Biotransformation of the diterpenes epicandicandiol and candicandiol by Mucor plumbeus

Article abstract of DOI:10.1021/np020420x

The microbiological transformation of the diterpene epicandicandiol (1) with Mucor plumbeus gave foliol (3), sideritriol (5), and 7β,16α,17,18-tetrahydroxy-ent-kaurane (7), while the incubation of candicandiol (2) gave 7α,9β,18-trihydroxy-ent-kaur-16-ene (10), canditriol (11), and 7α,16α,17,18-tetrahydroxy-entkaurane (12). Thus, the main difference observed in both feedings, resulting from the spatial change in the orientation of the 7-hydroxyl, from axial in the substrate 1 to equatorial in 2, was the formation of a 3α- and a 9β-hydroxylated derivative, respectively.

Full text of DOI:10.1021/np020420x

J . Nat. Prod. 2003, 66, 327-331
327
Full Papers
Biotr a n sfor m a tion of th e Diter p en es Ep ica n d ica n d iol a n d Ca n d ica n d iol by
Mu cor plu m beu s
Braulio M. Fraga,*,† Laura Alvarez,‡ and Sergio Sua´rez§
Instituto de Productos Naturales y Agrobiologı´a, PO Box 195, CSIC, 38206-La Laguna, Tenerife, Canary Islands, Spain,
Instituto Universitario de Bio-Orga´nica ‘Antonio Gonza´lez’, Universidad de La Laguna, Tenerife, Spain, and Centro de
Investigaciones Quı´micas, Universidad Auto´noma del Estado de Morelos, Avenida Universidad 1001, Cuernavaca 62210,
Morelos, Me´xico
Received September 6, 2002
The microbiological transformation of the diterpene epicandicandiol (1) with Mucor plumbeus gave foliol
(3), sideritriol (5), and 7â,16R,17,18-tetrahydroxy-ent-kaurane (7), while the incubation of candicandiol
(2) gave 7R,9â,18-trihydroxy-ent-kaur-16-ene (10), canditriol (11), and 7R,16R,17,18-tetrahydroxy-ent-
kaurane (12). Thus, the main difference observed in both feedings, resulting from the spatial change in
the orientation of the 7-hydroxyl, from axial in the substrate 1 to equatorial in 2, was the formation of
a 3R- and a 9â-hydroxylated derivative, respectively.
For several years, we have been interested in the study
of the microbiological transformation of terpenoids with
fungi. One of our aims was the biotransformation of ent-
kaurene derivatives by Gibberella fujikuroi, the fungus that
produces the gibberellin plant hormones. These studies
have mainly been directed to preparing new gibberellin
analogues and to obtaining information about the substrate
specificity of the enzymes involved in the biosynthesis of
gibberellins.1-3 In this research we have used as substrates
natural ent-kaurene diterpenes or synthetic derivatives
prepared from them. To continue with these studies and
to prepare new substrates, we have initiated the biotrans-
formation of ent-kaurene derivatives with Mucor plumbeus,
a fungus that has been used for the functionalization of
unactivated carbons in sesqui-4-9 and diterpenes.10,11 On
the other hand, these biotransformations should permit the
development of models to rationalize diterpenoid micro-
biological hydroxylation by M. plumbeus. In this context,
we have previously carried out incubations of labdane
diterpenes with this fungus.12-14
isolation of candicandiol (2) from Sideritis huber-morathii,19
but its 13C NMR spectrum was different from that of 2.
Therefore, the structure had been erroneously assigned.
We have now identified it with epicandicandiol (1). Its
spectrum, conveniently reassigned, was identical with that
of 1.
Resu lts a n d Discu ssion
The incubation of epicandicandiol (1) with M. plumbeus
for 6 days afforded three hydroxylation products, 3, 5, and
7. The metabolite 5 was obtained as its triacetate 6, while
7 was isolated as the triacetate 8 and the tetraacetate 9,
by acetylation of the fractions containing them.
