Microbial transformation of two 15α-hydroxy-ent-kaur-16-ene diterpenes by Mucor plumbeus

Article abstract of DOI:10.1016/j.tet.2009.10.105

The microbiological transformation of candidiol (15α,18-dihydroxy-ent-kaur-16-ene) by Mucor plumbeus led to 3β,15α,18-trihydroxy-ent-kaur-16-ene, 6α,15α,18-trihydroxy-ent-kaur-16-ene, 3α,15α,18-trihydroxy-ent-kaur-16-ene, 11β,15α,18-trihydroxy-ent-kaur-16

Full text of DOI:10.1016/j.tet.2009.10.105

Tetrahedron 66 (2010) 227–234
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Microbial transformation of two 15a-hydroxy-ent-kaur-16-ene diterpenes
by Mucor plumbeus
,
a
a
b
Braulio M. Fraga a , Ignacio de Alfonso , Victoria Gonzalez-Vallejo , Ricardo Guillermo
*
a
´
Instituto de Productos Naturales y Agrobiolog´ıa, CSIC, Avda. Astrof´ısico F. Sanchez, La Laguna, 38206 Tenerife, Canary Islands, Spain
Instituto Universitario de Bioorganica ‘Antonio Gonzalez’, Universidad de La Laguna, Tenerife, Spain
b
´
´
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 candidiol (15a,18-dihydroxy-ent-kaur-16-ene) by Mucor plumbeus led
Received 21 September 2009
Received in revised form 28 October 2009
Accepted 29 October 2009
to 3
kaur-16-ene, 11
whilst the incubation of 15
,15 ,19-trihydroxy-ent-kaur-16-ene, 11
kaur-16-ene, 15 ,17,19-trihydroxy-11 ,16
kaur-16-ene and 19-( -glucopyranosyl)-15-oxo-ent-kaur-16-ene. An interesting rearrangement in dilute
acid medium of 9 ,15 ,19-trihydroxy-ent-kaur-16-ene into 16-oxo-19-hydroxy-ent-abiet-8(9),15-diene, is
also described in this work.
b
,15
a
,18-trihydroxy-ent-kaur-16-ene, 6
,15 ,18-trihydroxy-ent-kaur-16-ene and 15
,19-dihydroxy-ent-kaur-16-ene gave 9
,15 ,19-trihydroxy-ent-kaur-16-ene,
-epoxy-ent-kaurane, 19-( -glucopyranosyl)-15
a
,15
a
,18-trihydroxy-ent-kaur-16-ene, 3
,17,18-trihydroxy-11 ,16
,15 ,19-trihydroxy-ent-kaur-16-ene,
,15 ,19-trihydroxy-ent-
-hydroxy-ent-
a
,15
a,18-trihydroxy-ent-
b
a
a
b
b-epoxy-ent-kaurane,
a
b
a
Available online 1 November 2009
3a
a
b
a
6
a
a
a
b
b
b
-D
a
Keywords:
b-D
Mucor plumbeus
Biotransformations
Diterpenes
b
a
Ó 2009 Elsevier Ltd. All rights reserved.
Candidiol
15
15
a
a
,18-Dihydroxy-ent-kaur-16-ene
,19-Dihydroxy-ent-kaur-16-ene
Glucosidation
Abiet-8(9),15-diene
Rearrangement
1. Introduction
species of this genus,8,9 whilst 15
a,19-dihydroxy-ent-kaur-16-ene
(8) was obtained by lithium aluminium hydride reduction of
grandiflorolic acid methyl ester. This alcohol 8 had been prepared
Over the last few years, we have been interested in the study of
the microbiological transformation of diterpenes by the fungus
Mucor plumbeus, which possesses a broad specificity in the sub-
strate. The aim of these studies has been to develop models to
explain the hydroxylation of these compounds by this microor-
ganism. Firstly, we carried out the biotransformation with this
fungus of labdane1 and labdane oxide derivatives, of the normal and
enantio series.2,3 Later, we incubated dehydroabietene,4 stemo-
dane5 and ent-kaur-16-ene diterpenes6 with this fungus.
In this work, we describe the results of the microbiological
transformation by M. plumbeus of two ent-kaurene derivatives,
15a,18-dihydroxy-ent-kaur-16-ene (1) and 15a,19-dihydroxy-ent-
kaur-16-ene (8), which are epimeric at C-4. In this way, we can also
determine whether a spatial change in the orientation of the
by reduction of the corresponding acid, 15a-hydroxy-ent-kaur-16-
en-19-oic acid, isolated from Espeletia grandiflora.10
2. Results and discussion
The incubation of candidiol (1) with M. plumbeus led to five
hydroxylated products 2–6 (Scheme 1). The last metabolite was
obtained as the 18-monoacetate (7a) and 17-monoacetate (7b) by
acetylation and chromatography of the fractions containing it.
The least polar metabolite 2 showed in the HRMS the molecular
ion at m/z 320.2341, in accordance with the molecular formula
C20H32O3, indicating that a new oxygen was introduced into the
molecule. This must be a part of a secondary hydroxyl group, be-
cause a new geminal hydrogen to an alcoholic group was observed
hydroxymethylene group at this carbon, from
the former to -axial in the latter, causes any effect on the
biotransformation.
b-equatorial in
3.70 (br s) in the 1H NMR spectrum. This form of resonance is
-equatorial proton at C-1, C-3 or C-12, or
a
at
characteristic of an
-equatorial at C-6. The corresponding alcohol was located at C-3
because it affected the resonances of the hydroxymethylene hy-
drogens ( 3.42 and 3.55, each doublet) in comparison with those of
the substrate 1 (
3.11 and 3.43). Moreover, in the 13C NMR spec-
trum or -gauche effects, due to the 3 -alcohol in 2, were
d
a
15a,18-Dihydroxy-ent-kaur-16-ene (candidiol) (1) was isolated
a
b
for the first time from Sideritis candicans7 and later from other
d
d
* Corresponding author. Tel.: þ34 922251728; fax: þ34 922260135.
