The microbiological transformation of the diterpenes dehydroabietanol and teideadiol by Mucor plumbeus

Article abstract of DOI:10.1016/S0031-9422(03)00291-7

The microbiological transformation of dehydroabietanol (18-hydroxy-dehydroabietane) by Mucor plumbeus led to 2α,18-dihydroxy-abieta-8,11,13,15-tetraene, 2α,15-dihydroxy-dehydroabietanol, 2α-hydroxy-15-methoxy-dehydroabietanol, 7β-hydroxy-2-oxo-dehydroabietanol, 15-hydroxy-2-oxo-dehydroabietanol and 15,16-dihydroxy-2-oxo-dehydroabietanol, whilst that of teideadiol (1α,18-dihydroxy-dehydroabietane) gave 2α-hydroxy-teideadiol, 7α-hydroxy-teideadiol and 7β-hydroxy-teideadiol. Thus, 2α- and 7β-hydroxylation occur in both biotransformations and the 15-hydroxylation is inhibited in the biotransformation of teideadiol by the presence of a 1α-alcohol.

Full text of DOI:10.1016/S0031-9422(03)00291-7

Phytochemistry 63 (2003) 663–668
www.elsevier.com/locate/phytochem
The microbiological transformation of the diterpenes
dehydroabietanol and teideadiol by Mucor plumbeus
a,
a
b
Braulio M. Fraga *, Melchor G. Hernandez , Jose M. Artega , Sergio Suarez
´ ´
aInstituto de Productos Naturales y Agrobiologı´a, CSIC, Avda. Astrofı´sico F. S a´ nchez 3, La Laguna, 38206-Tenerife, Canary Islands, Spain
bInstituto Universitario de Bio-Org a´ nica ‘‘Antonio Gonz a´ lez’’, Universidad de La Laguna, Tenerife, Spain
Received 27 January 2003; received in revised form 11 April 2003
Abstract
The microbiological transformation of dehydroabietanol (18-hydroxy-dehydroabietane) by Mucor plumbeus led to 2ꢀ,18-dihy-
droxy-abieta-8,11,13,15-tetraene, 2ꢀ,15-dihydroxy-dehydroabietanol, 2ꢀ-hydroxy-15-methoxy-dehydroabietanol, 7ꢁ-hydroxy-2-
oxo-dehydroabietanol, 15-hydroxy-2-oxo-dehydroabietanol and 15,16-dihydroxy-2-oxo-dehydroabietanol, whilst that of teideadiol
(
1ꢀ,18-dihydroxy-dehydroabietane) gave 2ꢀ-hydroxy-teideadiol, 7ꢀ-hydroxy-teideadiol and 7ꢁ-hydroxy-teideadiol. Thus, 2ꢀ- and
ꢁ-hydroxylation occur in both biotransformations and the 15-hydroxylation is inhibited in the biotransformation of teideadiol by
the presence of a 1ꢀ-alcohol.
2003 Elsevier Ltd. All rights reserved.
7
#
Keywords: Mucor plumbeus; Biotransformations; Diterpenes; Dehydroabietanol; Teideadiol
1
. Introduction
with its 4-epimer, from Juniperus phoeniceae (San Feli-
ciano et al., 1992) and later from Salvia pomifera, and
named pomiferin A(Ulubelen and Topcu, 1992), whilst
the latter has been isolated from Nepeta teydea (Breton
et al., 1970; Fraga et al., 1994). We have prepared 1 by
lithium aluminum hydride reduction of dehydroabietic
acid methyl ester (3) (Fraga et al., 1994).
For the last 20 years we have been interested in the
study of the microbiological transformation of diter-
penes by fungi. One of the microorganisms employed
has been Mucor plumbeus, which possesses a broad spe-
cificity in the substrate. The aim of these studies was the
preparation of analogues of the bioactive diterpene for-
skolin, using as substrates manoyl oxide derivatives of
the normal and enantio series (Fraga et al., 1998, 2001).
Now we have continued these works, but using sub-
strates with a different carbon skeleton, with a view to
developing models to explain these diterpene micro-
biological hydroxylations by this fungus. On the other
hand, some dehydroabietane diterpenes have been
shown to possess antimicrobial activity against bacteria
and fungi (Tapia et al., 1997; Gigante et al., 2002).