The least polar compound 3 was isolated as colorless
needles in 7.3% yield. Its MS was in accordance with the
formula C20H32O3, indicating that a new oxygen was
introduced in the molecule of 1. This must form a part of
a secondary hydroxyl group, because a new geminal proton
was observed in the 1H NMR spectrum resonating at δ 3.68
(dd, J ) 8.9, 7.7 Hz). The form of resonance was typical of
a â-axial hydrogen at C-1, C-3, or C-12, but the effect
produced by the new alcohol in the resonance of the
hydroxymethylene group (δ 3.35 and 3.59, each 1H, d) with
respect to the substrate (δ 2.94 and 3.46) permitted it to
be located at C-3(R). This location was confirmed by
assignments of its 13C NMR spectrum, and that of its
acetate 4, using 2D NMR data. Thus, the structure of this
metabolite was determined as 3R,7â,18-trihydroxy-ent-
kaur-16-ene. This compound has been isolated previously
as a natural product from Sideritis linearifolia and named
foliol (3).20
In this work we describe the results of the biotransfor-
mation of the two ent-kaurene derivatives, epicandicandiol
(7â,18-dihydroxy-ent-kaur-16-ene) (1) and candicandiol
(7R,18-dihydroxy-ent-kaur-16-ene) (2), which are epimeric
at C-7. Thus, we can also determine whether a spatial
change in the orientation of the hydroxyl group at this
carbon, from â-axial in the first to R-equatorial in the
second, has any effect on the biotransformation.
Compounds 1 and 2 had been obtained from Sideritis
candicans15,16 and other species of this genus.17 We have
1
assigned the H and 13C NMR spectra of these substrates
utilizing 2D NMR data (COSY, HSQC, and HMBC). The
carbon spectra of 1 and 2 are included in Table 1. Previ-
ously we had reported the spectrum of 1 in deuterated
pyridine.18 Italian and Turkish authors have reported the
The second compound obtained in this biotransformation
with a 0.93% yield was 5, identified as its triacetate 6 by
acetylation of the fraction containing it. Spectroscopic data
of 6 were the same as those described in the literature for
the triacetate of sideritriol.21 We have now assigned the
1H and 13C NMR spectra of this compound utilizing two-
dimensional NMR data. The corresponding alcohol side-
ritriol (5) had been isolated from the corollas of Sideritis
sicula.21,22
* To whom correspondence should be addressed. Tel: 34-922-251728.
Fax: 34-922-260135. E-mail: bmfraga@ipna.csic.es.
† Instituto de Productos Naturales y Agrobiolog´ıa, CSIC.
‡ Centro de Investigaciones Qu´ımicas, Universidad Auto´noma de Morelos.
§ Instituto de Bio-Orga´nica, Universidad de La Laguna.
10.1021/np020420x CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/28/2003

328 J ournal of Natural Products, 2003, Vol. 66, No. 3
Fraga et al.
Ta ble 1. 13C NMR Data of Compounds 3, 4, 6, 8-11, 13, and 14
carbon
1
2
3
4
6
8
9
10
11
13
14
1
2
3
4
5
6
7
8
39.9
17.7
35.1
37.0
38.5
26.9
77.3
48.3
50.7
38.9
17.7
33.6
43.7
38.6
45.1
155.0
103.6
71.0
17.7
18.0
39.6
17.8a
34.9
37.3
45.8
29.0
74.7
49.7
55.2
39.1
17.9a
33.5
43.2
30.5
43.1
154.9
103.4
71.6
17.4
18.1
38.5
26.9
74.1
41.6
38.6
27.0
77.0
48.2
50.4
39.0
17.8
33.5
43.6
38.5
45.0
154.6
103.8
68.3
11.7
68.3
37.7
23.0a
73.8
40.3
39.6
24.2a
79.2
46.7
50.8
38.6
18.0
33.2
43.4
38.0
44.9
154.0
103.8
64.9
12.8
17.7
39.4
18.0
35.4
36.0
41.3
23.8
77.0
51.6
43.9
39.1
17.0
25.1
41.3
41.8
133.3
142.1
62.1
72.3
17.1
17.5
39.4
17.6
35.4
36.1
41.5
24.7
79.6
47.2
51.6
38.9
17.6
26.2
45.8
35.7
49.4
79.3
68.2
72.4
17.2
17.9
39.3
17.7a
35.3
36.0
41.4
24.5
79.6
46.7
51.3
38.0
17.5a
25.9
43.0
35.6
47.6
89.9
63.1
72.2
17.2
17.8
31.9
17.8
34.7
37.5
38.8
29.4
70.5
54.6
78.5
43.7
30.9
34.1
41.6
30.0
37.3
154.6
103.4
71.9
17.6
19.7
39.6
17.8
34.8
37.3
44.9
28.5
73.6
52.4
52.9
39.1
17.8
33.1
41.2
27.5
84.0
160.2
107.8
71.2
17.5
18.1
39.5
17.6a
35.6
36.2
46.4
25.7
76.1
48.9
55.7
38.9
17.7a
26.2
44.8
29.4
47.7
79.2
68.3
72.1
17.4
18.2
39.5
17.6a
35.6
36.2
46.3
25.6
76.4
48.4
55.6
38.9
17.8a
25.9
42.0
29.3
45.3
89.9
63.4
72.1
17.4
18.2
9
10
11
12
13
14
15
16
17
18
19
20
a
These values can be interchanged.
