Combined biotransformations of 4(20),11-taxadienes

Article abstract of DOI:10.1016/j.tet.2005.03.136

Taxuyunnanine C (1) and its analogs (2 and 3), the C-14 oxygenated 4(20), 11-taxadienes from callus cultures of Taxus sp., were regio- and stereo-selectively hydroxylated at the 7β position by a fungus, Abisidia coerulea IFO 4011, and it was interesting that the longer the alkyl chain of the acyloxyl group at C-14 became, the higher the yield of 7β-hydroxylated product was. Besides the three 7β-hydroxylated products (5, 9, 17), other nine new products (7, 11, 12, 14, 15, 16, 18, 20 and 21) and six known products (4, 6, 8, 10, 13 and 19) were obtained. Subsequently, the acetylated derivatives (24 and 27) of 7β-and 9α-hydroxylated products of 1 were regio- and stereo-specifically hydroxylated at the 9α position by Ginkgo cells and 7β position by A. coerulea, respectively. Thus, the two specific oxidations have been combined. These bioconversions would provide not only valuable intermediates for the semi-synthesis of paclitaxel or other bioactive taxoids from 1 and its analogs, but also some useful hints for the biosynthetic pathway of taxoid in the natural Taxus plant.

Full text of DOI:10.1016/j.tet.2005.03.136

Tetrahedron 61 (2005) 5507–5517
Combined biotransformations of 4(20),11-taxadienes
b
c
c
Jungui Dai,a, Lin Yang, Jun-ichi Sakai and Masayoshi Ando
*
aInstitute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 1 Xian Nong Tan Street,
Beijing 100050, People’s Republic of China
bCollege of Life and Environmental Sciences, The Central University for Nationalities, 27 South Zhongguancun Street, Beijing 100081,
People’s Republic of China
cDepartment of Chemistry and Chemical Engineering, Niigata University, Ikarashi 2-8050, Niigata 950-2181, Japan
Received 7 January 2005; revised 28 March 2005; accepted 30 March 2005
Available online 29 April 2005
Abstract—Taxuyunnanine C (1) and its analogs (2 and 3), the C-14 oxygenated 4(20), 11-taxadienes from callus cultures of Taxus sp., were
regio- and stereo-selectively hydroxylated at the 7b position by a fungus, Abisidia coerulea IFO 4011, and it was interesting that the longer
the alkyl chain of the acyloxyl group at C-14 became, the higher the yield of 7b-hydroxylated product was. Besides the three 7b-hydroxylated
products (5, 9, 17), other nine new products (7, 11, 12, 14, 15, 16, 18, 20 and 21) and six known products (4, 6, 8, 10, 13 and 19) were
obtained. Subsequently, the acetylated derivatives (24 and 27) of 7b-and 9a-hydroxylated products of 1 were regio- and stereo-specifically
hydroxylated at the 9a position by Ginkgo cells and 7b position by A. coerulea, respectively. Thus, the two specific oxidations have been
combined. These bioconversions would provide not only valuable intermediates for the semi-synthesis of paclitaxel or other bioactive
taxoids from 1 and its analogs, but also some useful hints for the biosynthetic pathway of taxoid in the natural Taxus plant.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
skin, head and neck cancers with encouraging results.5
Since the discovery of paclitaxel in the late 1960s, its unique
chemical structure, significant biological activity, as well as
its novel mechanism of action6,7 have led to research by
scientists from different fields.8,9
The diterpenoid paclitaxel (Taxolw, Scheme 1), originally
isolated from the Pacific yew (Taxus brevifolia Nutt.) in
1971,1 exhibited remarkably high cytotoxicity and strong
antitumor activity against different tumors resistantly
treated by existing anticancer drugs.2 It has been approved
for the treatment of advanced ovarian and breast cancers,3,4
and it is currently in clinical trials for treatment of lung,
The source of paclitaxel has been a serious problem all the
time since its only approved source was the bark of
T. brevifolia, which yielded very low amount of paclitaxel
(approximately 0.01% of the dry weight). Nowadays, the
demand in clinics increases largely because its anticancer
spectrum has been broadened and the wild Taxus trees have
been forbidden to cut for the supply of paclitaxel by the
governments of most countries in the world. This situation
illustrates a serious resource crisis that has to be alleviated
by using various approaches to produce alternate sources of
paclitaxel, such as total synthesis, semisynthesis, nursery
production of Taxus trees, fungal production, plant cell
culture, etc.10 Among them, plant tissue and cell culture of
Taxus species is considered as one of the most promising
approaches to produce paclitaxel and related taxanes. In the
past decade, there have been a lot of successful reports and
patents on the production of paclitaxel by callus or cell
culture of various Taxus species, but the paclitaxel content
was substantially different according to the particular Taxus
species being investigated.11–15 However, for most of these
cases, only paclitaxel accumulation was reported, and its
content was generally too low to scale-up industrially.
Scheme 1. The structures of paclitaxel, docetaxel and taxanes from cell
cultures of T. chinensis.
Keywords: Ginkgo biloba L.; Abisidia coerulea IFO 4011; Cell suspension
cultures; 4(20), 11-Taxadiene; Biotransformation; Combination of 7b-and
9a-oxidations.
Corresponding author. Tel.: C86 10 63165195; fax: C86 10 63017757;
e-mail: jgdai@imm.ac.cn
*
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.03.136

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J. Dai et al. / Tetrahedron 61 (2005) 5507–5517
More than 10 years ago, a high and stable taxane-yielding
callus strain of T. chinensis, (ca. 5–6% of the dry weight of
cell cultures) was screened out of various callus strains of
different Taxus species in our laboratory, which lost the
ability to produce paclitaxel and other C-13 oxygenated
taxoids, however, possessed the ability to produce C-14
oxygenated 4(20), 11-taxadienes only.16,17 The three major
taxanes are taxuyunnanine C [2a,5a,10b,14b-tetraacetoxy-
taxa-4(20),11-diene, 1], 2a,5a,10b-triacetoxy- 14b-(2-
methylbutyryl)oxytaxa-4(20),11-diene (2), and yunnanxane
{2a,5a,10b-triacetoxy-14b-[3 (s)-hydroxy-2 (R)-methyl-
butyryl]oxytaxa- 4(20),11-diene, 3} (Scheme 1). Their
high contents in the cultures and their taxane-skeleton
endow them with valuable potential for the semi-synthesis
of paclitaxel or other bioactive taxoids. Unfortunately, these
compounds have fewer functional groups with the skeleton
in comparison with paclitaxel and other bioactive taxoids,
such as at C-1, C-7, C-9 and C-13 positions. A number of
studies on their structural modification by chemical and
biocatalytic approaches were reported, and have achieved a
lot of intriguing results.18–25 However, the regio- and stereo-
selective introduction of oxygen functional groups at these
positions seems to be very difficult through conventional
chemical methods. In this context, the enzymatic conversion
by employing microorganisms or plant cell suspension
cultures is an alternative. In our previous investigation, 9a
and 7b selectively hydroxylated products of these
taxadeines were obtained successfully by cell suspension
cultures of G. biloba and the fungus A. coerulea,
respectively.22–27 However, could these two selective
oxidations be combined by the above two biocatalysts?
So, as a part of our ongoing investigations, herein, we report
the successful combination of the two reactions by
subsequent biotransformation with the aid of chemical
modification of the substrates. In addition, in our recent
communication, only specific 7b-hydroxylations of 1, 2 and
3 by A. coerulea were reported,24 however, there are other
fifteen products yielded from those bioprocesses, and nine
of them are new compounds. These results will be presented
in detail, and the effects of different substituents on the
biotransformation will also be discussed.
Scheme 2. The transformed products of 1, 2 and 3 by A. coerulea.
absorption at 3620 cmK1 1H NMR spectrum of 7 was
.
similar to that of 1 except that the signals of H-6a or H-6b
(2H, d 1.82, m) in 1 had disappeared, while a new
oxymethine proton signal at d 3.92 (1H, ddd, JZ4.1, 5.1,
11.8 Hz) was observed, which was correlated with H-7 and
H-5 in 1H–1H COSY spectrum, suggesting that the
introduced OH group was at C-6 position. It was confirmed
by the signal of C-6 which was substantially shifted
downfield at d 69.11 (d) compared with d 28.88 (t) in 1,
and by the signal of H-6 which was correlated with C-5 and
C-7 in HMBC spectrum. The stereochemistry of 6-OH was
determined to be a-configuration by the NOE difference
spectrum, in which the integration values of H-5, H-7b and
H-19 were enhanced when H-6 was irradiated. Therefore,
the structure of 7 was determined as 6a-hydroxy-
2a,5a,10b,14b-tetraacetoxytaxa-4(20),11-diene. Although
6a hydroxylation of 1 occurred in the cases of fungus
Cunninghamella echinulata, cell suspension cultures of
Catharanthus roseus and Ginkgo biloba as the bio-
catalysts,19–21,23 but in all cases, the C-10 acetyl group
was simultaneously removed to afford 8, thus this was the
first time to get 7 from 1 by biotransformation.
2. Results and discussion
After incubation with cell cultures of fungus A. coerulea for
7 days, five more polar metabolites were obtained from 1 by
the combination of open silica gel chromatography and
semi-prep. HPLC (Scheme 2). On the basis, of the physical
and spectroscopic data, their structures were identified as
9a-hydroxy-2a,5a,10b,14b-tetraacetoxytaxa-4(20),11-
diene (4, 1%),22 7b-hydroxy-2a,5a,10b,14b-tetraacetoxy-
taxa-4(20),11-diene (5, 5%),24 10b-hydroxy-2a,5a,14b-
triacetoxytaxa-4(20),11-diene (6, 15%),19–21,26 6a-
hydroxy-2a,5a,10b,14b-tetraacetoxytaxa-4(20),11-diene
(7, 2%), 6a,10b-dihydroxy-2a,5a,14b-triacetoxytaxa-
4(20),11-diene (8, 1%).19–21,23 Among them, only 7 was a
new compound.