The incubation of dehydroabietanol (1) with M.
plumbeus gave the metabolites 4, 7–10 and 12. The
main compound was 4, which showed in its high reso-
lution MS the molecular ion at m/z 318.2200
(C H O ). The two new oxygens introduced in the
2
0
30
3
molecule were due to two alcoholic groups, one sec-
1
ondary and one tertiary. Thus, in the H NMR spec-
trum the geminal proton to the secondary hydroxyl
resonates as a broad multiplet at ꢂ 4.11 (W1/2=23 Hz),
which is characteristic of a ꢁ-axial hydrogen at C-2. The
tertiary alcohol was located at C-15, because in this
spectrum the resonance of H-15 does not appear and
the two methyls at C-15 resonate as a singlet at a low
field, ꢂ 1.55. The C-2 and C-15 bearing these new alco-
2
. Results and discussion
1
3
The substrates used were dehydroabietanol (1) and
teideadiol (15). The former has been obtained, mixed
hols appear in the C NMR spectrum at ꢂ 65.5 and
72.3, respectively (Table 1). In consequence, the struc-
ture of this metabolite was determined to be 2ꢀ,15-
dihydroxy-dehydroabietanol (4), which was confirmed
by 2D NMR data analysis of this compound and those
of their diacetate (5) and triacetate (6).
*
Corresponding author. Tel.: +34-922-251728; fax: 34-922-
60135.
E-mail address: bmfraga@ipna.csic.es (B.M. Fraga).
2
0
031-9422/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0031-9422(03)00291-7

664
B.M. Fraga et al. / Phytochemistry 63 (2003) 663–668
Table 1
13C NMR data of compounds 1 and 4–8
C
1
4
5
6
7
8
1
2
3
4
5
6
7
8
9
38.4
18.6
35.1
37.8
43.9
18.8
30.1
47.5
65.5
44.5
39.2
42.8
18.6
30.0
43.4
68.5
40.6
37.8
43.1
18.2
30.0
43.2
68.6
40.6
38.9
43.2
18.5
30.0
47.5
65.4
44.5
39.0
42.8
18.5
29.9
54.0
211.1
49.9
42.0
40.7
30.2
70.5
134.7
147.3
37.3
134.5
147.4
39.2
134.3
146.8
38.0
134.3
146.9
37.8
134.3
147.3
39.0
137.6
144.0
42.2
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
123.8
124.2
145.4
126.8
33.4
124.0
122.0
146.2
124.9
72.3
123.9
122.1
146.4
125.0
124.0
121.9
143.1
124.9
81.4
123.3
123.8
142.8
126.2
n.o
124.7
126.2
147.1
124.9
33.6
n.o.a
24.0
24.0
31.6
31.6
31.6
31.6
71.6
18.1
25.9
28.6
28.6
27.8
27.8
23.8
23.9
72.2
17.4
71.5
18.2
71.6
18.2
72.0
18.3
69.6
19.1
25.2
26.1
26.3
26.1
26.8
a
n.o., Not observed, overlapped with the solvent.
1
C H O . The H NMR spectrum showed the reso-
28
2
0
3
nances of H-7 as a double doublet at ꢂ 4.92 (J=10 and 7
Hz), of the two H-1 at 2.49 (d, J=13.1 Hz) and 2.95 (dd,
J=13.1 and 2 Hz), and of the two H-3 at ꢂ 2.15 (dd, 14
and 2 Hz) and 2.76 (d, J=14 Hz). Along distance W-
coupling (2 Hz) was observed between H-1(ꢁ) and H-
3(ꢁ). The carbon resonances of C-2 and C-7 appeared at
ꢂ 211.1 and 70.5, respectively. In the HMBC spectrum
correlations were observed of H-1 with C-2, of C-5 with
C-20, of H-3 with C-2, C-5, C-18 and C-19, and of H-7
with C-6, C-8 and C-9.
To a metabolite, isomer of 8, the structure 15-
hydroxy-2-oxo-dehydroabietanol (9) was assigned con-
sidering that in its 13C NMR spectrum the resonance of
1
the carbonyl group at C-2 appears at ꢂ 211.9. Its H
NMR spectrum showed a double doublet at ꢂ 2.14 and
a doublet at ꢂ 2.75, corresponding to the two H-3, a
doublet at ꢂ 2.50 and a double doublet at ꢂ 2.91 of the
two H-1, and a singlet at ꢂ 1.56, due to the methyls over
C-15. The structure was confirmed by assignment of its
1H and 13C NMR spectra using 2D NMR techniques.
One of the minor compounds isolated from the feed-
ing of 1 was 10, which was obtained in the form of its
triacetate 11, by acetylation and chromatography of the
fractions containing it. The molecular formula was
C H O determined by high resolution MS. Com-
The second of the metabolites obtained in this feeding
was 7 (C H O ), which showed a structure very simi-
2
1
32
3
lar to that of 4, the main difference between them being
the presence of a tertiary methoxyl group, whose signals
appear at ꢂ 3.05 and ꢂ 56.6 in the H and 13C NMR
spectra, respectively. Therefore, we assigned the struc-
ture 2ꢀ-hydroxy-15-methoxy-dehydroabietanol (7) to
this product, which was confirmed by study of its 2D
NMR spectra (COSY, HSQC and HMBC).