In addition to 3 and 5, a more polar metabolite 7 was
isolated from the M. plumbeus culture. The fraction
containing it was acetylated and chromatographed to give
the triacetate 8 and the tetraacetate 9 in 13.8% and 1.3%
yield, respectively. The NMR spectrum of 8 was similar to
that for 1 with the exception of carbon and proton signals
at positions 16 and 17. 1H and 13C spectra of 8 showed the
presence of a new acetoxymethylene group and a tertiary
hydroxyl. This observation was consistent with the absence
of vinylic hydrogen signals of H-17 and the appearance in
its 1H NMR spectrum of a 2H singlet at δ 4.18. The
existence of the tertiary hydroxyl group was reflected in
the 13C NMR spectrum by the resonance of a quaternary
carbinol carbon at δ 79.3. The MS spectrum of 8 was in
full accordance with these assignments with [M]+ at m/z
464.2786 (C26H40O7). On the other hand, HSQC and HMBC
spectral analysis showed that the tertiary carbinol carbon
was three-bond correlated with the protons at C-14 and
C-12, while the methylene carbon at C-17 correlated with
H-13 and H-15, thus confirming the hydroxylation pattern.
The stereochemistry C-16(R) for 8 followed from the
observation of a NOE effect between H-13 and H-17. This
interaction is only possible if the acetoxymethylene group
has a â-orientation. Thus, the structure of this triacetate
was determined as 7â,17,18-triacetoxy-16R-hydroxy-ent-
kaurane. A study of 2D NMR data of the tetraacetate 9
confirmed this assignment. The two H-17 appear now as a
pair of doublets at δ 4.43 and 4.86 (J ) 12.5 Hz); C-16
resonates at 89.9 and C-17 at δ 63.1. These carbon chemical
shifts are in accordance with a 16R-OH stereochemistry.23,24
Therefore, the original metabolite obtained in the feeding
was the corresponding alcohol 7â,16R,17,18-tetrahydroxy-
ent-kaurane (7).
The incubation of candicandiol (2) with M. plumbeus for
6 days afforded the hydroxylation products 10-12. This
last compound was obtained as the triacetate 13 and the
tetraacetate 14 by acetylation of the fractions containing
it.
The least polar metabolite 10 was isolated as colorless
crystals in 1.3% yield. The molecular formula was found
to be C20H30O2 on the basis of an ion peak at m/z 302.2194
mined from the HMBC spectrum to be located at C-9 due
to the presence of correlations between this carbon (δ 78.5)
and the methyl group protons at H-20 (δ 1.17, s), the
methylene protons at H-11 (δ 1.73, m), H-14 (1.26, m and
1.98, dd), and H-15 (δ 2.43, dt, and 2.46, d), and the
methine proton of H-5 (δ 1.89, dd). This was further
supported by the expected downfield shifts for C-8 (∆δ )
4.9 ppm), C-10 (∆δ ) 4.6 ppm), and C-11 (∆δ ) 13.0 ppm)
1
[M - H2O]+ and other spectroscopic data. The H and 13C
NMR spectra of 10 were similar to those of 2 except for
resonances attributed to rings B and C. The 13C NMR
spectrum revealed the presence of an additional quaternary
carbinol signal at δ 78.5. This hydroxyl group was deter-

Biotransformation of Diterpenes by Mucor
J ournal of Natural Products, 2003, Vol. 66, No. 3 329
and the upfield shift, due to γ-gauche effects, for C-1 (∆δ
) 7.7 ppm), C-5 (∆δ ) 7.0 ppm), C-7 (∆δ ) 4.2 ppm), and
C-15 (∆δ ) 7.8 ppm) relative to those of 2. Moreover, shift
displacements in the carbon resonances of C-8, C-9, C-10,
and C-11 in comparison with those of the diterpene
7R,9R,16â,17-tetrahydroxy-ent-kaurane24 are in accordance
with this assignment. Therefore the structure of 10 was
established as 7R,9â,18-trihydroxy-ent-kaur-16-ene.
the formation of the 7R-epimer of the diterpene sideritriol
(5), which was not isolated therefrom.
4. The hydroxylations produced in these incubations of
the substrates 1 and 2 with M. plumbeus resemble those
observed in the Sideritis genus, where these compounds
were transformed into the metabolites 3 and 11, respec-
tively, and from which compound 5 was also obtained.