Incubation of 2 with A. coerulea for 7 days yielded six
metabolites (Scheme 2). By combination of 1H NMR,
1H–1H COSY, 13C NMR, DEPT, HMQC, HMBC, NOE,
HREIMS and IR spectral analyses, their structures were
determined to be 7b-hydroxy-2a,5a,10b-triacetoxy-14b-(2-
methylbutyryl)oxytaxa-4(20),11-diene (9, 10%),24 9a-
hydroxy-2a,5a,10b-triacetoxy-14b-(2-methylbutyryl)oxy-
taxa-4(20),11-diene (10, trace),26 6a-hydroxy-2a,5a,10b-
triacetoxy-14b-(2-methylbutyryl)oxytaxa-4(20),11-diene
(11, 2%), 7b,10b-dihydroxy-2a,5a-diacetoxy-14b-(2-methyl-
butyryl)oxytaxa-4(20),11-diene (12, trace), 2a,5a,10b-tri-
acetoxy-14b-[3 (s)-hydroxy-2 (R)-methylbutyryl]oxytaxa-
The HREIMS, 1H and 13C NMR spectral data of 7 exhibited
an elemental composition of C28H40O9 (see Section 4.4.1),
suggesting that a hydroxyl group may be introduced. The
presence of an OH group in 7 was supported by the IR

J. Dai et al. / Tetrahedron 61 (2005) 5507–5517
5509
4(20),11-diene (13, yunnanxane28) and 2a,5a,10b-tri-
acetoxy-14b-[3(R)-hydroxy-2(R)-methylbutyryl]oxytaxa-
4(20),11-diene (14, 30-epimer of 13, obtained as a mixture
with 13, totally in 5% yield). Among them, 11, 12 and 14
were three new compounds.
yunnanxane) and d 4.04 (d 3.86 in yunnanxane), therefore,
the structure of 14 was determined to be 2a,5a,10b-
triacetoxy-14b-[3 (R)-hydroxy-2 (R)-methylbutyryl]oxy-
taxa- 4(20),11-diene, the 30-epimer of yunnanxane. The
result showed that 2 could not be specifically hydroxylated
at C-30 position by this fungus. To some extent, this result
indicated that 3 was biosynthesized from 2 through C-30
hydroxylation in the cell cultures of T. chinensis, however,
in a specific manner.
The HREIMS spectrum of 11 showed a molecular ion peak
[M]C at m/z 562.3154, consistent with the molecular
formula of C31H46O9, suggesting that an OH group may
be introduced in comparison with 2. The presence of an OH
group in 11 was supported by the IR absorption at
3 was administered to 2-day-old cell cultures of A. coerulea
and seven products were isolated by chromatographic
methods after additional 7 days of incubation. Based upon
the spectral and chemical data, their structures (Scheme 2)
were identified as 2a,5a,10b-triacetoxy-14b-(2-methyl-3-
keto)-butyryloxytaxa-4(20),11-diene (C-20 diastereo-
isomers, 15 and 16, totally in 10% yield), 7b-hydroxy-
2a,5a,10b-triacetoxy-14b-(3-hydroxy-2-methyl)-butyryl-
oxytaxa-4(20),11-diene (17, 15%),24 9a-hydroxy-
2a,5a,10b-triacetoxy-14b-(3-hydroxy-2-methyl)-butyryl-
oxytaxa-4(20),11-diene (18, 2%), 10b-hydroxy-2a,5a-
diiacetoxy-14b-(3-hydroxy-2-methyl)-butyryloxytaxa-
4(20),11-diene (19, 5%),29 6a-hydroxy-2a,5a,10b-tri-
acetoxy-14b-(3-hydroxy-2-methyl)-butyryloxytaxa-
4(20),11-diene (20, 2%), 7b,10b-dihydroxy-2a,5a-di-
acetoxy-14b-(3-hydroxy-2-methyl)-butyryloxytaxa-
4(20),11-diene (21, trace). Among them, 15, 16, 18, 20 and
21 were five new compounds.
1
3620 cmK1. H NMR spectrum of 11 was similar to that
of 2 except that the signals of H-6a or H-6b (2H, d 1.82, m)
had disappeared, while an additional oxymethine signal at d
3.96 (1H, ddd, JZ3.9, 5.1, 11.8 Hz) was observed,
suggesting that the OH group may be introduced at C-6
position. It was further supported by the signal of C-6 which
was significantly shifted downfield to d 68.16 (d) when
compared with d 28.88 (t) in 2, and the correlations of this
proton signal with C-5 and C-7 in HMBC spectrum. The
stereochemistry of 6-OH was determined to be a-configur-
ation by the NOE difference spectral experiment, in which
the integration values of H-5, H-7b and H-19 were enhanced
when H-6 was irradiated. Therefore, the structure of 11 was
determined as 6a-hydroxy-2a,5a,10b-triacetoxy-14b-(2-
methylbutyryl)oxytaxa-4(20),11-diene.
HRESIMS (negative) and HRESIMS (positive) of 12
displayed two quasi molecular ion peaks at m/z 519.3019
[MKH]C and 543.2963 [MCNa]C, respectively,
consistent with the molecular formula of C29H44O8. The
1H NMR spectrum of 12 was similar to that of 9, but only
two OAc signals were observed. The C-10 proton signal was
shifted upfield to 0.9 ppm as compared with that of 9,
strongly suggesting a free OH group at C-10 in 12. The 1H
NMR spectrum of 12 further showed that the resonances
corresponding to H-7a or 7b [d 1.24 (m); 1.98 (m)] in 2 had
disappeared, and one new oxygen-bearing methine signal
appeared at d 3.83 (dd, JZ5.1, 12.2 Hz), suggesting an
insertion of an OH group at C-7 position in 12. It was further
supported by the HMBC experiment that this proton was
correlated to C-5, C-6, C-8, C-9 and C-19. The stereo-
chemistry of 7-OH was determined to be b-configuration by
the NOE difference spectral analysis, in which the
integration values of H-3, H-6a, H-10 and H-18 were
enhanced when H-7 was irradiated. Thus, the structure of 12
was determined as 7b,10b-dihydroxy-2a,5a-diacetoxy-
14b-(2-methylbutyryl)oxytaxa-4(20),11-diene, which
could be biosynthesized from 9 via C-10 specific
deacetylation.
15 and 16 were obtained as a mixture of C-20 diastereo-
isomers. Normal and reverse phase HPLC had been used for
their separation, unfortunately, both efforts failed. The ratio
of this pair of isomers was 1:1 by the analysis of 1H NMR.
The HREIMS spectrum showed a molecular ion peak [M]C
at m/z 560.2987, consistent with the molecular formula of
C31H44O9. However, the OH absorption at 3572 cmK10 in 3
was not observed in IR spectrum, suggesting that C-3 OH
1
group probably oxidized to keto group. The H and 13C
NMR spectral data were very similar to those of 3 except
that the signals of H-30 at d 3.860(dq, JZ7.0, 6.8 Hz) had
disappeared, and the signal of C-3 was shifted downfield to
d 203.47 (s) as compared with that of 3 at d 69.47 (d), solidly
indicating the presence of C]O group at C-30 in 15 and 16
rather than an OH group. But how was 3 with a single
stereochemistry at C-20 converted to a pair of isomers with
20 (R) and 20 (s) configurations? There was no evidence for
the mechanism of this conversion yet.
HREI mass spectrum of 18 exhibited a molecular ion peak
[M]C at m/z 578.3102, consistent with the molecular
formula of C31H46O10, indicating the substitution of an
1
13 and 14 were obtained as a mixture, clearly as a pair of
additional OH group as compared with 3. The H NMR
1
isomers according to the H and 13C NMR spectra. The
spectral data were very similar to those of 3 except that the
signals corresponding to H-9a (d 1.64, dd, JZ5.6, 14.9 Hz)
or H-9b (d 2.38, m) in 3 had disappeared, while an oxygen-
bearing methine signal was observed at d 4.14 (d, JZ
9.8 Hz). And the signals of H-10a (d 6.06, dd, JZ5.6,
11.5 Hz) in 3 were shifted upfield to d 5.75 (d, JZ9.8 Hz).
All of these suggested that an OH group might be introduced
at C-9 position. It was confirmed by the signal of C-9 which
was significantly shifted downfield to d 76.23 (d) when
compared with d 43.88 (t) in 3, and the correlations of this
proton to C-3, C-7, C-8, C-10 and C-19 in HMBC spectrum.
separation of these two isomers was tried by normal and
reverse phase HPLC, however, it was troublesome and
failed to give positive results. The ratio of this pair of
isomers was 1:1 by the analysis of 1H NMR. The NMR and
IR spectral data of 13 were in good agreement with those of
yunnanxane {2a,5a,10b-triacetoxy-14b-[3 (s)-hydroxy-2
(R)-methylbutyryl]oxytaxa- 4(20),11-diene}, and it was
also supported by the HRMS experimental data.28 The
NMR data of 14 were very similar to those of 13 except that
the signals of C-30 and H-30 were at d 68.01 (d 69.47 in

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J. Dai et al. / Tetrahedron 61 (2005) 5507–5517
The stereochemistry of 9-OH was determined to be
a-configuration by the NOE difference spectrum, in which
the integration values of H-16 and H-19 were enhanced
when H-9 was irradiated. Accordingly, the structure of 18
was identified as 9a-hydroxy-2a,5a,10b-triacetoxy-14b-(3-
hydroxy-2-methyl)-butyryloxytaxa-4(20),11-diene.
biotransformation is an efficient approach to diversify
natural products.
Thus, both 7b and 9a hydroxylations of this type of taxanes
could be achieved by fungus A. coerulea and cell suspension
cultures of G. biloba,22,23 respectively. Clearly, 7b and 9a
hydroxylations of 1 were constituted two key steps to the
semi-synthesis of paclitaxel or other bioactive taxoids from
1 and /or its analogs, therefore, the combination of two
oxidations would be of interest and importance, and the
further efforts were carried out.
