Novel derivatives of ent-17,19-dihydroxy-16βH-kaurane obtained by biotransformation with Verticillium lecanii

Article abstract of DOI:10.1021/jf020009f

In the search for new routes in the synthesis of hydroxylated kaurane diterpenoids, Verticillium lecanii, grown in a mineral liquid medium for 48 h, was fed with ent-17,19-dihydroxy-16βH-kaurane, obtained by hydroboration/oxidation of kaurenoic acid, a natural product easily isolated from plants of the genera Xylopia (Annonaceae) and Wedelia (Asteraceae). After 14 days, the culture medium was extracted with ethyl acetate, and the metabolites were purified by column chromatography on silica gel. The results show that V. lecanii biotransformed the starting material into three novel compounds: ent-11α, 17,19-trihydroxy-16βH-kaurane; ent-7β, 17,19-trihydroxy-16βH-kaurane; and ent-7β, 17,19-trihy-droxy-16βH-kaurane, the structures of which were fully elucidated by using classical and modern two-dimensional NMR techniques.

Full text of DOI:10.1021/jf020009f

3704 J. Agric. Food Chem. 2002, 50, 3704−3707
Novel Derivatives of ent-17,19-Dihydroxy-16âH-kaurane
Obtained by Biotransformation with Verticillium lecanii
HENRIETE S. VIEIRA, JACQUELINE A. TAKAHASHI, AND
MARIA AM EÄ LIA D. BOAVENTURA*
Departamento de Qu ´ı mica, ICEx, Universidade Federal de Minas Gerais,
Avenida Ant oˆ nio Carlos 6627, Pampulha 31270-901, Belo Horizonte, MG, Brazil
In the search for new routes in the synthesis of hydroxylated kaurane diterpenoids, Verticillium lecanii,
grown in a mineral liquid medium for 48 h, was fed with ent-17,19-dihydroxy-16âH-kaurane, obtained
by hydroboration/oxidation of kaurenoic acid, a natural product easily isolated from plants of the genera
Xylopia (Annonaceae) and Wedelia (Asteraceae). After 14 days, the culture medium was extracted
with ethyl acetate, and the metabolites were purified by column chromatography on silica gel. The
results show that V. lecanii biotransformed the starting material into three novel compounds: ent-
11R,17,19-trihydroxy-16âH-kaurane; ent-7R,17,19-trihydroxy-16âH-kaurane; and ent-7â,17,19-trihy-
droxy-16âH-kaurane, the structures of which were fully elucidated by using classical and modern
two-dimensional NMR techniques.
KEYWORDS: ent-7r,17,19-Trihydroxy-16âH-kaurane; ent-11r,17,19-trihydroxy-16âH-kaurane; ent-7â,17,-
1
9-trihydroxy-16âH-kaurane; ent-17,19-dihydroxy-16âH-kaurane; Verticillium lecanii; microbial hydrox-
ylation
INTRODUCTION
of highly hydroxylated compounds, which are potentially
bioactive (6). This approach is safer and, in most cases, more
efficient for the production of many different novel compounds
Kaurane diterpenoids are a very important class of natural
products widespread in the plant kingdom. These compounds
are the biosynthetic precursors of gibberellins, the well-known
fungal plant growth hormones. A considerable number of
important biological activities have been associated with kaurane
diterpenoids, such as antimicrobial, antiparasitic, insect anti-
feedant, cytotoxic, anti-HIV, antifertility, hypotensive, and
antiinflammatory (1) activities, among others. Kaurenoic acid
(7).
Verticillium lecanii (former Cephalosporium aphidicola) is
a very versatile fungus, having the ability to transform several
diterpenoids into hydroxylated derivatives (8). This ability seems
to be related to its endogenous production of the antiviral and
antitumoral tetrahydroxylated diterpenoid aphidicolin (2) (9).