Exp er im en ta l Section
The second metabolite obtained in this feeding was
identified as 7R,15R,18-trihydroxy-ent-kaur-16-ene (can-
ditriol) (11). This triol had been described previously as a
natural product from Sideritis infernalis,25 and comparison
of spectroscopic data confirmed its identity. We have now
Gen er a l Exp er im en ta l P r oced u r es. Melting points 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 solutions at
500.13 MHz with a Bruker AMX2-500 spectrometer. 13C NMR
spectra were run in CDCl3 at 50.32 and 125.13 MHz with a
Bruker AC-200 or a Bruker AMX2-500, respectively. Chemical
shifts are given in ppm (δ). Mass spectra and HRMS were
taken at 70 eV in a Micromass Autospec spectrometer. HPLC
was performed using a Beckman System Gold 125P. Purifica-
tion by HPLC was achieved using a Si gel column (Ultrasphere
Si 5 µm, 10 × 250 mm). Dry column chromatographies were
made on Si gel Merck 0.2-0.065 mm. Epicandicandiol (1) and
candicandiol (2) were obtained from Sideritis candicans (La-
biatae), a species endemic to Tenerife (Canary Islands).15,16 The
acetylated compounds were prepared with Ac2O-pyridine (2:
1) at room temperature overnight.
Or ga n ism . The fungal strain Mucor plumbeus CMI 116688
was a gift from Dr. J . R. Hanson, School of Chemistry, Physics
and Environmental Sciences, University of Sussex, U.K.
In cu ba tion Exp er im en ts. The fungus M. plumbeus was
grown in shake cultures at 25 °C in conical flasks (250 mL),
each containing 50 mL of a sterile medium comprising (per
liter) glucose (80 g), NH4NO3 (0.48 g), KH2PO4 (5 g), MgSO4
(1 g), and trace elements solution (2 mL) containing (per 100
mL) Co(NO3)2 (0.01 g), CuSO4 (0.015 g), ZnSO4 (0.16 g), MnSO4
(0.01 g), and (NH4)6Mo7O24 (0.01 g). The substrate dissolved
in EtOH was equally distributed among 40 conical flasks, and
the fermentation was continued for a further 6 days before
the mycelium was filtered and the broth extracted with ethyl
acetate. The extracts were dried over Na2SO4 and the solvent
was evaporated to give a residue, which was chromatographed
on a Si gel column using a petroleum ether-EtOAc gradient.
Fractions collected were purified further by HPLC when
necessary.
1
assigned the H and 13C NMR spectra utilizing 2D NMR
data (COSY, HSQC, and HMBC).
The most polar compound, 12, of this bioconversion was
obtained as its triacetate 13 and its tetraacetate 14 by
acetylation and chromatography of the fractions containing
it. Comparison of the spectroscopic data of 13 with those
of 8 indicated that those compounds were very similar.
Thus, the absence in their 1H NMR spectrum of vinylic
hydrogen signals at C-17, the appearance of a pair of
doublets at δ 4.44 and 4.82 (J ) 12.4 Hz) of the 17-
acetoxymethylene group, and the quaternary carbinol at
C-16 resonating at δ 79.2 confirmed this assertion. The
HRMS of 13 showed the molecular ion at m/z 464.2786 [M]+
and was in accordance with the molecular formula C26H40O7.
The stereochemistry at C-16 was established as R on the
basis of the same arguments described above for 10. On
the other hand, the NMR spectrum of the tetraacetate 14
showed signals similar to those observed for 9. Therefore,
the original alcohol was assigned the structure 7R,16R,17,18-
tetrahydroxy-ent-kaurane (12).
Several conclusions can be obtained from these micro-
biological transformations:
1. A spatial change in the orientation of the hydroxyl
group at C-7 from axial in epicandicandiol (1) to equatorial
in candicandiol (2) affected the way in which these kau-
renes bind to the oxidative enzymes affording a different
hydroxylation pattern in the A and B rings. Thus, the main
difference in both feedings was the formation of a 3R-
hydroxylated derivative of 1 in the first and a 9â-hydroxy-
lated one of 2 in the second.
Ep ica n d ica n d iol (1): 1H NMR (CDCl3, 500 MHz) δ 0.71
(3H, s, H-19), 0.85 (1H, td, J ) 13, 3.3 Hz, H-1â), 1.06 (1H, s,
H-20), 1.17 (2H, m, H-3 and H-14), 1.82 (3H, m, H-1R, H-5
and H-14), 2.24 (2H, br s, H-15), 2.68 (1H, br s, H-13), 2.96
and 3.47 (each 1H, d, J ) 11 Hz, H-18), 3.59 (1H, t, J ) 2.8
Hz, H-7), 4.80 and 4.82 (each 1H, s, H-17); EIMS m/z (rel int)
304 (1), 286 (14), 255 (100), 241 (14), 213 (12), 199 (11), 185
(9), 173 (11), 159 (7), 145 (10); HREIMS m/z 304.2409 (calcd
for C20H32O2, 304.2402).
