Substrate specificity for the hydroxylation of polyoxygenated 4(20),11-taxadienes by Ginkgo cell suspension cultures

Article abstract of DOI:10.1016/S0045-2068(03)00063-4

Three C-14 oxygenated taxanes isolated from callus cultures of Taxus spp., 2α,5α,10β,14β-tetra-acetoxy-4(20),11-taxadiene 3, 2α,5α,10β-triacetoxy-14β-propionyloxy-4(20),11- taxadiene 4, 2α,5α,10β-triacetoxy-14β-(2-methylbutyryl)-oxy-4(20), 11-taxadiene 5, and three deacetylated derivatives of 3, 10β-hydroxy-2α,5α,14β-triacetoxy-4(20),11-taxadiene 6, 14β-hydroxy-2α,5α,10β-triacetoxy-4(20),11-taxadiene 7, 10β,14β-dihydroxy-2α,5α-diacetoxy-4(20),11-taxadiene 8, could all be regio- and stereo-selectively hydroxylated at the 9α-position by Ginkgo cell suspension cultures to yield a series of new 9α,14β-dihydroxylated taxoids. The effects of functional groups, especially at C-14 of the substrates, on the biotransformation were also investigated. The results revealed that substrates with an acetoxyl group at C-14 could be more efficiently 9α-hydroxylated than those with a longer ester chain or a hydroxyl group at C-14. An acetoxyl or hydroxyl group at C-10 had no effect on the conversion rates of the substrates, but substrates with the hydroxyl group (compared with the acetoxyl analogues) could be converted into 9α-hydroxylated products more easily.

Full text of DOI:10.1016/S0045-2068(03)00063-4

BIOORGANIC
CHEMISTRY
Bioorganic Chemistry 31 (2003) 345–356
www.elsevier.com/locate/bioorg
Substrate specificity for the hydroxylation of
polyoxygenated 4(20),11-taxadienes by
Ginkgo cell suspension cultures
Jungui Dai,a Min Ye,a Hongzhu Guo,a Weihua Zhu,b
Dayong Zhang,b Qiu Hu,b Junhua Zheng,a and Dean Guoa,*
a
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Xueyuan Road 38#, Beijing 100083, PR China
b
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, Xiannong Tan Street 1#, Beijing 100050, PRChina
Received 7 March 2003
Abstract
Three C-14 oxygenated taxanes isolated from callus cultures of Taxus spp., 2a,5a,10b,14b-
tetra-acetoxy-4(20),11-taxadiene 3, 2a,5a,10b-triacetoxy-14b-propionyloxy-4(20),11-taxadiene
4, 2a,5a,10b-triacetoxy-14b-(2-methylbutyryl)-oxy-4(20),11-taxadiene 5, and three deacetylated
derivatives of 3, 10b-hydroxy-2a,5a,14b-triacetoxy-4(20),11-taxadiene 6, 14b-hydroxy-2a,5a,
10b-triacetoxy-4(20),11-taxadiene 7, 10b,14b-dihydroxy-2a,5a-diacetoxy-4(20),11-taxadiene 8,
could all be regio- and stereo-selectively hydroxylated at the 9a-position by Ginkgo cell suspen-
sion cultures to yield a series of new 9a,14b-dihydroxylated taxoids. The effects of functional
groups, especially at C-14 of the substrates, on the biotransformation were also investigated.
The results revealed that substrates with an acetoxyl group at C-14 could be more efficiently
9a-hydroxylated than those with a longer ester chain or a hydroxyl group at C-14. An acetoxyl
or hydroxyl group at C-10 had no effect on the conversion rates of the substrates, but substrates
with the hydroxyl group (compared with the acetoxyl analogues) could be converted into 9a-hy-
droxylated products more easily.
Ó 2003 Elsevier Science (USA). All rights reserved.
Keywords: Taxane; 4(20),11-Taxadiene; Hydroxylation; Cell suspension cultures; Ginkgo biloba L.
* Corresponding author. Fax: +86-10-6209-2700.
E-mail address: gda@bjmu.edu.cn (D. Guo).
0045-2068/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.
doi:10.1016/S0045-2068(03)00063-4

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1. Introduction
The diterpenoid paclitaxel 1 (Scheme 1), first isolated by Wani and Wall from
the bark of the Pacific yew (Taxus brevifolia Nutt.) [1], is one of the most effective
anticancer drugs used in clinical treatment for various cancers [2]. The structural
complexity, significant biological activities of 1, as well as its unique mechanism
of action [3] have led to extensive research towards the synthesis of paclitaxel
and other new analogs. The chemical synthesis of paclitaxel has been achieved
both by semisynthesis [4], with 10-deacetyl baccatin III 2 as the starting material
(Scheme 1), and by total synthesis [5]. However, it seems that the most promising
approach for large-scale production of paclitaxel still relies on the combination of
biological and semisynthetic methods [6]. As a good example, the Bristol–Myers
Squibb Corporation reported recently the semisynthesis of paclitaxel in which bi-
ocatalysis was fully employed [6a]. Three novel enzymes were discovered that could
convert a variety of taxanes in the plant extracts into a single molecule, 10-deacetyl
baccatin III, an important synthetic precursor of paclitaxel. The concentration of
10-deacetyl baccatin III was increased by 5.5–24-folds in the extracts treated with
the enzymes, and the difficult separation procedure could be avoided. Biocatalysis
processes have also been reported for the preparation of C-13 paclitaxel side chain
synthons [6a].
