Biotransformation of 7α-hydroxy-and 7-oxo-ent-atis-16-ene derivatives by the fungus Gibberella fujikuroi

Article abstract of DOI:10.1016/j.phytochem.2010.04.024

The microbiological transformation of 7α,19-dihydroxy-ent-atis-16-ene by the fungus Gibberella fujikuroi gave 19-hydroxy-7-oxo-ent-atis-16-ene, 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene, 7α,11β,19-trihydroxy-ent- atis-16-ene and 7α,16β,19-trihydroxy-ent-atis-16-ene, while the incubation of 19-hydroxy-7-oxo-ent-atis-16-ene afforded 13(R),19-dihydroxy-7- oxo-ent-atis-16-ene and 16β,17-dihydroxy-7-oxo-ent-atisan-19-al. The biotransformation of 7-oxo-ent-atis-16-en-19-oic acid gave 6β-hydroxy-7- oxo-ent-atis-16-en-19-oic acid, 6β,16β,17-trihydroxy-7-oxo-19-nor-ent- atis-4(18)-ene and 3β,7α-dihydroxy-6-oxo-ent-atis-16-en-19-oic acid.

Full text of DOI:10.1016/j.phytochem.2010.04.024

Phytochemistry 71 (2010) 1313–1321
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Biotransformation of 7a-hydroxy- and 7-oxo-ent-atis-16-ene derivatives
by the fungus Gibberella fujikuroi
b
a
b
a
Braulio M. Fraga a, , Pedro Gonzalez , Victoria Gonzalez-Vallejo , Ricardo Guillermo , Luz N. Diaz
*
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:
The microbiological transformation of 7
gave 19-hydroxy-7-oxo-ent-atis-16-ene, 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene,
a
,19-dihydroxy-ent-atis-16-ene by the fungus Gibberella fujikuroi
Received 12 January 2010
Received in revised form 21 April 2010
Available online 20 May 2010
7a,11b,19-trihy-
droxy-ent-atis-16-ene and 7 ,16b,19-trihydroxy-ent-atis-16-ene, while the incubation of 19-hydroxy-
a
7-oxo-ent-atis-16-ene afforded 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene and 16b,17-dihydroxy-7-
oxo-ent-atisan-19-al. The biotransformation of 7-oxo-ent-atis-16-en-19-oic acid gave 6b-hydroxy-7-
Keywords:
oxo-ent-atis-16-en-19-oic acid, 6b,16b,17-trihydroxy-7-oxo-19-nor-ent-atis-4(18)-ene and 3b,7
droxy-6-oxo-ent-atis-16-en-19-oic acid.
a-dihy-
Gibberella fujikuroi
Biotransformations
Diterpenes
Ó 2010 Elsevier Ltd. All rights reserved.
ent-Atis-16-ene
7a,19-Dihydroxy-ent-atis-16-ene
19-Hydroxy-7-oxo-ent-atis-16-ene
7-Oxo-ent-atis-16-en-19-oic acid
1. Introduction
tion of the corresponding ent-kaur-16-ene analogues, which led to
the isolation of a 6,7-seco-acid, fujenoic acid (6), and other ent-
kaur-16-ene derivatives (Fraga et al., 2005, 2007).
Although all the known natural gibberellins belong to the
ent-kaur-16-ene series, the occurrence of gibberellin analogues of
other tetracyclic diterpenes with ent-phyllocladene, ent-beyerene
or ent-atisene skeleta is biosynthetically possible. The same applies
for the kaurenolides and 6,7-seco-ring ent-kaur-16-enes, produced
by the fungus Gibberella fujikuroi together with the gibberellins.
In a previous work we described the biotransformation of 7b-hy-
droxy-ent-atis-16-en-19-oic acid by this fungus, which led to the
formation of atisagibberellins A12 and A14 (Hanson et al., 1979).
However, oxygenated C-20 atisagibberellins or the corresponding
2. Results and discussion
The incubations were carried out in the presence of AMO 1618.
This substance inhibits the production of ent-kaur-16-ene and con-
sequently metabolites derived from it are not formed. However,
the post-kaurene metabolism is not affected, it being possible to
utilize the biosynthetic enzymes for the transformation of the arti-
ficial substrates (Dennis et al., 1965; Cross and Myers, 1969).
Substrate 1 was prepared by LiAlH4 reduction of methyl 7-oxo-
ent-atis-16-en-19-oate (4) (Fig. 1), which also afforded its 7-epimer
(7) in very low yield. Compound 4 had been prepared, with a global
yield 85%, by methylation, hydrolysis and oxidation of gummiferol-
ic acid (11), a diterpenic angelate, isolated from Margotia gummif-
era in good yield (Pinar et al., 1978).
C19-gibberellins were not isolated. Later, Beale et al. (1983) ob-
tained atisagibberellins, an atisenolide and a 6,7-seco-ring diacid
by incubation of ent-atis-16-en-19-oic acid and its 13(R)-hydro-
xy-derivative with the mutant B1–41A of G. fujikuroi. We also car-
ried out the preparation of atisenolides by incubation of 19-
hydroxy-ent-atis-6,16-diene and ent-atis-6,16-dien-19-oic acid
with this microorganism (Fraga et al., 1992).
Now, to complete our studies with this type of diterpenes we
have examined the biotransformation of the 7a-hydroxy- and 7-
oxo-ent-atis-16-ene derivatives 1–3 with the fungus G. fujikuroi.
The aims were to prepare seco-ring B compounds, such as 5, and
compare the results with those previously obtained in the incuba-
The incubation of 7
fungus G. fujikuroi gave 19-hydroxy-7-oxo-ent-atis-16-ene (2),
13(R),19-dihydroxy-7-oxo-ent-atis-16-ene (13), 7 ,11b,19-trihy-
,16b,19-trihydroxy-ent-atisane
a,19-dihydroxy-ent-atis-16-ene (1) with the
a
droxy-ent-atis-16-ene (14) and 7
a
(15) (Fig. 2).
The least polar metabolite isolated from this biotransformation
was 2, which showed in its HRMS the molecular ion at m/z
302.2253, corresponding to a molecular formula of C20H30O2.
Consequently, this metabolite had lost two hydrogens during the
* 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 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.04.024

1314
B.M. Fraga et al. / Phytochemistry 71 (2010) 1313–1321
12
8
also does not allow naming the stereochemistry of this alcohol as
or b. Consequently, the 13(R)-configuration assigned to this center
was given considering the
-gauche effect, À8.4 ppm, on the car-
a
17
16
13
11
9
20
14
c
1
4
15
bon resonance of C-11, which is produced by the presence of the
13-hydroxyl group. Thus, C-11 appears at dC 20.4 in 13 and 28.8
in 2. These facts indicated that the structure of this metabolite
was 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene (13), which is
formed via the 13(R)-hydroxylation of 2 (see also below). A com-
pound of this type, 13(R)-angeloxy-ent-atis-16-en-19-oic acid,
had been isolated from Helianthus decapetalus (Beale et al., 1983).
Another metabolite isolated (14) had the molecular formula
2
3
10
H
7
H
5
CO2H
O
O
6
H
H
CO2Me
18
O
19
O
5
4
C20H32O3 (m/z 320.2325), which indicated that a new hydroxyl
group had been introduced into the molecule of the substrate 1.