2. Incubations of both candicandiols 1 and 2 with M.
plumbeus afforded mainly 7 and 12 in 15.0% and 22.7%
yield, respectively. These 16,17-dihydroxylated compounds
can be formed by enzymatic epoxidation of the exocyclic
double bond to give the corresponding epoxides, followed
by opening of these epoxides in the medium. In this context,
we have reported that 16R,17-dihydroxy derivatives were
formed when 16R,17-epoxy-ent-kauranes were added with-
out the fungus to a medium prepared for growing Gibber-
ella fujikuroi.24
Ca n d ica n d iol (2): 1H NMR (CDCl3, 500 MHz) δ 0.72 (1H,
td, J ) 13, 4.2 Hz, H-1â), 0.74 (3H, s, H-19), 1.05 (3H, s, H-20),
1.08 (1H, d, J ) 7.1 Hz, H-9), 1.24 (1H, dd, J ) 11, 2 Hz, H-5),
1.26 (1H, m, H-3), 1.78 (1H, dt, J ) 13, 2 Hz, H-1R), 1.93 (1H,
d, J ) 16.4 Hz, H-15), 2.63 (1H, dt, J ) 16.9 Hz, H-15), 2.67
(1H, br s, H-13), 3.09 and 3.41 (each 1H, d, J ) 10.8 Hz, H-18),
3.50 (1H, br d, J ) 10.7 Hz, H-7), 4.75 and 4.81 (each 1H, s,
H-17); EIMS m/z (rel int) 304 (84), 286 (78), 268 (66), 255 (100),
240 (26), 225 (26), 211 (18), 199 (29), 185 (27), 169 (28), 157
(39), 145 (43); HREIMS m/z 304.2409 (calcd for C20H32O2,
304.2402).
In cu ba tion of Ep ica n d ica n d iol (1). The substrate 1 (140
mg) dissolved in EtOH (8 mL) was equally distributed among
40 conical flasks. After 6 days the mycelium was filtered and
the broth extracted with ethyl acetate. The solvent was
evaporated to give a residue (715 mg), which was chromato-
graphed on a Si gel column, using a n-hexane-EtOAc-MeOH
gradient as eluent, obtaining four fractions, which showed
biotransformation products F-1 (7:3:0, 15.4 mg), F-2 (3:7:0, 33.9
mg), F-3 (0:9:1, 58.6 mg), and F-4 (0:85:15, 206.3 mg). Fraction
3. The formation of sideritriol (5) in the incubation of 1
and of canditriol (11) in that of 2 should be due to the same
enzyme. Thus, 5 can be formed by enzymatic abstraction
of a hydrogen at C-15 in 1 with formation of a carbonium
ion, migration of the double bond to the 15,16-position, and
neutralization of the cation at C-17 by a -OH group,
probably originating from water. The presence of the 7â-
OH in the substrate can anchimerically assist this process.
Sideritriol (5) can also be formed by enzymatic opening of
the epoxide 15 and concomitant formation of the 15,16-
double bond. We think that this is not the mechanism of
formation in our case, because analogously in the biotrans-
formation of candicandiol (2) this process must also imply

330 J ournal of Natural Products, 2003, Vol. 66, No. 3
Fraga et al.
1 contained starting material 1 (1.5 mg). Fraction 2 was
purified by column chromatography on Si gel, using a n-hex-
ane-EtOAc gradient. Fractions eluted with n-hexane-EtOAc
(9:1) were purified by HPLC using a gradient of 35% EtOAc
to 55% in n-hexane, giving foliol 3 (4.8 mg). Fractions eluted
with n-hexane-EtOAc (85:15) were acetylated and purified by
HPLC using EtOAc as isocratic eluent to give foliol triacetate
(4) (2.3 mg). Chromatographic separation of the acetylated
fraction 3 afforded 4 (3.1 mg) and sideritriol triacetate 6 (1.3
mg). Fraction 4 was acetylated and purified by chromatogra-
phy on a Si gel column with a gradient of ethyl acetate-hexane
(4:1 to 1:1, v/v) and then by HPLC with a gradient of 30%
EtOAc to 50% in n-hexane to give the tetraacetate 9 (1 mg)
and the triacetate 8 (19.2 mg).
266 (58), 253 (100), 225 (40), 197 (19), 157 (25); HREIMS m/z
446.2726 [M - AcOH]+ (calcd for C26H38O6, 446.26683).
