Biotransformation of 2α,5α,10β,14β-tetra-acetoxy-4(20), 11-taxadiene by Ginkgo cell suspension cultures

Article abstract of DOI:10.1016/S0040-4039(01)00836-X

Ginkgo biloba cell suspension cultures were used to biotransform 2α,5α,10β,14β-tetra-acetoxy-4(20),11-taxadiene. Two novel compounds were obtained and their structures were identified as 9α-hydroxyl-2α,5α,10β,14β-tetra-acetoxy-4(20), 11-taxadiene 1 and 9α, 10β-dihydroxyl-2α,5α,14β-tri-acetoxy-4(20), 11-taxadiene 2, respectively, on the basis of their physical and chemical data. Compound 1 was subsequently used as a substrate for the bioconversion by Ginkgo cell cultures, and the product obtained was confirmed to be the same as 2, which suggested that 2 is biosynthesized from 1. Investigation on properties of the related enzymes responsible for the biotransformation reaction through the experimental techniques of cell-free culture and substrate/product concentration analysis revealed that the enzymes were extracellular and constitutive.

Full text of DOI:10.1016/S0040-4039(01)00836-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 4677–4679
Biotransformation of 2a,5a,10b,14b-tetra-acetoxy-4(20),
11-taxadiene by Ginkgo cell suspension cultures
Jungui Dai,a Hongzhu Guo,a Dandan Lu,a Weihua Zhu,b Dayong Zhang,b Junhua Zhenga and
Dean Guoa,*
aThe State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University,
Xueyuan Road c38, Beijing 100083, PR China
bInstitute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College,
Xiannong Tan Street c1, Beijing 100050, PR China
Received 19 February 2001; revised 4 May 2001; accepted 16 May 2001
Abstract—Ginkgo biloba cell suspension cultures were used to biotransform 2a,5a,10b,14b-tetra-acetoxy-4(20),11-taxadiene. Two
novel compounds were obtained and their structures were identified as 9a-hydroxyl-2a,5a,10b,14b-tetra-acetoxy-4(20), 11-taxa-
diene 1 and 9a, 10b-dihydroxyl-2a,5a,14b-tri-acetoxy-4(20), 11-taxadiene 2, respectively, on the basis of their physical and
chemical data. Compound 1 was subsequently used as a substrate for the bioconversion by Ginkgo cell cultures, and the product
obtained was confirmed to be the same as 2, which suggested that 2 is biosynthesized from 1. Investigation on properties of the
related enzymes responsible for the biotransformation reaction through the experimental techniques of cell-free culture and
substrate/product concentration analysis revealed that the enzymes were extracellular and constitutive. © 2001 Elsevier Science
Ltd. All rights reserved.
Sinenxan A, 2a,5a,10b,14b-tetra-acetoxy-4(20),11-taxa-
diene and its analogs (sinenxan B and C) (Scheme 1)
are three main taxane compounds isolated from the
callus cultures of Taxus yunnanensis, T. cuspidata,
T. chinesis and T. chinensis var. mairei.1 These taxane
compounds are abundant in the callus cultures of the
above plants, in which the total concentration of
sinenxans could reach 5–6% of the callus dry weight.
AcO
9
2
10
sinenxan A R=OCOCH3
sinenxan B R=OCOCH2CH3
sinenxan C R=OCOCH(CH3)CH2CH3
7
4
5
OAc
H
14
R
H
OAc
AcO
OAc
AcO
O
O
OH
Ph
NH O
OCH=CHPh
O
H
O
Ph
O
H
H
OAc
OCOPh
H
OH
taxol
OAc
taxinine
Scheme 1.
Keywords: biotransformation; taxane; cell suspension cultures; Ginkgo biloba.
* Corresponding author. Tel.: 86-10-62091516; fax: 86-10-62092700; e-mail: gda@mail.bjmu.edu.cn
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00836-X