2a,5a,10b,14b-tetra-acetoxy-4(20),11-taxadiene 3 and its analogs, 2a,5a,10b-
triacetoxy-14b-propionyloxy-4(20),11-taxadiene 4, and 2a,5a,10b-triacetoxy-14b-
(2-methylbutyryl)oxy-4(20),11-taxadiene 5 (Scheme 2) are major taxanes isolated
in high yields (ca. 4% of dry weight) from callus cultures of Taxus spp. [7]
The abundance of these 14b-hydroxylated taxanes makes them valuable for the
semisynthesis of paclitaxel or other structurally related taxoids [8]. A number
of studies on their structural modification by chemical and biocatalytic ap-
proaches were reported and encouraging progress has been achieved [9]. Recently,
we also reported the specific hydroxylation of 3 at the 9a-position by Ginkgo cell
suspension cultures [10]. That was the first report of 9a-hydroxylation of taxanes
by plant cultures, and it might play an important role in the semisynthesis of new
taxoids. Could the other closely related taxanes also be hydroxylated at 9a-posi-
tion by Ginkgo cells? And how do the various substituents on substrates affect the
biotransformation process? To confirm the capacity of the enzymes in Ginkgo
Scheme 1. Chemical structures of taxol and 10-deacetyl baccatin III.

J. Dai et al. / Bioorganic Chemistry 31 (2003) 345–356
347
Scheme 2. The chemical structures of 3–15.
cells responsible for the hydroxylation reactions, five taxadienes, 4, 5, 6, 7, and 8,
in addition to 3, were tested as substrates for biotransformation by Ginkgo cells
(Scheme 2). The effects of different substrates on the hydroxylation were investi-
gated by determining the biotransformation rates of the substrates and the yields
of the 9a-hydroxylated products.

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2. Results
Six substrates with different substitution profiles were obtained, three of which, 3,
4, and 5, were isolated from the callus cultures of Taxus spp. and characterized as
described in [7a]. The only difference of these compounds is the length of the ester
chain at C-14 position, wherein an acetoxyl group occurred in 3, a propionyloxy
group in 4, and a (2-methylbutyryl)oxy group in 5. Compound 3 was hydrolyzed un-
der basic conditions as described [9a] to yield three major products identified as 6 (in
10% yield), 7 (in 30% yield), and 8 (in 15% yield). These six compounds were used as
substrates in the biotransformation studies.
2.1. Biotransformation of 4 and 5 by Ginkgo cell suspension cultures
Ginkgo cell suspension cultures were verified to be able to convert 4 and 5. The
two substrates were, respectively, administered to the 15-day-old cell cultures, and
one more polar product was, respectively, obtained after additional 6 days incuba-
tion. FAB mass spectrum of the product (9) derived from 4 showed a quasi molec-
ular ion peak [M+ Na] þ at m/z 557, and HREIMS at 533.8720, consistent with the
molecular formula C29H42O9, suggesting the substitution of an additional hydroxyl
1
group as compared with 4. The H-NMR spectrum of 9 was similar to that of 4 ex-
cept that the signals corresponding to H-9a (d1.64, dd, J ¼ 5.6, 14.9 Hz) or H-9b(d
2.39, m) in 4 disappeared, while an oxygen-bearing methine signal was observed at d
4.21 (d, J ¼ 10.0 Hz). As a result, the signal of H-10a (d 6.06, dd, J ¼ 5.6, 12.1 Hz)
shifted to an upper field at d 5.82 (d, J ¼ 10.0 Hz), suggesting the existence of a hy-
droxyl group at C-9 position. This was confirmed by the signal of C-9 which signif-
1
icantly shifted downfield to d 76.2 when compared with d 43.9 in 4. The H-NMR
spectrum of 9 also showed that the coupling constant of H-9 and H-10 was 10
Hz, which indicates a position of the 9-hydroxyl group. Therefore, 9 was determined
to be the 9a-hydroxyl derivative of 4. All the signals of 10 derived from 5 in
1H-NMR, 13C-NMR and FAB mass spectrum were all in good agreement with those
of
9a-hydroxy-2a,5a,10b-triacetoxy-14b-(2-methylbutyryl)oxy-4(20),11-taxadiene
[11]. The results showed that 4 and 5 could be specifically hydroxylated at 9a posi-
tion by Ginkgo cell suspension cultures.