E-mail address: bmfraga@ipna.csic.es (B.M. Fraga).
b
g
b
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.10.105

228
B.M. Fraga et al. / Tetrahedron 66 (2010) 227–234
H
H
OH
OR
HO
RO
H
H
OH
OR
2
12
4
R = H
13
11
17
20
4a R = Ac
1
4
9
14
16
8
2
3
10
H
15
5
7
OH
6
H
18
19
OH
1
RO
H
H
H
OH
OR
H
OH
OR
OH
5
R = H
5a R = Ac
3
HO
O
OR2
O
H
H
H
OR1
OH
H
H
OR3
OH
6
7
R1 = R2 = R3 = H
7a R1 = R2 = H R3 = Ac
7b
1 = R3 = H R2 = Ac
7c R1 = R2 = R3 = Ac
R
Scheme 1.
observed in the chemical shifts of C-1, C-2, C-4 and C-5,
40.5 and 42.8, in comparison with the corresponding carbons of the
substrate, 39.9, 17.9, 37.5 and 49.1, respectively. In addition, a new
methine was also observed in this spectrum at 76.4 (Table 1). This
fact was confirmed by study of the 2D NMR spectra. Thus, in the
HMBC experiment correlations of H-3 with C-1 and C-5, and of
H-18 and H-19 with C-3, were observed. Consequently, the struc-
d 33.0, 26.3,
Table 1
13C NMR data of compounds 1–5, 4a, 5a and 7a
d
Carbon
1
2
3
4
4a
37.9
5
5a
39.3
7a
d
1
2
3
4
5
6
7
8
39.9
17.9
35.2
37.5
49.1
19.1
34.8
47.6
54.2
39.3
18.0
32.8
42.3
36.3
82.9
33.0
26.3
76.4
40.5
42.8
18.8
34.6
47.5
53.9
39.1
18.1
32.8
42.3
36.3
82.9
42.4
18.1
37.2
38.4
50.3
67.6
42.9
45.5
54.2
38.9
18.1
32.7
43.0
37.6
83.6
38.4
26.9
76.5
41.9
49.6
19.3
34.9
47.4
54.0
39.0
18.1
32.7
42.2
36.2
82.8
39.4
17.9
35.1
38.1
49.0
18.9
34.3
46.9
63.9
37.6
66.6
43.0
40.6
35.7
82.2
40.7
17.6
35.5
36.7
49.8
19.3
32.1
48.4
59.5
36.3
77.0
40.4
40.9
38.0
86.2
90.1
63.5
72.8
17.8
19.3
23.0
74.2
40.5
47.3
18.6
34.2
47.1
53.4
39.0
18.3
32.8
42.4
37.1
83.0
155.3
110.2
65.2
13.2
18.2
17.6
35.4
38.4
49.3
18.9
33.8
46.4
60.1
36.5
68.7
39.6
40.7
36.6
82.2
155.3
109.4
72.5
17.6
18.9
ture of this metabolite was determined as 3b,15a,18-trihydroxy-
ent-kaur-16-ene (2).
The second of the products isolated from this feeding was 3, an
isomer of 2. In comparison with the substrate 1 a new signal at
9
d
4.46 (br s) appeared in the 1H NMR spectrum, which is typical of
10
11
12
13
14
15
16
17
18
19
20
an equatorial proton geminal to a hydroxyl group. In this spectrum
shifting of the H-19 and H-20 methyls to lower field was also ob-
served, which permitted this alcohol group to be assigned to C-6(a).
The 2D NMR experiments confirmed this and allowed the assign-
ment of the 1H and 13C NMR spectra. Thus, the structure of this
160.2
108.3
72.1
160.0
108.4
71.3
160.6
108.1
72.4
160.2
108.4
72.0
160.6
109.4
72.0
compound was determined as 6a,15a,18-trihydroxy-ent-kaur-16-
ene (3).
17.5
17.8
19.7
11.4
17.6
17.7
The structure 3a,15a,18-trihydroxy-ent-kaur-16-ene (4) was
18.1
17.8
19.6
18.0
assigned to another metabolite obtained in this incubation, on the

B.M. Fraga et al. / Tetrahedron 66 (2010) 227–234
229
following basis. The HRMS of this compound indicated that it was
The most polar compound 7 was obtained by acetylation with
Ac2O–pyridine, at room temperature, of the fractions containing it.
In this way, two monoacetates 7a and 7b, the former in better yield,
were isolated with the molecular formula C22H34O5. Later, com-
pound 7a was acetylated under reflux affording the triacetate 7c.
This product was identical with another one, which had been
prepared by acetylation of a triol, formed in a feeding of candidiol
(1) with G. fujikuroi.14 Compound 7 may be an artefact formed
during the isolation procedure from the true biotransformed me-
tabolite 6. Thus, opening of the oxirane ring and neutralization of
an isomer of 2 and 3 (C20H32O3). The geminal hydrogen to the new
hydroxyl group in C-3 resonates in the 1H NMR spectrum at
d 3.63,
as a triplet with a coupling constant of 8.2 Hz. This alcohol also
affected the resonance of the AB system of the two H-18. The car-
bon bearing the new alcohol appeared in the 13C NMR spectrum at
d
76.5. This signal showed cross-peaks with H-2, H-18 and H-19 in
the HMBC experiment. Compound 4 has been isolated from Sure-
gada multiflora, showing appreciable anti-allergic activity.11
The fourth product 5 showed the molecular ion in its HRMS in
accordance with the formula C20H32O3. The 1H NMR spectrum was
very similar to that of the substrate 1, the main difference being the
the carbocation formed at C-16, by attack of the 11b-hydroxyl
group, led to the alcohol 7.
appearance at
droxyl group. This chemical shift and form of resonance are typical of
a hydrogen geminal to an 11
-hydroxyl group.12,13 Thus, we assigned
to this metabolite the structure of 11 ,15 ,18-trihydroxy-ent-kaur-
d
3.90 (d, J¼5.4 Hz) of a geminal proton to a new hy-
The incubation of 15a,19-dihydroxy-ent-kaur-16-ene (8) with M.
plumbeus led to the isolation of the metabolites 9, 11–13 and 15–17
(Scheme 2). The structure of the least polar compound was de-
termined to be 9b,15a,19-trihydroxy-ent-kaur-16-ene (9). Its HRMS
b
b
a
16-ene (5). We had obtained this product by biotransformationof the
showed the molecular ion at m/z 320.2361 (C20H32O3). In com-
same substrate 1 by the fungus Gibberella fujikuroi.12
parison with the substrate, no new signal appeared in the 1H NMR
H
H
OR
OR
RO
H
H
OH
OR
OR
11 R = H
11a R = Ac
13 R = H
13a R = Ac
H
H
OH
OH
8
RO
H
H
OR
OR
H
H
4'
OR
2'
3'
RO
OR
OR
6'
OR
O
O
5'
1'
OH
12 R = H
12a R = Ac
16 R = H
16a R = Ac
OH
H
OH
9
HO
H
H
H
O
O
H
OH
OR
O
OR
RO
H
OR
OR
O
O
H
OH
14
OR
H
17 R = H
17a R = Ac
OR
15 R = H
15a R = Ac
Scheme 2.

230
B.M. Fraga et al. / Tetrahedron 66 (2010) 227–234
spectrum. We observed that this product rearranges into 10 in the
NMR tube when CDCl3 is used as solvent (see below); consequently,
we ran the 13C NMR spectrum and the 2D experiments in C6D6. In
Another compound isolated from this fermentation was 12,
which is also characterized by the introduction in the molecule of
a new oxygen atom. Thus, in the 1H NMR spectrum the resonance
of a new geminal hydrogen to an alcohol group was located at
the first one, a new singlet appeared at
a carbon bearing a tertiary alcohol. This was assigned to C-9 con-
sidering the observed shift of H-15 to lower field ( 4.57) and of
C-15 to higher field ( 77.6). This location was confirmed in the
HMBC experiment by a correlation of the methyl group protons of
H-20 with C-9. The -stereochemistry of the 9-OH was determined
considering the H-15 resonance, which was identical with that
d 77.1, which is typical of
d
3.89 (d, J¼5.6 Hz). This chemical shift and coupling were similar
to those observed in compound 5, indicating that the new hy-
droxyl group must be situated at C-11( ), which was confirmed in
the HMBC spectrum with cross-peaks of H-11 with C-8 ( 46.9)
and C-13 ( 40.6), and of H-9 with C-11 ( 66.6). Thus, the structure
11 ,15 ,19-trihydroxy-ent-kaur-16-ene (12) was assigned to this
substance.