1
2
6
34
7
pound 11 was related with 9, because rings A–C were
identical, but now the 18-alcohol was in its acetate form
(ꢂ 3.73 and 3.98, each 1H doublet). The structural dif-
ference between both products was the presence of a
functionalized isopropyl with an acetoxymethylene
To another compound obtained in the feeding of
dehydroabietanol (1) the structure 7ꢁ-hydroxy-2-oxo-
dehydroabietanol (8) was assigned on the basis of the
following considerations: The molecular formula was
1
group, which appears in the H NMR spectrum as a
pair of doublets centred at ꢂ 4.25 and 4.33. This chemi-

B.M. Fraga et al. / Phytochemistry 63 (2003) 663–668
665
cal shift is characteristic of a dehydroabietane diterpene
with a similar side chain (Yano et al., 1994). In con-
sequence, the structure 10 (15,16-dihydroxy-2-oxo-
dehydroabietanol) was assigned to the corresponding
alcohol obtained in the fermentation. The stereo-
chemistry at C-15 remains undetermined.
Finally, from this fermentation we isolated a very
small amount of the metabolite 12, which was impur-
ified by a compound tentatively identified as 14. The
structure of the first was shown to be 2ꢀ, 18-dihydroxy-
abieta-8,11,13,15-tetraene (12) on the basis of the fol-
Fig. 1. Afragmentation of 2 a-hydroxy-teideadiol (16).
1
lowing considerations: Its H NMR spectrum showed
signals of the two hydrogens at C-16 as two singlets at ꢂ
318.2199 (C H O ). The new hydroxyl group was
20 32 3
1
located at C-7(ꢀ) considering the H NMR spectrum,
which showed the resonance of the geminal proton to
the new alcohol as a broad singlet at ꢂ 4.78. This rela-
tively low value of resonance and the changes observed
in the chemical shifts of the aromatic protons, with
respect to the substrate, also indicated that the alcohol
group must be allylic to the aromatic ring.
5
.00 and 5.30, whilst the methyl over C-15 appeared as a
singlet at ꢂ 2.11. The proton geminal to the 2ꢀ-alcohol
appears as a broad multiplet at ꢂ 4.12 (W1/2=24 Hz) and
the hydroxymethylene group at C-4 as a pair of doublets
at ꢂ 3.23 and 3.48. Dehydroabietane diterpenes with a
1
5-hydroxy or a 15,16-double bond have been isolated
from the leaves of Larix kaempferi (Tanaka et al., 1997).
The incubation of teideadiol (15) with M. plumbeus
led to the isolation of the metabolites 16, 18 and 19. The
structure of 16, which was obtained in good yield, was
determined to be 2ꢀ-hydroxy-teideadiol. Its HRMS
showed the molecular ion at m/z 318.2216 correspond-
ing to the molecular formula C H O . The new oxy-
The third metabolite 19, with a molecular formula
C H O , was the 7ꢁ-epimer of 18. The proton geminal
2
0
30
3
to the 7ꢁ-hydroxyl resonates as a double doublet at ꢂ
4.85 with coupling constants of 10.4 and 7.1 Hz, and H-
14 is now shifted to lower field (ꢂ 7.47), with respect to
1
3
18 (ꢂ 7.24). The assignment of the C NMR spectrum
(Table 2) confirmed the C-7 position and the ꢁ-stereo-
chemistry. Thus, C-5 now resonates at ꢂ 35.5 in com-
parison with 30.5 in the spectrum of 18, because in the
latter there exists a syn-diaxial interaction between the
7ꢀ-OH and H-5.
2
0
30
3
gen introduced in the molecule during the incubation
must form a part of a secondary hydroxyl group,
1
because in its H NMR spectrum there appears a new
geminal proton at ꢂ 4.21, resonating as a multiplet.
When this spectrum was run in CD OD this signal was
3
Several conclusions can be obtained from these bio-
transformations:
simplified appearing at ꢂ 4.15 as a double doublet of
doublets with coupling constants of 12.4, 4.7 and 2.5
Hz. This fact indicated that this geminal proton was
also coupled with the hydrogen of the alcoholic group,
which in deuterated methanol is interchanged for deu-
terium. This proton of the hydroxyl group resonates, in
Table 2
13C NMR data of compounds 9, 15, 16, 18 and 19
16a
C
9
15
71.7
16
74.9
18
71.6
19
71.7
CDCl , at ꢂ 2.23 as a doublet (J=9.1 Hz), which col-
3
1
2
3
4
5
6
7
8
9
53.9
76.0
lapses to a singlet by irradiation of its geminal hydrogen
at ꢂ 4.21. The observed couplings only can be explained
if this new alcohol is assigned to the C-2(ꢀ) position.