2.2. Biotransformation of 6, 7, and 8
Three products were obtained from 6 added on day 15 and incubated with Ginkgo
cells for additional six days. On the basis of the chemical and physical spectral data,
their structures were identified as 9a,10b-dihydroxy-2a,5a, 14b-triacetoxy-4(20), 11-
taxadiene (11, in 70% yield), [10] 6a,10b-dihydroxy-2a,5a, 14b-triacetoxy-4(20),
11-taxadiene (12, in 7.5% yield ) [9d], and 6a,9a,10b-trihydroxy-2a,5a,14b-triacet-
oxy-4(20),11-taxadiene (13, in 10% yield), respectively. Also, three products were
obtained from 7 under the same conditions, and characterized as 9a,14b-dihydroxy-
2a,5a,14b-triacetoxy-4(20),11-taxadiene (14, in 20% yield), 6a,10-dihydroxy-2a,

J. Dai et al. / Bioorganic Chemistry 31 (2003) 345–356
349
5a,14b-triacetoxy-4(20),11-taxadiene(12, in 3% yield ) [9d], and 6a,9a,10b-trihydroxy-
2a, 5a,14b-triacetoxy-4(20),11-taxadiene (13, in 3% yield), respectively. Interestingly,
14-acetylated products (12 and 13) were obtained from 14-deacetylated substrate (7),
which suggested that transfer of acetyl group from C-10 to C-14 or acetylation at C-14
accompanied with deacetylation at C-10 also occurred in the bioprocess.
From 8 was obtained one polar product and identified as 9a, 10b, 14b-trihydroxy-
2a, 5a-diacetoxy-4(20),11-taxadiene (15, in 20% þyield). The FAB mass spectrum
showed a quasi molecular ion peak [M+ Na] at m/z 459, and HREIMS at
436.2460, consistent with the molecular formula C24H36O7, suggesting the substitu-
1
tion of an additional hydroxyl group as compared with 8. The H-NMR spectrum
of 15 was similar to that of 8, except that the signals corresponding to H-9a (d
1.66, dd, J ¼ 6.0, 15.0 Hz) or H-9b (d 2.34, m) in 8 disappeared, while an additional
oxygen-bearing methine signal was observed at d 4.10 (d, J ¼ 9.5 Hz). The signal of
H-10a (d 5.10, dd, J ¼ 5.7, 11.7 Hz) shifted to an upper field at d 4.79 (d, J ¼ 9.5
Hz). The above evidence suggested the existence of a hydroxyl group at C-9 position.
This was confirmed by the signal of C-9 which significantly shifted downfield to d
78.3 compared with d 47.0 in 8. The 9-hydroxyl group was also determined to be
in the a position as described for 9 and 10. Therefore, 15 was characterized as the
9a-hydroxyl derivative of 8.
2.3. The effects of different substitution groups on the biotransformation reactions
According to the above results, all the six tested taxadienes could be hydroxylated at
9a-position by Ginkgo cells. Thus, in order to shed light on the effects of different sub-
stitution groups in substrates on the biotransformation process and the substrate-spec-
ificity of the responsible enzyme, the above six substrates were used for the
biotransformation by the same Ginkgo cell suspension cultures, and the amounts of re-
sidual substrates and yielded 9a-hydroxyl products were used as indicators. The six
substrates at the concentration of 35 mg/L were administered into the 15-day-old cell
cultures, respectively. After six additional days of co-incubation, the reactions were
stopped, and the extracts were analyzed by HPLC. The results (Table 1) showed that:
Table 1
The effects of different substitution groups of taxanes on the biotransformation by Ginkgo cell suspension
cultures
Substrates
Biotransformation rates (%)
Yields of 9a-OH derivatives (%)
3
4
5
6
7
8
100
70 ꢀ 6.5
60 ꢀ 3.8
20 ꢀ 1.2
75 ꢀ 3.3
40 ꢀ 2.5
30 ꢀ 1.8
80 ꢀ 5.8
50 ꢀ 3.5
100
60 ꢀ 6.0
50 ꢀ 4.6
Note. Biotransformation rate, the percentage of transformed substrate; yields, the percentage of 9a-OH
products, detected by HPLC. Each value represents mean ꢀ SD (n ¼ 3). For 3–8, see Scheme 2.

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(1) The length of ester chain at C-14 influenced the biotransformation significantly. The
substrates with a shorter ester chain were more effectively converted than those with a
longer one. For example, the biotransformation rate of 3 and the yield of its 9-hydrox-
ylated product were higher than those of 4, and 4 higher than those of 5. In addition, the
substrate with a hydroxyl group at C-14, such as 7 and 8, was less effectively converted
than those with an acetoxyl group, such as 3 and 6. In other words, an acetyl group
substituted at C-14 favored the bioprocess. (2) Either an acetoxyl group or a hydroxyl
group substituted at C-10 position makes no difference on the biotransformation rate
of the substrates, for example, both 3 and 6 were completely converted. However, sub-
stitution group at C-10 position influenced the yields of 9a-hydroxylated products, the
yield of the 9a-hydroxylated product from the substrate with an hydroxyl group at
C-10 (6) was higher than that with an acetoxyl group (3).