The 13C NMR spectrum of 14, in comparison with that of 1, showed
the presence of a new secondary carbon bearing a hydroxyl group
at dC 69.9, and the corresponding proton resonance at dH 3.70,
which is overlapped with a H-19. The relative low value of reso-
nance was indicative of a oxymethine situated between carbons
bearing hydrogen atoms, such as C-2, C-11 or C-13. On the other
hand, the form of resonance and coupling of the geminal proton
to the new hydroxyl group at dH 3.45 (t, J = 3.5 Hz), in C6D6, and
the high value of the C-9 resonance at dC 63.9, permitted us to as-
sign it to C-11(b). This assertion was confirmed in the HMBC exper-
iment with a cross-peak between H-9 and C-11. Other correlations
observed in this spectrum were: H-20 with C-9 and C-1; H-18 with
C-3, C-5 and C-19; H-5 with C-4, C-10 and C-19; H-6 with C-7; H-
15 with C-9 and C-16; and H-17 with C-12 and C-15. Thus, the
H
O
CO2H
H
O
H
R2
O
H
6
CH2OR1
7
8
9
R1 = H R2 = β-OH,H
R1 = Ac R2 = β-OH,H
R1 = Ac R2 = β-OAc,H
H
OR
10 R1 = Ac R2 = O
H
structure of this product was determined as 7a,11b,19-trihy-
droxy-ent-atis-16-ene (14).
CO2H
To the fourth compound isolated from this biotransformation
the structure of 7 ,16b,19-trihydroxy-ent-atis-16-ene (15) was as-
signed, on the basis of the following considerations: Its 1H and 13
11 R = Ang
12 R = H
a
C
NMR spectra showed the disappearance of the exocyclic double
bond signals and the presence in the molecule of a new methyl
group at dH 1.30 and dC 30.4. These values indicated that this methyl
should be geminal to a hydroxyl group, which was confirmed in the
HMBC experiment. The MS spectrum also explains this fact show-
ing the peak of higher mass at 304.2444 (C20H32O2), which is
formed from the molecular ion by loss of water. The b-stereochem-
istry of the alcohol at C-16 was assigned considering that the hydra-
tion of the exocyclic double bond in the ent-atis-16-ene derivatives
occurs by the less hindered b-face. The natural compounds of this
type normally have the 16b-configuration (Schmitz et al., 1983; Pia-
cenza et al., 1985; Hao and Nie, 1998; Tavares et al., 2007). This fact
was confirmed in the NOESY spectrum where correlations of H-17
Fig. 1. Structures of compounds 4–12.
incubation with respect to the substrate. The disappearances of the
geminal proton to the secondary alcohol group and of the oxyme-
thine in the 1H and 13C NMR spectra, respectively, and the presence
in the latter of a new signal at d 214.8, indicated that the secondary
alcohol had been oxidized to a 7-oxo group. Thus, the structure of
this substance was determined as 19-hydroxy-7-oxo-ent-atis-16-
ene (2). Partial synthesis of this compound confirmed the assigned
structure (see below).
The structure 13 was assigned to another metabolite obtained
from this fermentation. The HRMS spectrum showed the molecular
ion at 318.2185 (C20H30O3). As in the metabolite 2, the geminal
with H-13 and H-15
a, and of H-7 with H-15b were observed, which
indicated an -stereochemistry for the C-17 methyl group.
a
proton to the 7
a
-hydroxyl group in the 1H NMR spectrum, and
We also carried out the biotransformation of the two 7-oxo-ent-
atis-16-ene derivatives 2 and 3. The former had been obtained in
the previous feeding, but as this compound had been isolated in
low yield, we synthesized it as follows: The diol 7, obtained by
LiAlH4 reduction of gummiferolic acid (11) (Pinar et al., 1978),
was partially acetylated affording the 19-monoacetate 8, which
was oxidized to the corresponding 7-oxo derivative 10. Alkaline
hydrolysis of this compound gave 19-hydroxy-7-oxo-ent-atis-16-
ene (2). The global yield from 11 was 74%.
The microbiological transformation of 2 afforded the metabo-
lites 13 and 16 (Fig. 2). The less polar of these compounds was iden-
tified as 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene (13), which was
identical with a substance obtained in the incubation of 1.
The second metabolite was obtained in very low yield and its
structure determined as 16b,17-dihydroxy-7-oxo-ent-atisan-19-al
(16). In the HRMS the peak of higher mass appears at m/z
319.1910 (C19H27O4), which is formed from the molecular ion by
loss of a methyl group. Thus, the molecular formula of this
metabolite was C20H30O4. The 1H and 13C NMR spectra showed
the corresponding carbon in the 13C NMR spectrum, were not ob-
served. Alternatively, the presence in the molecule of a geminal
hydrogen to a new secondary hydroxyl group at dY 4.04 (dt,
J = 9.6 and 3.6 Hz), the corresponding oxymethine at dC 67.7, and
the signal of a new oxo group at dC 214.4, were observed in both
spectra. These two functions were located at C-13(R) and C-7 by
assignment of the 13C NMR spectrum using 2D NMR data (Table
1). Thus, the HMBC and NOESY experiments showed correlations
of H-11, H-12 and H-14 with C-13, and of H-13 with H-12 and
H-14, respectively. Double resonance experiments confirmed the
assignment of the 13-hydroxyl group: irradiation of H-12 modifies
the H-13 signal from a double triplet into a clean double doublet,
whilst irradiation of H-14 collapses H-13 to a doublet. The stereo-
chemistry of the 13-OH cannot be determined using the coupling
constant of its geminal proton, because both hydrogens in the
13(R) or 13(S) configuration, have the same J-coupling. This is
due to the symmetrical bridge that joins C-8 and C-12, which is
perpendicular to the paper plane in the atisane skeleton. This fact

B.M. Fraga et al. / Phytochemistry 71 (2010) 1313–1321
1315
H
OH
H
CH2OH
1
G. fujikuroi
First incubation
HO
H
HO
OH
H
H
+
+
OH
OH
O
H
H
H
CH2OH
CH2OH
CH2OH
13
14
15
+
CH2OH
OH
G. fujikuroi
H
H
13
+
O
Second
incubation
O
H
H
CH2OH
CHO
2
16
Fig. 2. Biotransformation of 1 and 2.
Table 1
13C NMR data of compounds 1, 2, 7, 8, 10 and 13–16.
Position
1
2
7
8
10
38.9
13
14
15
16
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
39.0
17.7
35.6
37.7
53.7
28.0
78.9
38.1
52.0
38.1
28.5
36.2
26.3
20.9
43.8
39.0
17.6
35.8
38.1
53.8
37.0
39.3
17.2
35.9
39.2
17.7
36.5
36.2
47.6a
26.9b
73.6
37.6
47.7a
37.6
27.0b
36.3
27.7b
28.3b
41.6
39.0
39.3
38.9
17.6
35.6
37.5
53.7
28.2
79.0
39.3
50.4
37.8
23.1a
37.6
22.9a
19.4
52.7
71.6
30.4
26.8
65.6
14.4
38.3
17.9
34.8
42.8
53.7
36.2
n.o.