Compound 13 was obtained as its diacetyl derivative 13a by
d
d
b
d
b
d
d
b
a
observed in the methyl ester of 9
b,15a-dihydroxy-ent-kaur-16-en-
19-oic acid.15,16
The molecular formula of 10, formed by rearrangement of 9,
acetylation of the fractions containing it. Its HRMS spectrum
showed the molecular ion at m/z 404.2552 (C24H36O5), which
corresponds to a triol of molecular formula C20H32O3. Conse-
quently, it is an isomer of 12. The 1H NMR spectrum of the diacetate
13a showed the signal of a new geminal proton to a secondary
was found to be C20H30O2. Its 1H NMR spectrum showed, in
comparison with that of 9, the signal of an aldehyde group at
d 9.52
(s), the downfield shift of the two H-17 at 5.99 and 6.17, due to
d
the conjugation with the oxo group of the aldehydic function, and
the disappearance of the geminal proton to the hydroxyl group at
C-15. In its 13C NMR spectrum two new carbons of a tetrasub-
hydroxyl group at
equatorial hydroxyl group, whose corresponding carbon resonates
at 66.5. This relatively low chemical shift is characteristic of
d 4.44 as a broad singlet, which is typical of an
stituted double bond appeared at
carbon of an aldehyde group resonated at
d
124.9 and 137.8, whilst the
194.8 (d). Signals of
d
d
carbons, such as C-6 or C-12, situated between a methylene and
a methine group. The existence of correlations of H-6 with C-4 and
C-8 permitted to this hydroxyl group to be assigned to the position
C-6. Therefore, the structure of the alcohol formed in the bio-
secondary carbons bearing hydroxyl groups were not displayed in
this spectrum. In the HMBC experiment correlations of H-14 with
C-8, of H-17 with C-13, C-15 and C-16, and of H-20 with C-9, were
observed. Therefore, the structure of this rearranged compound
was determined as 16-oxo-19-hydroxy-ent-abiet-8(9),15-diene
(10). The possible mechanism of formation of this 8,15-seco-ent-
kaurene diterpene is shown in Scheme 3. A compound of this
type, named hebeiabinin A (18), has been isolated from Isodon
rubescens.17
transformation was determined as 6
16-ene (13). The non-acetylation of the
a
,15
a
,19-trihydroxy-ent-kaur-
-alcohol can be
6
a
explained by the steric hindrance due to the C-20 methyl.
Another substance obtained from this feeding was 15, which
was isolated in the form of its triacetate 15a, an isomer of 7c,
indicating that two new oxygens had been introduced in the
molecule of the substrate 8. The disappearance in the 1H NMR
spectrum of 15a of the double bond resonances, the presence of
signals from a new –CH2OAc group and of a geminal proton to an
oxygen function, indicated that the C and D rings of 15a and 7c
should have a similar structure. This was confirmed in the HMBC
experiment where correlations of C-16 with H-12, H-14, H-15 and
H-17, of C-15 with H-9, H-14 and H-7, of C-11 with H-9 and H-13,
and of C-17 with H-13, were observed. Consequently, the struc-
17
13
15
CHO
16
14
8
OH
OH
H
OH
H
OH
H
ture of the corresponding alcohol was determined as 15
a,17,19-
trihydroxy-11 ,16 -epoxy-ent-kaurane (15). As in the case of 7,
b
b
9
10
this alcohol 15, corresponding to the triacetate 15a, must be an
artefact formed during the isolation procedure and, consequently,
the true biotransformed product of 8 must be the epoxide 14
(Table 2).
The most polar compounds of this feeding were two diterpene
glucosides, 16 and 17, which were separated as their peracetates 16a
and 17a, respectively, by acetylation of the fractions containing
CHO
HO
CHO
H
O
them. The first of them, with a molecular formula of C36H52O12
,
H
showed in its 1H NMR spectrum signals of five acetoxy groups. In the
13C NMR spectrum signals at 62.1 (t), 68.7 (d), 71.3 (d), 71.6 (d), 72.9
(d) and 101.2 (d) indicated the presence in the molecule of an
acetylated glucose. A correlation of H-19 with C-10 of the glucose
OH
Hebeiabinin A (18)
Scheme 3.
showed the union of C-19 with the anomeric carbon. The
figuration of this carbon was indicated by the H-10 coupling constant
-glucopyranosyl)-15
hydroxy-ent-kaur-16-ene (16)was assigned to the original metabo-
lite formed in the incubation.
The second of the acetylated glucosides (17a) showed in the
HRMS the molecular ion to m/z 632.3174, which loses a molecule of
acetylated glucose to give a peak at m/z 284. The 1H NMR spectrum
was very similar to compound 16a, but now the disappearance of
the H-15 and the downfield shift of the two H-17 were observed.
These facts indicated that the hydroxyl group at C-15 had been
oxidized to an oxo group, being now conjugated with the 16,17-
b-con-
The following product in polarity order was 11, which was
obtained as its triacetate 11a by acetylation and chromatography of
the fractions containing it. This compound possesses one more
oxygen than the substrate, considering the molecular formula of its
triacetate, C26H38O6. In the 1H and 13C NMR spectra of 11a new
signals at 4.53 (dd, J¼10.0 and 6.7 Hz) and
were observed. These resonances correspond to a geminal hydro-
gen to an -equatorial acetoxy group, which was located at C-3,
(J¼7.9 Hz). Therefore, the structure 19-(
b-D
a-
d
d 80.1, respectively,
a
taking into consideration its correlations with C-18 and C-19 in the
HMBC spectrum. Therefore, the structure of the original alcohol,
formed in the incubation, was determined as 3a,15a,19-trihydroxy-
double bond. The carbon of this new oxo group appeared at
d 210.5.
ent-kaur-16-ene (11).