3. Discussion
CH oxidation is one of the most versatile reactions in organic chemistry since po-
tentially useful oxyfunctionized synthons may be obtained from readily accessible
hydrocarbons. Only a few conventional synthetic methods, which required extreme
reaction conditions and yielded racemic products are, to date, available for this pur-
pose. For this reason, a lot of efforts have been previously expended to develop ef-
ficient catalytic oxidations of hydrocarbons. For example, in the past several years,
metal catalyzed asymmetric CH oxidations have been performed by employing chiral
auxiliaries and optically active oxidants to afford enantiomerically enriched products
[12]. Nevertheless, in most of the cases only a moderate enantio-selectivity has been
achieved for unactivated alkanes. In this context, biomimetic studies have been car-
ried out to gain insight into the mechanism of the catalytic CH oxidation and to en-
hance its stereo-selectivity. Despite all these efforts, the development of an effective
catalytic asymmetric hydroxylation of unfunctionalized hydrocarbons with broad
applicability remains a challenge [13]. Alternatively, microorganisms and plant cell
suspension cultures have been successfully applied to the selective oxygenation of un-
activated CH bonds in organic substrates [14]. However, it seems that the structures
of substrates could greatly influence the biotransformation processes. From our re-
sults, the six tested taxanes could all be regio- and stereo-selectively hydroxylated by
Ginkgo cells but in different yields. The substitution groups at C-14 could affect the
reaction significantly, which suggested that the enzyme responsible for the hydroxyl-
ation is highly substrate-specific, and that longer C-14 substitution chain could
inhibit the binding of substrates with the catalytic enzyme.
It is well known that 9a hydroxylation is probably one of the cytochrome P450-
dependent oxidative steps in paclitaxel biosynthetic pathway [15]. So far, the enzyme
and its encoding gene are still enigmatic, the biotransformation of taxanes by em-
ploying plant or microbial cells may biomimic some steps of taxoid biosynthesis,
such as extensive oxidation of the taxane skeleton, therefore, which might give some
hints to the paclitaxel biosynthesis.

J. Dai et al. / Bioorganic Chemistry 31 (2003) 345–356
351
In conclusion, six taxadienes with different substitution groups could be regio-
and stereo-selectively hydroxylated at 9a-position by Ginkgo cell suspension cultures.
This reaction not only provides a novel approach to prepare bioactive taxoids, but
also might, to some extent, assist the biosynthetic studies of paclitaxel.
4. Materials and methods
4.1. General
Optical rotations were measured with a Perkin–Elmer 243 B polarimeter. IR spec-
tra were obtained on a Perkin–Elmer 983 spectrophotometer (KBr). NMR spectra
1
(1H-NMR, 13C-NMR, DEPT, H-1H-COSY, HMQC, HMBC, and NOESY) were
recorded in CDCl3 with Bruker DRX-500 or Varian INOVA-500 instrument (1H-
NMR, 500 MHz; 13C-NMR, 125 MHz) and chemical shifts were recorded in d using
TMS as internal standard. FABMS spectra were measured on a KYKY-ZHP-5#
mass spectrometer in the positive mode, and HREIMS analyses were performed
on a JEOL JMX HX-110 spectrometer (The Instrumental Analysis Center for
Chemistry, Tohoku University, Japan). The Ginkgo suspension cells were cultured
in 500-mL flask with 150 mL medium in the darkness at 25 °C, the inoculum size
was 5 g/L of cell cultures (dry weight), shaking at the rate of 110 rpm. All media were
autoclaved for 20 min at 121 °C, pH values were adjusted to 5.8 prior to autoclaving
and the amount of sucrose addition in medium was 30 g/L [16]. All chemicals were
obtained from Beijing Chemical Factory.
4.2. Substrate
2a,5a,10b,14b-tetra-acetoxy-4(20),11-taxadiene(3), 2a,5a,10b-triacetoxy-14b-\pro-
pionyloxy-4(20),11-taxadiene(4)
and
2a,5a,10b-triacetoxy-14b-(2-methylbuty-
ryl)oxy-4(20),11-taxadiene(5) were isolated from callus cultures of Taxus spp. [7a].
10-Hydroxy-2a,5a,14b-triacetoxy-4(20),11-taxadiene(6) 14b-hydroxy-2a,5a,10b-tri-
acetoxy-4(20),11-taxadiene(7), and 10b,14b-dihydroxy-2a,5a-diacetoxy-4(20),11-
taxadiene(8) were prepared from the hydrolysis of 3 [9a]. All the substrates were
characterized by chemical and spectral methods.