4678
J. Dai et al. / Tetrahedron Letters 42 (2001) 4677–4679
as 2, which suggested that 2 was biosynthesized from 1
Their high concentration and their taxol skeleton vest
sinenxans have valuable potential for the semisynthesis
of taxol, an anti-cancer drug,2 or other structurally
related bioactive compounds, such as taxinine, a multi-
drug-resistance (MDR) reversal agent3 (Scheme 1).
Recently, attention has been paid to the rapid emer-
gence of MDR cancer cells in the chemotherapeutic
treatment of various types of advanced solid tumors.
This is mainly caused by the over-expression of P-gly-
coprotein (P-gp),4 a cell-membrane transporter for a
variety of hydrophobic substrates, during the course of
the clinical use of vincristine, taxol and etoposide which
are very useful hydrophobic antitumor drugs for cancer
chemotherapy. Kobayashi and his colleagues reported
that a number of new and known taxoids, such as
taxinine and its derivatives, isolated from the Japanese
yew, T. cuspidata,3,5,6 having neither an oxetane ring at
C-4 and C-5 nor an n-acylphenyl–lisoserine group at
C-13 could increase cellular accumulation of vincristine
(VCR) in MDR tumor cells, which is as potent as
verapamil, and efficiently inhibited [3H]-azidopine photo-
labeling of P-gp. Sinenxan A is structurally analogous
to taxinine, and therefore it was chosen for structural
modification by a biotransformation approach to syn-
thesize taxinine-type compounds or other potential
bioactive compounds.
through a deacetylation process (Scheme 2).
The fact that hydroxylation reaction took place at the
unactivated C-9 position makes it possible to synthesize
other taxoids, especially taxinine. In an endeavor to
optimize the biotransformation conditions, the growth
curve for G. biloba cell cultures and the biotransforma-
tion curves for the substrate, products 1 and 2 were
recorded (data not shown). The results disclosed that
the optimal time for substrate addition was at the
logarithmic-phase (9th–18th day) during the cell growth
period. Substrate addition at this phase, especially on
the 18th day, the late logarithmic phase or early sta-
tionary phase, resulted in an efficient bioconversion of
substrate and yield of products (the yields for 1 and 2
were 2-fold more than those of substrate addition at the
beginning of the cell growth period).
In general, the concentration of substrate added affects
the substrate to product biotransformation process.
Thus, the effects of five different concentrations of
substrates (15 mg/L, 30 mg/L, 45 mg/L, 60 mg/L and
75 mg/L) on the substrate bioconversion were investi-
gated and the results showed that the optimal amount
of substrate addition was 60 mg/L. At this concentra-
tion, the substrate was efficiently converted (100%), and
simultaneously the products (1 and 2) also reached the
highest yields (data not shown).
In this paper, we report the successful biotransforma-
tion of sinenxan A into two products by the cell
suspension cultures of Ginkgo biloba L. Those two
products were structurally identified as 9a-hydroxyl-
Based on the above results, the kinetics of the sub-
strate/product bioconversion were also investigated. 60
mg/L of substrate was added into the culture on the
18th day, and incubated for 24 h, 48 h and 72 h,
respectively. The HPLC analysis results revealed that
the substrate was transformed close to completion and
the yields of the products 1 and 2 reached the highest
levels after 72 h incubation (data not shown).
2a,5a,10b,14b-tetra-acetoxy-4(20),11(12)-taxadiene
1
and 9a,10b - dihydroxyl - 2a,5a,14b - tri - acetoxy - 4(20),
11(12)taxadiene 2 (Scheme 2) on the basis of their IR,
1H NMR, 13C NMR, 1H–1H-COSY, 13C–1H-COSY
and FABMS7,8 data. The biotransformation yields for 1
and 2 were determined to be 60 and 20%, respectively.
Compound 1 was subsequently used for further bio-
transformation by Ginkgo cell cultures, and it was
proved that the converted product was exactly the same
In order to shed light on the basic properties (constitu-
tive or inducible, extracellular or endocellular) of the
R2
R1
AcO
10 9
Ginkgo cell cultures
OAc
OAc
H
OAc
25¡æ, 6 days
H
H
H
AcO
OAc
AcO
1 R1=OH R2=AcO
2 R1=OH R2=OH
sinenxan A
HO
OH
AcO
OH
10 9
10 9
Ginkgo cell cultures
OAc
25¡æ, 6 days
OAc
H
OAc
H
H
H OAc
AcO
AcO
2
1
Scheme 2.