This location was confirmed by double resonance
experiments and 2D NMR data (COSY, NOESY,
HMQC and HMBC), that also permitted unambiguous
assignment of the H and 13C NMR spectra. In the MS
spectrum the base peak was observed at m/z 186
211.9
50.1
42.3
25.7
27.8
66.5
37.3
67.6
37.8
24.0
27.7
25.7
27.8
37.5
37.4
38.5
36.6
39.6
38.3
37.0
30.5
37.2
35.5
42.2
19.1
18.4
30.6
18.2
30.5
19.7
31.6
27.1
68.5
29.6
70.7
30.3
134.9
145.6
42.5
137.2
146.2
43.4
136.6
146.4
44.2
137.2
146.8
45.2
138.0
147.1
43.8
140.1
147.1
44.1
1
1
0
(
C H ). This fragment (Fig. 1) may be formed by the
18
11
12
124.5
122.4
147.8
124.9
72.3
124.5
123.7
142.1
127.9
33.5
124.6
123.6
141.7
127.9
33.5
126.6
124.9
144.3
128.1
34.9
127.3
124.1
142.4
129.0
33.5
125.9
123.5
142.0
126.4
33.7
1
4
cleavage of C-1, C-10 and C-4, C-5 bonds, indicating
that the new hydroxyl group must be situated on ring A.
Further proof of the composition of this fragment was
obtained in the mass spectra of the deuterated com-
pound 17 and of teideadiol (15), in which the base peak
was also at m/z 186.
1
1
1
1
3
4
5
6
31.6
31.6
23.9
23.9
23.8
23.8
24.5
24.5
23.8
23.9
23.8
23.6
17
18
70.1
17.6
72.0
17.5
71.6
18.2
72.3
18.7
70.9
17.6
71.5
17.4
1
2
9
0
The structure of the second product obtained in this
incubation was determined as 7ꢀ-hydroxy-teideadiol
26.5
25.9
25.1
28.3
24.4
24.0
a
(18). The MS spectrum showed the molecular ion at
3
Solvent: CD OD.

666
B.M. Fraga et al. / Phytochemistry 63 (2003) 663–668
1
. The C-2(ꢀ) and the C-7 positions are the main
sites of the hydroxylations produced in the
incubations of both substrates, dehydroabietanol
(80 g), NH NO (0.48 g), KH PO (5 g), MgSO (1 g)
4 3 2 4 4
and trace elements solution (2 ml). The trace-elements
solution contained (per 100 ml) Co(NO )NO (0.01 g),
3
2
(
2
1) and teideadiol (15), with M. plumbeus. The
ꢀ-hydroxylation was also the main reaction
CuSO , (0.015 mg), ZnSO (0.16 g), MnSO (0.01 g) and
4 4 4
(NH ) Mo O (0.01 g). The substrate dissolved in EtOH
4 6 7 24
observed by us in the incubation of 18-hydroxy-
manoyl oxide by this fungus (Fraga et al., 1998).
. The main difference between both bio-
transformations was the 15-hydroxylation of
dehydroabietanol (1), which does not occur in
teideadiol (15). This indicated that the 1ꢀ-alcohol
in the latter inhibits the C-15 functionalization.
. The 15- and 16-hydroxylations produced now in
the incubation of 1 with M. plumbeus have also
been observed in the biotransformation of
dehydroabietic acid (2) by Chaetomium
cochliodes (Yano et al., 1994), Fomes annosus
was evenly distributed among flasks after 1 day of growth.
After a further 6 days, the fermentation was harvested.
The mycelium was filtered, and the culture filtrate was
extracted with EtOAc. The extract was dried over Na SO
2
2
4
and the solvent evaporated to give a syrupy residue.
3.3.1. Incubation of dehydroabietanol (1)
3
The substrate (1, 250 mg) in 50 conical flasks was
incubated as above. Chromatography of the residue on
silica gel eluting with petrol–EtOAc mixtures gave:
2ꢀ,18-dihydroxy-abieta-8,11,13,15-tetraene (12) impur-
ified by 2ꢀ-hydroxy-dehydroabietanol (14) (0.7 mg), 2ꢀ-
hydroxy-15-methoxy-dehydro-abietanol (7, 4.2 mg), 15-
hydroxy-2-oxo-dehydroabietanol (9, 1 mg), 7ꢀ-hydroxy-
2-oxo-dehydroabietanol (8, 3.4 mg), 2ꢀ,15-dihydroxy-
dehydroabietanol (4, 18 mg) and 15,16-dihydroxy-2-
oxo-dehydroabietanol (10, 0.6 mg).
¨
(Ekman and Sjoholm, 1979) and Mortierella
isabellina (Kutney et al., 1981), which implies a
similar functionalization carried out by
enzyme(s) from four different genera of fungi.