Thus, the structure of the original compound formed in the

B.M. Fraga et al. / Tetrahedron 66 (2010) 227–234
231
Table 2
13C NMR data of compounds 8–10, 11a–13a, 12 and 15a–17a
Carbon
8
8c
9c
10
11a
38.4
12
40.0
12a
39.8
13a
43.0
15a
16a
40.2
17a
39.6
1
2
3
4
5
6
7
8
40.4
18.0a
35.5b
38.6
56.7
19.6
35.6b
47.6
54.4
39.4
18.2a
32.6
42.3
36.2
82.8
40.6
32.4
37.1
41.0
18.0
36.0
36.9a
56.3
19.3
32.7
48.6
59.2
36.7a
77.4
40.1
41.7
38.6
84.7
88.6
63.4
27.8
67.0
19.4
18.3a
35.9b
38.8
56.7
19.9
35.4b
47.9
54.6
39.5
18.7a
33.0
42.8
36.4
82.8
161.0
107.8
27.3
65.2
18.3
18.6
35.5
38.8
49.0
19.9
30.9
52.4
77.1
44.4
29.1
34.4
41.4
36.7
77.6
160.0
108.0
27.4
65.4
19.9
18.7a
35.5
23.6
80.1
41.1
55.4
20.1
35.0
47.2
53.6
39.0
18.4
32.7
42.4
36.9
82.9
155.2
110.2
22.8
65.3
17.5
18.2
35.1
37.9
56.5
19.4
35.4a
46.9
64.0
38.7
66.6
43.0
40.6
35.5a
82.1
160.6
109.4
27.0
65.6
17.8
18.0
36.0
37.0
56.0
19.2
36.5
46.5
60.2
38.3
68.6
39.5
40.7
34.5
82.2
155.2
109.4
27.5
67.0
18.0
18.7a
39.3
39.6
57.5
66.5
41.8
45.8
54.5
38.2
18.8a
33.1
43.5
38.9
84.4
155.3
110.2
27.9
69.4
19.6
18.2
36.2
37.7
56.1
19.6
35.1
47.3
54.0
39.4
18.2
32.8
42.5
37.2
83.1
155.4
110.1
27.5
73.2
18.1
101.2
71.3
72.9
68.7
71.6
62.1
18.0a
36.1
37.7
55.7
18.9
36.6
52.4
57.7
40.0
18.2a
32.4
38.1
33.9
210.5
149.4
114.5
27.5
73.0
18.0
101.2
71.4
72.9
68.8
71.7
62.1
38.7
52.7
19.0a
32.9
124.9
137.8
37.5
21.1
26.8
30.8
35.3
153.9
194.8
133.4
26.7
9
10
11
12
13
14
15
16
17
18
19
20
10
160.2
108.3
27.0
65.5
18.2
65.5
20.4
20
30
40
50
60
a
These values can be interchanged.
These values can be interchanged.
Solvent C6D6.
b
c
6. The rearrangement in dilute acid medium of 9
b,15a,19-tri-
biotransformation was determined as 19-(b-D-glucopyranosyl)-15-
hydroxy-ent-kaur-16-ene (9) to form the aldehyde 10, which
possesses an abietane skeleton, is now described for the first
time. This is an interesting reaction, because it permits the
preparation of abiet-8-ene derivatives functionalized at the iso-
propyl group.
oxo-ent-kaur-16-ene (17), which was confirmed by a study of the
2D NMR spectra. Diterpene glucosides derived from ent-kaurenoic
acid have been shown to possess trypanocidal activity.18
Several conclusions can be obtained from the results of these
biotransformations:
1. Hydroxylations at C-3(a), C-6(a) or C-11(b) occur in both bio-
3. Experimental
transformations. This means that a change in the spatial ori-
entation of the hydroxymethylene group at C-4, from equatorial
in 1 to axial in 8, does not affect the way in which these ent-
kaurenes bind to the oxidative enzymes. The preference for an
equatorial hydroxylation at C-3 by this fungus had been ob-
served with other diterpenic substrates.4,5,19
2. Epoxidation of the exocyclic double bond also occurred in both
fermentations. This type of reaction had also been observed in
the incubation of other diterpenes of the enantio and normal
series with this fungus.2,3,6
3.1. General experimental details
Mps were determined with a Reichert Thermovar apparatus and
are uncorrected. IR spectra were taken in a Thermo Nicolet Avatar
360 FT-IR spectrometer. 1H NMR and 13C NMR spectra were recor-
ded in CDCl3 soln at 500.13 and 125.77 MHz, respectively, with
a Bruker AMX2-500 spectrometer. Chemical shifts are given in
parts per million (d). Mass spectra were taken at 70 eV (probe) in
a Micromass Autospec spectrometer. Semipreparative HPLC was
carried out with a Beckman System Gold 125P. Dry column chro-
matographies were made on silica gel Merck 0.015–0.04 mm. The
substances were crystallised from petrol–EtOAc except where
otherwise indicated.
3. The 3b-axial hydroxylation, together with the 3a named above,
was also produced in the incubation of 1, which indicates a loss
of stereoselectivity at C-3 in this feeding. This fact had also been
observed at positions such as C-2, C-3, C-6, C-7 and C-11 in
other diterpenic biotransformations carried out with this
microorganism.
4. A C-9(
This reaction had also been observed in the feeding of candi-
candiol (7
,18-dihydroxy-ent-kaur-16-ene) with this fungus.6
Consequently, a 15 ,19-diol is equivalent to a 7 ,18-diol in the
b
) hydroxylation was produced in the incubation of 8.
3.2. Organism
a
The fungal strain, M. plumbeus CMI 116688, was a gift from Prof.
J.R. Hanson, Department of Chemistry, University of Sussex, UK.
a
a
molecular recognition of the substrate by the enzyme involved
in this reaction.
3.3. Substrates
5. Glucosides were formed in the feeding of 8 (
a-axial CH2OH),
but not in that of 1 ( -equatorial CH2OH), this being the first
b
3.3.1. 15
(500 MHz)
H-19), 1.07 (3H, s, H-20), 1.07 (1H, d, J¼7.9 Hz, H-9), 1.13 (1H, dd,
), 1.92
(1H, dd, J¼11.5 and 1.6 Hz, H-14), 2.74 (1H, br s, H-13), 3.11 and 3.43
(each 1H, d, J¼10.8 Hz, H-18), 3.81 (1H, s, H-15), 5.07 and 5.20 (each
a
,18-Dihydroxy-ent-kaur-16-ene (candidiol) (1). 1H NMR
0.76 (1H, td, J¼12.8 and 4.0 Hz, H-1 ), 0.77 (3H, s,
time that ent-kaurene derivatives of this type are formed in
a biotransformation by a Mucor species. Other glucosyl de-
rivatives had been obtained in the feeding of the mycotoxin
zearalenone20 and the steroid resibufogenin21 with other spe-
cies of Mucor.
d
b
J¼12.1 and 1.7 Hz, H-5), 1.82 (1H, dt, J¼12.8 and 3.3 Hz, H-1
a

232
B.M. Fraga et al. / Tetrahedron 66 (2010) 227–234
1H, s, H-17); EIMS m/z (rel int.) 304 [M]þ (14), 289 (17), 286 (11),
273 (100), 255 (54), 246 (40), 229 (8), 221 (9), 203 (9), 189 (10), 173
(18), 159 (15), 147 (22), 135 (24), 123 (42); HREIMS m/z 304.2354
(calcd for C20H32O2, 304.2402).