In the search for an efficient microbial route to prepare novel
polyhydroxylated kaurane diterpenoids to be evaluated in
biological screenings, the biotransformation of ent-17,19-
dihydroxy-16âH-kaurane (3) by whole cells of the fungus V.
lecanii was performed. Compound 3 was prepared from
kaurenoic acid (1) via hydroboration, followed by oxidation with
hydrogen peroxide. We report herein the successful biotrans-
formation of 3 by V. lecanii into three novel compounds: ent-
(1) is a typical kaurane representative possessing several
biological activities including the ability to promote leaf sheath
elongation in some Zea mays dwarf mutants (2). Kaurenoic acid
can be easily isolated from plants of Xylopia (Annonaceae) and
Wedelia (Asteraceae) genera in good yields (3, 4). Other known
sources of 1 are seedless sunflower heads, discarded as wastes
in the edible sunflower oil industry (5). The ready availability
of kaurenoic acid from natural sources makes this compound a
suitable starting material for the synthesis of its novel derivatives
for biological screening. The synthesis of hydroxylated kaurane
derivatives is of special interest because highly oxygenated
kaurane diterpenoids, scarcely available from natural resources,
usually have higher levels of biological activity than their less
hydroxylated precursors (1). However, the introduction of
functional groups into the inactive backbone of kaurenoic acid
is a nearly impossible task. Alternatively, fungal biotransfor-
mation is a method of considerable value for the partial synthesis
1
1
(
1R,17,19-trihydroxy-16âH-kaurane (4); ent-7R,17,19-trihydroxy-
6âH-kaurane (5); and ent-7â,17,19-trihydroxy-16âH-kaurane
6).
MATERIALS AND METHODS
General. Melting points were determined with a Kofler hot plate
apparatus and are uncorrected.
Infrared (IR) spectra were recorded with a Shimadzu/IR-408
spectrophotometer.
Nuclear magnetic resonance (NMR) spectra were recorded, in
pyridine, at room temperature, on a Bruker Avance DRX 400 MHz
spectrometer ( H NMR, 400 MHz; C NMR, 100 MHz). Two-
*
Author to whom correspondence should be addressed (telephone +55
1
13
3
1 34995754; fax +55 31 3499 5700; e-mail dianadb@dedalus.lcc.ufmg.br).
1
0.1021/jf020009f CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/23/2002

Biotransformation of ent-17,19-Dihydroxy-16âH-kaurane
J. Agric. Food Chem., Vol. 50, No. 13, 2002 3705
, 400 MHz), see Table 1; 13C NMR (pyridine-d
, 100 MHz),
(
pyridine-d
5
5
34 3
see Table 2. Elem Anal. Calcd for C20H O : C, 74.7%; H, 10.3%.
Found: C, 74.5%; H, 10.6%.
2
5
ent-7R,17,19-Trihydroxy-16âH-kaurane (5): oil; [R]
D
) -21° (c
OH); IR (KBr) νmax 3400, 2900 cm ; H NMR (pyridine-
, 400 MHz), see Table 1; 13C NMR (pyridine-d
, 100 MHz), see
: C, 74.7%; H, 10.3%. Found:
-1 1
0.002, in CH
3
d
5
5
34 3
Table 2. Elem Anal. Calcd for C20H O
C, 74.6%; H, 10.6%.
2
5
ent-7â,17,19-Trihydroxy-16âH-kaurane (6): oil; [R]
D
) -17° (c
-1 1
0
.002, in CH
3
OH); IR (KBr) νmax 3300, 2900 cm ; H NMR (pyridine-
Figure 1. Structures of kaurenoic acid (1) and aphidicolin (2).
13
d
5
, 400 MHz), see Table 1; C NMR (pyridine-d
5
, 100 MHz), see
: C, 74.7%; H, 10.3%. Found:
Table 2. Elem Anal. Calcd for C20
H
34
O
3
dimensional (2D) NMR experiments used were heteronuclear multiple-
quantum correlation (HMQC) and nuclear Overhauser effect spectros-
copy (NOESY). Chemical shifts are reported in parts per million (ppm),
with respect to tetramethylsilane as the internal standard. Multiplicities
were determined by DEPT pulse sequence.
Elemental analyses were performed on a Perkin-Elmer 2400, CHN
elemental analyzer instrument. The optical rotation was determined with
a Perkin-Elmer (model 341) polarimeter.
C, 74.4%; H, 10.6%.