The HREIMS spectrum of 20 showed a molecular ion peak
[M]C at m/z 578.3084, consistent with the molecular
formula of C31H46O9, suggesting that an OH group may
be introduced in comparison with 3. The 1H NMR spectrum
of 20 was similar to that of 3 except that the free methylene
signals of H-6a or H-6b (2H, d 1.80, m) had disappeared,
while a new oxymethine signal at d 3.96 (1H, ddd, JZ3.9,
5.1, 11.8 Hz) was observed. In addition, in its 13C NMR
spectrum, the signal of C-6 was shifted downfield to d 68.16
(d) as compared with that of 3 at d 28.88 (t). These indicated
that OH group was introduced at C-6 position. It was further
confirmed by the observed correlations of this proton to C-5
and C-7 in HMBC spectrum. The stereochemistry of 6-OH
was determined to be a-configuration by the NOE difference
spectrum experiment, in which the integration values of
H-5, H-7b and H-19 were enhanced when H-6 was
irradiated. Therefore, the structure of 20 was determined
as 6a-hydroxy-2a,5a,10b-triacetoxy-14b-(3-hydroxy-2-
methyl)-butyryloxytaxa-4(20),11-diene.
First, 9a and 7b hydroxylated products (4, 5) were prepared
as described before,22–24 and bioconverted directly as the
substrates by fungus A. coerulea and cell suspension
cultures of Ginkgo following the method described as in
Section 4, respectively. However, not as expected, the
desired reactions-7b or 9a hydroxylation did not occur
while 10-deacetylation occurred in the both cases and
yielded products 22 and 23 (Scheme 3). Their structures
were identified as 9a,10b-dihydroxy-2a,5a,14b-triacetoxy-
taxa-4(20), 11-diene (22) and 7b,10b-dihydroxy-
2a,5a,14b-triacetoxy-taxa-4(20),11-diene (23)4 by IR,
NMR, HRMS analyses. 22 was a known compound
obtained firstly from the biotransformation of 1 by Ginkgo
cells,22 and 23 was a new compound.
The HRFABMS spectrum of 21 exhibited a quasi molecular
ion [MCNa]C at m/z 559.2886, consistent with the
molecular formula of C29H44O9. The H NMR spectrum
1
of 21 was similar to that of 17 except that only two OAc
groups were observed. The C-10 proton signal was shifted
upfield to 0.9 ppm as compared with that of 17, strongly
suggesting the existence of a free OH group at C-10 in 21.
The 1H NMR spectrum of 21 further showed that the
resonances corresponding to H-7a or 7b [d 1.24 (m); 1.96
(m)] in 3 had disappeared, and one additional oxygen-
bearing methine signal appeared at d 3.82 (dd, JZ5.0,
11.4 Hz), indicating an insertion of an OH group at C-7
position in 21. This was further supported by the HMBC
experiment that this proton signal was correlated to C-5,
C-6, C-8 and C-19. The stereochemistry of 7-OH was
unambiguously determined to be b-configuration based on
the NOE difference spectrum, in which the integration
values of H-3, H-6a, H-10 and H-18 were enhanced when
H-7 was irradiated. Therefore, the structure of 21 was
determined to be 7b,10b-dihydroxy-2a,5a-diacetoxy-14b-
(3-hydroxy-2-methyl)-butyryloxytaxa-4(20),11-diene (21),
which might be biosynthesized from 17 by specific
deacetylation at C-10.
Scheme 3. Subsequent biotransformation of 4 and 5 by A. coerulea and
Ginkgo cells.
The HRFABMS spectrum of 23 exhibited a quasi molecular
ion peak [MCNa]C at m/z 501.2472, consistent with the
molecular formula of C26H38O8, suggesting the removal of
one acetyl group in comparison with the molecular of 5. The
presence of three acetoxyl groups in 1H and 13C NMR
spectra of 23 confirmed the deduction. The 1H NMR
spectrum of 23 was similar to that of 5 except that the signal
of H-10b was shifted to an upper field at d 5.08 (dd, JZ5.1,
12.0 Hz) as compared with that of 5 at d 5.97 (dd, JZ5.1,
12.0 Hz), suggesting the existence of an OH group rather
than an OAc group at C-10. It was supported by its 13C
NMR spectrum in which the signal of C-10 was shifted to an
upper field at d 66.90 (d) compared with that of 5 at d 69.58
(d). Therefore, 23 was elucidated to be 7b,10b-dihydroxy-
2a,5a,14b-triacetoxy-taxa-4(20),11-diene, the 10-deacetyl
derivative of 5, might be formed through specific
deacetylation.
From the above results, all of the substrates (1, 2 and 3)
could be regio- and stereo-selectively hydroxylated at 7b,
9a and 6a positions by the fungus A. coerulea, which
suggested that the enzymes responsible for these reactions
were highly substrate-specific. It was interesting that the
longer the alkyl chain of acyloxyl group at C-14 became, the
higher the yield of 7b-hydroxylated product was. Addition-
ally, some other reactions also occurred, such as selective
deacetylation at C-10 position for all of them, hydroxylation
at C-30 position for 2, oxidation of OH group to C]O group
for 3. These results indicated that there were several types of
enzymes involved in the bioprocess, in other words,
The above results indicated somewhat that the enzymes
responsible for 7b and 9a hydroxylations were strictly
substrate-specific. Considering the effects of the structure of

J. Dai et al. / Tetrahedron 61 (2005) 5507–5517
5511
the substrate (substituent, polarity, conformation, etc.) on
the biotransformation, we tried the following strategies. 4
and 5 were first acetylated by routinely chemical
method, their acetylated products (24, 27; Scheme 4) were
subsequently biotransformed by fungus A. coerulea and cell
suspension cultures of Ginkgo, respectively. Intriguingly,
the desired hydroxylations took place in both cases, and
their corresponding 7b and 9a hydroxylated products (25
and 28) were obtained in about 2 and 10% yields,
respectively. Also one byproduct, compound 26 (4%) was
formed in the former incubation. Their structures were
9.0 Hz) and d 68.87 (d), respectively. So, compound 26
was identified to be 14b-hydroxy-2a,5a,9a,10b-tetra-
acetoxy-taxa-4(20),11-diene, the 14-deacetyl derivative of
compound 24.
The HRFABMS spectrum of 28 displayed two quasi
molecular ion peaks [MCNa]C and [MCH]C at m/z
601.2626 and 579.2742, respectively, consistent with the
molecular formula of C30H42O11, implying that an OH
group may be introduced. The presence of an additional OH
group in 28 was confirmed by the IR absorption at
1
determined on the basis of the 1H NMR, 1H–1H COSY, 13
C
3620 cmK1. The H NMR of 28 was similar to that of 27
except that the signal of H-9a or H-9b (1H, d 2.10, m; 1H, d
1.98, m) in 27 had disappeared, however, a new oxymethine
signal at d 4.20 (1H, d, JZ11.5 Hz) was observed,
suggesting the introduction of an OH group at C-9 position.
It was supported by the fact that the signal of C-9 was
shifted downfield at d 76.24 (d) in 13C NMR spectrum of 28
as compared with that of 27 at d 37.18 (t), and by the
correlations of this activated carbon with H-3, H-7, H-10
and H-19 in HMBC spectrum. The stereochemistry of 9-OH
was determined to be a-configuration by the NOE difference
spectrum, in which the integration values of H-16 and H-19
were enhanced when H-9 was irradiated. Therefore, the
structure of 28 was elucidated to be 9a-hydroxy-
2a,5a,7b,10b,14b-pentaacetoxy-taxa-4(20),11-diene.
NMR, DEPT, HMQC, HMBC, NOE, HRMS and IR
spectral data.
The HRFABMS spectrum of 25 showed two quasi
molecular ion peaks [MCNa]C and [MCH]C at m/z
601.2630 and 579.2736, respectively, consistent with the
molecular formula of C30H42O11, suggesting that an OH
group may be introduced. The presence of an OH group in
25 was confirmed by the IR absorption at 3614 cmK1. The
1H NMR spectrum of 25 was similar to that of 24 except that
the signals of H-7a or H-7b [2H, d 1.68, m] in 24 had
disappeared, while an additional oxymethine signal at d 3.90
(dd, JZ5.4, 11.5 Hz) was observed, indicating an OH
introduction at C-7 position. It was supported by the signal
of C-7 which was shifted to a downfield at d 71.67 (d) as
compared with d 27.27 (t) in 24, and by the correlations of
this proton with C-5, C-6, C-8, C-9 and C-19 in HMBC. The
stereochemistry of 7-OH was determined to be b-configur-
ation by the NOE difference spectrum, in which the
integration values of H-3, H-6a, H-10 and H-18 were
enhanced when H-7 was irradiated. Thus, the structure of 25
was determined as 7b-hydroxy-2a,5a,9a,10b,14b-penta-
acetoxy-taxa- 4(20),11-diene.
Thus, with the aid of simple chemical acetylation, the two
selective 9a and 7b bio-oxidations of taxuyunanine C were
combined by the cell cultures of G. biloba and fungus A.
coerulea. The results suggested that the structure of the
substrate (substituent, polarity, etc.) had influenced on the
enzymatic process, and there have already been a great
many of reports on it.26,30–33 On the other hand, it was
implied that subtle modification to the structure of substrate
(i.e. substrate engineering) was one of the most efficient and
simplest methods for obtaining helpful changes (e.g.
biotransformation mode, yield, etc.) in biotransformation.
Furthermore, the biotransformation of taxanes by employ-
ing plant and/or microbial cells may biomimic some steps of
taxoid biosynthesis, not only extensive oxidations of the
taxane skeleton, but also the order(s) of these functionaliza-
tions. It is well known that 9a and 7b oxidations are the two
key steps in the taxoid biosynthesis, however, the order of
the two steps of taxoids biosynthesis still remains unclear.
Our results might somewhat provide a hypothesis that the 7b
hydroxylation would occur before 9a functionalization or
The HRFABMS spectrum of 26 exhibited two quasi
molecular ion peaks [MCNa]C and [MCH]C at m/z
543.2571 and 521.2751, respectively, consistent with the
molecular formula of C28H40O9. The 1H NMR spectrum of
26 was similar to that of 24 except that four OAc groups
were observed, indicating that one acetyl group may be
eliminated, and it was supported by the IR absorption at
3624 cmK1. It was further confirmed by which the signals of
1
H-14a and C-14 in H NMR and 13C NMR spectra of 26
were shifted upfield at d 4.08 (dd, JZ5.0, 9.0 Hz) and d
67.56 (d) as compared with those of 24 at d 4.97 (dd, JZ4.9,
Scheme 4. Combination of 9a and 7b oxidations of 1 by A. coerulea and Ginkgo cells.

