Biotransformation of ent-kaur-16-ene and ent-trachylobane 7β-acetoxy derivatives by the fungus Gibberella fujikuroi (Fusarium fujikuroi)

Article abstract of DOI:10.1016/j.phytochem.2012.05.024

Candol A (7β-hydroxy-ent-kaur-16-ene) (6) is efficiently transformed by Gibberella fujikuroi into the gibberellin plant hormones. In this work, the biotransformation of its acetate by this fungus has led to the formation of 7β-acetoxy-ent-kaur-16-en-19-oic acid (3), whose corresponding alcohol is a short-lived intermediate in the biosynthesis of gibberellins and seco-ring ent-kaurenoids in this fungus. Further biotransformation of this compound led to the hydroxylation of the 3β-positions to give 7β-acetoxy-3β- hydroxy-ent-kaur-16-en-19-oic acid (14), followed by a 2β- or 18-hydroxylation of this metabolite. The incubation of epicandicandiol 7β-monoacetate (7β-acetoxy-18-hydroxy-ent-kaur-16-ene) (10) produces also the 19-hydroxylation to form the 18,19 diol (20), which is oxidized to give the corresponding C-18 or C-19 acids. These results indicated that the presence of a 7β-acetoxy group does not inhibit the fungal oxidation of C-19 in 7β-acetoxy-ent-kaur-16-ene, but avoids the ring B contraction that leads to the gibberellins and the 6β-hydroxylation necessary for the formation of seco-ring B ent-kaurenoids. The biotransformation of 7β-acetoxy-ent- trachylobane (trachinol acetate) (27) only led to the formation of 7β-acetoxy-18-hydroxy-ent-trachylobane (33).

Full text of DOI:10.1016/j.phytochem.2012.05.024

Phytochemistry 81 (2012) 60–70
Contents lists available at SciVerse ScienceDirect
Phytochemistry
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Biotransformation of ent-kaur-16-ene and ent-trachylobane 7b-acetoxy
derivatives by the fungus Gibberella fujikuroi (Fusarium fujikuroi)
a
a
b
b
Braulio M. Fraga a, , Carlo Bressa , Victoria González-Vallejo , Pedro González , Ricardo Guillermo
⇑
a Instituto de Productos Naturales y Agrobiología, C.S.I.C., Avda. Astrofísico F. Sánchez 3, 38206-La Laguna, Tenerife, Canary Islands, Spain
b Instituto Universitario de Bioorgánica ‘‘Antonio González’’, Universidad de La Laguna, Tenerife, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Candol A (7b-hydroxy-ent-kaur-16-ene) (6) is efficiently transformed by Gibberella fujikuroi into the gib-
berellin plant hormones. In this work, the biotransformation of its acetate by this fungus has led to the
formation of 7b-acetoxy-ent-kaur-16-en-19-oic acid (3), whose corresponding alcohol is a short-lived
intermediate in the biosynthesis of gibberellins and seco-ring ent-kaurenoids in this fungus. Further
biotransformation of this compound led to the hydroxylation of the 3b-positions to give 7b-acetoxy-
3b-hydroxy-ent-kaur-16-en-19-oic acid (14), followed by a 2b- or 18-hydroxylation of this metabolite.
The incubation of epicandicandiol 7b-monoacetate (7b-acetoxy-18-hydroxy-ent-kaur-16-ene) (10) pro-
duces also the 19-hydroxylation to form the 18,19 diol (20), which is oxidized to give the corresponding
C-18 or C-19 acids. These results indicated that the presence of a 7b-acetoxy group does not inhibit the
fungal oxidation of C-19 in 7b-acetoxy-ent-kaur-16-ene, but avoids the ring B contraction that leads to
the gibberellins and the 6b-hydroxylation necessary for the formation of seco-ring B ent-kaurenoids.
The biotransformation of 7b-acetoxy-ent-trachylobane (trachinol acetate) (27) only led to the formation
of 7b-acetoxy-18-hydroxy-ent-trachylobane (33).
Received 13 March 2012
Received in revised form 21 May 2012
Available online 21 June 2012
Keywords:
Gibberella fujikuroi (Fusarium fujikuroi)
Biotransformations
Diterpenes
Candol A acetate
Epicandicandiol 7b-monoacetate
Foliol 7b-monoacetate
Trachinol acetate
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
this process was the C-19 oxidation of 6 forming 7b-hydroxy-ent-
kaur-16-en-19-oic acid (1), via the corresponding 19-alcohol and
The partial synthesis of the gibberellin plant hormones (GAs)
and their analogues, using microbiological (Fraga et al., 1980,
1992, 1995) or chemical methods (González et al., 1978; Fraga
et al., 1988, 1990a, 1994) has been carried out by our group for sev-
eral years. The synthesis and biosynthesis of gibberellins have been
reviewed. Recently, we needed 7b-hydroxy-ent-kaur-16-en-19-oic
acid (1) in a study of the chemical preparation of gibberellin
analogues (Fraga et al., 2011). This acid is also an important inter-
mediate in the biosynthesis of gibberellins in plants (Lew and
West, 1971) and in the fungus Gibberella fujikuroi (Hanson et al.,
1972), being rapidly and efficiently transformed into GA12 alde-
hyde (2) (Scheme 1). Thus, the first aim of this work was to prepare
the 7-acetate 3 of the acid 1 using the biosynthetic machinery of
this fungus. In this way, we firstly describe here the microbiologi-
aldehyde. Thus, we believe that the presence of a 7b-OAc group
in the substrate 6 should avoid the ring B contraction (Scheme 1)
and, consequently, the production of these plant hormones, stop-
ping the bioreaction after the formation of the C-19 acid, thus lead-
ing to the required acetate-acid 3 (Scheme 1).
This study was completed with the biotransformation by G.
fujikuroi of other 7b-acetoxy derivatives such as 7b-acetoxy-18-
hydroxy-ent-kaur-16-ene (epicandicandiol 7b-monoacetate) (10),
7b-acetoxy-3a,18-dihydroxy-ent-kaur-16-ene (foliol 7b-monoace-
tate) (23) and 7b-acetoxy-ent-trachylobane (trachinol acetate)
(27), with the aim of obtaining other intermediates and gaining
more knowledge about the specificity in the substrate of the en-
zymes involved in the metabolic processes of this fungus.
2. Results and discussion
cal transformation of 7b-acetoxy-ent-kaur-16-ene (candol
A
acetate) (7) by G. fujikuroi to obtain 7b-acetoxy-ent-kaur-16-en-
19-oic acid (3) and other derivatives 12–16. We have taken into
consideration that the diterpene candol A (6), although it is not a
natural precursor of gibberellins, was transformed by this fungus
into these plant hormones (Fraga et al., 1980). The early stage of
The incubations with G. fujikuroi were carried out in the pres-
ence of AMO 1618, a compound that inhibits the formation of
ent-kaur-16-ene, without perturbing the post-kaurene metabolism
(Dennis et al., 1965; Cross and Myers, 1969). The diterpenes epi-
candicandiol (8) and trachinodiol (28), utilized as starting material
in the preparation of the substrates 7, 10 and 27, were isolated
from several Sideritis species, endemic to the Canary Islands (Fraga
et al., 2009), whilst foliol (22) used in the preparation of 23 had
⇑
Corresponding author. Tel.: +34 922 251728; fax: +34 922 260135.
E-mail address: bmfraga@ipna.csic.es (B.M. Fraga).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.05.024

B.M. Fraga et al. / Phytochemistry 81 (2012) 60–70
61
4
seco-ring B
kaurenoids
H
OH
12
OH
H
17
CO2H
13
11
20
10
1
16
14
9
5
8
7
G. fujikuroi
2
3
6
H
15
4
5
OH
6
CO2H
18
GAs
19
1
CO2H CHO
2
G. fujikuroi
H
7
H
OAc
GAs
CO2H
HO
3
CO2H CHO
4
Scheme 1. Aspects of the biotransformation of candol A (6) and its acetate (7) by G. fujikuroi.
been isolated from the Spanish species Sideritis leucantha (De Ques-
ada et al., 1972).
and C-19; H-19 with C-3 and C-18; and H-20 with C-1, C-5, C-9
and C-10, were observed in the HMBC spectrum.
Substrate 7 was obtained in the following way: Partial hydroly-
sis of epicandicandiol diacetate (9) afforded the monoacetate 10,
which was treated with triphenylphosphine in carbon tetrachlo-
ride to form the 18-chloro derivative 11. The reaction of this with
tri-n-butyl tin hydride gave 7b-acetoxy-ent-kaur-16-ene (7).
The incubation of 7 with G. fujikuroi gave 7b-acetoxy-19-hydro-
xy-ent-kaur-16-ene (12), 7b-acetoxy-ent-kaur-16-en-19-al (13),
7b-acetoxy-ent-kaur-16-en-19-oic acid (3), 7b-acetoxy-3b-hydroxy
-ent-kaur-16-en-19-oic acid (14), 7b-acetoxy-2b,3b-dihydroxy-ent-
kaur-16-en-19-oic acid (15) and 7b-acetoxy-3b,18-dihydroxy-ent-
kaur-16-en-19-oic acid (16) (Scheme 2). The last two compounds
were isolated as their methyl esters 15m and 16m, respectively,
by methylation of a mixture of them with diazomethane, and subse-
quent chromatography. Thus, they were more easily separated by
decreasing their polarity.
The metabolite 13 was the least polar substance isolated from
this fermentation. Its HREIMS showed the peak of highest mass
at m/z 284.2136 (C20H28O), which is formed by loss of an acetic
acid fragment from the molecular ion. Consequently, the molecular
formula was determined as C22H32O3, indicating that this com-
pound had gained one oxygen atom and lost two hydrogens during
the incubation. The disappearance of a methyl group and the pres-
ence of a new proton at dH 9.71 and of a new oxo group at dC 205.8
indicated that a methyl had been oxidized to an aldehyde group.