RESULTS AND DISCUSSION
In this experiment, V. lecanii metabolized diol 3 into three
novel compounds hydroxylated in the remote methylene carbons
C-11 and C-7. On the basis of the formal similarity between
the structure of 3 and the steroidal skeleton, the formation of
compound 4 is of special interest, as the presence of an oxygen
function at C-11 position of the steroidal nucleus is an obligatory
structural requirement for steroidal carbohydrate-regulating
hormonal activity (11). The formation of 11-hydroxylated
steroidal derivatives by the biotransformation using V. lecanii
was previously reported by Boynton et al. (12). Hydroxylation
of C-7 (compounds 5 and 6) is also relevant as some antitumor
kaurane diterpenoids bear a C-7-hydroxyl function (13). The
oxidation in this position makes easier the preparation of novel
kaurane diterpenoids modified in ring B.
Starting material for this work, kaurenoic acid (1), was previously
isolated from green fruits of Xylopia frutescens (3).
Hydroboration and Oxidation of Kaurenoic Acid (1; Figure 1).
Diborane was generated in situ by the addition of NaBH
mmol) in dry tetrahydrofuran (THF, 10 mL) dropwise over 15 min to
BF O (5.0 mL, 41.0 mmol). To this solution was slowly added
4
(0.75 g, 19.8
3
‚Et
2
compound 1 (0.6 g, 1.9 mmol) in dry THF (10 mL), with stirring. After
an additional 0.5 h of stirring, 10 mL of a 2 M NaOH solution and 7.0
mL of 30% H O were added at 0 °C to the resulting reaction mixture
2 2
and stirred for 0.5 h. THF was then removed with a rotatory evaporator,
and the residue was extracted with EtOAc (3 × 50 mL). The combined
2 4
organic layers were dried over anhydrous Na SO and then concentrated
under reduced pressure. The recovered residue was submitted to flash
chromatography with 30% EtOAc in petroleum ether to yield ent-17,-
The preparation of 3 by the hydroboration of 1 was carried
out with a mixture of sodium borohydride and boron trifluoride
diethyl etherate (in situ borane generation) in tetrahydrofuran
under reflux, followed by oxidation with 30% hydrogen peroxide
(14). The reaction produced, after flash chromatography, ent-
17,19-dihydroxy-16âH-kaurane (3) (Figure 2), used as substrate
for the biotransformation by V. lecanii that furnished the novel
compounds 4-6. We show here their structures were completely
elucidated by using modern NMR techniques.
1
9-dihydroxy-16âH-kaurane (3) (0.516 g, 85% yield): mp 178 °C;
2
5
[R]
D
) -30° (c 0.007, in CH
3 34 2
OH). Elem Anal. Calcd for C20H O :
C, 78.6%; H, 11.4%. Found: C, 78.4%; H, 11.2%. IR (KBr) νmax 3400,
-
1
1
13
2
900 cm ; H NMR (pyridine-d
5
, 400 MHz), see Table 1; C NMR
25
(
)
pyridine-d , 100 MHz), see Table 2. {Lit. mp 180-181 °C; [R]
5
D
-66° (c 1.8, in EtOH). Elem. Anal.: C, 78.2%; H, 11.2% (10).}
Fermentation Conditions. A culture of V. lecanii (IMI 68689),
obtained from International Mycological Institute, Egham, Surrey, U.K.,
was grown at 25 °C on malt agar slopes (3 days) to furnish stock
cultures. After this period, they were stored at 7-10 °C. The liquid
culture used for biotransformation consisted of the following: glucose
1
The H NMR spectra of metabolites 4-6 showed that the
two compounds contain methyl groups at C-4 and C-10 and
two hydroxymethyl (CH2OH) groups at C-4 and C-16 as in
(
(
100 g/L), MnSO
2.0 g/L), and an aqueous solution of trace elements (2.0 mL) that
‚6H O (0.1 g), FeSO ‚7H O (1.0 g),
‚7H O (1.61 g), MnSO ‚4H O (0.1 g),
O (1.0 g). Sterilization of malt agar slopes and
4 2 4
(2.0 g/L), KCl (1.0 g/L), KH PO (5.0 g/L), glycine
starting material 3. Moreover, the presence of a signal at δ 2.04
1
(d, J ) 6.8 Hz) in the H NMR spectrum of 4 together with the
contained (per liter) Co(NO
CuSO ‚5H O (0.15 g), ZnSO
and (NH Mo
3
)
2
2
4
2
absence of a signal corresponding to the C-11 methylene carbon,
the appearance of a signal at δ 64.6 corresponding to a
hydroxylated methine carbon, and the downfield shift of signals
4
2
4
2
4
2
4
)
6
7
O
24‚4H
2
liquid medium was carried in an autoclave for 15 min.