5512
J. Dai et al. / Tetrahedron 61 (2005) 5507–5517
after 9a acetoxylation, while, 9a hydroxylation would occur
before 7b functionalization or after 7b acetoxylation. To
some extent, the results were in good accordance with the
results reported by Croteau and his colleagues recently.34
4.3. Organisms, media and cultivation conditions
The cell suspension cultures of G. biloba were cultivated in
500 mL Erlenmeyer flask with 150 mL of liquid MS
medium supplemented with 0.5 mg/L of naphthalene acetic
acid, 0.5 mg/L of 6-benzylaminopurine and 0.2 mg/L of
2,4-dichlorophenoxy acetic acid on the rotary shaker at
110 rpm at (25G2) 8C in the dark.35 The inoculum size was
5 g/L of cell cultures (dry weight) and subcultured every 21
days. The cell cultures were maintained in the above
conditions before use for the biotransformation. The fungus,
A. coerulea IFO4011was purchased from Institute for
Fermentation, Osaka, Japan (IFO), and kept on solid PDA
medium containing potato (200 g/L), sucrose (20 g/L) and
agar (2%) at 4 8C. The seed cultures were prepared in
500 mL flask with 150 mL of liquid medium (PDA medium
without agar) and incubated for 2 days. Seed cultures
(5 mL) was added to 500 mL flask and shaken at 110 rpm at
(25G2) 8C in the dark for the use of biotransformation.
3. Conclusion
In conclusion, three C-14 oxygenated taxanes with different
substitution groups at C-14 position could be regio- and
stereo-selectively hydroxylated at 7b position by fungus A.
coerulea in different yields. Moreover, nine new taxoids of
the 18 products were obtained from these biotransformatios.
Most importantly, the combination of 7b- and 9a-oxidations
by two different biocatalytic systems, A. coerulea and
Ginkgo cells, has been achieved with the aid of simple
chemical modification of the substrates. The results would
supply the very useful intermediates for the semi-synthesis
of paclitaxel and other bioactive taxoids from readily
available natural products—taxuyunanine C and/or its
analogs, although in low yields. Additionally, these results
might provide a useful tool to probe some important
biosynthetic steps of taxoids and/or the order of these steps
in Taxus plant.
4.4. Biotransformation of 1 with A. coerulea
1 (400 mg) was dissolved in EtOH (8.0 mL), distributed
among forty flasks of 2-day-old cultures and incubated for
additional 7 days, after which the cultures were filtered
under vacuum, and the filtrate was saturated with NaCl and
extracted 5 times with ethyl acetate. All the extracts were
pooled, dried with anhydrous Na2SO4, and concentrated
under vacuum at 40 8C to give 700 mg of residue. The dried
cell cultures were extracted thrice by sonication with ethyl
acetate, the resulting extracts were pooled and concentrated
under vacuum at 40 8C to afford 136.9 mg of residue. The
above two parts of extracts were combined and separated by
combination of open silica gel chromatography and normal
phase semi-prep. HPLC to afford 1 (260 mg, 65%; analyzed
4. Experimental
4.1. General
Optical rotations were obtained using a Horiba SEPA-200
polarimeter. 1H NMR (500 MHz) and 13C NMR (125 MHz)
spectra were recorded with a Varian Unity-PS instrument
using CDCl3 as solvent and internal standard. 1H NMR and
1
by TLC and H NMR), 4 (4.5 mg, ca. 1%; tRZ7.6 min;
mobile phase: hexane/ethyl acetateZ50/50, v/v), 5 (20 mg,
ca. 5%; tRZ11.3 min; mobile phase: hexane/ethyl acetateZ
50/50, v/v), 6 (60 mg, ca. 15%; tRZ8.1 min; mobile phase:
hexane/ethyl acetateZ50/50, v/v), 7 (9.0 mg, ca. 2%; tRZ
19.6 min; mobile phase: hexane/ethyl acetateZ50/50, v/v),
and 8 (4.0 mg, ca. 1%; tRZ11.7 min; mobile phase: hexane/
ethyl acetateZ30/60, v/v).
1
13C NMR assignments were determined by H–1H COSY,
DEPT, HMQC and HMBC experiments. HRFABMS were
carried out on a JEOL-HX 110 FAB-mate instrument and
HREIMS on a JEOL-HX 110 instrument. HRESIMS were
performed on a Bruker Daltonics APEX II with a 7 T
magnet, compounds formed sodiated ions under the ESI
condition with a resolution range from 31,000 to 60,000. IR
spectra were taken on a Hitachi 270-30 spectrometer in
CHCl3. Semi-preparative HPLC was performed on a
Hitachi L-6200 HPLC instrument with an Inertsil Prep-sil
(GL Science, 25 cm!10 mm i.d.) stainless steel column
and an YRU-883 RI/UV bi-detector, the flow rate was
5.0 mL/min unless otherwise mentioned. Silica gel (230–
300 mesh) was employed for flash column chromatography,
analytical TLC plates (silica gel 60 F254, Merck) were
visualized at UV254 and by spraying 5% H2SO4 (in EtOH)
followed by heating. Pyridine for acetylation was distilled
from CaH2, and the reactions were run under an atmosphere
of N2.
4.4.1. 6a-Hydroxy-2a,5a,10b,14b-tetraacetoxy-taxa-
4(20),11-diene (7). White powder; [a]2D0 C44.98 (c 0.5,
CHCl3); IR nmax (CHCl3): 3620, 2936, 1732, 1436, 1240,
1102, 1020 cmK1; 1H NMR (CDCl3, 500 MHz) d 1.90 (1H,
d, JZ2.0 Hz, H-1), 5.35 (1H, dd, JZ2.1, 6.4 Hz, H-2), 2.89
(1H, d, JZ6.3 Hz, H-3), 5.43 (1H, d, JZ3.7 Hz, H-5), 3.92
(1H, ddd, JZ4.1, 5.1, 11.8 Hz, H-6), 1.82 (1H, dd, JZ12.4,
12.4 Hz, H-7b), 1.56 (1H, dd, JZ5.1, 12.7 Hz, H-7a), 2.35
(1H, dd, JZ12.2, 14.6 Hz, H-9b), 1.68 (1H, dd, JZ5.4,
14.9 Hz, H-9a), 6.03 (1H, dd, JZ5.6, 12.2 Hz, H-10), 2.81
(1H, dd, JZ9.0, 19.0 Hz, H-13b), 2.44 (1H, dd, JZ4.9,
19.0 Hz, H-13a), 4.99 (1H, dd, JZ4.9, 9.3 Hz, H-14), 1.65
(3H, s, H-16), 1.12 (3H, s, H-17), 2.11 (3H, br s, H-18), 0.86
(3H, s, H-19), 5.38 (1H, s, H-20a), 4.94 (1H, s, H-20b), 2.24,
2!2.05, 2.02 [3H each, OAc (CH3)]; 13C NMR (CDCl3,
125 MHz) d 58.8 (d, C-1), 70.4 (d, C-2), 41.2 (d, C-3), 140.0
(s, C-4), 80.4 (d, C-5), 69.1 (d, C-6), 42.2 (t, C-7), 38.0 (s,
C-8), 43.5 (t, C-9), 69.9 (d, C-10), 135.4 (s, C-11), 134.9 (s,
C-12), 39.4 (t, C-13), 70.2 (d, C-14), 37.3 (s, C-15), 25.4 (q,
4.2. Substrates
Compounds 1–3 (purities: O95% by HPLC analyses) were
isolated from callus cultures (Ts-19 strain) of T. chinensis
and identified by chemical and spectral methods.16,17 the
substrates were dissolved in EtOH (50 mg/mL) before use.

J. Dai et al. / Tetrahedron 61 (2005) 5507–5517
5513
C-16), 31.8 (q, C-17), 21.0 (q, C-18), 23.4 (q, C-19), 119.7
(t, C-20), 21.7, 2!21.42, 21.4 [q, OAc (CH3)], 171.0,
170.1, 170.0, 169.9 [s, OAc (CO)]; HREIMS m/z 520.2666
[M]C (calcd 520.2672 for C28H40O9).
4.88 (1H, s,0 H-20b), 2.26–2.38 (1H, m, H-20), 1.64–1.72
(1H, m, H-3 a), 1.45–1.52 (1H, m, H-30b), 0.89 (3H, t, JZ
7.3 Hz, H-40), 1.12 (3H, d, JZ6.8 Hz, H-50), 2.18, 2.07 [3H
each, OAc (CH3)]; 13C NMR (CDCl3, 125 MHz) d 59.4 (d,
C-1), 70.1 (d, C-2), 40.4 (d, C-3), 140.4 (s, C-4), 77.5 (d,
C-5), 37.5 (t, C-6), 69.3 (d, C-7), 44.4 (s, C-8), 40.5 (t, C-9),
66.9 (d, C-10), 139.0 (s, C-11), 132.2 (s, C-12), 37.0 (t,
C-13), 70.2 (d, C-14), 37.4 (s, C-15), 25.4 (q, C-16), 32.0 (q,
C-17), 021.2 (q, C-18), 16.7 (q, C-19), 117.9 (t, C-20), 175.7
(s, C-1 ), 41.1 (d, C-20), 26.8 (t, C-30), 11.6 (q, C-40), 16.6 (q,
C-50), 21.8, 21.4 [q, OAc (CH3)], 169.8, 169.9 [s, OAc
(CO)]; HRESIMS (negative) m/z 519.3019 [M-H]C (calcd
519.2958 for C29H43O8) and HRESIMS (positive) m/z
543.2963 [MCNa]C (calcd 543.2934 for C29H44O8Na).
4.5. Biotransformation of 2 with A. coerulea
The procedures were performed as described in Section 4.4,
except that 540 mg of 2 was used, finally 1162 mg of extract
(472 mg for filtrate, 690 mg for cell cultures) was afforded.