This was assigned at C-19 by comparison with the resonance val-
ues, d 9.78 and 204.7, previously reported for an analogous com-
pound (Fraga et al., 2010). The correlations observed in the
HMBC experiment were: H-18 with C-3 and C-5; H-19 with C-5;
H-20 with C-10; and H-3b, H-5 and H-18 with C-19. Thus, the
structure of 7b-acetoxy-ent-kaur-16-en-19-al (13) was assigned
to this metabolite, which must be biosynthetically formed by oxi-
dation of 12.
Compound 12 showed in its 1H NMR spectrum, in comparison
with the substrate (7), the disappearance of a methyl group and
the presence of a new pair of doublets at d 3.42 and 3.72, which
is a characteristic of an axial hydroxymethyl group at C-4 (Bohl-
mann et al., 1982). This was also in accordance with the corre-
sponding carbon resonance at d 66.0. In previous report, an
equatorial group of this type at C-4 resonates at d 72.1 (González
et al., 1981). Thus, the structure of this compound was determined
as 7b-acetoxy-19-hydroxy-ent-kaur-16-ene (12), which was con-
firmed by assignation of their 1H and 13C NMR spectra, using 2D
NMR data. Correlations of H-5 with C-19; H-18 with C-3, C-4, C-5
In the HREIMS spectrum compounds 12 and 13 showed the loss
of an acetic acid fragment from the molecular ion. The same was
observed in the spectrum of the metabolite 3, where the peak of
highest mass found, m/z 300.2077 (C20H28O2), indicated its molec-
ular formula C22H32O4. Consequently, the molecule had gained two
oxygens and lost two hydrogens during the fermentation. The 1H
NMR spectrum of 3 showed the disappearance of a methyl signal,
in comparison with that of the substrate (7), which indicated that
C-19 had been oxidised to acid level. This was confirmed in the 13
C

62
B.M. Fraga et al. / Phytochemistry 81 (2012) 60–70
G. f ujikuroi
H
H
7
OAc
OAc
CH2OH
12
H
H
OAc
OAc
CHO
13
CO2H
3
HO
HO
H
H
OAc
HO
OAc
CO2R
CO2H
14
15 R = H
15m R = Me
H
HO
HOH2C CO2R
OAc
16 R = H
16m R = Me
Scheme 2. Biotransformation of candol A acetate (7) by G. fujikuroi.
NMR spectrum with a new signal at d 182.7, and in the HMBC
experiment by correlations of C-19 with H-3, H-5 and H-18. Thus,
the structure of this metabolite was determined as 7b-acetoxy-ent-
kaur-16-en-19-oic acid (3). This compound had been isolated from
Xylopia aethiopica (Lajide et al., 1995). Its hydrolysis with 5% KOH
in MeOH led to the gibberellins precursor 1, which had also been
obtained from Didimocarpus oblonga (Mitra et al., 1980) and Aristol-
ochia anguicida (Gaitán et al., 2002; Fraga, 2004), and synthesized
from 7-oxokaurenolide (Hanson and White, 1969).
en-19-oic acid (14). Its 1H and 13C NMR spectra were similar to
those of 3, except that the resonance of the geminal proton to a
new hydroxyl group now appears at dH 4.09 (br s) and that of the
corresponding oxymethine at dC 70.5. These facts were also in
accordance with the HREIMS, where the peak of higher mass was
at m/z 316.2046 (M+ꢀAcOH). The new hydroxyl group was as-
signed to C-3(b) considering the form of resonance of its geminal
proton and the correlations observed in the HMBC spectrum, H-3
with C-1, C-4 and C-5, and H-18 with C-3. This compound 14
should be formed by 3b-hydroxylation of 3. The corresponding
dihydroxy-acid had been obtained in the biotransformation of
To the fourth metabolite isolated in this fermentation was as-
signed as the structure of 7b-acetoxy-3b-hydroxy-ent-kaur-16-

B.M. Fraga et al. / Phytochemistry 81 (2012) 60–70
63
3b-hydroxy-ent-kaur-16-en-19-oic acid by a wild-type strain and
two different mutants of G. fujikuroi (Lunnon et al., 1977; Barrero
et al., 1999).
The isomeric metabolites 15 and 16 were obtained in the form
of their methyl esters 15m and 16m, as stated above. The first of
them 15m showed resonances at dH 4.22 (ddd) and dC 66.5 (d),
characteristic of an oxymethine group. The hydrogen showed cou-
pling with another vicinal –CH(OH)– group at dH 4.09 (d,
the carbon resonance of this acid at d 182.9 and formation of the
methyl ester 17m, after treatment with diazomethane. The metab-
olite 17 was identical to a compound obtained by oxidation of epi-
candicandiol 7b-monoacetate (10) during the partial synthesis of
sventenic acid (21), a diterpene isolated from S. sventenii (Fraga
et al., 1990b).
The second metabolite isolated in this feeding was 18. Its HRMS
data indicated that a new oxygen atom had been introduced in the
molecule of the substrate. The 1H and 13C NMR spectra showed the
absence of a methyl group and the presence of a new hydroxy-
methylene group as a pair of doublets at dH 3.69 and 3.90 and a
triplet at dC 64.8. This alcohol was assigned to C-19 considering
the HMBC spectrum, with crosspeaks of H-19 with C-3, C-5 and
C-18. Therefore, its structure was determined as 7b-acetoxy-
18,19-dihydroxy-ent-kaur-16-ene (18). The corresponding triol
had been obtained in the biotransformation of epicandicandiol
(8) with G. fujikuroi (Fraga et al., 1978).
Another two isomers 19 and 20, with the same functional
groups and epimeric at C-4, were also obtained from this incuba-
tion. These metabolites can be formed from 18 by the oxidation
of the C-19 or C-18 alcohol, respectively, although 20 can also be
originated from 17. Both isomers showed the resonance of a
hydroxymethylene group in the 1H and 13C NMR spectra, at dH
3.45 and 3.81 (each d, J = 11.3 Hz) and dC 70.3 the former isomer,
and at dH 3.45 and 3.64 (each d, J = 10.4 Hz) and dC 63.4 (t) the
latter. These chemical shifts are characteristic of an equatorial
and axial hydroxymethylene on C-4, respectively, as stated above.
Therefore, the structures 7b-acetoxy-18-hydroxy-ent-kaur-16-en-
19-oic acid (19) and 7b-acetoxy-19-hydroxy-ent-kaur-16-en-18-
oic acid (20) were assigned to these compounds. The two isomers
showed different fragmentations in the MS. The ion of higher mass
in the spectrum of 19 is at m/z 316.2034, formed from the molec-
ular ion by loss of acetic acid, while that of 20 is at m/z 270.1990,
originated by loss of acetic acid and formic acid (Table 2).
The biotransformation of foliol 7b-monoacetate (7b-acetoxy-
J = 3.0 Hz), indicating that both protons were situated at C-2(
a)
and C-3( ), respectively. Consequently, the structure of the corre-
a
sponding acid formed in the incubation was determined as 7b-
acetoxy-2b,3b-dihydroxy-ent-kaur-16-en-19-oic acid (15). The
second methyl ester 16m showed typical resonances of a hydroxy-
methylene group, dH 3.73 and 3.87 (each d, J = 10.9 Hz) and dC 68.1
(t). This alcohol was located at C-18 considering its HMBC spec-
trum, which showed correlations of H-3 with C-1, C-4, C-5 and
C-18; H-5 with C-4, C-6, C-7, C-10, C-18, C-19 and C-20; H-18 with
C-3, C-4, C-5 and C-19; and H-20 with C-1, C-5, C-8, C-9 and C-10.
Thus, the structure of this compound was determined as 7b-acet-
oxy-3b,18-dihydroxy-ent-kaur-16-en-19-oic acid (16). The metab-
olites 15 and 16 can be formed by 2b- and 18-hydroxylation,
respectively, of the hydroxy-acid 14 (Scheme 2, Table 1)).
Another substrate incubated with this fungus was epicandican-
diol 7b-monoacetate (10), a diterpene found in several species of
the genus Sideritis (González et al., 1973; Fraga et al., 2009). The
metabolites obtained in this biotransformation, indicated in
Scheme 3, were: 7b-acetoxy-ent-kaur-16-en-18-oic acid (17),
7b-acetoxy-18,19-dihydroxy-ent-kaur-16-ene (18), 7b-acetoxy-
18-hydroxy-ent-kaur-16-en-19-oic acid (19), 7b-acetoxy-19-hy-
droxy-ent-kaur-16-en-18-oic acid (20) and 7b-acetoxy-3b,
18-dihydroxy-ent-kaur-16-en-19-oic acid (16) (Scheme 3). This
last substance had previously been obtained in the incubation of 7.
The structure of the least polar metabolite was determined as
7b-acetoxy-ent-kaur-16-en-18-oic acid (17) on the basis of the fol-
lowing considerations: Its molecular formula, C22H32O4, indicated
that two hydrogens had been lost and a new oxygen had been
introduced in the molecule during the incubation, whilst the 1H
and 13C NMR spectra showed the permanence of the two methyls
and the disappearance of the hydroxymethyl group of the sub-
strate. These facts indicated that the primary hydroxyl had been
oxidized by the fungus to an acid group, which was confirmed by
3a,18-dihydroxy-ent-kaur-16-ene) (23) did not afford any metabo-
lite, which confirmed that a 3 -hydroxyl group in ent-kaur-16-ene
a
derivatives inhibits the oxidation of C-19 by G. fujikuroi (Fraga
et al., 1981). The substrate 23 was obtained from foliol acetonide
(24) by acetylation to give 25, and subsequent hydrolysis of the
acetonide.
Table 1
13NMR data of compounds 3, 7 and 12–16.