Biot r a n sfor m a t ion of Ca n d ica n d iol (2). Candicandiol
(7R,18-dihydroxy-ent-kaur-16-ene) (2) (230.8 mg) dissolved in
EtOH (8 mL) and 3 drops of Tween-80 was distributed among
40 conical flasks and incubated for a further 6 days. The
metabolites were isolated as above and chromatographed on
Si gel. Elution with n-hexane-EtOAc (3:2) gave starting
material 2 (84.6 mg). Further elution with n-hexane-EtOAc
(1:1) gave 7R,9â,18-trihydroxy-ent-kaur-16-ene (10) (2.5 mg).
Fractions eluted with n-hexane-EtOAc (1:4) were purified by
HPLC using EtOAc as isocratic eluent to give canditriol (11)
(3.4 mg). Acetylation of fractions eluted with EtOAc-MeOH
(1:1) and further chromatography on a Si gel column eluted
with n-hexane-EtOAc mixtures gave the tetraacetate 14 (9.1
mg) and the triacetate 13 (25.7 mg).
F oliol (3): colorless crystals; mp 194-197 °C (lit.20 198-
200 °C); IR (CHCl3) νmax 3350, 2930, 1045, 1020, 875, 755 cm-1
;
1H NMR (CDCl3, 500 MHz) δ 0.75 (3H, s, H-19), 0.97 (1H, dd,
J ) 12.7, 7.6, H-14â), 1.04 (3H, s, H-20), 1.15 (1H, dd, J )
11.4, 4.7 Hz, H-1â), 1.43 (1H, br s, H-9), 2.21 (2H, br s, H-15),
2.66 (1H, br s, H-13), 3.58 (1H, br s, H-7), 3.35 and 3.59 (each
1H, J ) 11.1 Hz, H-18), 3.68 (1H, dd, J ) 8.9 and 7.7 Hz, H-3),
4.77 and 4.81 (each 1H, s, H-17); EIMS m/z (rel int) 320 [M]+
(3), 302 (21), 284 (1), 272 (100), 253 (59), 211 (20), 199 (24),
157 (24); HREIMS m/z 320.23363 (calcd for C20H32O3 320.23514).
7r,9â,18-Tr ih ydr oxy-en t-kau r -16-en e (10): colorless prism;
mp 196-198 °C; IR (CHCl3) νmax 3400, 2920, 1460, 1380, 1290,
1
1055, 1025, 1005, 935, 860 cm-1; H NMR (CDCl3, 500 MHz)
δ 0.76 (3H, s, H-19), 1.17 (3H, s, H-20), 1.26 (1H, m, J ) 14
Hz, H-14), 1.50 (1H, m, H-1), 1.67 (2H, m, H-2 and H-12), 1.73
(2H, m, H-11), 1.89 (1H, dd, J ) 12.9, 1.9 Hz, H-5), 1.98 (1H,
dd, J ) 14.3, 5.7 Hz, H-14), 2.43 (1H, dt, J ) 17.6, 3 Hz, H-15â),
2.46 (1H, d, J ) 17.6 Hz, H-15R), 2.67 (1H, br s, H-13), 3.11
and 3.42 (each 1H, J ) 10.9 Hz, H-18), 3.81 (1H, dd, J ) 12,
4.3 Hz, H-7), 4.78 and 4.81 (each 1H, s, H-17); EIMS m/z (rel
int) 302 [M - H2O]+ (3), 290 (2), 284 (2), 271 (11), 253 (9), 163
(30), 123 (62), 109 (100), 93 (17), 81 (18); HREIMS m/z
302.2195 [M - H2O]+ (calcd for C20H30O2, 302.2246).
1
Tr ia ceta te (4): H NMR (CDCl3, 500 MHz) δ 0.80 (3H, s,
H-19), 1.06 (1H, m, H-1â), 1.08 (3H, s, H-20), 1.24 (1H, dd, J
) 11.6, 5.3 Hz, H-14), 1.48 (1H, d, J ) 6.8 Hz, H-9), 1.99 and
2.01 (each 3H, s, OAc), 2.09 (1H, dt, J ) 17, 3 Hz, H-15), 2.17
(1H, d, J ) 17 Hz, H-15), 2.68 (1H, br s, H-13), 3.51 and 3.90
(each 1H, d, J ) 11.6 Hz, H-18), 4.73 (1H, dd, J ) 12, 5.3 Hz,
H-3), 4.74 (1H, s, H-17), 4.78 (1H, d, J ) 2 Hz, H-17); EIMS
m/z (rel int) 446 [M]+ (0.6), 386 (6), 344 (7), 326 (57), 311 (5),
284 (9), 266 (100), 251 (69), 238 (12), 225 (21), 223 (21);
HREIMS m/z 446.2672 (calcd for C26H38O6, 446.2668).