The extract was fractionated and separated by combination
of open silica gel chromatography and normal phase semi-
prep. HPLC (in this experiment the flow rate was 4 mL/min)
to give 2 (360 mg, 66.7%; analyzed by TLC and 1H NMR),
9 (60 mg, ca. 10%; tRZ34.6 min; mobile phase: hexane/
ethyl acetateZ70/30, v/v), 10 (4 mg; tRZ23.8 min; mobile
phase: hexane/ethyl acetateZ75/25, v/v), 11 (12 mg, ca.
2%; tRZ28.1 min; mobile phase: hexane/ethyl acetateZ60/
40, v/v), 12 (3 mg; tRZ15.9 min; mobile phase: hexane/ethyl
acetateZ60/30,v/v), 13 and 14 (isomers, 28 mg, ca.5%; tRZ
31.7 min; mobile phase: hexane/ethyl acetateZ70/30, v/v).
4.5.3. 2a,5a,10b-Triacetoxy-14b-[3(R)-hydroxy-2(R)-
methyl]-butyryloxytaxa-4(20),11-diene (14). White
powder (mixed with 13); IR nmax (CHCl3): 3620, 2994,
2936, 1728, 1646, 1240, 1102, 1020 cmK1 1H NMR
;
(CDCl3, 500 MHz) d 1.88 (1H, d, JZ2.0 Hz, H-1), 5.34
(1H, dd, JZ2.2, 6.6 Hz, H-2), 2.93 (1H, d, JZ6.4 Hz, H-3),
5.29 (1H, br s, H-5), 1.76–1.86 (2H, m, H-6), 1.97 (1H, ddd,
JZ6.3, 13.0, 13.0 Hz, H-7a), 1.20–1.26 (1H, m, H-7b),
2.35–2.45 (1H, m, H-9b), 1.64 (1H, dd, JZ5.8, 13.9 Hz,
H-9a), 6.06 (1H, dd, JZ5.6, 11.9 Hz, H-10), 2.85 (1H, dd,
JZ9.3, 19.0 Hz, H-13b), 2.38–2.46 (1H, m, H-13a), 5.03
(1H, dd, JZ4.6, 9.0 Hz, H-14), 1.67 (3H, s, H-16), 1.13 (3H,
s, H-17), 2.10 (3H, s, H-18), 0.85 (3H, s, H-19), 5.280(1H, s,
H-20a), 4.83 (1H, s, H-20b), 2.40–2.45 (1H, m, H-2 ), 4.04
(1H, dq, JZ6.3, 5.6 Hz, H-30), 1.16 (3H, d, JZ7.3 Hz,
H-40), 1.17 (3H, d, JZ7.0 Hz, H-50), 2.18, 2.06, 2.04 [s, 3H
each, OAc (CH3)]; 13C NMR (CDCl3, 125 MHz) d 59.2 (d,
C-1), 70.5 (d, C-2), 42.1 (d, C-3), 142.2 (s, C-4), 78.2 (d,
C-5), 28.9 (t, C-6), 33.8 (t, C-7), 39.7 (s, C-8), 43.9 (t, C-9),
70.0 (d, C-10), 135.4 (s, C-11), 134.6 (s, C-12), 39.5 (t,
C-13), 70.8 (d, C-14), 37.3 (s, C-15), 25.4 (q, C-16), 31.7 (q,
C-17), 020.9 (q, C-18), 022.5 (q, C-19),0116.9 (t, C-20), 174.7
(s, C-1 ), 46.6 (d, C-2 ), 68.0 (d, C-3 ), 19.9 (q, C-40), 11.0
(q, C-50), 21.9, 21.4, 20.9 [q, OAc (CH3)], 170.2, 170.0,
169.8 [s, OAc (CO)]; HREIMS m/z 562.3143 [M]C (calcd
562.3142 for C31H46O9).
4.5.1. 6a-Hydroxy-2a,5a,10b-triacetoxy-14b-(2-methyl)-
butyryloxytaxa-4(20),11-diene (11). White powder; [a]2D0 C
47.98 (c 0.1, CHCl3); IR nmax (CHCl3): 3620, 2936, 1732,
1436, 1374, 1240, 1102, 1020 cmK1 1H NMR (CDCl3,
;
500 MHz) d 1.92 (1H, d, JZ2.0 Hz, H-1), 5.39 (1H, dd, JZ
2.2, 6.6 Hz, H-2), 2.94 (1H, d, JZ6.4 Hz, H-3), 5.47 (1H, d,
JZ3.9 Hz, H-5), 3.96 (1H, ddd, JZ3.9, 9.3, 16.1 Hz, H-6),
1.86 (1H, dd, JZ12.5, 12.5 Hz, H-7b), 1.59 (1H, dd, JZ5.6,
12.5 Hz, H-7a), 2.35–2.45 (1H, m, H-9b), 1.72 (1H, dd, JZ
5.9, 14.9 Hz, H-9a), 6.07 (1H, dd, JZ5.6, 12.2 Hz, H-10),
2.88 (1H, dd, JZ9.2, 19.0 Hz, H-13b), 2.42 (1H, dd, JZ4.4,
19.0 Hz, H-13a), 5.02 (1H, dd, JZ4.6, 9.0 Hz, H-14), 1.70
(3H, s, H-16), 1.16 (3H, s, H-17), 2.15 (3H, br s, H-18), 0.90
(3H, s, H-19), 5.41 (1H, s, H0-20a), 4.94 (1H, s, H-20b), 2.37
(1H, dq, JZ6.8, 7.3 Hz, H-2 ), 1.68 (1H,0dq, JZ6.8, 7.3 Hz,
H-30a), 1.49 (1H, dq, JZ7.1, 7.3 Hz, H-3 b), 0.92 (3H, t, JZ
7.3 Hz, H-40), 1.15 (3H, d, JZ7.3 Hz, H-50), 2.29, 2.09, 2.05
[3H each, OAc (CH3)]; 13C NMR (CDCl3, 125 MHz) d 59.1
(d, C-1), 70.2 (d, C-2), 41.3 (d, C-3), 140.0 (s, C-4), 80.3 (d,
C-5), 69.2 (d, C-6), 42.2 (t, C-7), 38.0 (s, C-8), 43.6 (t, C-9),
69.9 (d, C-10), 135.4 (s, C-11), 135.0 (s, C-12), 39.7 (t, C-13),
70.0 (d, C-14), 37.3 (s, C-15), 25.4 (q, C-16), 31.7 (q, C-17),
21.0 (q, C-18), 23.4 (q, C-19), 119.6 (t, C-20), 175.7 (s, C-10),
41.1(d, C-20), 26.8(t, C-30), 11.6(q, C-40), 16.6(q, C-50), 21.8,
21.3, 21.4 [q, OAc (CH3)], 171.0, 170.1, 169.9 [s, OAc (CO)];
HREIMS m/z 562.3154 [M]C (calcd 562.3142 for C31H46O9).
4.6. Biotransformation of 3 with A. coerulea
The procedures were carried out as described in Section 4.4,
except that 500 mg of 3 was used, finally 973 mg of extract
(500 mg for filtrate, 473 mg for cell cultures) was afforded.
The extract was fractionated and separated by combination
of open silica gel chromatography and normal phase semi-
prep. HPLC to yield 300 mg of 3 (ca. 60%; analyzed by
TLC and 1H NMR), 50 mg of the mixture of 15 and 16 (ca.
10%; isomers, tRZ13.1 min; mobile phase: hexane/ethyl
acetateZ7/3, v/v), 76 mg of 17 (ca. 15%; tRZ16.8 min;
mobile phase: hexane/ethyl acetateZ50/50, v/v), 2 mg of 18
(tRZ12.9 min, mobile phase: hexane/ethyl acetateZ50/50,
v/v), 25.0 mg of 19 (ca. 5%; tRZ14.7 min; mobile phase:
hexane/ethyl acetateZ50/50,v/v), 10 mg of 20 (ca. 2%;
tRZ18.0 min; mobile phase: hexane/ethyl acetateZ1/2,
v/v), and 2 mg of 21 (tRZ20.5 min; mobile phase: hexane/
ethyl acetateZ1/4, v/v).
4.5.2.