[M]þ (11), 305 (6), 302 (26), 289 (31), 287 (17), 272 (23), 271
(58), 269 (34), 257 (18), 253 (16), 201 (10), 187 (10), 175 (11), 159
(16), 151 (23), 145 (22); HREIMS m/z 320.2365 (calcd for
C20H32O3, 320.2351).
3.3.2. 15
obtained by lithium aluminium hydride reduction of grandiflorolic
acid methyl ester. 1H NMR (500 MHz)
0.77 (1H, td, J¼13.0 and
3.9 Hz, H-1 ), 0.93 (2H, m, H-3 and H-5), 0.96 (3H, s, H-18), 1.00
(3H, s, H-20), 1.02 (1H, d, J¼8.5 Hz, H-9), 1.33 (1H, m, H-14), 1.40
(1H, m, H-6), 1.45 (1H, m, H-12), 1.56 (1H, m, H-11), 1.63 (1H, ddd,
J¼12.7, 5.7 and 2.2 Hz, H-12), 1.72 (1H, m, H-6), 1.77 (1H, br d,
a
,19-Dihydroxy-ent-kaur-16-ene (8). This substrate was
3.5.3. 3
NMR (500 MHz)
H-19 and H-5, H-1
a
,15
a
,18-Trihydroxy-ent-kaur-16-ene (4). Mp 174–176 ꢀC; 1H
0.88 (3H, s, H-19), 0.89 (1H, m overlapped with
d
d
b
), 0.92 (1H, d, J¼10.8 Hz, H-5), 1.01 (1H, d,
b
b
J¼8.4 Hz, H-9), 1.07 (3H, s, H-20), 1.37 (1H, m, H-14), 1.56 (1H, dd,
J¼14.9 and 5.7 Hz, H-11), 1.64 (2H, m, H-2 and H-12),1.70 (1H, m, H-
7), 1.86 (1H, dt, J¼13.2 and 3.4 Hz, H-1 ), 1.89 (1H, d, J¼11.8 Hz, H-
a
14), 2.74 (1H, br s, H-13), 3.41 and 3.71 (each 1H, d, J¼10.3 Hz, H-
18), 3.63 (1H, t, J¼8.2 Hz, H-3), 3.79 (1H, s, H-15), 5.07 and 5.20
(each 1H, s, H-17); EIMS m/z (rel int.) 320 [M]þ (5), 305 (7), 302 (23),
287 (31), 284 (18), 272 (14), 271 (42), 269 (16), 253 (13), 242 (26),
229 (13), 185 (19), 145 (30). Triacetate (4a) HREIMS m/z 446.2674
J¼13.6 Hz, H-3
a
), 1.84 (1H, br d, J¼12.8 Hz, H-1
a), 1.86 (1H, dd,
J¼12.0 and 1.4 Hz, H-14), 2.72 (1H, br s, H-13), 3.44 and 3.73 (each
1H, d, J¼11.0 Hz, H-19), 3.79 (1H, s, H-15), 5.06 and 5.19 (each 1H, s,
H-17); 1H NMR (500 MHz, C6D6)
d
0.59 (1H, td, J¼13.1 and 4.0 Hz,
H-1
(3H, s, H-20), 1.73 (1H, dd, J¼11.8 and 1.7 Hz, H-14), 1.77 (1H, dt,
J¼13.5 and 2.3 Hz, H-1 ), 1.85 (1H, dtd, J¼13.5, 3.4 and 1.6 Hz, H-
b), 0.77 (1H, dd, J¼12.3 and 1.8 Hz, H-5), 0.84 (3H, s, H-18), 0.94
(calcd for C26H38O6, 446.2668); 1H NMR (500 MHz)
19), 1.00 (1H, td, J¼13.3 and 3.5 Hz, H-1
(1H, dt, J¼13.3 and 3.6 Hz, H-1
d
0.82 (3H, s, H-
), 1.09 (3H, s, H-20), 1.86
), 1.93 (1H, d, J¼11.7 Hz, H-14), 2.80
b
a
a
3
a
), 2.60 (1H, br s, H-13), 3.23 (1H, dd, J¼10.8 and 1.2 Hz, H-19),
(1H, br s, H-13), 3.71 and 3.80 (each 1H, d, J¼12.0 Hz, H-18), 4.76
(1H, dd, J¼11.9 and 5 Hz, H-3), 5.09 and 5.13 (each 1H, s, H-17), 5.26
(1H, s, H-15); EIMS m/z (rel int.) 446 [M]þ (2), 404 (4), 386 (72), 371
(20), 344 (31), 326 (100), 311 (58), 284 (60), 269 (30), 266 (57), 253
(46), 251 (56), 246 (30); HREIMS m/z 320.2347 (calcd for C20H32O3,
320.2351).
3.53 (1H, d, J¼10.8 Hz, H-19), 3.73 (1H, br s, H-15), 4.98 and 5.16
(each 1H, br s, H-17); EIMS m/z (rel int.) 304 [M]þ (24), 289 (29), 286
(12), 274 (22), 273 (68), 271 (16), 256 (21), 255 (94), 246 (33), 229
(8), 215 (8), 203 (8); HREIMS m/z 304.2410 (calcd for C20H32O2,
304.2402).
3.4. Fermentation and isolation conditions
3.5.4. 11
(500 MHz)
J¼13.1 and 3.6 Hz, H-1
(1H, br s, H-9), 1.45 (1H, m, H-14), 1.65 (1H, qt, J¼13.8 and 3.6 Hz,
H-2
), 1.79 (1H, dt, J¼12.8 and 2.9 Hz, H-7), 1.84 (1H, dd, J¼11.7
and 1.8 Hz, H-14), 1.92 (2H, m, H-1 and H-12 ), 2.01 (1H, ddd,
J¼14.7, 5.7 and 2.9 Hz, H-12 ), 2.78 (1H, br s, H-13), 3.14 and 3.44
b
,15
d
a
,18-Trihydroxy-ent-kaur-16-ene
0.77 (3H, s, H-19), 0.96 (3H, s, H-20), 1.04 (1H, td,
), 1.22 (1H, dd, J¼12.0 and 1.1 Hz, H-5), 1.41
(5). 1H
NMR
M. plumbeus was grown in shake culture at 25 ꢀC for two days in
20–22 conical flasks (250 ml), each containing sterile medium6
(50 ml). The substrate (see below) in EtOH (4–4.5 ml) was distrib-
uted equally between the flasks and the incubation allowed to
continue for a further 6 days. The mycelium was filtered and the
broth extracted with EtOAc in the usual way. The solvent was
evaporated to give a residue, which was chromatographed on
a silica gel column using a petroleum ether–EtOAc gradient. Frac-
tions collected were purified further by HPLC when necessary.
b
a
a
a
b
(each 1H, d, J¼10.8 Hz, H-18), 3.90 (1H, d, J¼5.4 Hz, H-11), 4.28
(1H, s, H-15), 5.24 and 5.27 (each 1H, s, H-17); EIMS m/z (rel int.)