47.8
50.1
37.7
23.2a
31.3
22.6a
26.8
44.1
73.5
68.2
23.8
204.7
13.1
17.6
36.6
37.4
53.7
36.9
214.4
47.5
52.8
36.7
28.4
35.8
26.0
27.6
40.0
150.0
106.3
26.8
66.9
13.9
17.6
35.8
37.5
53.2
37.5
214.4
47.9
52.5
38.0
20.4
44.6
67.7
37.3
39.3
146.7
109.1
26.4
65.2
14.8
17.6
35.6
37.2
53.5
28.2
78.3
38.2a
63.9
40.0a
69.9
44.4
23.7
19.5
43.3
145.7
110.8
26.9
65.6
15.8
37.6a
47.6b
27.0c
73.6
214.8
47.4
52.8
37.4
28.4
35.8
26.0
27.6
40.0
150.2
106.3
26.4
65.3
14.0
37.7a
47.7b
37.8a
27.0c
36.3
27.8c
28.3c
41.6
152.0
105.0
26.7
151.8
105.0
26.9
151.9
105.0
27.1
67.4
13.9
65.4
14.4
65.9
13.9
a,b,cThese values can be interchanged.
n.o., Not observed.

1316
B.M. Fraga et al. / Phytochemistry 71 (2010) 1313–1321
Table 2
the C-19 alcohol. In both spectra the disappearance of the signals
13C NMR data of compounds 3, 17, 19 and 21.
originated by the exocyclic double bond was observed, being
substituted by those due to a hydroxymethylene group, dH 3.50
and 3.60 (each d, J = 11.0 Hz) and dC 68.2. This primary alcohol
was geminal to a hydroxyl group situated at C-16 (dC 73.5). In
the HMBC experiment correlations of H-15 with C-17 and of
H-20 with C-1, C-5 and C-9 could be observed. The small amount
isolated, and the instability of this metabolite, did not permit us
to run a NOESY spectrum to determine the configuration at C-16.
However, comparison of the 13C NMR data of C-16 and other sur-
rounding carbons (C-12, C-13, C-14, C-15 and C-17) (Tables 1 and
2) of 16 with the corresponding of 19, where a NOESY spectrum
could be obtained, indicated that both metabolites had the same
stereochemistry at C-16. Moreover, the formation of the
16b,17-diol, in these compounds, must occur in the same way
(see below).
Position
3
17
39.9
19
21
32.9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
39.1
18.8
37.6
43.7
53.9
38.2
214.9
47.6
51.9
38.0
28.3
35.8
26.1
27.6
39.9
150.0
106.3
28.3
182.4
12.0
39.5
22.4
36.9
145.3
58.4
70.7
214.9
45.9
49.3
39.8
22.8
31.4
23.7
26.8
44.5
73.4
68.4
109.3
–
18.3
39.5
45.1
58.8
73.9
214.0
46.6
52.0
39.0
25.8
35.7
28.5
27.8
40.0
148.9
107.0
31.6
179.8
13.7
25.4
70.3
46.6a
58.4
219.3
82. 2
47.6a
49.6
46.9a
28.1b
35.8
26.3b
21.1
44.6
149.7
106.4
22.4
175.3
12.9
The third substrate, 7-oxo-ent-atis-16-en-19-oic acid (3), was
prepared by hydrolysis of gummiferolic acid (11) and subsequent
oxidation of the obtained 7b-alcohol (12), with a global yield of
82%. The biotransformation of 3 gave 6b-hydroxy-7-oxo-ent-atis-
16-en-19-oic acid (17), 6b,16b,17-trihydroxy-7-oxo-19-nor-ent-
13.5
a,bThese values can be interchanged.
atis-4(18)-ene (19) and 3b,7a-dihydroxy-6-oxo-ent-atis-16-en-
19-oic acid (21) (Fig. 3).
The least polar of these metabolites was 17. Its molecular for-
resonances at dH 9.78 and dC 204.7, respectively. These signals were
mula (C20H28O4) indicated that a new oxygen group had been
due to an unstable aldehyde group, which is formed by oxidation of
G. fujikuroi
H
H
Third
incubation
O
H
O
H
OH
CO2H
CO2H
3
17
H
H
O
HO
O
H
H
OH
CO2H
OH
18
20
OH
OH
H
H
HO
OH
O
H
CO2H
H
O
OH
21
19
Fig. 3. Biotransformation of 3.

B.M. Fraga et al. / Phytochemistry 71 (2010) 1313–1321
1317
OH
OH
G. fujikuroi
18
H
H
Enz+
O
O
H
H
OH
O
OH
H
O
H
19
17
G. fujikuroi
OH
OH
H
H
OH
OH
CH2OH
HOH2C
CH2OH
22
23
G. fujikuroi
G. fujikuroi
H
H
H
O
O
H
HOH2C
OH
O
O
24
25
H
O
H
OH
OH
H
H
R
O
O
O
26
27 R = CH2OH
28 R = CO2H
Fig. 4. Formation of 19, 23, 25, 27 and 28.
introduced into the molecule during the fermentation. This must
be a part of a secondary hydroxyl group, because a geminal hydro-
gen to this alcohol appeared at dH 4.95 (d, J = 12.6 Hz) in the 1H
NMR spectrum, and the corresponding oxymethine at dC 73.9 in
the 13C NMR one. The form of resonance and the coupling constant
in the proton spectrum indicated that the axial geminal proton was
of the 6b-hydroxyl was deduced from the NMR spectra. Thus, the
resonance of its geminal proton at dH 4.43 (d, J = 12.1 Hz) and of
the corresponding oxymethine at dC 70.7 were displayed. Other
functions observed in these spectra were the hydroxymethylene
group at dH 3.53 and 3.61 (each 1H, J = 11.0 Hz) and dC 68.4, respec-
tively, and the signals corresponding to the 4,18-double bond at dY
4.97, 5.07 and dC 145.3, 109.3, respectively. In the HMBC spectrum
the two H-18 showed correlations with C-3 and C-5. Other ob-
served cross-peaks were H-6 with C-5, and H-17 with C-16. The
situated at C-6(a). This position was confirmed by the unambigu-
ous assignment of the 13C NMR spectrum using 2D NMR data (Ta-
ble 2). The HMBC experiment showed correlations of H-6 with C-5
and C-7. Consequently, the structure of this metabolite was deter-
mined as 6b-hydroxy-7-oxo-ent-atis-16-en-19-oic acid (17).
The structure 6b,16b,17-trihydroxy-7-oxo-19-nor-ent-atis-4
(18)-ene (19) was assigned to the second metabolite of this feeding
on the following basis: In its HRMS, the peak of higher mass ap-
peared at m/z 289.1818 (C18H25O3), which is formed from the
molecular ion by loss of a hydroxymethylene group. The presence
a-stereochemistry of the C-17 hydroxymethylene group was
determined observing the NOESY spectrum, which showed a corre-
lation of H-17 with H-13. This configuration at C-16 is the one usu-
ally observed in natural compounds (Satti et al., 1987; Lal et al.,
1990; Gustafson et al., 1991). The precursor of this type of products
must be a 16b,17-epoxide, which is formed via the less hindered b-
face. Opening of the oxirane ring and neutralization by a hydroxyl

1318
B.M. Fraga et al. / Phytochemistry 71 (2010) 1313–1321
group, probably of water origin, led to the corresponding 16b,17-
diol. Compound 19 can be formed via the intermediate 18 by enzy-
matic hydrogen abstraction from the C-18 methyl, migration of the
two electrons of the 4,19-bond and concomitant decarboxylation
of the C-19 acid (Fig. 4). ent-Atisane derivatives with 16,17-dihydr-
oxyl groups have been shown to possess interesting biological
properties (Kurata et al., 2008; Tachibana et al., 2008).