7b,10b-Dihydroxy-2a,5a-diacetoxy-14b-(2-
methyl)-butyryloxytaxa-4(20),11-diene (12). White
powder; [a]D20 C59.58 (c 0.3, CHCl3); IR nmax (CHCl3):
3624, 2972, 2940, 1732, 1460, 1376, 1244, 1214, 1154,
1
1084, 1058, 1020 cmK1; H NMR (CDCl3, 500 MHz) d
1.87 (1H, d, JZ2.0 Hz, H-1), 5.42 (1H, dd, JZ2.2, 6.6 Hz,
H-2), 2.83 (1H, d, JZ6.6 Hz, H-3), 5.33 (1H, t, JZ3.4 Hz,
H-5), 2.05–2.15 (1H, m, H-6a), 1.58–1.62 (1H, m, H-6b),
3.83 (1H, dd, JZ5.1, 12.2 Hz, H-7), 2.25–2.35 (1H, m,
H-9b), 2.10–2.16 (1H, m, H-9a), 5.08 (1H, dd, JZ5.4,
11.7 Hz, H-10), 2.83 (1H, dd, JZ9.3, 19.0 Hz, H-13b), 2.37
(1H, dd, JZ5.1, 19.0 Hz, H-13a), 4.95 (1H, dd, JZ4.9,
9.3 Hz, H-14), 1.76 (3H, s, H-16), 1.19 (3H, s, H-17), 1.95
(3H, br s, H-18), 0.76 (3H, s, H-19), 5.28 (1H, s, H-20a),
4.6.1. 2a,5a,10b-Triacetoxy-14b-(2-methyl-3-keto)-
butyryloxytaxa-4(20),11-diene (isomers, 15 and 16, the

5514
J. Dai et al. / Tetrahedron 61 (2005) 5507–5517
1
ratio was 1:1 by H NMR analysis). White powder; IR
nmax (CHCl3): 3000, 2940, 1730, 1646, 1456, 1376, 1320,
3612, 2940, 1730, 1606, 1458, 1376, 1320, 1240, 1166, 1112,
1020 cmK1; 1H NMR (CDCl3, 500 MHz) d 1.91 (1H, d, JZ
2.2 Hz, H-1), 5.35 (1H, dd, JZ2.2, 6.6 Hz, H-2), 2.89 (1H, d,
JZ6.6 Hz, H-3), 5.43 (1H, d, JZ3.9 Hz, H-5), 3.92 (1H, ddd,
JZ3.9, 5.3, 12.0 Hz, H-6), 1.82 (1H, dd, JZ12.4, 12.4 Hz,
H-7b), 1.56(1H, dd, JZ5.1, 12.5 Hz, H-7a), 2.35(1H, dd, JZ
12.2, 14.9 Hz, H-9b), 1.68 (1H, dd, JZ5.6, 14.9 Hz, H-9a),
6.03 (1H, dd, JZ5.6, 12.0 Hz, H-10), 2.84 (1H, dd, JZ9.2,
19.0 Hz, H-13b), 2.39 (1H, dd, JZ4.6, 19.0 Hz, H-13a), 5.02
(1H, dd, JZ4.6, 9.3 Hz, H-14), 1.63 (3H, s, H-16), 1.12(3H, s,
H-17), 2.11 (3H, br s, H-18), 0.86 (3H, s, H-19), 5.38 (1H,0s,
H-20a), 4.91 (1H, s, H-20b), 2.39 (0 dq, JZ7.0, 7.3 Hz, H-2 ),
3.86 (1H, dq, JZ6.3, 7.0 Hz, H-3 ), 1.20 (3H, d, JZ6.3 Hz,
H-40), 1.15(3H, d,JZ7.3 Hz, H-50),2.25, 2.05, 2.02[3Heach,
OAc (CH3)]; 13C NMR (CDCl3, 125 MHz) d 58.9 (d, C-1),
70.2 (d, C-2), 41.3 (d, C-3), 134.0 (s, C-4), 80.3 (d, C-5), 69.1
(d, C-6), 42.2 (t, C-7), 38.0 (s, C-8), 43.5 (t, C-9), 69.8 (d,
C-10), 135.4 (s, C-11), 134.8 (s, C-12), 39.5 (t, C-13), 70.6 (d,
C-14), 37.2 (s, C-15), 25.3 (q, C-16), 31.7 (q, C-17), 22.0 (q,
C-18), 23.4 (q, C-19), 119.7 (t, C-20), 174.8 (s, C-10), 47.0 (d,
C-20), 69.5 (d, C-30), 20.9 (q, C-40), 14.0 (q, C-50), 21.8, 21.4,
21.4 [q, OAc (CH3)], 171.0, 170.1, 169.9 [s, OAc (CO)];
HREIMS m/z 578.3084 [M]C (calcd 578.3091 for
C31H46O10).
1246, 1230, 1156, 1106, 1072, 1018 cmK1 1H NMR
;
(CDCl3, 500 MHz) d 1.86 (1H, br s, H-1), 5.35 (1H, dd,
JZ2.4, 6.6 Hz, H-2), 2.910 and 2.904 (1H, d, JZ6.1 Hz, H-3,
15 and 16), 5.28(1H, br s, H-5), 1.75–1.85 (2H, m, H-6), 1.90–
2.02 (1H, m, H-7b), 1.20–1.28 (1H, m, H-7a), 2.32–2.44 (1H,
m, H-9b), 1.60–1.70 (1H, m, H-9a), 6.04 (1H, dd, JZ5.5,
11.9 Hz, H-10), 2.84 (1H, dd, JZ9.3, 19.3 Hz, H-13b), 2.36–
2.44 (1H, m, H-13a), 5.033 and 5.024 (1H, dd, JZ5.1, 9.8 Hz,
H-14, 15 and 16), 1.65 (3H, s, H-16), 1.08 and 1.07 (3H, s,
H-17, 15 and 16), 2.09 (3H, br s, H-18), 0.84 (3H, s, H-19),
3.471 and 3.465 (1H, q, JZ7.2 Hz, H-20, 15 and 16), 2.22 and
2.21 (3H, s, H-40, 15 and 16), 1.32 (3H, d, JZ7.3 Hz, H-50),
2.086(3H,s, 2-OAc), 2.176and2.168(3H, 5-OAc, 15and16),
2.043 and 2.035 (3H, s, 10-OAc, 15 and 16); 13C NMR
(CDCl3, 125 MHz) d 59.02 and 59.00 (d, C-1, 15 and 16),
70.39 and 70.33 (d, C-2, 15 and 16), 42.13 and 42.11 (d, C-3,
15 and 16), 142.19 and 142.14 (s, C-4, 15 and 16), 78.2 (d,
C-5), 29.0 (t, C-6), 33.80 and 33.77 (t, C-7, 15 and 16), 39.7 (s,
C-8), 43.8 (t, C-9), 70.00 and 69.98 (d, C-10, 15 and 16),
135.44 and 135.34 (s, C-11, 15 and 16), 134.53 and 134.44 (s,
C-12, 15 and 16), 39.27 and 39.16 (t, C-13, 15 and 16), 71.7 (d,
C-14), 37.20 and 37.18 (s, C-15, 15 and 16), 25.3 (q, C-16),
31.6 (q, C-17), 21.4 (q, C-18), 22.5 (q, C-19), 116.94 and
116.91 (t, C-20, 15 and 16),0169.43 and 169.35 (s, C-10, 15 and
16),053.59and53.57(d, C-2 , 15and16), 203.47and203.33(s,
C-30, 15 and 16), 28.64 and 28.57 (q, C-40, 15 and 16), 12.7 (q,
C-5 ), 21.4 (q, 2-OAc), 21.9 (q, 5-OAc), 20.92 and 20.90 (q,
10-OAc, 15 and 16), 169.98 and 169.91 (s, 2-OAc, 15 and 16),
169.76 and 169.73 (s, 5-OAc, 15 and 16), 170.23 and 170.21
(s, 10-OAc, 15 and 16); HREIMS m/z 560.2987 [M]C (calcd
560.2985 for C31H44O9).
4.6.4.
7b,10b-Dihydroxy-2a,5a-diacetoxy-14b-(3-
hydroxy-2-methyl)-butyryloxytaxa-4(20),11-diene (21).
White powder; [a]2D0 C45.08 (c 0.3, CHCl3); IR nmax
(CHCl3): 3624, 2992, 2944, 1732, 1644, 1456, 1376, 1320,
1242, 1220, 1168, 1114, 1048, 1004 cmK1 1H NMR
;
(CDCl3, 500 MHz) d 1.90 (1H, d, JZ2.0 Hz, H-1), 5.42
(1H, dd, JZ2.2, 6.6 Hz, H-2), 2.89 (1H, d, JZ6.4 Hz, H-3),
5.23 (1H, t, JZ3.9 Hz, H-5), 2.08–2.16 (1H, m, H-6a),
1.60–1.70 (1H, m, H-6b), 3.82 (1H, dd, JZ5.0, 11.4 Hz,
H-7), 2.29 (1H, dd, JZ5.4, 14.9 Hz, H-9b), 1.68 (1H, dd,
JZ10.7, 14.9 Hz, H-9a), 5.08 (1H, dd, JZ5.4, 10.7 Hz,
H-10), 2.80 (1H, dd, JZ9.3, 18.8 Hz, H-13b), 2.39 (1H, dd,
JZ4.9, 18.8 Hz, H-13a), 4.99 (1H, dd, JZ4.9, 9.3 Hz,
H-14), 1.75 (3H, s, H-16), 1.19 (3H, s, H-17), 1.95 (3H, br s,
H-18), 0.76 (3H, s, H-19), 5.29 (1H, s, H-200a), 4.89 (1H, s,
H-20b), 2.39 (1H, dq, JZ7.0, 7.3 Hz, H-2 ), 3.86 (1H, dq,
JZ7.0, 6.3 Hz, H-30), 1.21 (3H, d, JZ7.3 Hz, H-40), 1.16
(3H, d, JZ7.3 Hz, H-50), 2.19, 2.03 [3H each, OAc (CH3)];
13C NMR (CDCl3, 125 MHz) d 59.2 (d, C-1), 70.1 (d, C-2),
40.4 (d, C-3), 140.9 (s, C-4), 77.6 (d, C-5), 37.5 (t, C-6),
69.2 (d, C-7), 44.4 (s, C-8), 40.5 (t, C-9), 66.9 (d, C-10),
139.1 (s, C-11), 132.0 (s, C-12), 39.5 (t, C-13), 70.8 (d,
C-14), 37.4 (s, C-15), 25.4 (q, C-16), 31.9 (q, C-17), 21.1 (q,
C-18), 16.7 (q, C-19),0118.0 (t, C-20), 174.8 (s, C-10), 47.0
(d, C-20), 69.5 (d, C-3 ), 14.1 (q, C-40), 20.9 (q, C-50), 21.8,
21.4 [q, OAc (CH3)], 169.8, 169.6 [s, OAc (CO)];
HRFABMS m/z 559.2886 [MCNa]C (calcd 559.2883 for
C29H44O9Na).