Position
3
7
12
40.7
13
40.0
14
33.2
15m
16m
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
40.3
17.8a
37.6
42.9
48.3
26.3
79.5
46.8
50.0
39.2
19.0a
33.2
43.4
39.0
45.1
40.2
17.6a
41.9
32.5
47.3
24.8
80.0
46.9
51.5
39.1
18.6a
33.4
43.6
38.3
45.2
42.5
66.5
74.2
48.0
40.1
26.3
79.7
46.4
50.1
n.o.
18.2
33.5
43.8
38.8
45.4
n.o.
33.2
26.5
69.6
51.9
36.6
25.8
79.5
46.6
50.0
38.6
17.8
33.5
43.4
38.5
45.1
154.3
103.8
68.1
175.4
15.0
18.1a
36.1
38.4
48.3
25.4
80.1
47.2
52.0
39.4
18.6a
33.7
44.0
38.6
48.5
154.9
104.1
27.3
66.0
18.2
18.1a
34.7
48.0
48.7
24.7
79.5
47.2
50.2
39.4
18.7a
33.5
43.8
38.9
45.5
154.3
104.3
24.6
205.8
16.7
26.2
70.5
46.7
40.6
26.0
80.0
46.7
49.8
39.0
17.7
33.6
43.4
38.3
45.0
154.2
103.7
23.8
182.4
15.0
154.3
103.6
28.4
182.7
15.4
154.7
103.5
33.3
21.3
17.3
104.3
24.0
180.6
16.4
n.o., not observed.
a
These values can be interchanged.

64
B.M. Fraga et al. / Phytochemistry 81 (2012) 60–70
H
H
OR1
OR
R2H2C
8 R1 = H R2 = OH
6 R = H
7 R = Ac
9 R1 = Ac R2 = OAc
10 R1 = Ac R2 = OH
11 R1 = Ac R2 = Cl
H
H
HO
HOH2C
OR
OH
HO2C
22 R = H
23 R = Ac
21
H
O
OR
O
24 R = H
25 R = Ac
The last substrate incubated with G. fujikuroi was 7b-acetoxy-
The incubation of trachinol acetate (27) only gave one biotrans-
formation product, the 19-hydroxy derivative 33. Its molecular for-
mula was determined as C22H34O3, which was indicative of the
presence of a new oxygen atom in the molecule. This forms a part
of an –CH2OH group, because resonances of an AB system at d
3.40 and 3.68 were observed in the 1H NMR spectrum, confirmed
by the corresponding carbon resonance at d 65.7 (t). These are typ-
ical values of an axial hydroxymethylene group at C-4, as stated
above. Moreover, some of the correlations observed in the HMBC
spectrum were: H-5 with C-4, C-6, C-7, C-19 and C-20; H-18 with
C-3, C-4, C-5 and C-19; and H-19 with C-3, C-4, C-5 and C-18, whilst
in the NOESY experiment a crosspeak was observed between H-19
and H-20. Therefore, the structure of this metabolite was deter-
mined as 7b-acetoxy-19-hydroxy-ent-trachylobane (33).
ent-trachylobane (trachinol acetate) (27). Trachinol (26) and trach-
inodiol (28) are two diterpenes with a trachylobane skeleton,
which had been isolated from S. canariensis (González et al.,
1971). We have now prepared trachinol (26) and its acetate 27
from trachinodiol diacetate (29) in the following way: partial
hydrolysis of 29 with K2CO3 in MeOH afforded trachinodiol 7b-
monoacetate (30) and trachinodiol (28). The first was treated with
triphenylphosphine, iodine and imidazole in toluene (Garegg and
Samuelsson, 1980) to give 7b-acetoxy-18-iodo-ent-trachylobane
(31), which without purification was reduced with lithium alumin-
ium hydride to afford trachinol (26) and a very low proportion of a
dimer (34). Acetylation of 26 in the usual way afforded the sub-
strate 27. We have also observed that the direct iodination of
trachinodiol (28) gave a 18-iodo derivative (32) (Table 3).
The structure of the dimer was determined as 18,18-bis-7b-hy-
droxy-ent-trachylobane (34) on the basis of the following data: its
HRMS showed the fragment of higher mass at m/z 556.4632,
(C40H60O) formed from the molecular ion by loss of water. Its
1H and 13C NMR spectrum showed that it was a symmetrical
dimer, because duplicated signals were not observed. The pres-
ence in the HMBC spectrum of a correlation of the hydrogens
and carbon of the same methylene group indicated that dimeriza-
tion had occurred between two C-18, which can be explained by
homolytic cleavage of the carbon-iodo bond and subsequent rad-
ical dimerization.
3. Conclusions
Several conclusions can be deduced from the results obtained in
the biotransformation of the 7b-acetoxy derivatives 7, 10, 23 and
27 by the fungus G. fujikuroi:
(1) A 7b-acetoxy group does not inhibit the oxidation of C-19 in
ent-kaur-16-ene derivatives.
(2) The presence of a 7b-OAc avoids the ring B contraction that
leads to the gibberellins. It also inhibits the hydroxylation of

B.M. Fraga et al. / Phytochemistry 81 (2012) 60–70
65
H
H
OR1
OR
R2H2C
26 R = H
27 R = Ac
28 R1 = H R2 = OH
29 R1 = Ac R2 = OAc
30 R1 = Ac R2 = OH
31 R1 = Ac R2 = I
32 R1 = H R2 = I
H
OAc
CH2OH
33
H
OH
HO
H
34
C-6(b), which is necessary for the cleavage of ring B to form
fujenal and other seco-ring B ent-kaurenoid derivatives by G.
fujikuroi.
transformed by this fungus than the corresponding ent-
kaur-16-ene one (7).
(3) The incubation of candol A acetate (7) with this fungus led to
the formation of 7b-acetoxy-ent-kaur-16-en-19-oic acid (3).
Its hydrolysis led to the corresponding hydroxy-acid 1, an
important precursor of gibberellin plant hormones and
seco-ring B ent-kaur-16-ene derivatives.
(4) The enzyme involved in the 3b-hydroxylation of 3 to form
7b-acetoxy-3b-hydroxy-ent-kaur-16-en-19-oic acid (14) in
the incubation of 7 (Scheme 2) is probably the same multi-
4. Experimental
4.1. General procedures
Mps were determined with a Reichert Thermovar apparatus and
are uncorrected. 1H and 13C NMR spectra were recorded in CDCl3
solution at 500.13 and 125.03 MHz, respectively, with a Bruker
AMX-500 spectrometer. Mass spectra were taken at 70 eV (probe)
in a Micromass Autospec spectrometer. HPLC was performed using
a Beckman System Gold 125P. Purification by HPLC was achieved
functional P450-1 monooxygenase that transforms GA12
-
aldehyde (2) into GA14-aldehyde (4) (Scheme 1) (Rojas
et al., 2001). Moreover, this enzyme may also be responsible
for the 2b-hydroxylation of 14 affording the diol 15 (Scheme
2), because the 2b,3b-dihydroxyl group formed resembles
that of 6b,7b-diol of 5, which is produced by this monooxy-
genase in the biosynthesis of the seco-ring B ent-kaurenoids
in this fungus (Rojas et al., 2004).
using a silica gel column (Ultrasphere Si 5
lm, 10 ꢁ 250 mm).
Dry column chromatography was made on silica gel Merck
0.040–0.063 mm. The substances were crystallized from petrol-
EtOAc except where otherwise indicated.
4.2. Microorganism
(5) Epicandicandiol 7b-monoacetate (10) is better metabo-
lized by the fungus than epicandicandiol (8) (Fraga et al.,
1978). The main difference between both incubations is that
in the case of 10 there is a loss of regioselectivity of the
enzyme responsible for the C-19 oxidation, which possibly
also produces the oxidation of C-18 in 10 to form the
acid 17.
(6) The 19-hydroxy derivative 33 was the only metabolite
obtained in the incubation of trachinol acetate (27) indicat-
ing that this ent-trachylobane derivative is less efficiently
The fungal strain was G. fujikuroi MP-C (Fusarium fujikuroi) IMI
58289 and was a gift from Prof. J.R. Hanson, School of Chemistry
(University of Sussex, UK).
4.3. Incubation procedure
The fungus G. fujikuroi, inhibited with 5 ꢁ 10ꢀ5 M AMO 1618,
was grown on shake culture at 25 °C for 2 days in 70 conical flasks
(250 mL), each containing 50 mL of sterile medium comprising

66
B.M. Fraga et al. / Phytochemistry 81 (2012) 60–70
G. fujikuroi
H
H
OAc
OAc
HOH2C
RO2C
10
17 R = H
17m R = Me
G. fujikuroi
H
H
OAc
OAc
HOH2C CH2OH
18
HO2C CH2OH
20
H
H
OAc
HO
HOH2C CO2R
OAc
R1OH2C CO2R2
19 R1 = R2 = H
19m R1 = H R2 = Me
19am R1 = Ac R2 = Me
16 R = H
16m R = Me
Scheme 3. Biotransformation of epicandicandiol 7b-monoacetate (10) by G. fujikuroi.
(per dm3) glucose (80 g), NH4NO3 (0.48 g), KH2PO4 (5 g), MgSO4
(1 g), and trace elements solution (2 mL). The trace elements solu-
tion contained (per 100 mL) Co(NO3)2 (0.01 g), CuSO4 (0.015 g),
ZnSO4 (0.16 g), MnSO4 (0.01 g), (NH4)6Mo7O24 (0.01 g). The sub-
strate dissolved in EtOH (13 mL) and Tween 80 (three drops) was
evenly distributed between the flasks and the incubation allowed
to continue for a further 6 days. The broth was filtered and the cul-
ture filtrate extracted with EtOAc. The mycelium was treated with
liquid nitrogen, crushed in a mortar and extracted with EtOAc.