The last microorganism also produces the 2ꢀ-
hydroxylation of dehydroabietic acid (2). How-
ever, the formation of a 2ꢀ-alcohol and its sub-
sequent oxidation to a 2-oxo derivative, such as
3.3.1.1. 2ꢀ,15-Dihydroxy-dehydroabietanol (4). Colour-
ꢀ
1
less prisms, m.p. 100–102 C (MeOH); H NMR (500
MHz, CDCl ): ꢂ 0.91 (3H, s, H-19), 1.23 (3H, s, H-20),
8–10, has only now been produced in the bio-
transformation of 1 with M. plumbeus.
3
1.35 (1H, t, J=11.8 Hz, H-1), 1.42 (1H, t, J=11.8 Hz,
H-3), 1.55 (6H, s, H-16 and H-17), 2.62 (1H, ddd,
J=12.1, 3.9 and 2.1 Hz, H-1), 2.91 (2H, m, H-7), 3.22
and 3.48 (each 1H, d, J=11 Hz, H-18), 4.11 (1H, br m,
3
. Experimental
W1/2=23 Hz, H-2), 7.17 (1H, br s, H-14), 7.21 (2H, s,
1
H-11 and H-12); H NMR (500 MHz, CD OD): ꢂ 0.89
3
3
.1. General
(3H, s, H-19), 1.22 (3H, s, H-20), 1.24 (1H, m, H-1),
1.48 (6H, s, H-16 and H-17), 1.49 (1H, m, H-3), 1.66
1
H NMR spectra were recorded in CDCl solutions at
3
and 1.83 (each 1H, m, H-6), 2.59 (1H, ddd, J=12.1, 3.9
and 2.0 Hz, H-1), 2.90 (2H, m, H-7), 3.11 and 3.43 (each
1H, d, J=11 Hz, H-18), 4.05 (1H, br m, W1/2=23 Hz,
H-2), 7.12 (1H, d, J=2.1 Hz, H-14), 7.17 (1H, dd,
J=8.2 and 2.1 Hz, H-12), 7.19 (1H, d, J=8.2 Hz, H-
11); [M]+ at m/z 318.2200, C H O requires 318.2194.
5
00.13 MHz with a Bruker AMX2-500 spectrometer. 13
C
NMR spectra were run in CDCl at 50.32 and 125.13
3
MHz with a Bruker AC-200 or a Bruker AMX2-500,
respectively. Chemical shifts are given in ppm (ꢂ). Mass
spectra and HRMS were taken at 70 eV in a Micromass
Autospec spectrometer. Dry column chromatographies
were made on Si gel Merck 0.2–0.065 mm. Acommercial
sample of dehydroabietic acid (American Drugs, Cali-
fornia, USA) has been used in this work.
2
0
30
3
1
Diacetate (5): H NMR (500 MHz, CDCl ): ꢂ 1.01 (3H,
3
s, H-19), 1.28 (3H, s, H-20), 1.46 (1H, t, J=11.7, H-1ꢁ),
1.49 (1H, t, J=12.2 Hz, H-3ꢁ), 1.54 (6H, s, H-16 and H-
17), 1.67 (1H, br d, J=10.3 Hz, H-5), 1.74 and 1.78
(each 1H, m, H-6), 1.83 (1H, br d, H-3ꢀ), 2.04 and 2.07
3
.2. Organism
(each 3H, s,–OAc), 2.63 (1H, br d, J=11.7 and 1.9 Hz,
H-1ꢁ), 2.85 (1H, m, H-7ꢀ), 2.96 (1H, dd, J=17 and 6.1
Hz, H-7ꢁ), 3.71 and 3.99 (each 1H, d, J=11 Hz, H-18),
5.22 (1H, m, W1/2=26 Hz, H-2ꢀ), 7.19 (1H, br s, H-14),
7.21 (1H, d, J=8.4 Hz, H-11), 7.25 (1H, br d, J=8.4 Hz,
H-12); EIMS m/z (rel. int.): 402 [M]+ (10), 384 (27), 342
(5), 327 (42), 324 (13), 309 (25), 267 (100), 249 (89), 239
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, UK.