4.3. Biotransformation of 4
One hundred milligrams of 4 was dissolved in 10 mL EtOH and distributed into
10 flasks of cell cultures on day 15. After additional six days of incubation, the cell
cultures were filtered, the filtrate was collected, and extracted three times with
EtOAc. All the extracted solutions were pooled and concentrated in vacuum at
40 °C to give 150 mg residue. The residue was separated on a silica gel column, (silica
gel H, 5–40 mesh), eluted with acetone–petroleum ether (60–90 °C) (1:10–1:1) to
yield 50 mg of 9.

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9a-hydroxy-2a,5a,10-triacetoxy-14b-propionyloxy-4(20),11-taxadiene(9): color-
less needles. [aꢁ25 + 45.45° (c 0.0067, MeOH); IR mmax (KBr): 3432, 2940, 1726,
D
1636, 1373, 1236, 1023 cmꢂ1
;
1H-NMR d 5.82(1H, d, J ¼ 10.0 Hz, H-10),
5.34(1H, dd, J ¼ 2.5, 6.5 Hz, H-2), 5.31(2H, brs, H-5, H-20a), 4.97(1H, dd,
J ¼ 5.0, 9.0 Hz, H-14), 4.91(1H, brs, H-20b), 4.21(1H, d, J ¼ 10.0 Hz, H-9),
2.95(1H, d, J ¼ 6.0 Hz, H-3), 2.86(1H, dd, J ¼ 9.0, 19.0 Hz, H-13a), 2.42(1H,
dd, J ¼ 4.5, 19.5 Hz, H-13b), 2.28(2H, q, J ¼ 7.5 Hz, 14-OCOCH2CH3),
2.19(3H, s, 10-OCOCH3), 2.14(3H, brs, 18-CH3), 2.05(3H, s, 2-OCOCH3),
2.01(3H, s, 5-OCOCH3), 1.86(1H, d, J ¼ 2.0 Hz, H-1), 1.83(2H, m, H-6),
1.71(1H, m, H-7a), 1.59(3H, s, 16-CH3), 1.55(1H, m, H-7b), 1.11(3H, s, 17-
CH3), 1.11(3H, t, J ¼ 7.5 Hz, 14-OCOCH2CH3), 1.05(3H, s, 19-CH3); 13C-NMR
d 173.3(14-OCOCH2CH3), 170.5(10-OCOCH3), 169.9(2-OCOCH3), 169.7(5-OC-
OCH3), 141.8(C-20), 137.0(C-11), 133.2(C-12), 117.6(C-20), 78.6(C-5), 76.2(C-9),
76.0(C-10), 70.1(C-2),70.0(C-14), 58.7(C-1), 44.7(C-8), 44.0(C-3), 39.6(C-15),
37.1(C-13), 31.5(C-17), 28.4(C-6), 27.9(14-OCOCH2CH3), 26.1(C-16), 25.8(C-7),
21.9(C-18), 21.4(2-OCOCH3), 21.3(5-OCOCH3), 21.0(10-OCOCH3), 17.5(C-19),
9.14(14-OCOCH2CH3); FABMS (NBA) m/z: 557(M+ Na, 30), 401(3), 341(9),
299(18), 281(30), 135(100); HREIMS for C29H42O9 requires: 533.8714, found:
533.8720.
4.4. Biotransformation of 5
To 15-day-old cell cultures, 100 mg of 5 was added, and the procedure was per-
formed as for 4. Ten milligrams of 10 was obtained.
9a-hydroxy-2a,5a,10b-triacetoxy-14b-(2-methylbutyryl)oxy-4(20),11-taxadiene
25
D
(10): colorless needles. [aꢁ + 65.40° (c 0.0054, MeOH); IR mmax (KBr): 3456, 2940,
1726, 1620, 1373, 1236, 1023 cmꢂ1; H-NMR d 5.80(1H, d, J ¼ 10.0 Hz, H-10),
1
5.32(1H, dd, J ¼ 2.4, 6.3 Hz, H-2), 5.28(1H, brs, H-20a), 5.24(1H, brs, H-5),
4.93(1H, dd, J ¼ 4.4, 8.8 Hz, H-14), 4.84(1H, brs, H-20b), 4.19(1H, d, J ¼ 10.0
Hz, H-9), 2.93(1H, d, J ¼ 6.3 Hz, H-3), 2.87(1H, dd, J ¼ 9.2, 19.2, H-13a),
2.34(1H, m, 14-OCOCH(CH3)CH2CH3), 2.32(1H, dd, J ¼ 4.5, 19.2 Hz, H-13b),
2.18(3H, s, 10-OCOCH3), 2.12(3H, s, 18-CH3), 2.05(3H, s, 2-OCOCH3), 2.01(3H,
s, 5-OCOCH3), 1.83(2H, m, H-6), 1.82(1H, d, J ¼ 2.4 Hz, H-1), 1.71(1H, m,
H-7a), 1.58(3H, s, 16-CH3), 1.55(1H, m, H-7b), 1.46(3H, m, 14-OCOCH
(CH3)CH2CH3), 1.12(3H, s, 17-CH3), 1.11(3H, d, J ¼ 7.2 Hz, 14-OCOCH
(CH3)CH2CH3), 1.06(3H, s, 19-CH3), 0.85(2H, t, J ¼ 7.2 Hz, 14-OCOCH (CH3)
CH2CH3); 13C-NMR
d 175.6(14-OCOCH(CH3)CH2CH3), 170.1(2-OCOCH3),
169.8(5-OCOCH3), 169.6(10-OCOCH3), 141.8(C-4), 137.0(C-11), 133.2(C-12),
117.5(C-20), 78.6(C-5), 76.2(C-9), 76.0(C-10), 70.0(C-2), 69.9(C-14), 59.0(C-1),
44.8(C-8), 44.1(C-3), 41.0(14-OCOCH(CH3)CH2CH3), 39.7(C-13), 36.8(C-15),
31.5(C-17), 28.5(C-6), 26.7(14-OCOCH(CH3)CH2CH3), 26.2(C-16), 25.8(C-7),
22.4(C-19), 21.0(C-18), 21.3(10-OCOCH3), 21.3(2-OCOCH3), 20.9(10-OCOCH3),
11.5(14-OCOCH (CH3)CH2CH3), 13.9(14-OCOCH(CH3)CH2CH3); FABMS
(NBA) m/z: 585(M+ Na, 15), 401(6), 341(16), 281(30), 136(100); HREIMS for
C31H46O9 requires: 562.2142, found: 562.3147.