Both extracts were combined.
4.4.2. 7b-Acetoxy-18-hydroxy-ent-kaur-16-ene (10)
1H NMR (500 MHz) d 0.72 (3H, s, H-19), 0.85 (1H, dt, J = 13.2 and
3.8 Hz, H-1b), 1.07 (3H, s, H-20), 1.25 (2H, m, H-3 and H-14), 1.70
(1H, dd, J = 12.6 and 1.6 Hz, H-5), 1.76 (1H, ddd, J = 14.2, 3.5 and
1.6 Hz, H-6b), 1.82 (1H, br d, J = 12.3 Hz, H-1
J = 11.2 and 2.5 Hz, H-14), 2.04 (3H, s, OAc), 2.10 (1H, dt, J = 17.2
and 2.7 Hz, H-15 ), 2.20 (1H, dd, J = 17.2 and 2.5 Hz, H-15b), 2.69
a), 1.90 (1H, dd,
a
(1H, br s, H-13), 3.01 and 3.32 (each 1H, d, J = 10.8 Hz, H-18),
4.72 (1H, dd, J = 3.5 and 2.2 Hz, H-7), 4.76 and 4.81 (each 1H, br
s, H-17); EIMS m/z (rel. int.) 286 [MꢀH2O]+ (76), 268 (18), 255
(100), 239 (21), 227 (5), 225 (5), 213 (9), 199 (16), 187 (20), 185
(20), 173 (21), 159 (17), 145 (19); HREIMS m/z [M]+ 286.2292
(calcd. for C20H30O, 286.2297).
4.4. Preparation of the substrate 7b-acetoxy-ent-kaur-16-ene (acetate
of candol A) (7)
4.4.1. Partial hydrolysis of epicandicandiol diacetate (9)
The diacetate 9 (960 mg) in MeOH (40 ml) was treated with
K2CO3 (300 mg) with stirring for 3 h at room temp. Extraction in
the usual way and chromatography afforded starting material
(414 mg), the monoacetate 10 (385 mg) and epicandicandiol (8)
(58 mg).
4.4.3. 7b-Acetoxy-18-chloro-ent-kaur-16-ene (11)
Compound 10 (450 mg) in CCl4 (14 ml) was treated with tri-
phenylphosphine (1.35 g), at reflux, under nitrogen for 9 h. Usual
work up afforded 7b-acetoxy-18-chloro-ent-kaur-16-ene (11)
(475 mg), m.p. 110–113 °C (petroleum–EtOAc), 1H NMR
(500 MHz) d 0.85 (3H, s, H-19), 0.87 (1H, dt, J = 12.2 and 4.1 Hz,

B.M. Fraga et al. / Phytochemistry 81 (2012) 60–70
67
Table 2
13NMR data of compounds 10 and 17–20.
Position
10
39.7
17
39.3
17m
18
39.9
19
40.0
19m
19am
20
39.4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
39.3
17.5a
36.7
47.1
42.1
27.1
79.8
47.3
51.4
38.5
17.7a
33.3
43.5
38.1
45.2
154.4
103.7
178.8
16.3
17.6
40.0
18.5
31.8
49.3
42.7
26.1
79.6
46.6
50.1
38.8
17.8
33.2
43.4
38.4
45.2
154.3
103.8
70.1
175.9
15.4
39.8
18.3
32.2
49.3
43.7
26.5
79.0
46.6
50.0
38.8
17.8
33.2
43.4
38.3
45.0
154.1
103.8
71.0
174.9
15.6
17.7a
35.1
37.0
40.1
24.3
80.0
46.9
51.4
38.8
17.9a
33.4
43.6
38.3
45.2
154.6
103.6
71.4
17.4
17.7
17.5a
36.8
46.9
41.9
27.2
79.7
47.1
51.5
38.4
17.7a
33.3
43.5
38.2
45.2
154.4
103.7
182.9
16.0
17.5
17.7
30.2
41.4
44.2
25.1
79.6
46.7
51.5
38.8
17.7
33.3
43.6
38.2
45.0
154.3
103.7
72.9
64.8
17.6
18.4
31.8
49.1
42.9
26.0
79.7
46.6
50.1
39.0
17.7
33.2
43.4
38.4
45.1
154.3
103.8
70.3
180.5
15.6
19.1
36.4
50.9
48.2
23.7
78.9
48.2
50.8
39.4
17.8
33.2
43.5
38.2
45.0
154.1
103.8
n.o.
63.4
17.2
a
These values can be interchanged.
98:2) led to 7 (317 mg): 1H NMR (500 MHz) d 0.77 (3H, s, H-18),
0.78 (3H, s, H-19), 0.82 (1H, td, J = 13.0 and 3.6 Hz, H-1b), 1.03
(3H, s, H-20), 1.16 (1H, td, J = 13.3 and 4.2 Hz, H-3b), 1.29 (1H, br
d, J = 12.7 Hz, H-5), 1.81 (2H, m, H-1 and H-6), 1.90 (1H, dd,
J = 11.1 and 2.3 Hz, H-14), 2.04 (3H, s, OAc), 2.08 (1H, dt, J = 17.2
and 2.7 Hz, H-15), 2.18 (1H, br d, J = 17.2 Hz, H-15), 2.68 (1H, br
s, H-13), 4.75 (2H, br s, H-7 and H-17), 4.80 (1H, br s, H-17); EIMS
m/z (rel. int.) 330 [M]+ (1), 270 (80), 255 (44), 241 (10), 227 (17),
214 (8), 190 (25), 190 (99), 185 (31), 160 (24); HREIMS m/z [M]+
330.2557 (calcd. for C22H34O2, 330.2559).
Table 3
13NMR data of compounds 26, 27, 29 and 32–34.
Position
26
27
29
32
33
34
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
39.0
18.1
42.0
32.5
46.9
27.6
76.0
45.4
47.5
38.4
19.3
20.6
24.0
32.7
45.4
23.0
20.4
33.1
21.6
14.5
39.0
18.1
42.0
32.4
47.8
25.1
78.5
43.9
48.4
38.2
19.2
20.4
23.8
32.5
45.2
22.8
20.3
33.0
21.4
14.4
38.6
17.4
35.2
36.8
40.7
24.7
78.4
43.9
48.5
38.0
19.3
20.4
23.8
32.4
45.1
22.8
20.3
71.4
17.4
14.8
38.5
17.7
39.0
34.4
44.0
27.5
75.6
45.1
47.2
38.4
19.3
20.5
23.8
32.5
45.1
22.8
20.4
28.7
18.5
14.4
39.2
17.8
35.8
37.7
48.4
25.4
78.2
44.0
48.6
38.2
19.5
20.4
23.8
32.4
45.1
22.8
20.3
26.6
65.7
14.9
38.8
17.9
36.3
34.4
44.1
27.2
76.2
45.3
47.6
38.4
19.3
20.6
24.0
32.7
45.3
22.9
20.4
36.9
21.5
15.1
4.5. Incubation of 7b-acetoxy-ent-kaur-16-ene (7)
The substrate 7 (317 mg) in EtOH (19 ml) was distributed
between 94 conical flasks. Its biotransformation gave starting
material (202 mg), 7b-acetoxy-19-hydroxy-ent-kaur-16-ene (12)
(4 mg), 7b-acetoxy-ent-kaur-16-en-19-al (13) (12 mg), 7b-acet-
oxy-ent-kaur-16-en-19-oic acid (3) (15 mg), and 7b-acetoxy-3b-
hydroxy-ent-kaur-16-en-19-oic acid (14) (23 mg). The metabolites
7b-acetoxy-2b,3b-dihydroxy-ent-kaur-16-en-19-oic acid (15) and
7b-acetoxy-3b,18-dihydroxy-ent-kaur-16-en-19-oic acid (16) were
obtained in methyl ester form, 15m (1.5 mg) and 16m (1 mg)
respectively, by methylation with CH2N2 and chromatography of
the fractions containing a mixture of these acids.
H-1b), 1.07 (3H, s, H-20), 1.23 (2H, m, H-3b and H-14), 1.82 (1H, m,
H-1 overlapped with H-5), 1.83 (1H, dd, J = 12.8 and 2.0 Hz, H-5),
a
1.89 (1H, dd, J = 11.2 and 2.6 Hz, H-14), 2.05 (3H, s, OAc), 2.08 (1H,
dt, J = 17.2 and 2.6 Hz, H-15), 2.22 (1H, dd, J = 17.2 and 2.0 Hz, H-
15), 2.69 (1H, br s, H-13), 3.07 and 3.41 (each 1H, d, J = 11.2 Hz,
H-18), 4.70 (1H, dd, J = 3.2 and 2.5 Hz, H-7), 4.76 and 4.81 (each
1H, br s, H-17); EIMS m/z (rel. int.) 306 [MꢀAcOH]+ (30), 304
(100), 291 (17), 289 (41), 269 (20), 255 (48), 226 (14), 224 (36),
213 (12), 199 (20), 185 (37), 173 (25), 160 (33); HREIMS m/z
[MꢀAcOH]+ 306.1918 (calcd. for C20H2937Cl, 306.1918),
[MꢀAcOH]+ at 304.1966 (calcd. for C20H2935Cl, 304.1958).