4. In the incubation of 7-oxo-ent-atis-16-en-19-oic acid (3) a 6b-
hydroxylation took place, but the reaction did not progress to
form a 6,7-seco-ring compound 5, or the corresponding triacid,
as occurs in the feeding of 7-oxo-ent-kaur-16-en-19-oic acid,
which gave fujenoic acid (6) in very good yield (Fraga et al.,
2005). This difference in behaviour, and the difficulty in the
C-19 oxidation (see conclusion 2), can be attributed to steric
hindrance produced by the C/D rings in the ent-atis-16-ene
diterpenes. We must remember here that atisagibberellins
functionalized at C-20 could not be obtained by biotransforma-
tion, probably for the same reason (Hanson et al., 1979; Beale
et al., 1983).
5. The oxidative decarboxylation produced in the incubation of 3
to form the metabolite 19, probably via 18, must be highlighted.
This is the first time that this reaction has been observed in a
biotransformation by G. fujikuroi. Its biosynthesis implies an
enzymatic abstraction of H-18 (see above, Fig. 4). Previously,
we had obtained with this fungus the C-18 functionalized
metabolites 23 and 25 in the biotransformation of 22 (unpub-
lished work) and 24 (Fraga et al., 2005), respectively (Fig. 4).
Now, the same H-18 abstraction led to a decarboxylation due
to the presence of the C-19 acid group.
The last compound isolated 21 showed in its HRMS that two
oxygen atoms had been introduced in the molecule during the feed-
ing (m/z 348.1928, C20H28O5), which are part of two secondary hy-
droxyl groups. Thus, two geminal protons were observed in the 1H
NMR spectrum, the first at dH 4.18 (t, J = 2.5 Hz) being assigned at C-
3(a
). It showed in the 13C NMR spectrum gauche effects with C-1
and C-5, while in the HSQC experiment a cross-peak was observed
with the carbon at dC 70.3. In the HMBC experiment a correlation of
H-3 with C-2 and C-5 was displayed. The second geminal hydrogen
appeared at dH 4.19 (d, J = 1.4 Hz) and the corresponding carbon at
dC 82.2, which is more typical of an oxymethine at C-7 than at C-6.
The resonance of H-5 at dH 3.21 observed in the ketol 21, in compar-
ison with 1.36 in 17, and correlations of H-7 with C-6 and C-15 in
the HMBC experiment also confirmed this assertion. This means
that the oxo group at dC 219.3 must be at C-6 and the hydroxyl
group at C-7, which must be a consequence of the previous forma-
tion of 20 and subsequent isomerization to another ketol 21 (Fig. 3).
Other correlations observed in the HMBC experiment were: H-5
with C-3, C-4, C-6, C-9, C-18, C-19 and C-20; H-15 with C-9 and
C-17; H-9 with C-1, C-5, C-8, C-10, C-14 and C-15; H-18 with C-3,
6. The metabolites 17, 22 and 24 precursors of 19, 23 and 25,
respectively, possess a 6b-hydroxyl group, which suggests that
its presence in the molecule is a prerequisite to carry out the
oxidation of C-18 by this fungus. We must also indicate that
these levels of oxidation have also been observed in another
two metabolites of G. fujikuroi, such as 7b,18-dihydroxykauren-
olide (27) and the corresponding 18-acid (28) (Fig. 4), which are
C-4, C-5 and C-19; and H-20 with C-1, C-5 and C-9. The a-equatorial
stereochemistry was assigned to the C-7 alcohol considering the J-
coupling through a carbonyl of 1.4 Hz, observed between the axial
H-5 and the axial H-7 (Bhacca and Williams, 1964). These facts
were confirmed in the NOESY experiment where cross-peaks of
H-7 with H-5, H-9 and H-15, and of H-5 with H-7, H-9 and H-18,
were displayed. Consequently, the structure of this compound
was determined as 3b,7a-dihydroxy-6-oxo-ent-atis-16-en-19-oic
acid (21). The observed isomerization perhaps occurs after the 3b-
hydroxylation, via the intermediate 20. The occurrence in plants
of isomeric a-ketols in the beyerane class of diterpenes has been re-
also functionalized at C-6 but with the
a-stereochemistry
(Cross et al., 1963; Hanson and Sarah, 1979).
7. The major dihydroxykaurenolides produced by the fungus
G. fujikuroi are 7b,18- and 3b,7b-dihydroxykaurenolide, which
are formed from 7b-hydroxy-kaurenolide (26) (Cross et al.,
1968; Rojas et al., 2001). This functionalization, at C-18 and
C-3(b), is the same now observed in the ent-atisane derivatives
19 and 21, respectively, formed from the ketol 17 (Fig. 3), which
could indicate that the same enzyme is working in both pro-
cesses. This hypothesis is also in accordance with the results
obtained from studies on the gibberellin P450-1 monooxygen-
ase of this fungus, which catalyses the oxidation of C-7, C-6,
C-3 and C-18 in the biosynthetic pathway of kaurenolides and
seco-ring B acids, in addition to the GA14 synthesis (Rojas
et al., 2001, 2004). If the same enzyme acts on the functionali-
zation of C-3 and C-18, we also can conclude that the 3b-
hydroxylation to form 21 is produced before, and not after,
the ketol isomerization (Fig. 3).
ported (Connolly and Harding, 1972; Ansell et al., 1993).
3. Conclusions
Several conclusions can be deduced from the results obtained in
the microbiological transformations of the ent-atis-16-ene deriva-
tives 1–3:
1. We had indicated that the oxidation by this fungus of a 7a-
hydroxyl to a 7-oxo group, in the ent-kaur-16-ene series, prob-
ably required the presence in the molecule of a 19-acid group
(Fraga et al., 2007). However, the biotransformation of 1 indi-
cated that a 19-hydroxyl group is sufficient to carry out this
7-oxidation.
4. Experimental
4.1. General procedures
Mps were determined with a Reichert Thermovar apparatus and
are uncorrected. IR spectra were recorded in a Perkin Elmer 1600
FT. 1H NMR spectra were recorded in CDCl3 solution at
500.13 MHz with a Bruker AMX-500 spectrometer, and the 13C
NMR at 125.03 MHz in a Bruker AMX-500, except those of 1 and
15, which were recorded at 50 MHz in a Bruker AC-200. Mass spec-
tra were taken at 70 eV (probe) in a Shimadzu Q2000, and high res-
olution mass spectra in a Micromass Autospec spectrometer.
Optical rotations have been measured at 25 °C in a Perkin Elmer
343 Plus. HPLC was performed using a Beckman System Gold
125P. Purification by HPLC was achieved using a silica gel column
2. A 7a-hydroxy- or a 7-oxo group in ent-atis-16-ene derivatives
hinders the oxidation of C-19, which is characteristic of the bio-
synthesis of gibberellins, kaurenolides and seco-ring B com-
pounds. However, this does not occur in the biotransformation
by G. fujikuroi of analogous substrates of the ent-kaur-16-ene
series, which are efficiently transformed into seco-ring B deriva-
tives (Fraga et al., 2005, 2007).
3. The formation of a 6b-hydroxy-7-oxo intermediate in the bio-
transformation by G. fujikuroi of 7a-hydroxy or 7-oxo-ent-
kaur-16-ene derivatives (Fraga et al., 2005) into 6,7-seco-ring
compounds, such as fujenoic acid (6) or the corresponding tri-
acid, has now been confirmed in the ent-atis-16-ene series with
the biotransformation of 3.