J. Dai et al. / Bioorganic Chemistry 31 (2003) 345–356
353
4.5. Biotransformation of 6
The procedure was similar to that described for 4, except that 20 mg of 6 was
used, and yielded 10 mg of 11, 1.5 mg of 12, and 2.0 mg of 13.
25
9a,10b-dihydroxyl-2a,5a,14b-tri-acetoxy-4(20),11-taxadiene(11): needles; [aꢁD
+
40.60° (c 0.0061, MeOH); IR mmax (KBr): 3432, 2940, 1726, 1636, 1373, 1236, 1023
cmꢂ1; HNMR (CDCl3, 500MHz) d 5.37(1H, dd, J ¼ 6.5, 2.3 Hz, H-2), 5.31(2H,
1
brs, H-5, H-20), 4.97(1H, dd, J ¼ 4.5, 8.5 Hz, H-14), 4.90(1H, brs, H-20),
4.80(1H, d, J ¼ 9.5 Hz, H-10), 4.09(1H, d, J ¼ 9.6 Hz, H-9), 2.93(1H, d, J ¼ 6.5
Hz, H-3), 2.82(1H, dd, J ¼ 8.5, 9.5 Hz, H-13), 2.46(1H, dd, J ¼ 5.0, 19.0 Hz, H-
13), 2.18(3H, s, 18-CH3), 2.05(3H, s, OCOCH3), 2.02(3H, s, OCOCH3), 2.00(3H,
s, OCOCH3), 1.86(1H, d, J ¼ 2.1 Hz, H-1), 1.81(1H, m, H-6), 1.77(1H, m, H- 7),
1.75(1H, m, H-6), 1.65(3H, s, 16-CH3), 1.50(1H, m, H-7), 1.20(3H, s, 17-CH3),
1.03(3H, s, 19-CH3); 1 3CNMR (CDCl3, 500MHz) d 169.9(2, 5, 14-OCOCH3),
142.1(C-4), 136.7(C-11), 134.4(C-12), 117.6(C-20), 78.8(C-9), 78.5(C-5), 72.2(C-10),
70.6(C-14), 70.2(C-2), 59.0(C-1), 44.4(C-8), 44.2(C-3), 39.6(C-13), 37.4(C- 15),
31.8(C-17), 28.5(C-6), 26.5(C-16), 26.0(C-7), 21.8(C-18), 21.3(2, 10- OCOCH3),
21.2(5-OCOCH3), 17.6(C-19). FABMS (m/z): 501(M + Na, 6), 401(2), 341(1),
299(2), 154(100), 136(94); HREIMS for C26H38O8 requires: 478.2567, found:
478.2575.
6a,10b-dihydroxyl-2a,5a,14b-triacetoxy-4(20),11-taxadiene(12): white powder.