J. Dai et al. / Tetrahedron Letters 42 (2001) 4677–4679
4679
enzymes responsible for the biotransformation, a series
of experiments were designed to characterize the
enzymes through the methods of cell-free culture and
substrate/product concentration analysis. The results
showed that the enzymes are extracellular and constitu-
tive (data not shown). Plant cells in vitro could convert
the exogenous substrates to new products, but in
essence it is the enzymes produced by plant cells that
play the role. If the enzymes’ properties and additional
information were well investigated, the biotransforma-
tion rate and yield could be optimized through adjust-
ing their activities. Subsequently, the enzymes could be
extracted, purified and immobilized for large-scale pro-
duction of the desired products.
5. Kobayashi, J.; Hosoyama, H.; Wang, X. X.; Shigemori,
H.; Koiso, Y.; Iwasaki, S.; Sasaki, T.; Naito, M; Tsuruo,
T. Bioorg. Med. Chem. Lett. 1997, 7, 393–398.
6. Hosoyama, H.; Shigemori, H.; Tomida, A.; Tsuruo, T.;
Kobayashi, J. Biorg. Med. Chem. Lett. 1999, 9, 389–394.
7. 9a-Hydroxyl-2a,5a,10b,14b-tetra-acetoxy-4(20),11(12)-taxa-
diene 1: prism-like; [h]2D5+44.64 (c 0.0083, MeOH); IR umax
1
(KBr): 3432, 2940, 1726, 1636, 1373, 1236, 1023 cm−1; H
NMR (CDCl3, 500 MHz) l 5.83 (1H, d, J=10, H-10),
5.34 (1H, dd, J=6.5, 2.3, H-2), 5.31 (2H, brs, H-5, H-20),
4.98 (1H, dd, J=5.0, 9.5, H-14), 4.91 (1H, s, H-20), 4.21
(1H, d, J=9.5, H-9), 2.94 (1H, d, J=5.5, H-3), 2.85 (1H,
dd, J=10, 20, H-13), 2.44 (1H, dd, J=5.0, 19.5, H-13),
2.18 (3H, d, J=2.0, H-18), 2.14 (3H, s), 2.12 (3H, s), 2.06
(3H, s), 2.02 (3H, s), 1.87 (1H, m, H-1), 1.83 (2H, m, H-6,
H-7), 1.72 (1H, m, H-6), 1.60 (3H, s, H-16), 1.54 (1H, m,
H-7) 1.12 (3H, s, H-17), 1.05 (3H, s, H-19); 13CNMR
(CDCl3, 500 MHz) l 170.51, 169.98, 169.98, 169.76,
141.84 (C-4), 136.41 (C-11), 133.26 (C-12), 117.69 (C-20),
78.67 (C-5), 76.22 (C-9), 76.01 (C-10), 70.34 (C-2), 70.03
(C-14), 58.66 (C-1), 44.78 (C-8), 44.07 (C-3), 39.56 (C-13),
37.12 (C-15), 31.54 (C-17), 28.47 (C-6), 26.19 (C-16), 25.84
(C-7), 21.87 (C-18), 21.40, 21.40, 21.40, 21.02, 17.55 (C-
19). FABMS (m/z): 543 (M+Na, 21), 461 (8), 419 (23), 401
(14), 341 (7), 299 (12), 281 (28), 281 (28), 135 (100).
In conclusion, we have obtained a powerful method for
preparation of C-9 hydroxylated derivatives by Ginkgo
cell cultures from sinenxan A, a readily available
product from tissue and cell cultures of Taxus sp. This
might provide a useful tool to prepare bioactive taxoids
and to probe some important biosynthetic steps in
taxoid biosynthesis in Taxus sp. To the best of our
knowledge, this is also the first example of 9-hydroxyla-
tion of taxoids by cultured plant cells.
8. 9a,10b-Dihydroxyl-2a,5a,14b-tri-acetoxy-4(20),11(12)-taxa-
Acknowledgements
diene 2: needle; [h]D25 +40.60 (c 0.0061, MeOH); IR umax
(KBr): 3432, 2940, 1726, 1636, 1373, 1236, 1023 cm−1
;
1HNMR (CDCl3, 500 MHz) l 5.37 (1H, dd, J=6.5, 2.3,
H-2), 5.31 (2H, brs, H-5, H-20), 4.97 (1H, dd, J=4.5, 8.5,
H-14), 4.90 (1H, brs, H-20), 4.80 (1H, d, J=9.5, H-10),
4.09 (1H, d, J=9.6, H-9), 2.93 (1H, d, J=6.5, H-3), 2.82
(1H, dd, J=8.5, 9.5, H-13), 2.46 (1H, dd, J=5.0, 19.0,
H-13), 2.18 (3H, s, H-18), 2.05 (3H, s), 2.02 (3H, s), 2.00
(3H, s), 1.86 (1H, d, J=2.1, H-1), 1.81 (1H, m, H-6), 1.77
(1H, m, H-7), 1.75 (1H, m, H-6), 1.65 (3H, s, H-16), 1.50
(1H, m, H-7), 1.20 (3H, s, H-17), 1.03 (3H, s, H-19). 13C
NMR (CDCl3, 500 MHz) l 169.91, 169.91, 169.91, 142.12
(C-4), 136.70 (C-11), 134.48 (C-12), 117.60 (C-20), 78.84
(C-9), 78.52 (C-5), 72.21 (C-10), 70.62 (C-14), 70.21 (C-2),
59.05 (C-1), 44.46 (C-8), 44.27 (C-3), 39.63 (C-13), 37.48
(C-15), 31.89 (C-17), 28.53 (C-6), 26.57 (C-16), 26.09 (C-7),
21.80 (C-18), 21.33, 21.33, 21.29, 17.60 (C-19). FABMS
(m/z): 501 (M+Na, 6), 401 (2), 341 (1), 299 (2), 154 (100),
136 (94).
We thank The National Outstanding Youth Founda-
tion by NSF of China and Trans-Century Training
Program Foundation for the Talents by the Ministry of
Education for financial support.
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