3
.3. Incubation experiments
(
7), 209 (20), 207 (11), 173 (44), 157 (18), 134 (36); [M]+
at m/z 402.2397. C H O requires 402.2406. Triacetate
The fungus Mucor plumbeus was grown in shake cul-
ꢀ
2
4
34
5
1
ture at 25 C, in conical flasks (250 ml), each containing
0 ml of a sterile medium comprising (per litre) glucose
(6): H NMR (500 MHz, CDCl ): ꢂ 1.02 (3H, s, H-19),
3
5
1.28 (3H, s, H-20), 1.45 (1H, t, J=11.7 Hz, H-1ꢀ), 1.51

B.M. Fraga et al. / Phytochemistry 63 (2003) 663–668
667
(
1
(
1H, t, J=12.3 Hz, H-3ꢀ), 1.67 (1H, br d, J=11.6, H-5),
.70 (1H, m, H-6), 1.73 (6H, s, H-16 and H-17), 1.87
2H, m, H-3ꢁ and H-6), 2.03, 2.06 and 2.07 (each 3H,
s,–OAc), 2.60 (1H, dd, J=11.7 and 1.9 Hz, H-1ꢁ), 2.83
1H, m, H-7ꢀ), 2.92 (1H, dd, J=16.7 and 6.2 Hz, H-7ꢁ),
.72 and 3.99 (each 1H, d, J=11.2 Hz, H-1ꢁ), 5.23 (1H, m,
(20), 197 (48), 183 (80), 181 (62), 165 (58), 157 (30), 141
(62), 129 (61), 115 (60); [M]+ at m/z 316.2055. C H O
requires 316.2038.
2
0
28
3
(
3
3.3.1.5. 15,16-Dihydroxy-2-oxo-dehydroabietanol (10).
Obtained in the form of its triacetate by acetylation,
W1/2=23 Hz, H-2ꢀ), 7.00 (1H, br s, H-14), 7.09 (1H, br d,
J=8.4 Hz, H-12), 7.17 (1H, d, J=8.4 Hz, H-11); EIMS m/
z (rel. int.): 318 [M]+ (26), 303 (78), 300 (46), 285 (27), 267
with Ac O–pyridine (2:1) at reflux for 6 h, and chroma-
2
tography, of the fractions containing it. Triacetate (11):
1
H NMR (500 MHz, CDCl ): ꢂ 1.02 (3H, s, H-19), 1.26
3
(
96), 249 (100), 237 (24), 209 (35), 181 (36) 171 (52). [M]+
at m/z 444.2493, C H O requires 444.2511.
(3H, s, H-20), 1.55 (3H, s, H-17), 2.07 (9H, s, 3-OAc),
2.12 (1H, dd, J=14 and 2 Hz, H-3ꢁ), 2.26 (1H, dd,
J=12 and 2 Hz, H-5), 2.53 (1H, d, J=13.5 Hz, H-1ꢀ),
2.65 (1H, d, J=14 Hz, H-3ꢀ), 2.99 (1H, dd, J=13.5 Hz
and 2 Hz, H-1ꢁ), 3.73 and 3.98 (each 1H, d, J=11. 5
Hz, H-18), 4.25 and 4.33 (each 1H, d, J=11.6 Hz, H-
16), 7.00 (3H, s, H-14), 7.08 (2H, s, H-11 and H-12);
EIMS m/z (rel. int.): 458 [M]+ (3), 398 (33), 370 (42),
356 (57), 343 (35), 323 (44), 296 (52), 281 (75), 267 (38),
249 (60), 197 (46), 179 (62), 165 (100), 155 (55), 152 (55),
141 (63), 128 (73), 115 (81); [M]+ at m/z 458.2339.
C H O requires 358.2304.
2
6
36 6
3
Agum; H NMR (500 MHz, CD OD): ꢂ 0.93 (3H, s, H-
.3.1.2. 2ꢀ-Hydroxy-15-methoxy-dehydroabietanol (7).
1
3
19), 1.26 (3H, s, H-20), 1.36 (1H, t, J=11.8 Hz, H-1ꢀ),
1.44 (1H, t, J=11.8 Hz, H-3ꢀ), 1.49 (6H, s, H-16 and H-
17), 1.72 (1H, m, H-6), 1.77 (1H; ddd, J=12, 3.8 and 1.8
Hz, H-3ꢁ), 1.81 (1H, m, H-6), 2.64 (1H, ddd, J=11.8,
3.9 and 1.7 Hz, H-1ꢁ), 2.91 (2H, m, H-7), 3.05 (3H, s,
–OMe), 3.25 and 3.50 (each 1H, d, J=11 Hz, H-18),
4.14 (1H, m, W1/2=23 Hz, H-2ꢀ), 7.06 (1H, d, J=1.7
26
34
7
Hz, H-14), 7.15 (1H, dd, J=8.3 and 1.7 Hz, H-12), 7.22
(
(
1H, d, J=8.3 Hz, H-11); EIMS m/z (rel. int.): 332 [M]+
3), 317 (100), 303 (8), 301 (5), 285 (3), 267 (4), 203 (3),
3.3.1.6. 2ꢀ,18-Dihydroxy-abieta-8,11,13,15-tetraene
1
(12). H NMR (500 MHz, CDCl ): ꢂ 0.93 (3H, s, H-19),
3
2
C H O requires 332.2351.
01 (3), 183 (3), 171 (4); [M]+ at m/z 332.2336.