25
D
[aꢁ + 47.1° (c 0.0072, MeOH); IR mmax (KBr): 3432, 2940, 1726, 1636, 1373,
1236, 1023 cmꢂ1; H-NMR d 5.41(1H, s, H-20a ), 5.36(1H, dd, J ¼ 2.0, 6.0 Hz,
1
H-2), 5.06 (1H, dd, J ¼ 6.0, 12.0 Hz, H-10), 5.04(1H, brs, H-5), 5.01(1H, dd,
J ¼ 9.5, 5.5 Hz, H-14), 4.97(1H, s, H-20b), 3.89(1H, m, H-6), 2.95(1H, d, J ¼ 6.0
Hz, H-3), 2.80(1H, dd, J ¼ 9.0, 19.0 Hz, H-13a), 2.42(1H, dd, J ¼ 3.5, 19.0 Hz,
H-13b), 2.39(1H, m, H-9a), 2.20(3H, s, 18-CH3), 2.06(3H, s, OCOCH3), 2.02(3H,
s, OCOCH3), 1.98(1H, dd, J ¼ 4.5, 14.5 Hz, H-7a), 1.94(3H, s, OCOCH3),
1.91(1H, d, J ¼ 2.5 Hz, H-1), 1.72(3H, s, 16-CH3), 1.67(1H, dd, J ¼ 5.5, 15.0 Hz,
H-9b), 1.48(1H, d, J ¼ 14.5 Hz, H-7), 1.19(3H, s, 17-CH3), 1.04(3H, s, 19-CH3);
1 3C-NMR d 169.9(OCOCH3), 169.9(OCOCH3), 169.5(OCOCH3), 138.6(C-4),
138.5(C-11), 132.3(C-12), 120.4(C-20), 81.6(C-5), 70.6(C-2), 70.2(C-6, C-14),
67.3(C-10), 58.8(C-1), 47.4(C-9), 41.5(C-3), 41.3(C-7), 39.6(C-15), 39.4(C-8),
37.5(C-13), 32.1(C-17), 25.4(C-16, C-19), 21.5(C-18), 21.5(OCOCH3), 21.4(OC-
OCH3), 21.0(OCOCH3); FABMS (NBA) m/z: 501(M+ Na, 28), 401(4), 341(4),
299(8), 281 (12), 154(94), 136(100); HREIMS for C26H38O8 requires: 478.2567,
found: 478.2570.
6a,9a,10b-trihydroxy-2a,5a,14b-triacetoxy-4(20),11-taxadiene(13): colorless nee-
1
dles. IR mmax (KBr): 3432, 2940, 1726, 1636, 1373, 1236, 1023 cmꢂ1; H-NMR d
5.46(1H, s, H-20a), 5.37(1H, dd, J ¼ 2.5, 6.0 Hz, H-2), 5.08(1H, brs, H-5),
5.01(1H, s, H-20b), 4.98(1H, dd, J ¼ 5.1, 9.3 Hz, H-14), 4.75(1H, d, J ¼ 9.5 Hz,
H-10), 4.01(1H, d, J ¼ 9.5 Hz, H-9), 3.93(1H, m, H-6), 2.96(1H, d, J ¼ 6.0 Hz,
H-3), 2.82(1H, dd, J ¼ 8.6, 18.6 Hz, H-13a), 2.44(1H, dd, J ¼ 5.1, 18.6 Hz,
H-13b), 2.20(3H, s, 18-CH3), 2.06(3H, s, OCOCH3), 2.02(3H, s, OCOCH3), 2.00
(1H, m, H-7a), 1.96 (3H, s, OCOCH3), 1.90 (1H, d, J ¼ 2.0 Hz, H-1), 1.64(3H, s,

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J. Dai et al. / Bioorganic Chemistry 31 (2003) 345–356
16-CH3), 1.62(1H, d, J ¼ 4.0 Hz, H-7b), 1.24 (3H, s, 17-CH3), 1.20(3H, s,
19-CH3); 13C-NMR
170.0(OCOCH3), 169.8(OCOCH3), 169.4(OCOCH3),
d
138.2(C-4), 136.7(C-11), 134.8(C-12), 121.1(C-20), 82.0(C-5), 79.3(C-9), 72.1(C-10),
70.5(C-14), 69.8(C-2), 69.8(C-6), 58.7(C-1), 44.1(C-8), 43.6(C-3), 39.5(C-15),
37.4(C-13), 34.2(C-7), 31.8(C-16), 26.0(C-17), 21.6(18-CH3, 19-CH3), 21.3(OC-
OCH3), 21.3(OCOCH3), 20.8(OCOCH3); FABMS (NBA) m/z: 517 (M+ Na, 7),
154(100), 136(82); HREIMS for C26H38O9 requires: 494.2616, found: 494.2523.
4.6. Biotransformation of 7
The procedure was similar to that of described above as 4, except that 300 mg of 7
was added, and gave 50 mg of 14, 10 mg of 12, and 10 mg of 13.
9a,14b-dihydroxy-2a,5a,10b-triacetoxy-4(20),11-taxadiene(14): colorless needles.