Biotransformation of ent-17,19-Dihydroxy-16âH-kaurane (3).
Small sections of the stock cultures were cut under sterile conditions
and transferred to a 500 mL Erlenmeyer flask containing 200 mL of
liquid medium, which was incubated for 48 h at 25 °C on an orbital
shaker (150 rpm). The resulting culture was used to inoculate 10 250-
mL conical flasks containing the same culture medium (110 mL/flask).
All of the cultures were incubated for an additional 48 h (two-stage
culture), and then the substrate, ent-17,19-dihydroxy-16âH-kaurane (3)
corresponding to C-9 (from δ 56.7 to 68.2) and C-12 (from δ
13
2
5.9 to 36.1) observed in the C NMR spectrum suggested the
presence of a hydroxyl group on C-11 in 4. Two-dimensional
NMR experiments were used to establish stereochemistry at
1
1
13
C-11. On the basis of the J correlation among H and C nuclei
observed in the 2D-HMQC spectrum of 4, the signals at δ 1.14
and 1.97 were assigned to H-1 protons, whereas signals at δ
0.92 and 2.04 were assigned to H-20 and H-11, respectively.
Moreover, the corresponding 2D-NOESY-NMR spectrum (see
Table 3 and Figure 3) showed that spatial correlation among
H-11, H-20, and the signal at δ 1.97, corresponding to H-1R,
is compatible only with â-stereochemistry for hydroxyl group
at C-11. In addition, the multiplicity for the signals of H-17 in
(450 mg) in DMSO (5 mL), was evenly distributed among the cultures,
except one that was used as a cell control. The same culture medium
in a flask containing only the substrate was prepared as a substrate
control. All culture media were shaken for 14 days at 25 °C. The
resulting mycelium was separated from the broth by filtration. The broth
was extracted with EtOAc (3 × 500 mL), and the resulting EtOAc
2 4
solution was dried over anhydrous Na SO . After filtration, the solvent
1
the CH2OH group at C-16 in the H NMR spectrum of 4 differed
was removed by evaporation under reduced pressure to furnish a
yellowish residue (470 mg). The residue was purified by silica gel flash
chromatography (hexane/EtOAc, 3:7 v/v) to give products 4 (10 mg,
1
with that of 3, indicating that the resonance of H nuclei in the
CH2 group at C-16 is partially affected by the neighboring
hydroxyl group at C-11 via long-range coupling. This together
with the absence of γ-gauche effect over C-10 relative to 3 is
in good agreement with the proposed stereochemistry.
2
.1%), 5 (12 mg, 2.5%), and 6 (15 mg, 3.2%).
25
ent-11R,17,19-Trihydroxy-16âH-kaurane (4): oil; [R]
D
) -23°
-
(c 0.004, in CH
3
OH); IR (KBr) νmax 3300, 2900 cm 1; 1H NMR

3706 J. Agric. Food Chem., Vol. 50, No. 13, 2002
Vieira et al.
