H-15), 2.78 (1H, t,
Jˆ4.2 Hz, H-15), 3.09 (1H, t, Jˆ3.6 Hz, H-14), 4.47 (1H,
dt, Jˆ12, 4.7 Hz, H-3).
3
Ê
difference map was 0.345 e/A. Crystallographic data of
32a, including atomic coordinates, have been deposited
with the Cambridge Crystallographic Data Centre. Copies
of the data can be obtained free of charge, on application to
the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ,
U.K. (Fax: 144-(0)1223-336033, e-mail: deposit@ccdc.
cam.ac.uk).
4.2.26. 3a-Acetoxy-14S,15-epoxy-ent-13-epi-manoyl oxide
(32a). Colourless needles, mp 200±201 8C, (petroleum
ether±EtOAc), [a]Dˆ228 (c 0.15, CHCl3); IR (®lm) nmax
1
2945, 1730, 1480, 1455, 1365, 1250, 1105, 1020, 985; H
Acknowledgements
NMR (500 MHz) d: 0.76 (3H, s, H-20), 0.82 (3H, s, H-19),
0.85 (3H, s, H-18), 1.01 (1H, dd, Jˆ12.2, 2.4 Hz, H-5), 1.06
(1H, td, Jˆ13.6, 3.6 Hz, H-1b), 1.19 (3H, s, H-16), 1.28
(3H, s, H-17), 1.42 (1H, td, Jˆ12.8, 3.6 Hz, H-7b), 1.47
(1H, m, H-11), 1.80 (1H, dt, Jˆ12.1, 3.3 Hz, H-7a), 2.77
(1H, dd, Jˆ4.8, 3.2 Hz, H-15), 2.79 (1H, t, Jˆ4.7, H-15),
2.89 (1H, t, Jˆ3.1 Hz, H-14), 4.45 (1H, dd, Jˆ11.7, 5.0 Hz,
H-3); EIMS m/z (rel. int.): 364 [M]1 (0.2), 349 (7), 321 (56),
261 (86), 243 (100), 203 (24), 201 (32), 191 (12), 189 (11),
187 (17), 173 (9), 147 (9), 135 (52), 121 (19); [M]1 at m/z
364.2613, C22H36O4 requires 364.2614.
This work has been supported by the SEUID, Ministry of
Â
Education and Culture, Spain (PB98-0540). S. Suarez
thanks the University of La Laguna and Santander Bank
for a fellowship.
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