1
IR mmax (KBr): 3432, 2940, 1726, 1636, 1373, 1236, 1023 cmꢂ1; H-NMR d 5.82(1H,
d, J ¼ 10.0 Hz, H-10), 5.41(1H, dd, J ¼ 6.5, 2.3 Hz, H-2), 5.31(1H, brs, H-20a),
5.28(1H, brs, H-5), 4.95(1H, brs, H-20b),4.22(1H, d, J ¼ 10.0 Hz, H-9) 4.09(1H,
dd, J ¼ 5.0, 9.0 Hz, H-14), 2.88(1H, d, J ¼ 6.0 Hz, H-3), 2.67(1H, dd, J ¼ 9.0,
19.0 Hz, H-13a), 2.53(1H, dd, J ¼ 5.0, 19.0 Hz, H-13b), 2.15(3H, s, 10-OCOCH3),
2.11(6H, s, 18-CH3, 2-OCOCH3), 2.08(3H, s, 5-OCOCH3), 2.00(1H, brs, H-1),
1.81(2H, m, H-6), 1.77(1H, m, H-6a), 1.59(3H, s, 16-CH3), 1.54(1H, m, H-7b),
1.17(3H, s, 17-CH3), 1.04(3H, s, 19-CH3). 1 3C-NMR d 170.5(2-OCOCH3),
169.7(5-OCOCH3), 169.6(10-OCOCH3), 142.3(C-4), 137.5(C-11), 133.3(C-12),
117.4(C-20), 79.0(C-5), 76.2(C-10), 76.0(C-9), 70.6(C-2), 67.5(C-14), 63.2(C-1),
44.7(C-8),44.0(C-3), 42.3(C-13), 37.5(C-15), 31.2(C-17), 28.4(C-6), 26.4(C-7),
25.6(C-16), 21.9(C-18), 21.5(2-OCOCH3), 21.3(10-OCOCH3), 21.1(5-OCOCH3),
17.4(C-19); FABMS (NBA) m/z: 501(M+ Na, 15), 441(1), 419(2), 401(3), 341(4),
299(6), 154(77), 136(100); HREIMS for C26H38O8 requires: 478.2567, found:
478.2566.
4.7. Biotransformation of 8
The procedure was similar to that of described above as 4, also, 100 mg of 7 was
added, and yielded 20 mg of 15.
9a,10b,14b-trihydroxy-2a,5a-diacetoxy-4(20),11-taxadiene(15): colorless prism.
25
D
[aꢁ + 33.6° (c 0.0029, MeOH); IR mmax (KBr): 3432, 2940, 1726, 1636, 1373, 1236,
1023 cmꢂ1; H-NMR d5.42(1H, dd, J ¼ 2.0, 6.0 Hz, H-2), 5.30(1H, brs, H-20a),
1
5.26(1H, t, J ¼ 2.5 Hz, H-5), 4.94(1H, brs, H-20b), 4.79(1H, d, J ¼ 9.5 Hz, H-10),
4.10(1H, d, J ¼ 9.5 Hz, H-9), 4.09(1H, dd, J ¼ 5.5, 14.0 Hz, H-14), 2.87(1H, d,
J ¼ 6.0 Hz, H-3), 2.64(1H, dd, J ¼ 9.0, 19.0 Hz, H-13a), 2.52(1H, dd, J ¼ 4.5,
19.0 Hz, H-13b), 2.14(3H, s, OCOCH3), 2.08(3H, s, 18-CH3), 2.01(3H, s, OCOCH3),
1.80(2H, m, H-6), 1.72(1H, d, J ¼ 2.0 Hz, H-1), 1.70(1H, m, H-H-7a), 1.64(3H, s,
16-CH3), 1.49(1H, m, H-7b), 1.20(3H, s, 17-CH3), 1.02(3H, s, 19-CH3); 13C-NMRd
169.7(OCOCH3), 169.6(OCOCH3), 142.4(C-4), 136.5(C-11), 135.1(C-12), 117.3(C-
20), 79.1(C-5), 78.3(C-9), 72.0(C-2), 70.7(C-10), 67.7(C-14), 63.4(C-1), 44.4(C-8),

J. Dai et al. / Bioorganic Chemistry 31 (2003) 345–356
355
44.0(C-3), 42.3(C-13), 37.8(C-15), 31.5(C-7), 30.9(C-17), 28.5(C-6), 26.2(C-16),
25.9(C-19), 21.9(C-18), 21.5(OCOCH3), 21.3(OCOCH3); FABMS (NBA) m/z:
459(M+ Na, 8), 299(4), 281(3), 154(100), 136(94); HREIMS for C 24H36O7 required:
436.2461, found: 436.2460.
4.8. The effects of different substitution groups on the biotransformation
Thirty-five mg/L of the six substrates were separately administered to the 15-day-
old cell cultures and the enzymatic reactions were stopped and analyzed by HPLC
after additional six days of incubation. The residues were dissolved in the HPLC mo-
bile phase and diluted with the same solution to give 2.0 mL. The amounts of resid-
ual substrates and their 9-hydroxylated products were determined by HPLC. HPLC
analyses were performed by using Zorbax C18 column (25 cm ꢃ 4.6 mm ID, 5 lm),
eluted with methanol–acetonitrile–water (50:15:35, v/v/v) at a flow rate of 1.0 mL/
min and detected at 227 nm.
Acknowledgments
We thank The National Outstanding Youth Foundation by NSF of China
(39925040) and Trans-Century Training Program Foundation for the Talents by
the Ministry of Education for financial support. We also thank Dr. Masayoshi Ando,
Faculty of Engineering, Niigata University, Japan, for the help of HREIMS analyses.
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