Table 1. 1H NMR (Pyridine-d
, 400 MHz, δ) Chemical Shift Values for Diterpenes 3−6
a
5
3
4
5
6
H
δ HR
δ Hâ
δ HR
δ Hâ
δ HR
δ Hâ
δ HR
δ Hâ
1
2
3
5
6
7
9
1.74 br d (12.8)
1.45−1.60 m
2.15 br d (12.8)
0.75 dt (3.7; 12.8)
1.65−1.75 m
0.93−0.99 m
0.95−1.05 m
1.32−1.45 m
1.20 ddd (1.2; 7.0; 12.0)
0.85−0.95 m
1.32−1.40 m
1.97 br d (12.8)
1.35−1.37 m
2.15 br d (13.1)
1.13−1.15 m
1.65−1.67 m
0.97 dt (4.0; 13.1)
1.03−1.05 m
1.30−1.50 m
1.68−1.70 m
1.55−1.60 m
1.70−1.80 m
1.35−1.45 m
2.10−2.20 m
0.87−0.97 m
1.70−1.80 m
1.00−1.08 m
2.10−2.20 m
2.07−2.08 m
1.74−1.76 m
1.37−1.39 m
2.14−2.16 m
0.73−0.75 m
1.59−1.61 m
0.92−1.05 m
1.12−1.14 m
2.25 dd (3.4; 11.9)
3.70−3.72 m
1.12−1.14 m
1.59−1.61 m
1.89−1.91 m
1.65−1.75 m
1.65 dd (1.2; 12.0)
1.68−1.70 m
2.27−2.29 m
1.83−1.85 m
3.80−3.82 m
2.14−2.16 m
1.74−1.76 m
1.55−1.65 m
1.83−1.84 m
1
1
1.65−1.75 m
1.45−1.60 m
2.36 br s
2.04 d (6.8)
1.86−1.88 m
2.48 br s
1.60−1.70 m
1.60−1.62 m
2.43 br s
1.74−1.76 m
1.59−1.61 m
2.45 br s
1
2
1.75−1.85 m
2.40−2.42 m
13
1
4
1.97 d (11.3)
0.94 br d (11.3)
1.45−1.55 m
1.93 d (11.2)
1.03 br d (11.2)
1.93 d (11.3)
1.51−1.53 m
2.49−2.51 m
1.14−1.16 m
2.42−2.43 m
2.14−2.16 m
2.74 t (12.3)
2.55 br s
1.74−1.76 m
1.10−1.12 m
1
5
1.55−1.57 m
16
2.35−2.47 m
2.46 br s
17
3.95 d (7.4)
4.35 dd (7.6; 10.4);
.58 dd (7.6; 10.4)
3.99 d (7.5)
4.06 d (7.6)
4
1
19
8
1.18 s
3.63 d (10.4);
.97 d (10.4)
0.99 s
1.18 s
3.63 d (10.8);
3.97 d (10.8)
0.92 s
1.27 s
3.71 d (10.6);
4.09 d (10.6)
1.08 s
1.20 s
3.70 d (10.4);
4.00 d (10.4)
1.08 s
3
20
a
Chemical shift values are in parts per million, and J values in hertz are presented in parentheses. All signals were assigned by COSY, HMQC, and NOESY spectra.
Table 2. 13C NMR (Pyridine-d
for Diterpenes 3−6
Table 3. Significant Long-Range NOE Peaks Observed for NOESY
Spectra of 4−6
a
5
, 100 MHz, δ) Chemical Shift Values
C
3
4
5
6
C
3
4
5
6
4
5
6
1
40.5 40.4 40.9 40.8
18.9 18.7 19.1 19.2
35.6 36.3 37.8 36.3
38.6 38.5 36.5 39.2
57.6 56.9 49.0 53.9
20.8 20.9 28.9 30.7
42.5 42.0 76.7 75.6
44.2 43.3 44.2 50.7
56.7 68.2 52.5 57.5
39.2 39.3 38.9 39.2
11 18.2 64.6 18.9 18.8
12 25.9 36.1 26.9 27.0
13 37.1 38.3 39.5 37.5
14 40.3 40.2 39.9 32.0
15 43.7 43.3 41.2 39.6
16 43.2 44.6 46.8 44.4
17 64.3 63.6 63.4 63.6
18 27.0 27.9 27.9 28.0
19 65.5 64.1 64.6 64.3
20 18.2 18.3 18.5 18.7
H-1R/H-20; H-12R; H-11R
H-1â/H-3â; H-5â; H-9â
H-3R/H-18;
H-1R/H-20
H-1R/H-20
2
3
4
5
6
7
8
9
H-1â/H-5â
H-1â/H-3â; H-5â; H-9â
H-3R/H-18;
H-3R/H-18;
H-3â/H-5â
H-3â/H-5â
H-3â/H-5â
H-5â/H-9â; H-7â
H-6R/H-20
H-7R/H-15R; H-14â
H-7â/H-9â; H-15â
H-11R/H-20
H-12â/H-17
H-14R/H-20
H-14â/H-16R; H-15R
H-15â/H-17
H-19R/H-20
H-5â/H-9â
H-5â/ H-7â
H-6R/H-20
H-6â/H-18
H-6â/H-18
H-7â/H-9â; H-15â
H-11â/H-17
H-7R/H-15R; H-14â
H-9â/H-15â
H-11â/H-17
H-12â/H-17
H-14R/H-20
H-14â/H-16R; H-15R
H-15â/ H-17
H-19R/ H-20
H-12R/H-20
H-12â/H-17
H-13R/H-16â
H-14R/H-20
H-14â/H-16R; H-15R
H-15â/H-17
H-19R/H-20
10
a
Excluding NOE cross-peaks between geminal or vicinal pairs of protons.