1.25 (3H, s, H-20), 2.11 (3H, s, H-17), 3.23 and 3.48
(each 1H, d, J=11 Hz, H-18), 4.12 (1H, m, W1/2=24
Hz, H-2ꢀ), 5.00 and 5.30 (1H, br s, H-16), 7.16 (1H, br
s, H-14), 7.21 (1H, d, J=8.1 Hz, H-11), 7.26 (1H, br d,
J=8 Hz, H-12); [M]+ at m/z 300.2134. C H O
2
2
1
32
3
3
gum; H NMR (500 MHz, CDCl ): ꢂ 0.93 (3H, s, H-19),
1
H-20), 1.73 (1H, dt, J=12.6 and 10.5, H-6ꢁ), 2.15 (1H,
dd, J=14 and 2 Hz, H-3ꢁ), 2.28 (1H, dd, J=12.2 and
7
.3.1.3. 7ꢁ-Hydroxy-2-oxo-dehydroabietanol (8).
1
A
3
20 28
1
.24 and 1.25 (each 3H, s, H-16 and H-17), 1.32 (3H, s,
requires 300.2089. Acetate (13): H NMR (500 MHz,
CDCl ): ꢂ 1.04 (3H, s, H-19), 1.25 (3H, s, H-20), 2.03
3
and 2.07 (each 3H, s, 2-OAc), 2.12 (3H, s, H-17), 2.63
(1H, ddd, J=12, 3.8 and 2.2 Hz, H-1ꢁ), 2.85 (1H, m, H-
7ꢀ), 2.96 (1H, br dd, J=16 and 5 Hz, H-7ꢁ), 3.72 and
4.00 (each 1H, d, J=11 Hz, H-18), 5.04 and 5.33 (each
1H, s, H-16), 7.16 (1H, br s, J=8 Hz, H-14), 7.20 (1H,
d, J=8 Hz, H-11), 7.26 (1H, br d, J=8, H-12); [M]+ at
m/z 384.2351. C H O requires 384.2301.
.0 Hz, H-6ꢀ), 2.39 (1H, dd, J=12.6 and 1.3 Hz, H-5),
.49 (1H, d, J=13.1 Hz, H-1ꢀ), 2.76 (1H, d, J=14 Hz,
2
H-3ꢀ), 2.89 (1H, sept, J=7 Hz, H-15), 2.95 (1H, dd,
J=13.1 and 2 Hz, H-1ꢁ), 3.16 and 3.66 (each 1H, d,
J=10.9 Hz, H-18), 4.92 (1H, dd, J=10.0 and 7.0 Hz, H-
7
2
), 7.07 (1H, d, J=8.3, H-11), 7.13 (1H, dd, J=8.3 and
.0 Hz, H-12), 7.42 (1H, d, J=2 Hz, H-14); EIMS m/z
2
4
32
4
(
(
(
(
rel. int.): 316 [M]+ (53), 301 (36), 283 (5), 265 (12), 249
7), 237 (28), 223 (8), 197 (23), 195 (21), 193 (12), 183
33), 181 (20), 171 (10), 169 (10), 162 (100), 155 (14), 149
12), 143 (24), 141 (16), 129 (12); [M]+ at m/z 316.2081.
3.3.2. Incubation of teideadiol (15)
The substrate (15, 290 mg) in 60 conical flasks was
incubated as above. Chromatography of the extract on
silica gel eluting with petrol–EtOAc mixtures gave
7ꢁ-hydroxy-teideadiol (19, 2 mg), 2ꢀ-hydroxy-teideadiol
C H O requires 316.2038.
2
0
28
3
(
16, 33.6 mg) and 7ꢀ-hydroxy-teideadiol (18, 3.5 mg).
3
.3.1.4. 15-Hydroxy-2-oxo-dehydroabietanol (9). 1H
NMR (500 MHz, CDCl ): ꢂ 0.92 (3H, s, H-19), 1.25
3.3.2.1. 2ꢀ-Hydroxy-teideadiol (16). Colourless prisms,
1
3
(
3H, s, H-20), 1.56 (6H, s, H-16 and H-17), 2.14 (1H, dd,
m.p. 214–216 (petrol-EtOAc); H NMR (500 MHz,
CDCl ): ꢂ 0.93 (3H, s, H-19), 1.22 and 1.23 (each 3H, 5,
J=13.7 and 2 Hz, H-3ꢁ), 2.31 (1H, dd, J=12.2 and 2.2
Hz, H-5), 2.50 (1H, d, J=13 Hz, H-1ꢀ), 2.75 (1H, d,
J=13.7 Hz, H-3ꢀ), 2.91 (1H, dd, J=13 and 2 Hz, H-
3
H-16 and H-17), 1.26 (3H, s, H-20), 1.50 (1H, dd,
J=12.4 and 4.9, H-3), 1.89 (1H, t, J=12.4 Hz, H-3),
2.10 (lH, dd, J=2 and 12.4, H-5), 2.23 (1H, d, J=9.1
Hz,-OH), 2.82 (1H, sept., H-5), 2.88 (1H, m, H-7), 3.26
and 3.51 (each 1H, d, J=11 Hz, H-18), 4.21 (1H, m, H-
2), 4.40 (1H, s, H-1), 6.94 (1H, d, J=1.9 Hz, H-14), 7.0
(1H, dd, J=8 and 1.9 Hz, H-12), 7.20 (1H, d, J=8 Hz,
1
ꢁ), 2.94 (1H, m, H-7), 3.15 and 3.64 (each 1H, d, J=11
Hz, H-18), 7.12 (1H, d, 8.6 Hz, H-11), 7.21 (1H, br s, H-
4), 7.24 (1H, br d, J=8 Hz, H-12) ; EIMS m/z (rel.