4.6.2. 9a-Hydroxy-2a,5a,10b-triacetoxy-14b-(3-hydroxy-
2-methyl)-butyryloxytaxa-4(20),11-diene (18). White
powder; [a]D20 C45.88 (c 0.4, CHCl3); IR nmax (CHCl3):
3624, 3540, 3036, 2988, 1726, 1644, 1456, 1374, 1248, 1178,
1108, 1020 cmK1; 1H NMR (CDCl3, 500 MHz) d 1.81 (1H, d,
JZ2.0 Hz, H-1), 5.28 (1H, dd, JZ2.2, 6.6 Hz, H-2), 2.87 (1H,
d, JZ6.6 Hz, H-3), 5.25 (1H, br s, H-5), 1.73–1.79 (2H, m,
H-6), 1.60–1.70 (1H, m, H-7b), 1.49 (1H, dd, JZ5.5, 13.6 Hz,
H-7a), 4.14 (1H, d, JZ9.8 Hz, H-9), 5.75 (1H, d, JZ9.8 Hz,
H-10), 2.81 (1H, dd, JZ9.0, 19.0 Hz, H-13b), 2.28–2.35 (1H,
m, H-13a), 4.94 (1H, dd, JZ4.6, 9.0 Hz, H-14), 1.53 (3H, s,
H-16), 1.05 (3H, s, H-17), 2.07 (3H, br s, H-18), 0.99 (3H, s,
H-19), 5.25 (1H, s, H-20a), 4.81 (1H, s, H-20b), 2.32 (1H, dq,
JZ7.0, 7.4 Hz, H-20), 3.790(1H, dq, JZ7.0, 6.4 Hz, H-300),
1.13 (3H, d, JZ6.4 Hz, H-4 ), 1.08 (3H, d, JZ7.4 Hz, H-5 ),
2.12, 2.05, 1.96 [3H each, OAc (CH3)]; 13C NMR (CDCl3,
125 MHz)d58.9(d, C-1), 70.0(d, C-2), 44.1(d, C-3), 141.8(s,
C-4), 78.6 (d, C-5), 28.5 (t, C-6), 25.9 (t, C-7), 44.1 (s, C-8),
76.2 (d, C-9), 76.0 (d, C-10), 136.8 (s, C-11), 133.3 (s, C-12),
39.6 (t, C-13), 70.5 (d, C-14), 37.1 (s, C-15), 26.2 (q, C-16),
31.5 (q, C-17), 21.0 (q, C-18), 17.5 (q, C-19), 117.7 (t, C-20),
174.8 (s, C-10), 47.0 (d, C-20), 69.5 (d, C-30), 20.9 (q, C-40),
14.0 (q, C-50), 21.9, 21.4, 21.3 [q, OAc (CH3)], 170.5, 169.9,
170.0 [s, OAc (CO)]; HREIMS m/z 578.3102 [M]C (calcd
578.3091 for C31H46O10).
4.7. Biotransformation of 4 by A. coerulea
The procedures were followed as described in Section 4.4,
except that 200 mg of 4 (prepared from biotransformation of
1 by Ginkgo cells23) was used, finally 373 mg of extract
(200 mg for filtrate, 173 mg for cell cultures) was resulted.
The extract was fractionated and separated by combination
of open silica gel chromatography and normal phase semi-
prep. HPLC. 4 (160 mg) (80%; tRZ11.9 min) and 4.0 mg of
4.6.3. 6a-Hydroxy-2a,5a,10b-triacetoxy-14b-(3-hydroxy-
2-methyl)-butyryloxytaxa-4(20),11-diene (20). White
powder; [a]D20 C44.48 (c 1.0, CHCl3); IR nmax (CHCl3):

J. Dai et al. / Tetrahedron 61 (2005) 5507–5517
5515
22 (ca. 2%; tRZ15.3 min) were obtained, the HPLC mobile
phasewas the mixtureofhexaneandethyl acetate (70/30, v/v).
4.9.1. 2a,5a,9a,10b,14b-Pentaacetoxy-taxa-4(20),11-
diene (24). White powder; [a]2D0 C50.88 (c 1.2, CHCl3);
IR nmax (CHCl3): 2964, 1734, 1436, 1374, 1236, 1224, 1114,
1022 cmK1; 1H NMR (CDCl3, 500 MHz) d 1.93 (1H, d, JZ
2.2 Hz, H-1), 5.40 (1H, dd, JZ2.4, 6.6 Hz, H-2), 2.96 (1H,
d, JZ6.6 Hz, H-3), 5.30 (1H, t, JZ3.6 Hz, H-5), 1.78–1.85
(1H, m, H-6a), 1.65–1.75 (1H, m, H-6b), 1.64–1.72 (2H, m,
H-7), 5.79 (1H, d, JZ10.3 Hz, H-9), 6.01 (1H, d, JZ
10.5 Hz, H-10), 2.84 (1H, dd, JZ9.0, 19.0 Hz, H-13b), 2.43
(1H, dd, JZ4.9, 19.0 Hz, H-13a), 4.97 (1H, dd, JZ4.9,
9.0 Hz, H-14), 1.71 (3H, s, H-16), 1.12 (3H, s, H-17), 2.14
(3H, br s, H-18), 0.84 (3H, s, H-19), 5.32 (1H, s, H-20a),
4.87 (1H, s, H-20b), 2.17, 2.04, 2.03, 2.01, 2.00 [3H each, s,
OAc (CH3)]; 13C NMR (CDCl3, 125 MHz) d 58.5 (d, C-1),
70.2 (d, C-2), 44.1 (d, C-3), 141.4 (s, C-4), 78.3 (d, C-5),
28.3 (t, C-6), 27.3 (t, C-7), 44.4 (s, C-8), 72.5 (d, C-9), 76.8
(d, C-10), 137.3 (s, C-11), 132.8 (s, C-12), 39.4 (t, C-13),
68.9 (d, C-14), 37.0 (s, C-15), 25.8 (q, C-16), 31.6 (q, C-17),
21.8 (q, C-18), 17.3 (q, C-19), 118.1 (t, C-20), 21.4, 21.3,
21.1, 21.0, 20.8 [q, OAc (CH3)], 170.0, 169.9, 169.8, 169.7,
169.6 [s, OAc(CO)]; HRFABMS m/z 585.2676 [MCNa]C
(calcd 585.2676 for C30H42O10Na); 563.2857 [MCH]C
(calcd 563.2856 for C30H43O10).
4.8. Biotransformation of 5 by cell suspension cultures of
G. biloba
Compound 5 (20 mg) was dissolved in EtOH (0.4 mL),
distributed between 2 flasks of 15-day-old cell cultures and
incubated for 6 days, after which the cultures were filtered
under vacuum and the filtrate was saturated with NaCl and
extracted 5 times with ethyl acetate. All the extracts were
pooled, dried with anhydrous Na2SO4, and concentrated
under vacuum at 40 8C to give 30 mg of residue. The dried
cell cultures were extracted 3 times by sonication with ethyl
acetate, the resulting extracts were pooled and concentrated
under vacuum at 40 8C to afford 40 mg of residue. 12 mg of
5 (60%; tRZ12.3 min) and 2.0 mg of 23 (ca. 10%; tRZ
14.2 min) were obtained by combination of open silica gel
column chromatography and semi-prep. HPLC (mobile
phase: hexane/ethyl acetateZ70/30, v/v).
4.8.1. 7b,10b-Dihydroxy-2a,5a,14b-triacetoxy-taxa-
4(20),11-diene (23). White powder; [a]2D0 C42.58 (c 0.1,
CHCl3); IR nmax (CHCl3): 3624, 2992, 2944, 1732, 1644,
1
1456, 1376, 1242, 1114, 1048 cmK1; H NMR (CDCl3,
4.9.2. 2a,5a,7b,10b,14b-Pentaacetoxy-taxa-4(20),11-
diene (27). White powder; [a]2D0 C35.58 (c 1.4, CHCl3);
IR nmax (CHCl3): 3004, 2940, 1730, 1650, 1432, 1374, 1250,
500 MHz) d 1.87 (1H, d, JZ2.0 Hz, H-1), 5.42 (1H, dd, JZ
2.2, 6.6 Hz, H-2), 2.83 (1H, d, JZ6.1 Hz, H-3), 5.33 (1H, t,
JZ3.4 Hz, H-5), 2.06–2.14 (1H, m, H-6a), 1.58–1.64 (1H,
m, H-6b), 3.83 (1H, dd, JZ5.1, 11.7 Hz, H-7), 2.30 (1H, dd,
JZ5.1, 15.1 Hz, H-9b), 2.09–2.24 (1H, m, H-9a), 5.08 (1H,
dd, JZ5.1, 12.0 Hz, H-10), 2.83 (1H, dd, JZ9.3, 19.3 Hz,
H-13b), 2.37 (1H, dd, JZ4.9, 19.0 Hz, H-13a), 4.95 (1H,
dd, JZ4.9, 9.3 Hz, H-14), 1.76 (3H, s, H-16), 1.19 (3H, s,
H-17), 1.95 (3H, br s, H-18), 0.75 (3H, s, H-19), 5.28 (1H, s,
H-20a), 4.88 (1H, s, H-20b), 2.18, 2.07, 2.03 [3H each, s,
OAc (CH3)]; 13C NMR (CDCl3, 125 MHz) d 59.3 (d, C-1),
70.1 (d, C-2), 40.4 (d, C-3), 140.9 (s, C-4), 77.5 (d, C-5),
37.6 (t, C-6), 69.2 (d, C-7), 44.4 (s, C-8), 40.5 (t, C-9), 66.9
(d, C-10), 139.2 (s, C-11), 132.3 (s, C-12), 39.6 (t, C-13),
70.2 (d, C-14), 37.4 (s, C-15), 25.5 (q, C-16), 31.9 (q, C-17),
21.2 (q, C-18), 16.7 (q, C-19), 118.0 (t, C-20), 21.8, 21.5,
21.4 [q, OAc (CH3)], 170.2, 169.8, 169.6 [s, OAc (CO)];
HRFABMS m/z 501.2472 [MCNa]C (calcd 501.2464 for
C26H38O8Na).
1
1222, 1216, 1182, 1104, 1028 cmK1; H NMR (CDCl3,
500 MHz) d 1.90 (1H, d, JZ2.2 Hz, H-1), 5.39 (1H, dd, JZ
2.2, 6.6 Hz, H-2), 2.85 (1H, d, JZ6.6 Hz, H-3), 5.34 (1H, t,
JZ2.4 Hz, H-5), 2.04–2.12 (1H, m, H-6a), 1.65–1.74 (1H,
m, H-6b), 2.05–2.15 (1H, m, H-9b), 1.94–2.12 (1H, m,
H-9a), 5.85 (1H, dd, JZ6.0, 11.5 Hz, H-10), 2.82 (1H, dd,
JZ9.3, 19.0 Hz, H-13b), 2.42 (1H, dd, JZ4.9, 19.0 Hz,
H-13a), 4.95 (1H, dd, JZ4.9, 9.0 Hz, H-14), 1.66 (3H, s,
H-16), 1.14 (3H, s, H-17), 2.08 (3H, br s, H-18), 0.83 (3H, s,
H-19), 5.32 (1H, s, H-20a), 4.94 (1H, s, H-20b), 2.19, 2.10,
2.06, 2.02, 2.01 [3H each, s, OAc (CH3)]; 13C NMR (CDCl3,
125 MHz) d 58.8 (d, C-1), 69.7 (d, C-2), 41.0 (d, C-3), 140.5
(s, C-4), 76.8 (d, C-5), 33.8 (t, C-6), 70.6 (d, C-7), 43.1 (s,
C-8), 37.2 (t, C-9), 69.8 (d, C-10), 135.2 (s, C-11), 134.9 (s,
C-12), 39.4 (t, C-13), 70.4 (d, C-14), 37.4 (s, C-15), 25.4 (q,
C-16), 31.6 (q, C-17), 21.3 (q, C-18), 17.8 (q, C-19), 118.4
(t, C-20), 21.7, 21.5, 2!21.4, 21.0 [q, OAc (CH3)], 170.4,
170.0, 169.9, 169.9, 169.5 [s, OAc(CO)]; HRFABMS m/z
585.2676 [MCNa]C (calcd 585.2676 for C30H42O10Na);
563.2857 [MCH]C (calcd 563.2856 for C30H43O10).