4.5.1. 7b-Acetoxy-ent-kaur-16-en-19-oic acid (3)
1H NMR (500 MHz) d 0.90 (1H, m, H-1), 0.97 (3H, s, H-20), 1.05
(1H, td, J = 13.4 and 4.3 Hz, H-3b), 1.13 (3H, s, H-18), 1.28 (1H, m,
H-14), 1.45 (1H, br s, H-9), 1.90 (1H, dd, J = 11.2 and 2.1 Hz, H-
14), 2.07 (3H, s, –OAc), 2.10 (2H, m, H-5 and H-6), 2.19 (1H, m,
H-3), 2.68 (1H, br s, H-13), 4.76 (1H, br s, H-17), 4.81 (2H, br s, H-
7 and H-17); EIMS m/z (rel. int.) 300 [MꢀAcOH]+ (100), 285 (18),
255 (17), 220 (22), 199 (15), 185 (23), 173 (18), 159 (13), 145
(16), 133 (22); HREIMS m/z [MꢀAcOH]+ 300.2077 (calcd. for
4.4.4. 7b-Acetoxy-ent-kaur-16-ene (7)
C20H28O2, 300.2089).
To the chloroacetate 11 (411 mg), in dry toluene (20 ml), tri-n-
butyl tin hydride (900 lL) and azobisisobutyronitrile (traces) were
added. The solution was heated to reflux for 15 h under nitrogen.
After removing the solvent under vacuum, the residue was dis-
solved in diethyl ether (15 ml) and a saturated solution of KF
was added, and stirred for 30 min. The precipitate of tin salts
was filtered and the liquid was evaporated under reduced pressure.
Purification by column chromatography (petroleum ether–EtOAc
4.5.2. 7b-Acetoxy-19-hydroxy-ent-kaur-16-ene (12)
1H NMR (500 MHz) d 0.87 (3H, s, H-18), 0.98 (1H, td, J = 13.6 and
4.5 Hz, H-3b), 1.03 (3H, s, H-20), 1.44 (1H, m, H-9 overlapped with
H-5), 1.46 (1H, dd, J = 13.3 and 1.8 Hz, H-5), 1.79 (1H, br d,
J = 13.6 Hz, H-3
a), 1.86 (2H, m, H-1a and H-14), 1.92 (1H, ddd,
J = 14.3, 3.6 and 1.6 Hz, H-6), 2.06 (3H, s, –OAc), 2.10 (1H, m, H-

68
B.M. Fraga et al. / Phytochemistry 81 (2012) 60–70
15), 2.18 (1H, br dd, J = 17.1 and 2.0 Hz, H-15), 2.68 (1H, br s, H-13),
3.42 and 3.72 (each 1H, d, J = 10.9 Hz, H-19), 4.74 (1H, dd, J = 3.5
and 2.4 Hz, H-7), 4.75 and 4.80 (each 1H, br s, H-17); EIMS m/z
(rel. int.) 286 [MꢀAcOH]+ (33), 271 (10), 255 (98), 239 (9), 206
(16), 185 (18); HREIMS m/z [MꢀAcOH]+ 286.2288 (calcd. for
kaur-16-en-19-oic acid (16), identified as its methyl ester (16m)
(5 mg).
4.6.1. 7b-Acetoxy-ent-kaur-16-en-18-oic acid (17)
1H NMR (500 MHz) d 0.96 (1H, td, J = 12.4 and 3.6 Hz, H-1b),
1.07 (3H, s, H-20), 1.15 (3H, s, H-19), 1.73 (1H, m, H-3), 1.84 (1H,
C20H30O, 286.2297).
m, H-1a), 1.88 (1H, dd, J = 12.3 and 2.3 Hz, H-14), 2.04 (3H, s,
–OAc), 2.09 (1H, dt, J = 17.4 and 2.7 Hz, H-15b), 2.21 (1H, dd,
J = 12.5 and 1.7 Hz, H-5), 2.24 (1H, br dd, J = 17.4 and 2.2 Hz, H-
4.5.3. 7b-Acetoxy-ent-kaur-16-en-19-al (13)
1H NMR (500 MHz) d 0.88 (1H, m, H-1), 0.90 (3H, s, H-18), 0.92
(3H, s, H-20), 1.03 (1H, m, H-3b), 1.86 (1H, m, H-1), 1.88 (1H, dd,
15a), 2.69 (1H, br s, H-13), 4.63 (1H, dd, J = 3.2 and 2.2 Hz, H-7),
4.77 and 4.81 (each 1H, s, H-17); EIMS m/z (rel. int.) 360 [M]+
(0.1), 300 (100), 285 (24), 271 (9), 255 (21), 239 (18), 220 (13),
199 (16), 185 (26), 149 (41); HREIMS [M]+ at m/z 360.2316 (calcd.
for C22H32O4, 360.2301). Methyl ester (17m): 1H NMR (500 MHz) d
0.95 (1H, td, J = 13.0 and 3.8 Hz, H-1b), 1.06 (3H, s, H-20), 1.14 (3H,
s, H-19), 1.39 (1H, ddd, J = 14.4, 3.5 and 1.9 Hz, H-6b), 1.75 (1H, td,
J = 11.1 and 2.0 Hz, H-14), 1.96 (1H, dt, J = 13.6 and 2.4 Hz, H-6a),
2.08 (3H, s, –OAc), 2.17 (2H, m, H-3 and H-15), 2.70 (1H, br s, H-
13), 4.77 (1H, br s, H-17), 4.83 (2H, br s, H-7 and H-17), 9.71 (1H,
s, H-19); EIMS m/z (rel. int.) 284 [MꢀAcOH]+ (100), 255 (75), 241
(28), 213 (17), 204 (25), 185 (40), 176 (41), 145 (35); HREIMS m/
z [MꢀAcOH]+ 284.2136 (calcd. for C20H28O, 284.2140).
J = 12.5 and 4.1 Hz, H-3a), 1.84 (1H, br d, J = 13.0 Hz, H-1a), 1.87
(1H, dd, J = 11.4 and 2.7 Hz, H-14), 2.08 (3H, s, –OAc), 2.09 (1H,
dt, J = 17.2 and 2.9 Hz, H-15b), 2.18 (1H, dd, J = 12.8 and 1.7 Hz,
H-5), 2.23 (1H, dd, J = 17.2 and 1.9 Hz, H-15a), 2.69 (1H, br s, H-
13), 3.60 (3H, s, –OMe), 4.61 (1H, t, J = 2.8 Hz, H-7), 4.77 and 4.81
(each 1H, s, H-17); EIMS m/z (rel. int.) 314 [MꢀAcOH]+ (100), 299
(17), 285 (5), 254 (77), 239 (32), 234 (11), 211 (9), 199 (16), 185
(18), 173 (23), 159 (14), 149 (19); HREIMS m/z [MꢀAcOH]+
314.2259 (calcd. for C21H30O2, 314.2246).
4.5.4. 7b-Acetoxy-3b-hydroxy-ent-kaur-16-en-19-oic acid (14)
1H NMR (500 MHz) d 0.97 (3H, s, H-20), 1.24 (3H, s, H-18), 1.53
(1H, d, J = 6.2 Hz, H-9), 1.89 (1H, br d, J = 11.2 Hz, H-14), 1.98 (1H,
dd, J = 14.0 and 3.5 Hz, H-6), 2.03 (1H, d, J = 14.0 Hz, H-5), 2.07
(3H, s, –OAc), 2.10 (2H, m, H-6 and H-15), 2.17 (1H, br d,
J = 15.8 Hz, H-15), 2.68 (1H, br s, H-13), 4.09 (1H, br s, H-3), 4.76
(1H, br s, H-17), 4.81 (2H, br s, H-7 and H-17); EIMS m/z (rel. int.)
316 [MꢀAcOH]+ (67), 301 (10), 298 (40), 283 (14), 270 (6), 253
(23), 225 (19), 217 (25), 199 (45), 185 (28), 171 (29), 143 (30),
131 (39); HREIMS m/z [MꢀAcOH]+ 316.2046 (calcd. for C20H28O3,
316.2038).
4.6.2. 7b-Acetoxy-18,19-dihydroxy-ent-kaur-16-ene (18)
1H NMR (500 MHz) d 0.87 (1H, m, H-1b), 1.03 (3H, s, H-20), 1.05
(1H, m, H-3b), 1.23 (1H, dd, J = 11.3 and 5.2 Hz, H-14), 1.46 (1H, br t,
J = 4.0 Hz, H-9), 1.85 (2H, m, H-1
H-6), 2.06 (3H, s, –OAc), 2.08 (1H, dt, J = 17.5 and 2.7 Hz, H-15b),
2.17 (1H, br dd, J = 17.5 and 2.0 Hz, H-15 ), 2.68 (1H, br s, H-13),
3.31 and 3.72 (each 1H, d, J = 10.6 Hz, H-18), 3.69 and 3.90 (each
1H, d, J = 10.8 Hz, H-19), 4.71 (1H, dd, J = 3.4 and 2.2 Hz, H-7),
4.75 and 4.80 (each 1H, br s, H-17); EIMS m/z (rel. int.) 302
[MꢀAcOH]+ (49), 284 (64), 271 (26), 253 (100), 239 (27), 225
(12), 211 (14), 199 (18), 185 (31), 173 (24), 159 (28), 145 (36);
HREIMS m/z [MꢀAcOH]+ 302.2247 (calcd. for C20H30O2, 302.2246).
a and H-14), 1.94 (2H, m, H-3a and
4.5.5. 7b-Acetoxy-2b,3b-dihydroxy-ent-kaur-16-en-19-oic acid
methyl ester (15m)
a
1H NMR (500 MHz) d 0.87 (3H, s, H-20), 1.25 (3H, s, H-18), 1.83
(1H, dd, J = 11.2 and 2.4 Hz, H-14), 1.90 (1H, dd, J = 12.4 and 4.5 Hz,
H-1), 2.00 (1H, br s, H-5), 2.08 (3H, s, –OAc), 2.18 (1H, br d,
J = 17 Hz, H-15), 2.69 (1H, br s, H-13), 3.65 (3H, s, –OMe), 4.09
(1H, d, J = 3.0 Hz, H-3), 4.22 (1H, ddd, J = 12.0, 5.0 and 3.0 Hz, H-
2), 4.77 and 4.82 (each 1H, br s, H-17), 4.80 (1H, t, J = 2.4 Hz, H-
7); EIMS m/z (rel. int.) 346 [MꢀAcOH]+ (22), 328 (28), 310 (19),
302 (13), 286 (10), 268 (25), 251 (15), 229 (14), 197 (15), 183
(18); HREIMS m/z [MꢀAcOH]+ 346.2151 (calcd. for C21H30O4,
346.2144).