Figure 3. Phase-sensitive NOESY responses of ent-11R,17,19-trihydroxy-
16âH-kaurane (4).
Figure 2. Synthesis of ent-17,19-dihydroxy-16âH-kaurane (3) and its
biotransformations by V. lecanii.
1
1
formation of 3 to 5. On the basis of J correlation among H
1
13
The H spectrum of 5 showed a new one-proton signal
and C nuclei observed in the HMQC spectrum of 5, signals
13
centered at δ 3.80 (in the C NMR spectrum at δ 76.7), whereas
the 13C NMR spectrum of 5 showed the absence of a signal for
C-7 at δ 42.5 and the downfield shift of signal for C-6 from δ
centered at δ 1.15 and 1.93 were assigned to H-14 protons,
whereas those at δ 1.08 and 3.80-3.82 were assigned to H-20
and H-7, respectively. Furthermore, the corresponding 2D-
NOESY-NMR spectrum (see Table 3 and Figure 4) showed
that signals at δ 1.93 and 1.15 are correlated to those of H-20
20.8 to 28.9, as compared to that of the starting material 3. These
indicate the occurrence of hydroxylation at C-7 in the biotrans-

Biotransformation of ent-17,19-Dihydroxy-16âH-kaurane
J. Agric. Food Chem., Vol. 50, No. 13, 2002 3707
group in 6, which is also confirmed by the upfield shift of C-14
and C-15 signals, the γ-gauche effect relative to 3.
Compounds 4-6 are new compounds with no known natural
sources. Despite the low yields, the biotransformation of 3 into
its hydroxylated derivatives by V. lecanii is a very useful tool
for the preparation of such compounds and related ones for
biological testing or for submission to further chemical or
biological modifications. Improvement in the yields may be
achieved by evaluation of different biotransformation conditions
as well as by using immobilized cells or genetic engineering
tools. From a natural products chemistry point of view, this route
is of high interest because the chemical synthesis of 4-6 is
considerably difficult. The results so far are encouraging because
this methodology for the preparation of the same kind of novel
compounds is not only rapid and inexpensive but also ecologi-
cally compatible.
Figure 4. Phase-sensitive NOESY responses of ent-7R,17,19-trihydroxy-
16âH-kaurane (5).
LITERATURE CITED
(
1) Ghisalberti, E. L. The biological activity of naturally occurring
kaurane diterpenes. Fitoterapia 1997, 68, 303-325.
(
2) Katsumi, M.; Phinney, B. O.; Jefferies, P. C.; Henrick, C. A.
Growth Response of the d-5 and an-1 Mutants of Maize to Some
Kaurene Derivatives. Science 1964, 144, 849-850.
(
(
(
(
3) Takahashi, J. A.; Boaventura, M. A. D.; Bayma, J. C.; Oliveira,
A. B. Frutoic acid, a dimeric kaurane diterpene from Xylopia
frutescens. Phytochemistry 1995, 40, 607-609.
4) Vieira, H. S.; Takahashi, J. A.; Boaventura, M. A. D. Constituents
of aereal parts of Wedelia paludosa. Fitoterapia 2001, 72, 854-
8
56.
5) Panizo, M. F.; Rodriguez, B. Algunos componentes diterpenicos
del girasol (Helianthus annuus L.). An. Quim. 1979, 75, 428-
4
31.
6) Boaventura, M. A. D.; Hanson, J. R.; Hitchcock, P. B.;
Figure 5. Phase-sensitive NOESY responses of ent-7â,17,19-trihydroxy-
6âH-kaurane (6).