(Ultrasphere Si 5
was made on silica gel Merck 0.040–0.063 mm. The substances
were crystallized from petrol–EtOAc except where otherwise indi-
l
m, 10 Â 250 mm). Dry column chromatography

B.M. Fraga et al. / Phytochemistry 71 (2010) 1313–1321
1319
cated. Molecular mechanics calculations were carried out with the
program Hyperchem 7.0 (Hypercube).
fraction 19-hydroxy-7-oxo-ent-atis-16-ene (2) (1 mg), starting
material (160 mg), 13(R),19-dihydroxy-7-oxo-ent-atis-16-ene (13)
(9 mg), 7a,11b,19-trihydroxy-ent-atis-16-ene (14) (3.5 mg) and
4.2. Microorganism
7a,16b,19-trihydroxy-ent-atisane (15) (12 mg). Transformed prod-
ucts were not obtained in the acid fraction.
The fungal strain was G. fujikuroi MP-C (Fusarium fujikuroi) IMI
58289 and was a gift from Prof. J.R. Hanson, Department of Chem-
istry (University of Sussex, UK).
4.5.1. 19-Hydroxy-7-oxo-ent-atis-16-ene (2)
Colourless crystal, m.p. 119–120 °C; [
a
]D: À7.6° (c, 0.58); IR
(CCl4) mmax 3640, 2932, 1702 cmÀ1 1H NMR (500 MHz, CDCl3): d
;
4.3. Incubation procedure
0.92 (1H, td, J = 13.2 and 3.6 Hz, H-1b), 0.96 (3H, s, H-18), 0.99
(1H, td, J = 13.7 and 3.7 Hz, H-3b), 1.14 (3H, s, H-20), 1.30 (1H, m,
H-14), 1.39 (1H, dd, J = 14.6 and 2.7 Hz, H-5), 1.45 (1H, m, H-2),
1.48 (2H, m, H-9 and H-11), 1.57 (1H, ddt, J = 13.7 and 3.3 Hz, H-
The fungus G. fujikuroi, inhibited with 5 Â 10À5 M AMO 1618,
was grown on shake culture at 25 °C for two days in 54–80 conical
flasks (250 ml), each containing 50 ml of sterile medium compris-
ing (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 (11–16 ml) and Tween 80 (three drops)
was evenly distributed between the flasks and the incubation al-
lowed to continue for a further 6 days. The broth was filtered
and the culture filtrate extracted with EtOAc. The mycelium was
treated with liquid nitrogen, crushed in a mortar and extracted
with EtOAc. Both extracts were combined and separated into
‘acidic’ and ‘neutral’ fractions with aqueous NaHCO3. The acidic
fraction was methylated with CH2N2.
2
a
), 1.65 (3H, m, H-11 and 2H-13), 1.68 (1H, m, H-1
br d, J = 13.7 Hz, H-3 ), 1.96 (1H, m, H-14), 2.10 (1H, br d,
J = 17.2 Hz, H-15), 2.31 (1H, br s, H-12), 2.45 (1H, dd, J = 15.5 and
2.7 Hz, H-6b), 2.55 (1H, dd, J = 15.5 and 14.6 Hz, H-6 ), 2.65 (1H,
a), 1.80 (1H,
a
a
br d, J = 17.2 Hz, H-15), 3.54 and 3.74 (each 1H, d, J = 10.8 Hz, H-
19), 4.67 and 4.79 (each 1H, br s, H-17); EIMS m/z (rel. int.): 302
[M]+ (100), 284 (2), 271 (9), 189 (4), 175 (5), 162 (56), 147 (13);
HRMS: [M]+ at m/z 302.2253. C20H30O2 requires 302.2246.
4.5.2. 13(R),19-Dihydroxy-7-oxo-ent-atis-16-ene (13)
A gum; 1H NMR (500 MHz, CDCl3): d 0.93 (1H, td, J = 13.2 and
3.9 Hz, H-1b), 0.96 (3H, s, H-18), 0.99 (1H, td, J = 13.8 and 3.8 Hz,
H-3b), 1.20 (3H, s, H-20), 1.40 (1H, dd, J = 13.5 and 4.2 Hz, H-5),
1.46 (3H, m, H-2b, H-9 and H-11), 1.58 (1H, ddt, J = 13.8 and
4.4. Preparation of the substrate 7
a,19-dihydroxy-ent-atis-16-ene (1)
3.4 Hz, H-2
a), 1.74 (1H, br d, J = 13.2 Hz, H-1
a), 1.77 (1H, dd,
J = 9.6 and 1.2 Hz, H-14), 1.80 (2H, m, H-3
a
and H-14), 2.03 (2H,
Methyl 7-oxo-ent-atis-16-en-19-oate (4) (330 mg) in dry THF
(50 ml) was treated with LiAlH4 (165 mg) at reflux for 3 h. The ex-
cess of reagent was destroyed 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 va-
cuo and chromatographed on SiO2, eluting with a petrol–EtOAc
gradient to afford 7 (11 mg) (Pinar et al., 1978) and 1 (280 mg).
m, H-11 and H-15), 2.34 (1H, br s, H-12), 2.48 (1H, dd, J = 15.9
and 4.2 Hz, H-6b), 2.52 (1H, br d, J = 17.1 Hz, H-15), 2.53 (1H, dd,
J = 15.9 and 13.5 Hz, H-6a), 3.53 (1H, dd, J = 10.9 and 0.6 Hz, H-
19), 3.75 (1H, d, J = 10.9 Hz, H-19), 4.04 (1H, dt, J = 9.6 and 3.6 Hz,
H-13), 4.79 (1H, dd, J = 3.9 and 2.0 Hz, H-17), 4.91 (1H, m, H-17);
EIMS m/z (rel. int.): 318 [M]+ (100), 300 (15), 289 (11), 273 (40),
269 (17), 243 (9), 178 (29), 160 (14), 145 (18), 134 (26); HRMS:
[M]+ at m/z 318.2185. C20H30O3 requires 318.2195.
4.4.1. 7b,19-Dihydroxy-ent-atis-16-ene (7)
1H NMR (500 MHz, CDCl3): d 0.90 (1H, td, J = 13.6 and 4.5 Hz, H-
1b), 0.96 (3H, s, H-20), 0.97 (3H, s, H-18), 1.02 (2H, m, H-3b and H-
11), 1.40 (2H, m, H-2 and H-11), 1.77 (2H, m, H-3a and H-6), 1.94
(1H, dt, J = 16.7 and 2.1 Hz, H-15), 2.24 (1H, m, H-12), 2.42 (1H, ddd,
J = 16.7, 5.8 and 2.6 Hz, H-15), 3.46 (1H, t, J = 2.9 Hz, H-7), 3.49 and
3.75 (each 1H, d, J = 10.9 Hz, H-19), 4.62 (1H, dd, J = 4.2 and 2.1 Hz,
H-17), 4.75 (1H, dd, J = 4.6 and 2.3 Hz, H-17); EIMS m/z (rel. int.):
304 [M]+ (5), 286 (39), 273 (11), 268 (4), 255 (100), 243 (4), 199
(8), 173 (10), 164 (11); HRMS: [M]+ at m/z 304.2401. C20H32O2 re-
quires 304.2402.