4.6.3. 7b-Acetoxy-18-hydroxy-ent-kaur-16-en-19-oic acid (19)
1H NMR (500 MHz) d 0.90 (1H, td, J = 13.8 and 4.5 Hz, H-1b),
1.00 (3H, s, H-20), 1.21 (1H, td, J = 13.2 and 4.5 Hz, H-3b), 1.28
(1H, dd, J = 11.0 and 5.0 Hz, H-14), 1.51 (1H, br s, H-9), 1.79 (1H,
dd, J = 9.6 and 5.5 Hz, H-5), 1.89 (2H, m, H-1 and H-14), 2.06 (3H,
s, –OAc), 2.10 (1H, dt, J = 16.5 and 2.7 Hz, H-15b), 2.18 (1H, br d,
4.5.6. 7b-Acetoxy-3b,18-dihydroxy-ent-kaur-16-en-19-oic acid
methyl ester (16m)
1H NMR (500 MHz) d 0.88 (3H, s, H-20), 1.30 (1H, m, H-14), 1.57
(1H, s, H-9), 1.62 (1H, m, H-11), 1.86 (1H, dd, J = 11.5 and 2.7 Hz, H-
14), 1.96 (1H, m, H-6), 2.05 (3H, s, –OAc), 2.10 (1H, dt, J = 17.2 and
J = 16.5 Hz, H-15a), 2.30 (1H, br d, J = 13.2 Hz, H-3a), 2.69 (1H, br
s, H-13), 3.45 and 3.81 (each 1H, d, J = 10.4 Hz, H-18), 4.76 (2H,
br s, H-7 and H-17), 4.81 (1H, br s, H-17); EIMS m/z (rel. int.) 316
[MꢀAcOH]+ (100), 301 (14), 298 (27), 286 (25), 270 (26), 256
(19), 253 (24), 239 (32), 225 (11), 199 (17), 185 (33), 171 (19),
157 (25), 149 (43); HREIMS [MꢀAcOH]+ at m/z 316.2034 (calcd.
for C20H28O3, 316.2038). Methyl ester (19m): 1H NMR (500 MHz)
d 0.89 (3H, s, H-20), 0.90 (1H, m, H-1), 1.21 (1H, td, J = 13.5 and
4.9 Hz, H-3b), 1.27 (1H, ddt, J = 11.2, 5.1 and 1.5 Hz, H-14), 1.42
(1H, t, J = 6.0 Hz, –OH), 1.48 (1H, br s, H-9), 1.78 (1H, dd, J = 12.8
and 2.4 Hz, H-5), 1.86 (1H, dd, J = 11.2 and 2.5 Hz, H-14), 1.89
2.7 Hz, H-15a), 2.20 (1H, br d, J = 17.2 Hz, H-15b), 2.32 (1H, dd,
J = 11.5 and 3.3 Hz, H-5), 2.68 (1H, br s, H-13), 3.67 (3H, s, –
OMe), 3.73 and 3.87 (each 1H, d, J = 10.9 Hz, H-18), 4.51 (1H, dd,
J = 5.6 and 3.0 Hz, H-3a), 4.75 (1H, t, J = 2.7 Hz, H-7), 4.77 and
4.81 (each 1H, br s, H-17); EIMS m/z (rel. int.) 346 [MꢀAcOH]+
(31), 328 (32), 310 (100), 298 (45), 283 (23), 268 (15), 251 (18),
239 (15), 230 (23), 199 (40), 183 (28), 157 (33); HREIMS m/z
[MꢀAcOH]+ 346.2153 (calcd. for C21H30O4, 346.2144).
(2H, m, H-1 and H-2), 1.96 (1H, ddd, J = 15.0, 12.8 and 2.2, H-6
2.04 (1H, m, H-6b), 2.05 (3H, s, –OAc), 2.09 (1H, dt, J = 17.3 and
2.7 Hz, H-15b), 2.18 (1H, br dd, J = 17.3 and 2.0 Hz, H-15 ), 2.29
(1H, br d, J = 13.3 Hz, H-3 ), 2.68 (1H, br s, H-13), 3.43 and 3.74
a),
4.6. Incubation of 7b-acetoxy-18-hydroxy-ent-kaur-16-ene (10)
a
The substrate 10 (280 mg) in EtOH (16 ml) was distributed
between 78 conical flasks. Its biotransformation gave in the neutral
fraction starting material (96 mg), 7b-acetoxy-18,19-dihydroxy-
ent-kaur-16-ene (18) (6 mg), 7b-acetoxy-ent-kaur-16-en-18
-oic acid (17) (10 mg), 7b-acetoxy-18-hydroxy-ent-kaur-16-en-
19-oic acid (19) (16 mg), 7b-acetoxy-19-hydroxy-ent-kaur-16
-en-18-oic acid (20) (2 mg), and 7b-acetoxy-3b,18-dihydroxy-ent-
a
(each 1H, dd, J = 10.4 and 6.0 Hz, H-18), 3.68 (3H, s, –OMe), 4.75
(1H, dd, J = 3.7 and 2.2 Hz, H-7 solapado con H-17), 4.76 and 4.81
(each 1H, br s, H-17); EIMS m/z (rel. int.) 330 [MꢀAcOH]+ (100),
315 (9), 312 (26), 285 (9), 270 (34), 253 (22), 239 (44), 225 (8),
209 (12), 199 (13), 185 (27), 171 (18), 157 (26); HREIMS
[MꢀAcOH]+ at m/z 330.2194 (calcd. for C21H30O3, 330.2195). Ace-

B.M. Fraga et al. / Phytochemistry 81 (2012) 60–70
69
tate methyl ester (19am): 1H H NMR (500 MHz) d 0.89 (3H, s, H-
20), 1.11 (1H, m, H-3), 1.47 (1H, br s, H-9), 1.77 (1H, dd, J = 11.5
and 3.9 Hz, H-5), 1.86 (1H, dd, J = 11.6 and 2.1 Hz, H-14), 1.89
(1H, m, H-1), 2.00 (1H, m, H-6), 2.02 and 2.07 (each 3H, s, –OAc),
2.12 (1H, m, H-15), 2.29 (1H, m, H-3), 2.68 (1H, br s, H-13), 3.68
(3H, s, –OMe), 3.86 and 4.26 (each 1H, d, J = 10.6 Hz, H-18), 4.76
and 4.81 (each 1H, br s, H-17), 4.83 (1H, t, J = 2.8 Hz, H-7); EIMS
m/z (rel. int.) 432 [M]+ (0.4), 372 (23), 358 (3), 340 (6), 330 (5),
312 (100), 298 (14), 252 (31), 239 (13), 209 (12), 199 (10), 185
(219), 171 (15), 157 (17); HREIMS: m/z [M]+ 432.2506 (calcd. for
(100), 239 (15), 227 (5), 213 (8), 199 (22), 185 (25), 173 (28); HRE-
IMS m/z [M]+ 346.2509 (calcd. for C22H34O3, 346.2508).
4.8.3. Iodination of 30
Compound 30 (312 mg) in dry toluene (20 mL) was treated with
triphenylphosphine (283 mg), imidazole (123 mg) and I2 (275 mg)
at 80 °C for 30 min. The excess of I2 was destroyed with Na2S2O3
saturated solution. Extraction with CH2Cl2 in the usual way led
to crude 31 (730 mg).
C25H36O6, 432.2512).
4.8.4. Reduction of 31
Crude compound 31, in dry THF (25 mL), was treated with
LiAlH4 (500 mg) at reflux for 2 h. The excess of reagent was de-
stroyed with drops of EtOAc. The reaction mixture was poured over
aqueous HCl (3%) (120 ml) and extracted with EtOAc, in the usual
way. The solution was concentrated in vacuo and chromato-
graphed on SiO2, eluting with a petrol–EtOAc gradient to afford
trachinol (26) (208 mg) and dimeric compound 34 (3 mg).
4.6.4. 7b-Acetoxy-19-hydroxy-ent-kaur-16-en-18-oic acid (20)
1H NMR (500 MHz) d 0.93 (1H, td, J = 12.9 and 4.5 Hz, H-1b),
0.98 (3H, s, H-20), 1.12 (1H, m, H-3), 1.76 (1H, dd, J = 13.1 and
1.8 Hz, H-5), 1.84 (2H, m, H-1 and H-14), 2.07 (3H, s, –OAc), 2.11
(3H, m, H-3, H-6 and H-15b), 2.19 (1H, br d, J = 17.0 Hz, H-15a),
2.69 (1H, br s, H-13), 3.45 and 3.64 (each 1H, d, J = 11.3 Hz, H-
19), 4.76 (1H, br s, H-17), 4.81 (2H, br s, H-7 and H-17); EIMS m/z
(rel. int.) 270 [MꢀH2CO2ꢀAcOH]+ (20), 257 (53), 239 (24), 189
(5), 161 (14), 151 (7), 143 (10), 129 (7); HREIMS m/z
[MꢀH2CO2ꢀAcOH]+ 270.1990 (calcd. for C19H26O, 270.1984).