1
+
int.): 316 [M] (15), 301 (70), 283 (86), 281 (11), 280
47), 265 (98), 251 (49), 239 (42), 237 (100), 223 (77), 209
(

668
B.M. Fraga et al. / Phytochemistry 63 (2003) 663–668
1
1
1-H); H NMR (500 MHz, CH OD): ꢂ 0.90 (3H, s, H-
3
Acknowledgements
19), 1.67 (3H, s, H-20), 1.81 and 1.90 (each 3H, s, H-16
and H-17), 1.36 (lH, dd, J=12.4 and 4.6, H-3), 1.70 and
This work has been supported by the SEUID, Minis-
try of Education, Spain (PB98-0540).
1.77 (each 1H, m, H-6), 1.89 (1H, t, J=12.5 Hz, H-3),
2.06 (1H, dd, J=12.4 and 2.1 Hz, H-5), 2.76 (1H, m, H-
15), 2.84 (2H, m, H-7), 3.18 and 3.40 (each 1H, d, J=11
Hz, H-18), 4.15 (1H, ddd, J=12.4, 4.7 and 2.5 Hz, H-2),
4
6
J=8.1 Hz); EIMS m/z (rel. int.): 318 [M] (64), 303
References
.28 (1H, d, J=2.5, H-1), 6.84 (1H, d, J=1.9 Hz, H-14),
.97 (1H, dd, J=8.1 and 1.9 Hz, H-12), 7.20 (1H, d,
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lez, A.G., De Leon, G., 1970. Estructura del tei-
¨
(
(
(
12), 300 (29), 285 (26), 282 (38), 269 (55), 267 (60), 264
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´
´
Microbiological transformation of manoyl oxyde derivatives by
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(
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2
0
30
3
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2
3
(
.3.2.2. 7ꢀ-Hydroxy-teideadiol (18). Agum; 1H NMR
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1
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4
H-11 and H-12), 7.24 (1H, br s, H-14); EIMS m/z (rel.
.34 (1H, br s, H-1), 4.78 (1H, br s, H-7), 7.21 (2H, br s,
int.): [M]+ 318 (7), 307 (12), 300 (6), 282 (12), 267 (21),
2
2
64 (16), 251 (100), 237 (11), 209 (39), 193 (20), 184 (38),
64 (16), 167 (24), 165 (24), 149 (52), 143 (45); [M]+ at
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´
m/z 318.2199. C H O requires 318.2195.
2
0
30
3
cos de las hojas de Juniperus phoeniceae. Anales de Quimica 88,
512–516.
3
(
1
.3.2.3. 7ꢁ-Hydroxy-teideadiol (19). Agum; 1H NMR
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H, s, H-16 and H-17), 1.31 (3H, s, H-20), 2.90 (1H,
sept., H-15), 3.26 and 3.52 (each 1H, d, J=10.8, H-18),
.35 (1H, br s, H-1), 4.85 (1H, dd, J=10.4 and 7.1 Hz,
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Fusarium species. Phytochemistry 46, 131–133.
4
H-7), 7.15 (1H, dd, J=8 and 1.9 Hz, H-12), 7.22 (1H, d,
J=8 Hz, H-11), 7.47 (1H, d, J=1.9 Hz, H-14); EIMS
m/z (rel. int.): [M]+ 318 (3), 300 (3), 282 (8), 267 (13),
2
1
Ulubelen, A., Topcu, G., 1992. Abietane diterpenoids from Salvia
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Yano, S., Nakamura, T., Uemura, T., Takeshi, F., Takahashi, A.,
51 (100), 236 (6), 221 (7), 209 (24), 193 (12), 184 (19),
67 (14), 155 (6), 143 (19); [M]+ at m/z 318.2200.
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1995, 123, 138444).
C H O requires 318.2195.
0
2
30
3