4.5.3. 7a,11b,19-Trihydroxy-ent-atis-16-ene (14)
A gum; 1H NMR (500 MHz, CDCl3): d 0.86 (1H, d, J = 3.3 Hz, H-9),
0.96 (3H, s, H-20), 0.97 (1H, td, J = 13.6 and 3.6 Hz, H-3b), 0.99 (3H,
s, H-18), 1.05 (1H, dd, J = 12.7 and 1.7 Hz, H-5), 1.08 (1H, m, H-1b),
1.35 (1H, m, H-14), 1.87 (1H, br d, J = 17.0, H-15b), 2.29 (1H, td,
J = 3.8 and 1.9 Hz, H-12), 2.64 (1H, dt, J = 17.0 and 2.2 Hz, H-15a),
3.31 (1H, dd, J = 11.4 and 4.8 Hz, H-7), 3.49 and 3.72 (each 1H, d,
J = 10.9 Hz, H-19), 3.70 (1H, overlapped with H-19, H-11), 4.90
and 4.93 (each 1H, dd, J = 4.0 and 2.1 Hz, H-17); 1H NMR
(500 MHz, C6D6): d 0.62 (1H, d, J = 3.3 Hz, H-9), 0.68 (3H, s), 0.73
(1H, dd, J = 12.7 and 1.7 Hz, H-5), 0.89 (3H, s), 1.66 (1H, br d,
J = 17.0 Hz, H-15b), 2.08 (1H, br s, H-12), 2.58 (1H, dt, J = 17.0 and
4.4.2. 7a,19-Dihydroxy-ent-atis-16-ene (1)
Colourless crystal, m.p. 79–80 °C; 1H NMR (500 MHz, CDCl3): d
0.77 (1H, td, J = 13.2 and 4.8 Hz, H-1b), 0.92 (1H, td, J = 13.7 and
4.2 Hz, H-3b), 0.94 (3H, s, H-20), 0.96 (3H, s, H-18), 1.00 (1H, d,
J = 11.4 Hz, H-5), 1.11 (1H, dd, J = 11.4 and 6.7 Hz, H-9), 1.77 (2H,
2.2 Hz, H-15a), 2.91 (1H, dd, J = 11.4 and 4.7 Hz, H-7), 3.18 and
3.39 (each 1H, d, J = 10.5 Hz, H-19), 3.45 (1H, t, J = 3.5 Hz, H-11),
4.86 and 4.90 (each 1H, dd, J = 4.3 and 2.4 Hz, H-17); EIMS m/z
(rel. int.): 320 [M]+ (28), 302 (25), 287 (33), 284 (35), 271 (42),
253 (37), 243 (14), 225 (9), 210 (8), 197 (10), 189 (11), 185 (13),
181 (17), 171 (17), 161 (11), 148 (36), 137 (31), 131 (39), 123
(97), 81 (100); HRMS: [M]+ at m/z 320.2325. C20H32O3 requires
320.2351.
m, H-3a and H-6), 1.81 and 2.59 (each 1H, br d, J = 16.8 Hz,
H-15), 2.23 (1H, br s, H-12), 3.25 (1H, dd, J = 11.5 and 4.3 Hz,
H-7), 3.46 and 3.69 (each 1H, d, J = 11.0 Hz, H-19), 4.58 and 4.73
(each 1H, br s, H-17); EIMS m/z (rel. int.): 304 [M]+ (84), 286
(25), 273 (11), 261 (100), 256 (82), 255 (61), 253 (16), 243 (13),
241 (26), 199 (14), 187 (12), 185 (11), 173 (13), 164 (36), 159
(17), 147 (21); [M]+ at m/z 304.2396. C20H32O2 requires 304.2402.
4.5.4. 7a,16b,19-Trihydroxy-ent-atisane (15)
A gum; 1H NMR (500 MHz, CDCl3): d 0.84 (1H, td, J = 12.8 and
4.0 Hz, H-1b), 0.95 (3H, s, H-20), 0.97 (3H, s, H-18), 1.02 (1H, br
d, J = 10.8 Hz, H-5), 1.13 (1H, br d, J = 13.5 Hz, H-15), 1.21 (1H, m,
4.5. Incubation of 7a,19-dihydroxy-ent-atis-16-ene (1)
H-9), 1.30 (3H, s, H-17), 1.66 (1H, br d, J = 12.8 Hz, H-1a), 1.77
The substrate 1 (270 mg) in EtOH (16 ml) was distributed be-
(2H, m, H-3 and H-6), 1.92 (1H, d, J = 13.5 Hz, H-15), 2.03 (1H, m,
tween 80 conical flasks. Its biotransformation gave in the neutral
H-13), 3.26 (1H, dd, J = 11.5 and 4.7 Hz, H-7), 3.47 and 3.72 (each

1320
B.M. Fraga et al. / Phytochemistry 71 (2010) 1313–1321
1H, d, J = 10.9 Hz, H-19); EIMS m/z (rel. int.): 304 [M-H2O]+ (45),
291 (8), 289 (13), 286 (9), 273 (63), 261 (27), 256 (48), 255 (44),
246 (9), 215 (10), 164 (11), 159 (12), 151 (11), 147 (13), 145
(12), 135 (10), 134 (13), 131 (14), 123 (100); HRMS: [MÀH2O]+
at m/z 304.2444. C20H32O2 requires 304.2402.
4.7.1. 16b,17-Dihydroxy-7-oxo-ent-atisan-19-al (16)
Colourless oil; 1H NMR (500 MHz, CDCl3): d 1.01 (1H, m, H-1b),
1.02 (6H, s, H-18 and H-20), 1.11 (2H, m, H-3b and H-14), 1.26 (1H,
m, H-11), 1.37 (1H, dd, J = 14.6 and 3.4 Hz, H-15), 1.59 (1H, dd,
J = 14.8 and 3.0 Hz, H-5), 1.65 (1H, m, H-2), 1.74 (2H, m, H-1
a
and H-9), 1.77 (1H, d, J = 14.6 Hz, H-15), 1.93 (1H, br s, H-12),
1.97 (1H, m, H-14), 2.05 (1H, m, H-11), 2.16 (1H, br d, J = 13.6 Hz,
4.6. Preparation of the substrate 19-hydroxy-7-oxo-ent-atis-16-ene
(2)
H-3a), 2.65 (1H, dd, J = 15.3 and 3.0 Hz, H-6b), 2.83 (1H, dd,
J = 15.3 and 14.8 Hz, H-6
a
), 3.50 and 3.60 (each 1H, d, J = 11.0 Hz,
H-17), 9.78 (1H, s, H-19); EIMS m/z (rel. int.): 334 [M]+ (1), 319
(100), 303 (14), 291 (10), 286 (11), 275 (13), 255 (5), 229 (6),
217 (6), 178 (8), 161 (14), 137 (26); HRMS: [MÀCH3]+ at m/z
319.1910. C19H27O4 requires 319.1909.
4.6.1. Partial acetylation of 7b,19-dihydroxy-ent-atis-16-ene (7)
The diol 7 (330 mg) in pyridine (2 ml) at 0 °C was treated with
Ac2O (2 ml) and stirred for 90 min. Usual work-up and chromatog-
raphy afforded starting material (7) (138 mg), 19-acetoxy-7b-hy-
droxy-ent-atis-16-ene (8) (213 mg) and 7b,19-diacetoxy-ent-atis-
16-ene (9) (traces).