4.8.5. Trachinol (26)
Colourless crystal, m.p. 108–110 °C; 1H NMR (500 MHz) d 0.58
(1H, dt, J = 7.7 and 2.7 Hz, H-12), 0.77 (1H, td, J = 12.9 and 3.4 Hz,
H-1b), 0.78 (3H, s, H-19), 0.83 (3H, s, H-18), 0.87 (1H, dd, J = 7.7
and 3.2 Hz, H-13), 0.92 (3H, s, H-20), 1.15 (3H, s, H-17), 1.17 (1H,
td, J = 13.0 and 4.3 Hz, H-3b), 1.20 (1H, ddd, J = 11.7, 3.2 and
4.7. Preparation of the substrate 7b-acetoxy-3a,18-dihydroxy-ent-
kaur-16-ene (foliol 7b-monoacetate) (23)
1.7 Hz, H-14), 1.36 (3H, m, H-2, H-3
1H, d, J = 11.0 Hz, H-15), 1.45 (1H, dd, J = 9.1 and 1.6 Hz, H-9), 1.48
(1H, m, H-1 ), 1.52 (1H, m, H-6 ), 1.55 (1H, m, H-2), 1.62 (1H, dt,
J = 14.0 and 2.7 Hz, H-6b), 1.67 (1H, ddd, J = 14.7, 6.4 and 2.6 Hz,
H-11), 1.90 (1H, ddd, J = 14.7, 11.6 and 2.9 Hz, H-11), 1.97 (1H, d,
J = 11.7 Hz, H-14), 3.52 (1H, t, J = 2.9 Hz, H-7); EIMS m/z (rel. int.)
288 [M]+ (8), 270 (100), 255 (40), 242 (5), 227 (12), 214 (13), 199
(18), 185 (26), 173 (19), 157 (46); HREIMS m/z [M]+ 288.2457
(calcd. for C20H32O, 288.2453).
a and H-5), 1.41 and 1.51 (each
The acetonide 24 (300 mg) (Fraga et al., 1981) by reaction with
Ac2O (4 ml) and pyridine (2 ml) for 15 h at room temp., and usual
work-up, afforded the acetate-acetonide 25. This crude product
(334 mg) dissolved in methanol (20 ml) was treated with a solu-
tion (5 ml) of aqueous methanolic hydrochloric acid (0.25%) for
11 h at 40 °C with stirring. Usual workup gave 23 (295 mg), 1H
NMR (500 MHz) d 0.80 (3H, s, H-19), 0.98 (1H, dt, J = 13.0 and
5.6 Hz, H-1b), 1.06 (3H, s, H-20), 1.23 (1H, m, H-14), 1.42 (1H, d,
J = 6.9 Hz, H-9), 1.50 (1H, m, H-5), 1.57 (1H, m, H-11), 1.63 (1H,
tt, J = 14.5 and 2.5 Hz, H-6), 1.84 (1H, dt, J = 13.0 and 3.5 Hz, H-1
overlapped with H-14), 1.86 (1H, dd, J = 11.1 and 2.5 Hz, H-14),
2.03 (3H, s, –OAc), 2.08 (1H, dt, J = 17.2 and 2.5 Hz, H-15), 2.17
(1H, dd, J = 17.2 and 1.5 Hz, H-15), 2.68 (1H, br s, H-13), 3.22 and
3.58 (each 1H, d, J = 10.4 Hz, H-18), 3.65 (1H, dd, J = 6.5 and
4.1 Hz, H-3b), 4.69 (1H, t, J = 2.5 Hz, H-7), 4.75 and 4.80 (each 1H,
s, H-17); EIMS m/z (rel. int.) 302 [MꢀAcOH]+ (29), 284 (71), 271
(34), 253 (96), 239 (18), 226 (37), 199 (33), 185 (32), 171 (30),
157 (32), 145 (46), 126 (44), 119 (60), 105 (74), 91 (80); HREIMS:
m/z [MꢀAcOH]+ 302.2254 (calcd. for C20H30O2, 302.2246).
a
a
4.8.6. 18,18-bis-7b-Hydroxy-ent-trachylobane (34)
Colourless oil; 1H NMR (500 MHz) d 0.57 (2H, dt, J = 7.8 and
2.6 Hz, H-12 and H-120), 0.75 (2H, td, J = 13.3 and 3.3 Hz, H-1b
and H-1b0), 0.76 (6H, s, H-19 and H-190), 0.85 (2H, dd, J = 7.8 and
3.1 Hz, H-13 and H-130), 0.93 (6H, s, H-20 and H-200), 0.94 (2H,
td, J = 10.2 and 3.1 Hz, H-3b and H-3b0), 1.14 (6H, s, H-17 and H-
170), 1.18 (8H, m, H-3
(2H, m, H-2 and H-20), 1.38 and 1.52 (each 2H, d, J = 11.3 Hz, H-
15 and H-150), 1.46 (8H, m, H-1 , H-1a0, H-5, H-50, H-6, H-60, H-9
a
, H-3a0, H-14, H-140, H-18 and H-180), 1.36
a
and H-90), 1.60 (2H, m, H-6 and H-60), 1.65 (2H, ddd, J = 14.7, 6.2
and 2.6 Hz, H-11 and H-110), 1.88 (2H, ddd, J = 14.7, 11.5 and
2.8 Hz, H-11 and H-110), 1.96 (2H, d, J = 11.7 Hz, H-14 and H-140),
3.50 (2H, t, J = 2.5 Hz, H-7 and H-70); EIMS m/z (rel. int.) 556
[MꢀH2O]+ (5), 538 (33), 523 (4), 283 (2), 255 (100), 239 (5), 227
(4), 201 (8), 185 (9), 173 (9); HREIMS m/z [MꢀH2O]+ 556.4632
(calcd. for C40H60O, 556.4644).
4.8. Preparation of the substrate trachinol acetate (27)
4.8.1. Partial hydrolysis of 7b,18-diacetoxy-ent-trachilobane (29)
Compound 29 (385 mg) in MeOH (25 mL) was treated with
K2CO3 (545 mg) with stirring at room temperature for 9 h. Usual
work-up afforded trachinodiol 7b-monoacetate (30) (312 mg)
and trachinodiol (28) (9 mg).
4.8.7. 7b-Hydroxy-18-iodo-ent-trachylobane (32)
Trachinodiol (28) (9 mg) was treated with triphenylphosphine
(10 mg), imidazole (4 mg) and I2 (9 mg) in the same way than for
30 affording: 7b-hydroxy-18-iodo-ent-trachylobane (32) (8.5 mg),
colourless crystal, m.p. 136–137 °C, 1H NMR (500 MHz) d 0.56
(1H, dt, J = 7.7 and 2.7 Hz, H-12), 0.78 (1H, td, J = 13.0 and 3.7 Hz,
H-1b), 0.85 (1H, dd, J = 7.7 and 3.2 Hz, H-13), 0.93 (3H, s, H-20),
0.94 (3H, s, H-19), 1.14 (3H, s, H-17), 1.18 (1H, ddd, J = 11.7, 3.2
and 1.7 Hz, H-14), 1.31 (2H, m, H-2 and H-3), 1.42 and 1.55 (each
4.8.2. Trachinodiol 7b-monoacetate (30)
Colourless crystal, m.p. 157–159 °C, 1H NMR (500 MHz) d 0.58
(1H, dt, J = 7.7 and 2.7 Hz, H-12), 0.70 (3H, s, H-19), 0.80 (1H, td,
J = 12.9 and 3.2 Hz, H-1b), 0.87 (1H, dd, J = 7.7 and 3.2 Hz, H-13),
0.97 (3H, s, H-20), 1.12 (3H, s, H-17), 1.23 (1H, br d, J = 12.6 Hz,
H-3b), 1.30 (1H, ddd, J = 11.7, 3.2 and 1.7 Hz, H-14), 1.32 and 1.45
(each 1H, d, J = 11.4 Hz, H-15), 1.61 (1H, dd, J = 11.4 and 1.5 Hz,
H-5), 1.63 (1H, m, H-6 overlapped with H-5 and H-11), 1.68 (1H,
ddd, J = 14.6, 6.7 and 2.5 Hz, H-11), 1.90 (1H, ddd, J = 14.6, 11.5
and 2.9 Hz, H-11), 2.00 (1H, d, J = 11.7 Hz, H-14), 2.02 (3H, s,
–OAc), 3.00 and 3.29 (each 1H, d, J = 10.9 Hz, H-18), 4.68 (1H, br
s, H-7); EIMS m/z (rel. int.) 346 [M]+ (3), 286 (91), 271 (15), 255
1H, d, J = 11.3 Hz, H-15), 1.43 (2H, m, H-1a and H-6), 1.48 (1H, m,
H-6), 1.57 (1H, d, J = 11.6 Hz, H-9), 1.62 (1H, dd, J = 12.7 and
2.1 Hz, H-5), 1.65 (1H, ddd, J = 14.7, 6.3 and 2.7 Hz, H-11), 1.90
(1H, ddd, J = 14.7, 11.6 and 2.8 Hz, H-11), 1.92 (1H, d, J = 11.7 Hz,
H-14), 3.14 and 3.19 (each 1H, d, J = 10.0 Hz, H-18), 3.51 (1H, t,

70
B.M. Fraga et al. / Phytochemistry 81 (2012) 60–70
J = 2.9 Hz, H-7); EIMS m/z (rel. int.) 414 [M]+ (1), 396 (6), 381 (4),
286 (11), 268 (100), 253 (68), 239 (30), 225 (10), 213 (14), 199
(23); HREIMS m/z [M]+ 414.1418 (calcd. for C20H31OI, 414.1420).
Cross, B.E., Myers, P.L., 1969. The effect of plant growth retardants on the
biosynthesis of diterpenes by Gibberella fujikuroi. Phytochemistry 8, 79–83.
De Quesada, T.G., Rodríguez, B., Valverde, S., Huneck, S., 1972. Six new diterpenes
from Sideritis leucantha Cav. and Sideritis linearifolia Lam.. Tetrahedron Lett. 22,
2187–2190.
Dennis, D.T., Uppper, C.D., West, C.A., 1965. An enzymic site of inhibition of
gibberellin biosynthesis by AMO 1618 and other plant growth retardants. Plant
Physiol. 40, 948–952.