320 [M]þ (12), 302 (23), 289 (13), 287 (13), 272 (26), 271 (62), 269
(13), 253 (21), 239 (12), 215 (12), 213 (10), 189 (12), 177 (13), 167
(15), 159 (14), 149 (53), 145 (21); HREIMS m/z 320.2361 (calcd for
3.5. Incubation of 15
a
,18-dihydroxy-ent-kaur-16-ene (1)
C20H32O3, 320.2351). Triacetate (5a) 1H NMR (500 MHz)
), 1.02 (3H, s, H-
d 0.82
(3H, s, H-19), 0.92 (1H, td, J¼13.0 and 3.6 Hz, H-1
b
The biotransformation of 1 (253 mg) gave 3
ent-kaur-16-ene (2) (14 mg), 6 ,15 ,18-trihydroxy-ent-kaur-16-ene
(3) (2 mg), ,15 ,18-trihydroxy-ent-kaur-16-ene (4) (20 mg),
11 ,15 ,18-trihydroxy-ent-kaur-16-ene (5) (5 mg) and 15 ,17,18-
trihydroxy-11 ,16 -epoxy-ent-kaurane (7), which was obtained as
b
,15
a
,18-trihydroxy-
20), 1.16 (1H, d, J¼11.4 Hz, H-5), 1.49 (1H, br s, H-9), 1.50 (2H, m
a
a
overlapped with H-9, H-2 and H-14), 1.61 (1H, m, H-2), 1.68 (1H,
3a
a
dd, J¼10.4 and 3.2 Hz, H-7
a), 1.95, 2.07 and 2.08 (each 3H, s), 2.77
b
a
a
(1H, br s, H-13), 3.62 and 3.85 (each 1H, d, J¼11.0 Hz, H-18), 5.04
and 5.09 (each 1H, s, H-17), 5.08 (1H, m, H-11), 5.64 (1H, s, H-15);
EIMS m/z (rel int.) 446 [M]þ (0.4), 404 (5), 386 (48), 371 (8), 344
(56), 326 (81), 313 (37), 311 (100), 284 (36), 269 (19), 266 (31), 253
(80), 251 (45), 211 (13); HREIMS m/z 446.2675 (calcd for C26H38O6,
446.2668).
b
b
its 18-acetate (7a) (1.6 mg) and its 17-acetate (7b) (0.5 mg) by
acetylation and chromatography of the fractions containing it.
3.5.1. 3
NMR (500 MHz)
J¼8.3 Hz, H-9), 1.26 (1H, td, J¼13.6 and 3.6 Hz, H-1
b
,15
a
,18-Trihydroxy-ent-kaur-16-ene (2). Mp 210–212 ꢀC; 1H
0.71 (3H, s, H-19), 1.07 (3H, s, H-20), 1.18 (1H, d,
), 1.39 (1H, dd,
d
b
3.5.5. 18-Acetoxy-15
a
,17-dihydroxy-11
b
,16
b
-epoxy-ent-kaurane
J¼11.8 and 5.9 Hz, H-14), 1.50 (1H, m, H-2), 1.70 (2H, m, H-5 and H-
7), 1.91 (1H, d, J¼11.8 Hz, H-14), 1.96 (1H, m, H-2), 2.75 (1H, br s, H-
13), 3.42 and 3.55 (each 1H, d, J¼11.4 Hz, H-18), 3.70 (1H, br s, H-3),
3.84 (1H, s, H-15), 5.08 and 5.22 (each 1H, s, H-17); EIMS m/z (rel
int.) 320 [M]þ (20), 302 (36), 287 (31), 272 (36), 271 (66), 269 (30),
253 (26), 229 (16), 213 (16), 185 (18),159 (27),145 (37); HREIMS m/z
320.2341 (calcd for C20H32O3, 320.2351).
(7a). IR (CHCl3) nmax 3500, 1730, 1241 cmꢁ1
;
1H NMR (500 MHz)
d
0.87 (3H, s, H-19), 1.17 (3H, s, H-20), 1.50 (1H, d, J¼3 Hz, H-9), 1.90
(1H, d, J¼3.1 Hz, H-14), 2.38 (1H, br s, H-13), 3.62 and 3.87 (each 1H,
d, J¼11.0 Hz, H-18), 3.68 (1H, s, H-15), 3.81 and 3.92 (each 1H, d,
J¼11.7 Hz, H-17), 4.36 (1H, t, J¼3.3 Hz, H-11); EIMS m/z (rel int.) 378
[M]þ (3), 360 (2), 347 (17), 332 (26), 329 (13), 318 (19), 305 (27), 301
(32), 287 (37), 272 (42), 269 (36), 257 (34), 229 (42); HREIMS m/z
378.2388 (calcd for C22H34O5, 378.2406).
3.5.2. 6
1H NMR (500 MHz)
(1H, d, J¼8.0 Hz, H-9), 1.16 (3H, s, H-19), 1.20 (1H, m, H-3), 1.42
(3H, s, H-20), 1.51 (1H, m, H-14), 1.78 (2H, m, H-1 and H-7),
a,15
a
,18-Trihydroxy-ent-kaur-16-ene (3). Mp 246–248 ꢀC;
0.76 (1H, td, J¼12.8 and 3.5 Hz, H-1 ), 1.14
d
b
3.5.6. 17-Acetoxy-15
a
,18-dihydroxy-11
b,16b-epoxy-ent-kaurane
(7b). 1H NMR (500 MHz)
d
0.86 (3H, s, H-19), 1.16 (3H, s, H-20), 2.29
a
(1H, t, J¼6.3 Hz, H-13), 3.10 and 3.39 (each 1H, d, J¼11.2 Hz, H-18),
3.49 (1H, s, H-15), 4.01 and 4.74 (each 1H, d, J¼11.8 Hz, H-17), 4.40
(1H, m, H-11); EIMS m/z (rel int.) 378 [M]þ (3), 360 (2), 347 (17), 332
(26), 329 (13), 318 (19), 305 (27), 301 (32), 287 (37), 272 (42), 269
2.28 (1H, d, J¼11.5 Hz, H-14), 2.77 (1H, br s, H-13), 3.21 and 3.59
(each 1H, d, J¼10.8 Hz, H-18), 3.84 (1H, s, H-15), 4.46 (1H, br s,
H-6), 5.09 and 5.22 (each 1H, s, H-17); EIMS m/z (rel int.) 320

B.M. Fraga et al. / Tetrahedron 66 (2010) 227–234
233
(36), 257 (34), 229 (42); HREIMS m/z 378.2403 (calcd for C22H34O5,
378.2406).
5.03 and 5.10 (each 1H, s, H-17), 5.07 (1H, d, J¼4.2 Hz, H-11), 5.64
(1H, s, H-15); EIMS m/z (rel int.) 446 [M]þ (0.9), 404 (5), 386 (29), 344
(30), 326 (36), 313 (26), 311 (25), 284 (12), 271 (11), 266 (13), 253
(46), 251 (14); HREIMS m/z 446.2658 (calcd for C26H38O6, 446.2668).