4.8. Preparation of the substrate 7-oxo-ent-atis-16-en-19-oic acid (3)
Compound 12 (238 mg) in acetone (10 ml) was treated with
Jones reagent as above affording 7-oxo-ent-atis-16-en-19-oic acid
(3) (230 mg). Colourless crystal; m.p. 175–176 °C; 1H NMR
(500 MHz, CDCl3): d 0.94 (1H, td, J = 13.4 and 4.1 Hz, H-1b), 1.05
(1H, td, J = 13.6 and 4.1 Hz, H-3b), 1.09 (3H, s, H-20), 1.24 (3H, s,
H-18), 1.35 (1H, ddd, J = 14.9, 6.5 and 1.5 Hz, H-14), 1.48 (1H, dd,
J = 14.6 and 2.8 Hz, H-5), 1.49 (3H, m, H-2b, H-9 and H-11), 1.65
4.6.1.1. 19-Acetoxy-7b-hydroxy-ent-atis-16-ene (8). Colourless crys-
tal, m.p. 101–103 °C; 1H NMR (500 MHz, CDCl3): d 0.90 (1H, td,
J = 13.6 and 3.8 Hz, H-1b), 0.95 (3H, s, H-18), 0.98 (3H, s, H-20),
1.06 (2H, m, H-3b and H-11), 1.40 (2H, m, H-2 and H-11), 1.94
(1H, dt, J = 16.7 and 2.2 Hz, H-15a), 2.05 (3H, s, OAc), 2.24 (1H,
m, H-12), 2.42 (1H, ddd, J = 16.7, 5.7 and 2.6 Hz, H-15b), 3.47 (1H,
t, J = 2.8 Hz, H-7), 3.92 and 4.22 (each 1H, d, J = 10.9 Hz, H-19),
4.62 and 4.75 (each 1H, dd, J = 4.4 and 2.2 Hz, H-17); EIMS m/z
(rel. int.): 346 [M]+ (23), 328 (39), 273 (26), 255 (92), 240 (10),
225 (16), 173 (19), 164 (41), 123 (77), 109 (60), 93 (92), 81
(100); HRMS: [M]+ at m/z 346.2498. C22H34O3 requires 346.2508.
(3H, m, H-11 and 2H-13), 1.72 (1H, br d, J = 13.4 Hz, H-1
(1H, ddt, J = 13.9 and 3.6 Hz, H-2
br d, J = 17.2 Hz, H-15), 2.23 (1H, br d, J = 13.6 Hz, H-3
a
), 1.87
a
), 2.05 (1H, m, H-14), 2.11 (1H,
a), 2.32
(1H, br s, H-12), 2.62 (1H, dd, J = 15.6 and 2.8 Hz, H-6b), 2.65 (1H,
dt, J = 17.2 and 2.5 Hz, H-15), 3.07 (1H, dd, J = 15.6 and 14.6 Hz,
H-6a), 4.68 and 4.79 (each 1H, dd, J = 4.0 and 2.0 Hz, H-17); EIMS
4.6.2. Oxidation of 8
m/z (rel. int.): 316 [M]+ (100), 270 (16), 255 (11), 227 (3), 215
(8), 201 (7), 174 (11), 162 (69), 147 (42), 119 (24). HRMS: [M]+
at m/z 316.2044. C20H28O3 requires 316.2038.
Compound 8 (270 mg) in acetone (15 ml) was treated with
Jones reagent in the usual way affording 19-acetoxy-7-oxo-ent-
atis-16-ene (10) (264 mg): colourless crystal, m.p. 116–117 °C;
1H NMR (500 MHz, CDCl3): d 0.91 (1H, td, J = 13.2 and 3.8 Hz, H-
1b), 0.95 (3H, s, H-18), 1.05 (1H, td, J = 13.8 and 4.0 Hz, H-3b),
1.15 (3H, s, H-20), 1.31 (1H, m, H-14), 1.39 (1H, dd, J = 13.9 and
3.6 Hz, H-5), 1.44 (1H, m, H-2), 1.48 (2H, m, H-9 and H-11), 1.57
(1H, ddt, J = 13.7 and 3.5 Hz, H-2
13), 1.70 (2H, m, H-1 and H-3 ), 1.96 (1H, m, H-14), 2.05 (3H, s,
OAc), 2.10 (1H, br d, J = 17.2 Hz, H-15b), 2.31 (1H, br s, H-12),
2.46 (1H, dd, J = 15.4 and 3.6 Hz, H-6b), 2.52 (1H, dd, J = 15.4 and
4.9. Incubation of 7-oxo-ent-atis-16-en-19-oic acid (3)
The substrate 3 (225 mg) in EtOH (12 ml) was distributed be-
tween 58 conical flasks. Its biotransformation gave starting material
(92 mg), 6b-hydroxy-7-oxo-ent-atis-16-en-19-oic acid (17) (8 mg),
6b,16b,17-trihydroxy-7-oxo-19-nor-ent-atis-4(18)-ene (19) (1 mg)
and 3b,7a-dihydroxy-6-oxo-ent-atis-16-en-19-oic acid (21)
(11 mg), in the neutral fraction. Transformed products were not ob-
a), 1.65 (3H, m, H-11 and 2H-
a
a
13.9 Hz, H-6a), 2.65 (1H, dt, J = 17.2 and 2.6 Hz, H-15a), 3.94 and
tained in the acid fraction.
4.23 (each 1H, d, J = 11.0 Hz, H-19), 4.66 and 4.78 (each 1H, dd,
J = 4.2 and 2.1 Hz, H-17); EIMS m/z (rel. int.): 344 [M]+ (81), 284
(18), 271 (6), 256 (4), 241 (3), 213 (5), 201 (8), 189 (13), 175
(24), 162 (24), 147 (46); HRMS: [M]+ at m/z 344.2349. C22H32O3 re-
quires 344.2351.
4.9.1. 6b-Hydroxy-7-oxo-ent-atis-16-en-19-oic acid (17)
Colourless oil; 1H NMR (500 MHz, CDCl3): d 0.92 (1H, td, J = 13.3
and 3.8 Hz, H-1b), 1.08 (1H, td, J = 13.7 and 4.1 Hz, H-3b), 1.19 (3H,
s, H-20), 1.36 (1H, d, J = 12.6 Hz, H-5), 1.41 (1H, m, H-14), 1.49 (3H,
m, H-2b, H-9 and H-11), 1.51 (3H, s, H-18), 1.68 (4H, m, H-1
2H-13), 1.84 (1H, ddt, J = 13.9 and 3.6 Hz, H-2 ), 2.09 (1H, m, H-14),
2.24 (2H, m, H-3 and H-15), 2.37 (1H, br s, H-12), 2.69 (1H, dt,
J = 17.3 and 2.5 Hz, H-15), 4.11 (1H, br s, OH), 4.72 and 4.83 (each
1H, dd, J = 4.2 and 2.1 Hz, H-17), 4.95 (1H, d, J = 12.6 Hz, H-6); EIMS
m/z (rel. int.): 332 [M]+ (6), 314 (28), 286 (41), 271 (17), 258 (13),
243 (16), 231 (20), 215 (18), 189 (12), 181 (11), 153 (100); HRMS:
[M]+ at m/z 332.1992. C20H28O4 requires 332.1988.
a, H-11,
4.6.3. Hydrolysis of 19-acetoxy-7-oxo-ent-atis-16-ene (10)
Compound 10 (263 mg) in MeOH (15 ml) was treated with
Na2CO3 (315 mg) with stirring at room temperature for 2 h and
then at 40 °C for 30 min. The solvent was evaporated off and the
syrupy oil neutralized with HCl solution (3%). Extraction with
EtOAc in the usual way afforded 19-hydroxy-7-oxo-ent-atis-16-
ene (2) (215 mg).
a
a
4.7. Incubation of 19-hydroxy-7-oxo-ent-atis-16-ene (2)
4.9.2. 6b,16b,17-Trihydroxy-7-oxo-19-nor-ent-atis-4(18)-ene (19)
Colourless oil; 1H NMR (500 MHz, CDCl3): d 1.10 (3H, s, H-20),
1.15 (1H, td, J = 13.2 and 4.7 Hz, H-1b), 1.21 (1H, m, H-14), 1.38
The substrate 2 (213 mg) in EtOH (11 ml) was distributed be-
tween 54 flasks. Its microbiological transformation and usual
work-up gave acid (79 mg) and neutral fractions (617 mg). Chro-
matography of the latter afforded starting material 2 (32 mg),
13(R),19-dihydroxy-7-oxo-ent-atis-16-ene (13) (13 mg) (identical
with that obtained in the feeding of 1) and 16b,17-dihydroxy-7-
oxo-ent-atisan-19-al (16) (1 mg). Transformed products were not
obtained in the acid fraction.