Ca n d itr iol (11): 1H NMR (CDCl3, 500 MHz) δ 0.71 (1H,
td, J ) 13.2, 3.4 Hz, H-1â), 0.74 (H-19), 1.04 (1H, d, J ) 9 Hz,
H-9), 1.29 (1H, dd, J ) 12.6, 1.0 Hz, H-5), 1.63 (1H, dd, J ) 2
Hz, H-14), 1.79 (1H, dt, J ) 13.2, 3 Hz, H-1R), 2.03 (1H, dd, J
) 12.1, 5.3 Hz, H-14), 2.82 (1H, br s, H-13), 3.06 and 3.47 (each
1H, d, J ) 11.0 Hz, H-18), 3.90 (1H, dd, J ) 11.1, 4.5 Hz, H-7),
4.08 (1H, br s, H-15), 5.08 and 5.15 (each 1H, s, H-17); EIMS
m/z (rel int) 320 [M]+ (87), 302 (63), 287 (29), 275 (32), 271
(61), 262 (61), 257 (27), 253 (19), 201 (16), 162 (35), 152 (28),
147 (30), 145 (27), 131 (26), 129 (21), 123 (66), 109 (100);
HREIMS m/z 320.2351 (calcd for C20H30O3, 320.2351).
Sid er itr iol (5). Acetylation and chromatography of the
fraction containing this metabolite gave the triacetate 6: 1H
NMR (CDCl3, 500 MHz) δ 0.79 (3H, s, H-19), 0.81 (1H, dd J )
13, 2 Hz, H-1â), 1.06 (3H, s, H-20), 1.29 (1H, dd, J ) 10.3, 3.6
Hz, H-3â), 1.36 (1H, d, J ) 6 Hz, H-9), 1.47 (1H, m, H-12),
1.56 (1H, d, J ) 12.2 Hz, H-6â), 1.6 (1H, m, H-5), 1.75 (1H,
dd, J ) 12.2, 3.1 Hz, H-6R), 1.80 (1H, dt, J ) 13, 3 Hz, H-1R),
2.00 (1H, d, J ) 8.2 Hz, H-14), 2.03, 2.05 and 2.06 (each 3H,
s, OAc), 2.58 (1H, br s, H-13), 3.63 and 3.70 (each 1H, J )
12.0 Hz, H-18), 4.55 and 4.65 (each 1H, J ) 14.0 Hz, H-17),
4.73 (1H, br s, H-7), 5.56 (1H, s, H-15); EIMS m/z (rel int) 446
[M]+ (1), 404 (3), 386 (13), 371 (7), 344 (74), 326 (86), 313 (21),
284 (16), 266 (69), 253 (100), 238 (21), 225 (30), 223 (29), 209
(18), 197 (23), 183 (25), 169 (23), 157 (26), 145 (41); HREIMS
m/z 446.2666 (calcd for C26H38O6, 446.2668).
7r,16r,17,18-Tetr a h yd r oxy-en t-k a u r a n e (12). This com-
pound was obtained as its triacetate 13 and its tetraacetate
14 by acetylation and chromatography of the fraction contain-
ing it (see above).
Tr ia ceta te 13: colorless crystals; mp 139-141 °C; IR
(CHCl3) νmax 3500, 2935, 1735, 1375, 1250, 1040, 755 cm-1
;
1H NMR (CDCl3, 500 MHz) δ 0.71 (1H, td, J ) 13.3, 4.3 Hz,
H-1â), 0.78 (3H, s, H-19), 1.05 (3H, s, H-20), 1.07 (1H, d, J )
7.6 Hz, H-9), 1.25 (1H, d, J ) 12.1 Hz, H-5), 1.61 (2H, m, H-14
and H-15), 1.74 (1H, dt, J ) 12.9, 2.8 Hz, H-1R), 2.01 (1H, m,
H-13), 2.13 (1H, dd, J ) 11.9, 5.2 Hz, H-14), 3.55 and 3.79
(each 1H, J ) 11 Hz, H-18), 4.64 (1H, dd, J ) 11.4, 4.3 Hz,
H-7), 4.13 and 4.22 (each 1H, J ) 11.5 Hz, H-17); EIMS m/z
(rel int) 446 (1), 386 (24), 344 (100), 326 (65), 284 (71), 269
(29), 266 (76), 253 (96), 237 (20); 223 (31); HREIMS m/z
446.2706 (calcd for C26H38O6, 446.2668).