Fraga, B.M., 2004. On the structure of an ent-kaurene diterpene from Aristolochia
anguicida. Rev. Latinoam. Quim. 32, 76–79.
Fraga, B.M., Hanson, J.R., Hernández, M.G., 1978. The microbiological transformation
of epicandicandiol, 7b,18-dihydroxy-ent-kaur-16-ene, by Gibberella fujikuroi.
Phytochemistry 17, 812–814.
Fraga, B.M., Hanson, J.R., Hernández, M.G., Sarah, F.Y., 1980. The microbiological
transformation of some ent-kaur-16-ene 7-, 15-, and 18-alcohols by Gibberella
fujikuroi. Phytochemistry 19, 1087–1091.
4.8.8. Trachinol acetate (27)
Trachinol (26) (208 mg) in pyridine (1.5 mL) at room tempera-
ture was treated with Ac2O (1.5 mL) and stirred for 12 h. Usual
work-up and chromatography afforded trachinol acetate (27)
(233 mg): colourless crystal, m.p. 119–120 °C; 1H NMR (500 MHz)
d 0.57 (1H, dt, J = 7.7 and 2.7 Hz, H-12), 0.74 (3H, s, H-18), 0.76
(3H, s, H-19), 0.78 (1H, td, J = 12.9 and 3.8 Hz, H-1b), 0.86 (1H, dd,
J = 7.7 and 3.2 Hz, H-13), 0.93 (3H, s, H-20), 1.12 (3H, s, H-17), 1.14
(1H, td, J = 13.5 and 3.7 Hz, H-3b), 1.23 (1H, dd, J = 12.9 and 2.0 Hz,
H-5), 1.29 (1H, ddd, J = 11.7, 3.3 and 1.8 Hz, H-14), 1.31 and 1.44
Fraga, B.M., González, A.G., Hanson, J.R., Hernandez, M.G., 1981. The microbiological
transformation of some ent-3b-hydroxykaur-16-enes by Gibberella fujikuroi.
Phytochemistry 20, 57–61.
(each 1H, d, J = 11.6 Hz, H-15), 1.36 (2H, m, H-2b and H-3
(1H, ddd, J = 14.4, 12.9 and 2.5 Hz, H-6 ), 1.54 (1H, ddt, J = 13.5,
13.5 and 3.5 Hz, H-2 ), 1.66 (1H, ddd, J = 14.6, 6.7 and 2.5 Hz, H-
a), 1.46
Fraga, B.M., Guillermo, R., Arraez, J.D., Luis, J.G., Perales, A., 1988. Partial synthesis of
4-epi-trachylobagibberellin A12. J. Chem. Soc., Perkin Trans. I, 1513–1516.
Fraga, B.M., Guillermo, R., Hanson, J.R., 1990a. The partial syntheses of 7b-
hydroxyatisenolide and atisagibberellin dimethyl ester. J. Chem. Res. 99.
Fraga, B.M., Hernández, M.G., Santana, J.M.H., Arteaga, J.M., 1990b. Diterpenes from
Sideritis sventenii and S. cystosiphon. Phytochemistry 29, 591–593.
Fraga, B.M., García-Tellado, F., González, P., Hernández, M.G., 1992. The chemical
and microbiological synthesis of 14-hydroxy-gibberellins. Tetrahedron Lett. 39,
8491–8504.
Fraga, B.M., Hernández, M.G., Arraez, J.D., Luis, J.G., 1994. The transformation of
epicandicandiol into a gibberellin isomer. Tetrahedron 44, 12643–12648.
Fraga, B.M., González, P., Guillermo, R., Hernández, M.G., Perales, A., 1995. The
chemical and microbiological preparation of 15-oxo-gibberellin derivatives.
Tetrahedron 51, 10053–10064.
a
a
11), 1.68 (1H, ddd, J = 14.4, 3.3 and 2.0 Hz, H-6b), 1.88 (1H, ddd,
J = 14.6, 11.5 and 3.0 Hz, H-11), 2.00 (1H, d, J = 11.7 Hz, H-14), 2.03
(3H, s, -OAc), 4.71 (1H, dd, J = 3.3 and 2.9 Hz, H-7); EIMS m/z (rel.
int.) 330 [M]+ (5), 270 (100), 255 (37), 227 (9), 214 (6), 199 (12),
185 (19), 173 (14), 157 (49), 146 (26); HRMS: m/z [M]+ 330.2551
(calcd. for C22H34O2, 330.2559).
4.9. Incubation of trachinol acetate (27)
Fraga, B.M., Hernández, M.G., Fernández, C., Santana, J.M.H., 2009.
A
chemotaxonomic study of nine Canarian Sideritis species. Phytochemistry 70,
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Fraga, B.M., González, P., Gonzalez-Vallejo, V., Guillermo, R., Díaz, L.N., 2010.
The substrate 27 (230 mg) in EtOH (13 mL) was distributed be-
tween 65 conical flasks. Its biotransformation gave in the neutral
fraction starting material (212 mg) and 7b-acetoxy-19-hydroxy-
ent-trachilobane (33) (3 mg).
Biotransformation of 7a- and 7-oxo-ent-atis-16-ene derivatives by the fungus
Gibberella fujikuroi. Phytochemistry 71, 1313–1321.
Fraga, B.M., Bressa, C., Gonzalez-Vallejo, V., Suárez, S., Guillermo, R., 2011.
Neighbouring group participation in the reaction of 7-oxo-ent-kaur-16-ene
derivatives with diacetoxyiodobenzene. Synthesis of gibberellin analogues.
Tetrahedron Lett. 52, 7138–7140.
Gaitán, R., Gómez, H.A., Tapia, S., Villadiego, A.M., Méndez, D., 2002. (ꢀ)-Acido 11-
hidroxi-kaur-16-en-19-oico, un nuevo kaureno aislado de Aristolochia anguicida
Jacq. Rev. Latinoam. Quim. 30, 83–86.
Garegg, P.J., Samuelsson, B., 1980. Novel reagent system for converting a hydroxy-
group into an iodo-group in carbohydrates with inversion of configuration: Part
2. J. Chem. Soc., Perkin Trans. I, 2866–2869.
González, A.G., Bretón, J.L., Fraga, B.M., Luis, J.G., 1971. Trachinodiol and trachinol,
two new diterpenes from Sideritis canariensis Ait. Tetrahedron Lett. 12, 3097–
3100.
4.9.1. 7b-Acetoxy-19-hydroxy-ent-trachilobane (33)
Colourless oil; 1H NMR (500 MHz) d 0.59 (1H, dt, J = 7.8 and
2.7 Hz, H-12), 0.83 (1H, td, J = 13.7 and 3.9 Hz, H-1b), 0.85 (3H, s,
H-18), 0.88 (1H, dd, J = 7.8 and 3.3 Hz, H-13), 0.93 (3H, s, H-20),
0.97 (1H, td, J = 13.6 and 4.3 Hz, H-3b), 1.12 (3H, s, H-17), 1.29
(1H, ddd, J = 11.7, 3.3 and 1.7 Hz, H-14), 1.32 and 1.44 (each 1H,
d, J = 11.7 Hz, H-15), 1.37 (1H, m, H-2), 1.39 (1H, dd, J = 13.1 and
1.6 Hz, H-5), 1.48 (1H, m, H-9), 1.53 (1H, td, J = 14.4 and 2.5 Hz,
H-6
a
), 1.66 (1H, ddd, J = 14.7, 6.5 and 2.5 Hz, H-11), 1.76 (1H, br
González, A.G., Fraga, B.M., Hernández, M.G., Luis, J.G., 1973. New diterpenes from
Sideritis candicans. Phytochemistry 12, 2721–2723.
González, A.G., Fraga, B.M., Hernández, M.G., Luis, J.G., 1978. Synthesis of 4-epi-
d, J = 13.6 Hz, H-3
a
), 1.80 (1H, ddd, J = 14.4, 3.3 and 1.8 Hz, H-6b),
1.91 (1H, ddd, J = 14.7, 11.5 and 2.9 Hz, H-11), 1.97 (1H, d,
J = 11.7 Hz, H-14), 2.05 (3H, s, –OAc), 3.40 (1H, dd, J = 10.9 and
0.8 Hz, H-19), 3.68 (1H, d, J = 10.9 Hz, H-19), 4.70 (1H, t,
J = 2.4 Hz, H-7); EIMS m/z (rel. int.) 346 [M]+ (3), 286 (81), 271
(14), 255 (100), 239 (7), 227 (4), 213 (6), 199 (16), 185 (21), 173
(24); HREIMS m/z [M]+ 346.2513 (calcd. for C22H34O3, 346.2508).
gibberellin A12 from ent-7a,18-dihydroxy-kaur-16-ene. Tetrahedron Lett. 19,
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González, A.G., Fraga, B.M., Hernández, M.G., Hanson, J.R., 1981. The 13C NMR
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Hanson, J.R., Hawker, J., White, A.F., 1972. Studies in terpenoid biosynthesis: Part IX.
The sequence of oxidation on ring B in kaurene–gibberellin biosynthesis. J.
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This work has been supported by Grant CTQ2009-14629-C02-
01, MICINN, Spain. C.B. thanks Ministry of Science and Technology
(Spain) for a FPI grant. V.G. thanks the Spanish Research Council
(CSIC) and the European Social Foundation for an I3P fellowship.
gibberellin biosynthesis. Phytochemistry 10, 2065–2076.
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Mitra, S.R., Das, A.K., Kirtaniya, C.L., Adityachaudhury, N., Patra, A., Mitra, A.K., 1980.
Didymooblongin, a new kaurenoid diterpene from Didymocarpus oblonga Wall.
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Kaurenolides and fujenoic acids are side products of the gibberellin P450-1
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