4.9. Acetylation of 4 and 5
Into 50 mL of egg-plant flask, 195 mg of 4 (0.375 mmol)
and 274.5 mg (2.25 mmol) of 4-(dimethylamino)-pyridine
(DMAP) were added and dissolved with 7.5 mL of dry
pyridine at ambient temperature by stirring, then 0.352 mL
of Ac2O (3.75 mmol) was added. After 7 h incubation at
50 8C, the reaction was quenched by adding 20 mL of sat.
NaCl aq and extracted with ethyl acetate (4!20 mL). The
extract was washed with 2 M HCl (3!20 mL) until the
pH!5, followed with sat. NaHCO3 aq (2!15 mL) until
the pH 7–8, then with sat. NaCl aq (2!10 mL), dried over
anhydrous NaSO4 and concentrated to afford 257 mg of
residue. The resulting residue was applied to an open silica
gel column and eluted with the mixture of hexane and ethyl
acetate (7/3, v/v) to afford 200 mg of 24. The same
procedure was performed in the acetylation of 5 (50 mg,
0.096 mmol), finally 50 mg of 27 was obtained.
4.10. Biotransformation of 24 by A. coerulea
Compound 24 (200 mg) was used as substrate in this
experiment, and the procedures were performed as
described in Section 4.4, finally 321 mg of extract was
obtained (210 mg for filtrate, 101 mg for cells). 162 mg of
24 (81%; tRZ9.6 min), 4.2 mg of 25 (ca. 2%; tRZ11.1 min)
and 8.5 mg of 26 (ca. 4%; tRZ15.16 min) were obtained by
combination of open silica gel chromatography and normal
phase semi-prep. HPLC (mobile phase: hexane/ethyl
acetateZ70/30, v/v).
4.10.1. 7b-Hydroxy-2a,5a,9a,10b,14b-pentaacetoxy-
taxa-4(20),11-diene (25). White powder; [a]2D0 C38.48 (c
0.2, CHCl3); IR nmax (CHCl3): 3614, 2972, 2884, 1732,

5516
J. Dai et al. / Tetrahedron 61 (2005) 5507–5517
1646, 1436, 1374, 1240, 1102, 1020 cmK1
;
1H NMR
H-2), 2.93 (1H, d, JZ6.5 Hz, H-3), 5.33 (1H, t, JZ2.6 Hz,
H-5), 2.05–2.15 (1H, m, H-6a), 1.60–1.70(1H, m, H-6b),
5.40 (1H, dd, JZ5.4, 11.5 Hz, H-7), 4.20 (1H, d, JZ
11.0 Hz, H-9), 5.85 (1H, d, JZ11.5 Hz, H-10), 2.80 (1H, dd,
JZ9.0, 19.0 Hz, H-13b), 2.38 (1H, dd, JZ4.9, 19.0 Hz,
H-13a), 4.94 (1H, dd, JZ4.9, 9.0 Hz, H-14), 1.65 (3H, s,
H-16), 1.16 (3H, s, H-17), 2.13 (3H, s, H-18), 1.25 (3H, s,
H-19), 5.25 (1H, s, H-20a), 4.88 (1H, s, H-20b), 2.19, 2.15,
2.06, 2.01, 2.00 [3H each, s, OAc (CH3)]; 13C NMR (CDCl3,
125 MHz) d 59.0 (d, C-1), 70.2 (d, C-2), 43.6 (d, C-3), 142.1
(s, C-4), 77.2 (d, C-5), 31.3 (t, C-6), 70.8 (d, C-7), 44.3 (s,
C-8), 76.2 (d, C-9), 76.0 (d, C-10), 136.5 (s, C-11), 133.3 (s,
C-12), 39.4 (t, C-13), 68.7 (d, C-14), 37.2 (s, C-15), 26.2 (q,
C-16), 31.7 (q, C-17), 21.9 (q, C-18), 12.0 (q, C-19), 118.6
(t, C-20), 21.4, 2!21.3, 21.1, 20.6 [q, OAc (CH3)], 170.5,
2!170.0, 169.9, 168.6 [s, OAc (CO)]; HRFABMS m/z
601.2626 [MCNa]C (calcd 601.2625 for C30H42O11Na);
579.2806 [MCH]C (calcd 579.2805 for C30H43O11).
(CDCl3, 500 MHz) d 1.88 (1H, d, JZ2.2 Hz, H-1), 5.41
(1H, dd, JZ2.2, 6.0 Hz, H-2), 2.81 (1H, d, JZ6.2 Hz, H-3),
5.31 (1H, t, JZ2.6 Hz, H-5), 2.09–2.15 (1H, m, H-6a),
1.67–1.73 (1H, m, H-6b), 3.90 (1H, dd, JZ5.4, 11.5 Hz,
H-7), 5.80 (1H, d, JZ11.0 Hz, H-9), 6.05 (1H, d, JZ
11.5 Hz, H-10), 2.86 (1H, dd, JZ9.0, 19.0 Hz, H-13b), 2.40
(1H, dd, JZ4.9, 19.0 Hz, H-13a), 4.95 (1H, dd, JZ4.9,
9.0 Hz, H-14), 1.71 (3H, s, H-16), 1.21 (3H, s, H-17), 2.15
(3H, br s, H-18), 1.31 (3H, s, H-19), 5.30 (1H, s, H-20a),
4.88 (1H, s, H-20b), 2.17, 2.04, 2.03, 2.01, 2.00 [3H each, s,
OAc (CH3)]; 13C NMR (CDCl3, 125 MHz) d 58.9 (d, C-1),
70.4 (d, C-2), 45.2 (d, C-3), 140.2 (s, C-4), 83.2 (d, C-5),
31.3 (t, C-6), 71.7 (d, C-7), 45.6 (s, C-8), 73.6 (d, C-9), 70.3
(d, C-10), 135.5 (s, C-11), 133.2 (s, C-12), 39.4 (t, C-13),
68.9 (d, C-14), 37.4 (s, C-15), 26.1 (q, C-16), 31.7 (q, C-17),
21.9 (q, C-18), 12.7 (q, C-19), 117.7 (t, C-20), 21.4, 21.3,
21.1, 21.0, 20.8 [q, OAc (CH3)], 170.4, 170.1, 169.9, 169.3,
168.8 [s, OAc (CO)]; HRFABMS m/z 601.2630 [MCNa]C
(calcd 601.2625 for C30H42O11Na); 579.2796 [MCH]C
(calcd 579.2805 for C30H43O11).
Acknowledgements
4.10.2. 14b-Hydroxy-2a,5a, 9a,10b-tetraacetoxy-taxa-
4(20),11-diene (26). White powder; [a]2D0 C35.88 (c 0.1,
CHCl3); IR nmax (CHCl3): 3624, 2940, 1736, 1376, 1226,
1022 cmK1; 1H NMR (CDCl3, 500 MHz) d 1.80 (1H, d, JZ
2.0 Hz, H-1), 5.48 (1H, dd, JZ2.2, 6.3 Hz, H-2), 2.92 (1H,
d, JZ6.3 Hz, H-3), 5.27 (1H, t, JZ2.9 Hz, H-5), 1.80–1.88
(1H, m, H-6a), 1.65–1.71 (1H, m, H-6b), 1.68–1.75 (2H, m,
H-7), 5.81 (1H, d, JZ10.3 Hz, H-9), 6.02 (1H, d, JZ
10.5 Hz, H-10), 2.76 (1H, dd, JZ9.0, 18.5 Hz, H-13a), 2.54
(1H, dd, JZ4.4, 18.0 Hz, H-13b), 4.08 (1H, dd, JZ5.0,
9.0 Hz, H-14), 1.72 (3H, s, H-16), 1.18 (3H, s, H-17), 2.16
(3H, s, H-18), 0.85 (3H, s, H-19), 5.33 (1H, s, H-20a), 4.91
(1H, s, H-20b), 2.16, 2.08, 2.04, 2.02 [3H each, s, OAc
(CH3)]; 13C NMR (CDCl3, 125 MHz) d 63.1 (d, C-1), 70.5
(d, C-2), 44.1 (d, C-3), 141.9 (s, C-4), 78.7 (d, C-5), 28.4 (t,
C-6), 27.2 (t, C-7), 44.4 (s, C-8), 76.8 (d, C-9), 72.5 (d,
C-10), 137.9 (s, C-11), 132.8 (s, C-12), 42.2 (t, C-13), 67.6
(d, C-14), 37.5 (s, C-15), 26.1 (q, C-16), 31.3 (q, C-17), 20.8
(q, C-18), 17.3 (q, C-19), 118.0 (t, C-20), 22.0, 21.6, 21.5,
21.1 [q, OAc (CH3)], 170.1, 170.0, 169.7, 169.4 [s, OAc
(CO)]; HRFABMS m/z 543.2571 [MCNa]C (calcd
543.2570 for C28H40O9Na); 521.2751 [MCH]C (calcd
521.2751 for C28H41O9).
This work is supported by the National Natural Science
Foundation of China (Project No. 30100230) and the Japan
Society for the Promotion of Science (Project No. 1300127).
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