7â,16r,17,18-Tetr a h yd r oxy-en t-k a u r a n e (7). This com-
pound was obtained as the triacetate 8 and tetraacetate 9 by
acetylation and chromatography of the fraction containing it.
Tr ia ceta te 8: colorless crystals; mp 155-158 °C; IR (CHCl3)
1
νmax 3495, 2935, 1735, 1370, 1250, 1040, 755 cm-1; H NMR
(CDCl3, 500 MHz) δ 0.78 (3H, s, H-19), 0.79 (1H, td, J ) 13.3,
3 Hz, H-1â), 1.04 (3H, s, H-20), 1.28 (1H, dd, J ) 11, 3.5 Hz,
H-14), 1.41 (1H, d, J ) 7.02 Hz, H-9), 1.57 (2H, m, H-15), 1.83
(1H, br d, J ) 11 Hz, H-14), 2.01, 2.08 and 2.14 (each 3H, s,
OAc), 2.06 (1H, m, H-13), 3.61 and 3.68 (each 1H, J ) 11.2
Hz, H-18), 4.18 (2H, s, H-17), 4.79 (1H, br s, H-7R); EIMS m/z
(rel int) 464 [M]+ (6), 446 (15), 404 (23), 344 (90), 331 (79),
313 (29), 284 (26), 271 (100), 253 (42), 225 (26), 145 (19), 119
(21), 109 (32); HREIMS m/z 464.27869 (calcd for C26H40O7,
464.27740).
Tetr a a ceta te 14: colorless needles; mp 169-171 °C; 1H
NMR (CDCl3, 500 MHz) δ 0.73 (1H, td, J ) 13.3, 3.8 Hz, H-1â),
0.77 (3H, s, H-19), 1.04 (3H, s, H-20), 1.10 (1H, d, J ) 8.1 Hz,
H-9), 1.24 (1H, d, J ) 14.8 Hz, H-5), 1.52 and 2.08 (each 1H,
d, J ) 14.8 Hz, H-15), 1.74 (1H, dt, J ) 12.9, 2 Hz, H-1R),
1.94 (1H, dd, J ) 14.7 and 4.3 Hz, H-14), 1.96, 2.00, 2.03, 2.05
(each 3H, s, -OAc), 2.53 (1H, d, J ) 2.9 Hz, H-13), 3.54 and
3.80 (each 1H, J ) 11.0 Hz, H-18), 4.58 (1H, d, J ) 11.1, 4.0
Hz, H-7â), 4.44 and 4.82 (each 1H, J ) 12.4 Hz, H-17); EIMS
m/z (rel int) 446 [M - H2O]+ (3), 404 (5), 386 (80), 373 (12),
344 (72), 326 (100), 313 (45), 284 (30), 266 (68), 253 (74), 225
(21), 197 (14), 157 (29); HREIMS m/z 446.26379 [M - AcOH]+
(calcd for C26H38O6, 446.26683).
Tetr a a ceta te 9: 1H NMR (CDCl3, 500 MHz) δ 0.78 (3H, s,
H-19) 0.84 (1H, td, J ) 13, 3.3 Hz, H-1â), 1.04 (3H, s, H-20),
1.29 (1H, m, H-3), 1.44 (1H, d, J ) 6.8 Hz, H-9), 1.52 (1H, dd,
J ) 11, 4.6 Hz, H-14), 1.70 (1H, dd, J ) 16.2, 3.8 Hz, H-15),
1.79 (1H, dd, J ) 13, 3 Hz, H-1R), 1.86 (1H, br d, J ) 11 Hz,
H-14), 1.96, 2.01, 2.02, 2.04 (each 3H, s, OAc), 1.98 (1H, m,
H-15), 2.53 (1H, d, J ) 2.8 Hz, H-13), 3.60 and 3.69 (each 1H,
d, J ) 11.1 Hz, H-18), 4.43 and 4.86 (each 1H, d, J ) 12.0 Hz,
H-17), 4.70 (1H, br s, H-7R); EIMS m/z (rel int) 446 [M -
AcOH]+ (6), 404 (15), 386 (49), 344 (85), 326 (92), 313 (62),
Ack n ow led gm en t. This work has been supported by the
SEUID, Ministry of Education and Culture, Spain (PB98-
0540). We are highly obliged to UAEM-PROMEP (Mexico) for
granting a postdoctoral fellowship to L. Alvarez and to

Biotransformation of Diterpenes by Mucor
J ournal of Natural Products, 2003, Vol. 66, No. 3 331
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NP020420X