3.6. Incubation of 15a,19-dihydroxy-ent-kaur-16-ene (8)
The biotransformation of 8 (338 mg) gave starting material
(102 mg), 9 ,15 ,19-trihydroxy-ent-kaur-16-ene (9) (5 mg), 3 ,15 ,19-
trihydroxy-ent-kaur-16-ene (11) (2 mg), 11 ,15 ,19-trihydroxy-ent-
kaur-16-ene (12) (2 mg), ,15 ,19-trihydroxy-ent-kaur-16-ene
(13) (5 mg), 15 ,17,19-trihydroxy-11 ,16 -epoxy-ent-kaurane (15)
(3 mg),19-( -glucopyranosyl)-15 -hydroxy-ent-kaur-16-ene (16)
(10 mg), 19-( -glucopyranosyl)-15-oxo-ent-kaur-16-ene (17)
(1 mg). Compound 9 rearranges into aldehyde 10 in the NMR tube.
Compounds 11, 13 and 15–17 were characterized as their acetates
11a, 13a and 15a–17a, by acetylation and chromatography of the
fractions containing them.
3.6.5. 15
164 ꢀC; 1H NMR (500 MHz)
0.99 (3H, s, H-18),1.05 (1H, s, H-5),1.09 (1H, td, J¼13.7 and 4.4 Hz, H-
), 1.30 (1H, d, J¼7.6 Hz, H-9), 1.39 (3H, s, H-20), 1.44 (1H, m, H-2),
1.56 (1H, dd, J¼14.4 and 3.4 Hz, H-7), 1.61 (1H, dd, J¼6.7 and 2.0 Hz,
H-14),1.68 (1H, m, H-3
),1.74 (1H, m, H-12),1.79 (1H, dd, J¼14.4 and
3.4 Hz, H-7),1.83 (1H, br d, H-1
), 2.34 (1H, dd, J¼12.0 and 1.5 Hz, H-
a
,19-Diacetoxy-6
a
-hydroxy-ent-kaur-16-ene(13a). Mp 162–
b
a
a
a
d
0.82 (1H, td, J¼13.0 and 3.9 Hz, H-1 ),
b
b
a
6a
a
3b
a
b
b
b
-
D
a
a
b
-D
a
14), 2.81 (1H, br s, H-13), 4.37 and 4.49 (each 1H, d, J¼11.6 Hz, H-19),
4.44 (1H, brs, H-6), 5.11 and5.12 (each1H, s, H-17), 5.30 (1H, s, H-15);
EIMS m/z (rel int.) 404 [M]þ (7), 386 (5), 362 (9), 344 (25), 326 (12),
311 (9), 302 (6), 284 (35), 269 (19), 253 (16), 251 (22); HREIMS m/z
404.2552 (calcd for C24H36O5, 404.2563).
3.6.1. 9
1.01 (3H, s, H-18) 1.14 (3H, s, H-20), 2.74 (1H, br s, H-13), 3.49 and
3.76 (each 1H, d, J¼10.7 Hz, H-19), 4.55 (1H, br s, H-15), 5.10 and
5.22 (each 1H, s, H-17); 1H NMR (C6D6, 500 MHz)
0.89 (3H, s, H-
b,15a
,19-Trihydroxy-ent-kaur-16-ene (9). 1H NMR (400 MHz)
d
3.6.6. 15
(CHCl3) nmax 3430,1733,1248 cmꢁ1; 1H NMR (500 MHz)
H-18), 0.98 (1H, d, J¼11.9 Hz, H-5), 1.03 (1H, m, H-3 ), 1.12 (3H, s, H-
20), 1.13 (1H, td, J¼12.7 and 3.7 Hz, H-1 ),1.21 (1H, m, H-6), 1.43 (1H,
m, H-2), 1.49 (2H, m, H-7), 1.63 (1H, m, H-6), 1.66 (1H, br s, H-9), 1.72
(1H, br d, J¼13.4 Hz, H-3 ), 1.80 (2H, m, H-1 and H-14), 1.88 (1H, m,
a
,17,19-Triacetoxy-11
b
,16
b
-epoxy-ent-kaurane (15a). IR
d
0.94 (3H, s,
d
b
20), 0.92 (3H, s, H-18), 1.01 (1H, ddd, J¼14.6, 13.3 and 7.8 Hz, H-11),
b
1.41 (2H, m, H-5 and H-14), 1.59 (1H, m, H-6), 1.67 (1H, dd, J¼14.6
and 2.3 Hz, H-11), 1.68 (1H, m, H-6), 1.80 (1H, br d, J¼13.5 Hz, H-3
a
),
a
a
1.84 (1H, dd, J¼12.0 and 1.5 Hz, H-14), 2.53 (1H, br s, H-13), 3.22 and
3.51 (each 1H, d, J¼10.7 Hz, H-19), 4.57 (1H, s, H-15), 4.97 and 5.14
(each 1H, s, H-17); EIMS m/z (rel int.) 320 [M]þ (2), 302 (4), 289 (42),
271 (38), 253 (17),161 (40), 149 (22), 123 (100), 109 (51), 95 (47);
HREIMS m/z 320.2361 (calcd for C20H32O3, 320.2351).
H-12),1.94 (1H, dd, J¼12.0 and 1.3 Hz, H-14), 2.15 (1H, d, J¼11.5 Hz, H-
12), 2.39 (1H, t, J¼6.4 Hz, H-13), 3.87 and 4.24 (each 1H, d, J¼11.0 Hz,
H-19), 3.99and4.53(each1H, d,J¼11.9 Hz, H-17), 4.42(1H, brs,H-11),
4.99 (1H, d, J¼1.3 Hz, H-15); EIMS m/z (rel int.) 462 [M]þ (7), 444 (6),
420 (11), 402 (14), 384 (12), 360 (18), 342 (70), 329 (30), 300 (9), 287
(12), 282 (20), 269 (34); HREIMS m/z 462.2631 (calcd for C26H38O7,
462.2618).
3.6.2. 16-Oxo-19-hydroxy-ent-abiet-8(9),15-diene (10). 1H NMR
(500 MHz)
d
0.96 (3H, s, H-20), 1.00 (3H, s, H-18), 1.30 (1H, dd, J¼9.0
and 7.3 Hz, H-5), 1.68 (1H, m, H-12), 1.94 (2H, m, H-7 and H-11), 2.08
(1H, dd, J¼17.0 and 6.0 Hz, H-14), 2.82 (1H, br s, H-13), 3.48 and 3.79
(each 1H, d, J¼10.9 Hz, H-19), 5.99and6.17 (each1H, s, H-17), 9.52 (1H,
s, H-16); EIMS m/z (rel int.) 302 [M]þ (48), 287 (36), 284 (14), 272 (27),
271 (100), 269(25), 254 (9), 253 (30), 251(10), 239 (9), 211 (7), 201(13),
189 (12); HREIMS m/z 302.2249 (calcd for C20H30O2, 302.2246).