(1H, ddd, J = 13.3, 7.8 and 2.2 Hz, H-11
and 3.5 Hz, H-15), 1.60 (4H, m, 2H-2 and 2H-13), 1.72 (1H, br d,
J = 13.2 Hz, H-1 ), 1.82 (1H, d, J = 14.7 Hz, H-15), 1.87 (1H, ddd,
a), 1.51 (1H, dd, J = 14.7
a
J = 10.9, 7.8 and 1.5 Hz, H-9), 1.96 (2H, m, H-3 and H-12), 2.02
(1H, d, J = 12.1 Hz, H-5), 2.08 (1H, m, H-14), 2.15 (1H, m, H-11b),
2.37 (1H, br d, J = 13.1 Hz, H-3), 3.53 and 3.61 (each 1H, d,
J = 11.0 Hz, H-17), 4.43 (1H, d, J = 12.1 Hz, H-6), 4.97 (1H, br s, H-

B.M. Fraga et al. / Phytochemistry 71 (2010) 1313–1321
1321
Cross, B.E., Galt, R.H.B., Hanson, J.R., Curtis, P.J., Grove, J.F., Morrison, A., 1963. New
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metabolites. J. Chem. Soc. 2937–2943.
Cross, B.E., Galt, R.H.B., Norton, K., 1968. The biosynthesis of gibberellins-II.
Tetrahedron 24, 231–237.
18), 5.07 (1H, dd, J = 2.8 and 1.4 Hz, H-18); EIMS m/z (rel. int.): 289
[M-CH2OH]+ (100), 271 (9), 243 (4), 215 (2), 137 (16); HRMS:
[MÀCH2OH]+ at m/z 289.1818. C18H25O3 requires 289.1804.
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., Guillermo, R., Hanson, J.R., 1992. Microbiological preparation of
atisenolides by Gibberella fujikuroi. Phytochemistry 31, 503–506.
Fraga, B.M., González, P., Hernández, M.G., Suárez, S., 2005. Biotransformation of 7-
oxo-ent-kaurene derivatives by Gibberella fujikuroi. Tetrahedron 61, 5623–5632.
Fraga, B.M., Bressa, C., González, P., Guillermo, R., Hernández, M.G., Suárez, S., 2007.
4.9.3. 3b,7a-Dihydroxy-6-oxo-ent-atis-16-en-19-oic acid (21)
A gum; 1H NMR (500 MHz, CDCl3): d 0.95 (3H, s, H-20), 1.27 (1H,
m, H-14), 1.34 (3H, s, H-18), 1.79 (1H, dddd, J = 13.5, 11.6, 2.9 and
2.9 Hz, H-11), 1.93 (1H, ddd, J = 11.6, 6.0 and 1.2 Hz, H-9), 2.18
(1H, m, H-2), 2.22 (1H, br d, J = 16.7 Hz, H-15b), 2.32 (1H, br s, H-
12), 2.74 (1H, dt, J = 16.7 and 2.2 Hz, H-15a), 3.21 (1H, d,
The microbiological transformation of 7a-hydroxy-ent-kaur-16-ene derivatives
by Gibberella fujikuroi. Phytochemistry 68, 1557–1563.
J = 1.4 Hz, H-5), 4.18 (1H, t, J = 2.5 Hz, H-3), 4.19 (1H, d, J = 1.4 Hz,
H-7), 4.68 (1H, dd, J = 3.9 and 2.1 Hz, H-17) and 4.82 (1H, dd,
J = 4.3 and 2.1 Hz, H-17); 1H NMR (500 MHz, C6D6): d 0.71 (3H, s,
H-20), 0.87 (1H, m, H-14), 1.10 (3H, s, H-18), 1.77 (1H, ddd,
J = 16.7, 5.6 and 2.6 Hz, H-15b), 2.01 (1H, m, H-12), 2.26 (1H, m,
Gustafson, K.R., Munro, M.H.G., Blunt, J.W., Cardellina II, J.H., McMahon, J.B.,
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Relationships between the kaurenolides and the seco-ring B metabolites of
Gibberela fujikuroi. J. Chem. Soc. Perkin Trans. 1, 3151–3154.
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H-2a), 2.62 (1H, dt, J = 16.7 and 2.2 Hz, H-15a), 2.82 (1H, d,
J = 1.4 Hz, H-5), 3.46 (1H, d, J = 1.4 Hz, H-7), 4.00 (1H, t, J = 2.5 Hz,
H-3), 4.67 (1H, dd, J = 3.9 and 2.1 Hz, H-17), 4.82 (1H, dd, J = 4.3
and 2.1 Hz, H-17); EIMS m/z (rel. int.): 348 [M]+ (41), 320 (9),
315 (6), 312 (6), 302 (62), 284 (23), 269 (20), 257 (12), 227 (11),
213 (14), 197 (32), 181 (23), 169 (23), 151 (85); HRMS: [M]+ at
m/z 348.1928. C20H28O5 requires 348.1937.
Kurata, S., Oshima, Y., Ueda, K., Kikuchi, H., Tachibana, Y., 2008. Diterpenes having
atisane skeleton, their manufacture, and pharmaceutical composition, cytokine
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Acknowledgements
1985.
A new atisane diterpene: ent-16a-hydroxyatis-13-en-3-one from
We thank the DGI, Ministry of Science and Technology, Spain,
for financial support (BQU2002-00765), Prof. Benjamin Rodríguez,
Instituto de Química Orgánica (CSIC, Spain), for a generous sample
of gummiferolic acid, and Prof. J.R. Hanson, School of Chemistry
(University of Sussex, UK) for his interest and comments on this
work. V.G.V. thanks the Spanish Research Council (CSIC) and the
European Social Foundation for an I3P fellowship.
Androstachys johnsonii Prain. J. Chem. Soc. Perkin Trans. I, 703–709.
Pinar, P., Rodríguez, B., Alemany, A., 1978. Gummiferolic acid, a new ent-atis-16-ene
diterpenoid from Margotia gummifera. Phytochemistry 17, 1637–1640.
Rojas, M.C., Hedden, P., Gaskin, P., Tudzynski, B., 2001. The P-450-1 gene of
Gibberella fujikuroi encodes
a
multifunctional enzyme in gibberellin
biosynthesis. Proc. Natl. Acad. Sci. USA 98, 5828–5834.
Rojas, M.C., Gaskin, P., Tudzynski, B., Hedden, P., 2004. Kaurenolides and fujenoic
acids are side products of the gibberellin P-450-1 monooxygenase in Gibberella
fujikuroi. Phytochemistry 65, 821–830.
Satti, N.K., Suri, O.P., Thaper, R.K., Kachroo, P.L., 1987. ent-Atisane-3b,16
a,17-triol, a
diterpene from Euphorbia acaulis. Phytochemistry 27, 1530–1531.
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