Takahashi, J. A. The biotransformation of ent-19-hydroxykaur-
1
1
1
6-en-15-one by Cephalosporium aphidicola. Phytochemistry
994, 37, 387-389.
at δ 1.08 and H-7 at δ 3.80-3.82, respectively. Therefore, the
signals at δ 1.93 and 1.15 correspond to H-14R and H-14â.
These data are compatible only with a â-stereochemistry for
the hydroxyl group at C-7. Furthermore, the upfield shift of
(
7) Ramachandra, Rao S.; Tripathi, U.; Ravishankar, G. A. Biotrans-
formation of codeine to morphine in freely suspended cells and
immobilized cultures of Spirulina platensis. World J. Microbiol.
Biotechnol. 1999, 15, 465-469.
1
3
signals assigned to C-5, C-9, and C-15, in the C NMR
spectrum, the γ-gauche effect relative to 3, is additional evidence
supporting the proposed stereochemistry.
(8) Hanson, J. R. Diterpenoids. Nat. Prod. Rep. 1995, 12, 207-
18.
2
(
9) Bucknall, R. A.; Moores, H.; Simms, R.; Hesp, B. Antiviral
effects of aphdicolin, a new antibiotic produced by Cephalospo-
rium aphidicola. Antimicrob. Agents Chemother. 1973, 4, 294-
1
In the H NMR spectrum of 6, an overlapped one-proton
signal centered at δ 3.70 (in the 13C NMR spectrum at δ 75.6)
indicated that compound 6 contains an additional hydroxyl group
as compared to the starting compound 3. Furthermore, the
presence of a hydroxyl group at C-7 in 6 is evidenced by the
absence of a signal at δ 42.5 corresponding C-7 in the starting
2
98.
(10) Henrick, C. A.; Jefferies, P. R. The chemistry of the Euphorbi-
aceae VII. The diterpenes of Ricinocarpus stylosus Diels. Aust.
J. Chem. 1964, 17, 915-933.
11) Dutta, T. K.; Samanta, T. B. Bioconversion of progesterone by
activated immobilized conidia of Aspergillus ochraceus TS. Curr.
Microbiol. 1999, 39, 309-312.
12) Boynton, J.; Hanson, J. R.; Hunter, A. C. The hydroxylation of
some 13R-methylsteroids by Cephalosporium aphidicola. Phy-
tochemistry 1997, 45, 951-956.
13) Nagao, Y.; Ito, N.; Kohno, T.; Kuroda, H.; Fujita, E. Antitumor
activity of Rabdosia and Teucrium diterpenoids against P 388
lymphocytic leukemia in mice. Chem. Pharm. Bull. 1982, 30,
(
(
(
1
3
material 3 and the downfield shifts observed in the C NMR
spectrum of 6. The signals for C-6 and C-8 of 6 are shifted
from δ 20.8 to 30.7 and from δ 44.2 to 50.7 relative to those of
1
3
, respectively. Here again, on the basis of J correlation among
1
13
H and C nuclei observed in the HMQC spectrum of 6, signals
at δ 1.75 and 2.15 were assigned to H-14 protons, whereas those
at δ 1.08 and 3.70 were assigned to H-20 and H-7, respectively.
Furthermore, the corresponding 2D-NOESY-NMR spectrum
see Table 3 and Figure 5) showed that the signal at δ 2.15 is
correlated with H-20. Consequently, the signals at δ 2.15 and
.75 correspond to H-14R and H-14â. Because the latter did
7
27-729.
(
14) Castellaro, S. J.; Dolan, S. C.; Macmillan: J.; Willis, C. L.
Deuterium labeling of kaur-16-19-oic acid at carbon-6 and -7.
Phytochemistry 1990, 29, 1823-1831.
(
1
not correlate to H-7, it is evident that 6 has a 7R-hydroxyl group
at C-7. In addition, an overlapped signal at δ 1.12-1.14 was
correlated to the signal at δ 3.70 (H-7â), and then the signal is
assigned to H-5â and H-9â. The spatial correlation elucidated
leads to the proposal of an R-stereochemistry for the 7-hydroxyl
Received for review January 3, 2002. Revised manuscript received
March 29, 2002. Accepted March 29, 2002. We acknowledge CNPq,
FAPEMIG, CAPES, and FINEP for grants and financial support.
JF020009F