3.6.7. 19-(20,30,40,60-Tetra-O-acetyl-
toxy-ent-kaur-16-ene (16a). 1H NMR (500 MHz)
), 0.89 (3H, s, H-18), 0.92 (2H, m, H-3b
b
-
D
-glucopyranosyl)-15
0.78 (1H, td,
and
a-ace-
d
J¼13.2 and 3.7 Hz, H-1
b
H-5), 1.00 (3H, s, H-20), 1.20 (1H, d, J¼7.2 Hz, H-9), 1.26 (2H, m, H-6
and H-7), 1.39 (1H, dd, J¼11.0 and 4.4 Hz, H-14), 1.52 (2H, m, H-2
and H-12), 1.59 (1H, m, H-7), 1.64 (1H, br d, J¼11.9 Hz, H-12), 1.72
(1H, br d, J¼13.3 Hz, H-3
a
),1.83 (1H, br d, J¼13.2 Hz, H-1
a),1.91 (1H,
3.6.3. 3
1H NMR (500 MHz)
1.04 (1H, m, H-1
a,15
a
,19-Triacetoxy-ent-kaur-16-ene (11a). Mp 143–145 ꢀC;
1.00 (1H, d, J¼12.8 Hz, H-5), 1.00 (3H, s, H-18),
dd, J¼11.8 and 1.1 Hz, H-14), 2.78 (1H, br s, H-13), 3.20 and 3.93
(each 1H, d, J¼9.3 Hz, H-19), 3.67 (1H, ddd, J¼10.0, 4.9 and 2.6 Hz,
H-50), 4.13 (1H, dd, J¼12.2 and 2.6 Hz, H-60), 4.27 (1H, dd, J¼12.2
and 5.0 Hz, H-60), 4.41 (1H, d, J¼7.9 Hz, H-10), 4.99 (1H, dd, J¼9.7
and 7.9 Hz, H-20), 5.07 (1H, t, J¼9.6 Hz, H-40), 5.08 and 5.09 (each
1H, s, H-17), 5.19 (1H, t, J¼9.6 Hz, H-30), 5.25 (1H, s, H-15); EIMS m/z
(rel int.) 676 [M]þ (0.5), 661 (0.4), 643 (0.4), 616 (0.8), 556 (0.4), 331
(52), 328 (7), 269 (14), 255 (9), 169 (100), 109 (36), 91 (7); HREIMS
m/z 676.3478 (calcd for C36H52O12, 676.3459).
d
b), 1.06 (3H, s, H-20), 1.20 (1H, d, J¼6.7 Hz, H-9),
1.27 (1H, m, H-7), 1.44 (2H, m, H-6 and H-14), 1.50 (1H, m, H-12),
1.58 (1H, m, H-11), 1.69 (3H, m, H-6, H-7 and H-12), 1.91 (2H, m, H-
1
a and H-14), 2.80 (1H, br s, H-13), 4.13 and 4.33 (each 1H, d,
J¼11.7 Hz, H-19), 4.53 (1H, dd, J¼10.0 and 6.7 Hz, H-3) 5.10 and 5.11
(each 1H, s, H-17), 5.25 (1H, s, H-15); EIMS m/z (rel int.) 446 [M]þ
(8), 404 (17), 386 (71), 371 (24), 344 (31), 329 (16), 326 (67), 311
(43), 284 (45), 269 (26), 266 (74), 253 (46), 251 (74), 211 (18), 209
(27); HREIMS m/z 446.2675 (calcd for C26H38O6, 446.2668).
3.6.8. 19-(20,30,40,60-Tetra-O-acetyl-
kaur-16-ene (17a). Mp 128–130 ꢀC; 1H NMR (500 MHz)
td, J¼13.3 and 4.0 Hz, H-1 ), 0.92 (3H, s, H-18), 0.93 (1H, m, H-3
b
-
D
-glucopyranosyl)-15-oxo-ent-
0.79 (1H,
),
d
3.6.4. 11
1H NMR (500 MHz)
H-18),1.08 (1H, m, H-1
1.80 (each 1H, m, H-14),1.89 (1H, m, H-12),1.96 (1H, br d, J¼12.5 Hz,
H-1
b
,15
a
,19-Trihydroxy-ent-kaur-16-ene (12). Mp 229–231 ꢀC;
0.91 (3H, s, H-20), 0.98 (1H, m, H-5),1.00 (3H, s,
),1.38 (1H, s, H-9),1.44 (1H, m, H-2),1.49 and
b
b
d
1.07 (3H, s, H-20), 2.36 (1H, d, J¼11.6 Hz, H-14), 3.04 (1H, br s, H-13),
3.19 and 3.96 (each 1H, d, J¼9.4 Hz, H-19), 3.68 (1H, ddd, J¼10.0, 4.9
and 2.6 Hz, H-50), 4.14 (1H, dd, J¼12.3 and 2.8 Hz, H-60), 4.27 (1H,
dd, J¼12.3 and 4.9 Hz, H-60), 4.42 (1H, d, J¼7.9 Hz, H-10), 5.00 (1H,
dd, J¼9.6 and 7.9 Hz, H-20), 5.08 (1H, t, J¼9.7 Hz, H-40), 5.20 (1H, t,
J¼9.5 Hz, H-30), 5.25 and 5.94 (each 1H, s, H-17); EIMS m/z (rel int.)
632 [M]þ (15), 331 (43), 284 (26), 271 (31), 211 (3), 169 (100), 127
(12), 109 (40), 91 (31); HREIMS m/z 632.3174 (calcd for C34H48O11,
632.3197).
b
a
), 2.01 (1H, ddd, J¼14.7, 5.7 and 2.9 Hz, H-12), 2.78 (1H, br s, H-
13), 3.47 and 3.72 (each 1H, d, J¼10.9 Hz, H-19), 3.89 (1H, d, J¼5.6 Hz,
H-11), 4.27 (1H, s, H-15), 5.24 and 5.27 (each 1H, s, H-17); EIMS m/z
(rel int.) 320 [M]þ (42), 302 (18), 289 (19), 287 (8), 275 (25), 271 (64),
259 (14), 253 (12), 246 (30), 229 (7), 215 (12), 213 (9), 187 (11);
HREIMS m/z 320.2353 (calcd for C20H32O3, 320.2351). Triacetate
(12a)1H NMR (500 MHz)
(3H, s, H-20),1.02 (1H, m, H-3
and H-14),1.46 (2H, m, H-2 and H-7),1.48 (1H, s, H-9),1.71 (3H, m, H-
3, H-6 and H-14), 1.90 (3H, m, H-1 , H-7 and H-12), 1.99 (1H, m, H-
12), 2.78 (1H, br s, H-13), 3.88 and 4.17 (each 1H, d, J¼11.1 Hz, H-19),
d
0.95 (3H, s, H-18), 0.95 (1H, m, H-1
b), 0.99
b
),1.04 (1H, br s, H-5),1.33 (2H, m, H-6
Acknowledgements
a
This work has been supported by the SGPCT, Ministry of Edu-
cation and Culture, Spain (BQU2002-765). V.G.V. thanks the

234
B.M. Fraga et al. / Tetrahedron 66 (2010) 227–234
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