Biological degradation of taxol by action of cultured cells on 7-acetyltaxol-2″-yl glucoside

Article abstract of DOI:10.1246/cl.2008.362

Biodegradation pathways of taxol in cultured cells of Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, Marchantia polymorpha, Nicotiana tabacum, and Glycine max were investigated using a water-soluble taxol derivative, 7-ace-tyltaxol-2″-yl glucoside, as the substrate. Although cyanobacteria, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942, and a lower plant, M. polymorpha, catalyzed the epimerization at 7-position of taxol skeleton, no epimerization occurred with higher plants, N. tabacum and G. max. On the other hand, Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, M. polymorpha, and N. tabacum catalyzed hydrolysis at 13-position of taxol to give baccatin III and 10-deacetyl baccatin III. Both cyanobacteria cells also deacetylated 7-epi-baccatin III at its 10-position. M. polymorpha and G. max deacetylated at 10-position of taxol. Copyright

Full text of DOI:10.1246/cl.2008.362

362
Chemistry Letters Vol.37, No.3 (2008)
Biological Degradation of Taxol by Action of Cultured Cells on 7-Acetyltaxol-200-yl Glucoside
Kei Shimoda,ꢀ1 Katsuhiko Mikuni,2 Kiyoshi Nakajima,3 Hatsuyuki Hamada,4 and Hiroki Hamadaꢀ5
1Department of Pharmacology and Therapeutics, Faculty of Medicine, Oita University,
1-1 Hasama-machi, Oita 879-5593
2Ensuiko Sugar Refining Co., Ltd., 1-1-1 Fukuura, Kanazawa-ku, Yokohama 236-0004
3Tokyo Supply Ltd., 1-23-2 Toranomon, Minato-ku, Tokyo 105-0001
4National Institute of Fitness and Sports in Kanoya, 1 Shiromizu-cho, Kagoshima 891-2390
5Department of Life Science, Faculty of Science, Okayama University of Science,
1-1 Ridai-cho, Okayama 700-0005
(Received December 20, 2007; CL-071413; E-mail: shimoda@med.oita-u.ac.jp, hamada@das.ous.ac.jp)
Biodegradation pathways of taxol in cultured cells of
OH
HO
OH
OH
O
Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942,
Marchantia polymorpha, Nicotiana tabacum, and Glycine max
were investigated using a water-soluble taxol derivative, 7-ace-
tyltaxol-200-yl glucoside, as the substrate. Although cyanobacte-
ria, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC
7942, and a lower plant, M. polymorpha, catalyzed the epimeri-
zation at 7-position of taxol skeleton, no epimerization occurred
with higher plants, N. tabacum and G. max. On the other hand,
Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, M.
polymorpha, and N. tabacum catalyzed hydrolysis at 13-position
of taxol to give baccatin III and 10-deacetyl baccatin III. Both
cyanobacteria cells also deacetylated 7-epi-baccatin III at its
10-position. M. polymorpha and G. max deacetylated at 10-
position of taxol.
O
R2
O19
R4O
GOAO:
TSC:
O
9
R1
6
1''
O
Bz
Ph
10
2''
18
11
17
16
7
4
8
5
12
13
15
14
3
O
2
NH
O
OAc20
1
R3O
OBz
2'
HO
3'
1'
OH
1: R1 = GOAO, R2 = H, R3 = TSC, R4 = Ac; 2: R1 = OH, R2 = H, R3 = TSC, R4 = Ac;
3: R1 = OH, R2,R4 = H, R3 = TSC; 4: R1 = H, R2 = OH, R3 = TSC, R4 = Ac;
5: R1 = OH, R2,R3 = H, R4 = Ac; 6: R1 = OH, R2,R3,R4 = H;
7: R1,R3 = H, R2 = OH, R4 = Ac; 8: R1,R3,R4 = H, R2 = OH.
Figure 1. Structures of substrate 1 and biotransformation prod-
ucts 2–8.
(5, 33%), 10-deacetylbaccatin III (6, 16%), 7-epi-baccatin III (7,
27%), and 10-deacetyl-7-epi-baccatin III (8, 10%) (Table 1). No
products which retained the (glucosyloxy)acetyl (GOA) group at
their 7-position were detected. These suggest that the GOA
group of 1 was readily hydrolyzed to give taxol (2), and that
other hydrolysis and epimerization products 4–8 were produced
by the action of Synechocystis sp. PCC 6803 cells on 2. Feeding
experiment of 7-epi-taxol (4) or baccatin III (5) established that
7-epi-baccatin III (7) was produced from both 4 and 5. These
findings revealed that Synechocystis sp. PCC 6803 hydrolyzed
10-acetoxy group of baccatin III and 7-epi-baccatin III, and
13-TSC (taxol side chain) group of taxol skeleton, and that epi-
merization of taxol and baccatin III occurred at their 7-positions.
No formation of ketone intermediates was found during the
incubation period. Also Synechococcus sp. PCC 7942 converted
1 into 2 (31%), 4 (24%), 5 (23%), 6 (4%), 7 (15%), and
8 (2%).
Taxol, a diterpenoid from the Pacific Yew Taxus brevifiolia,
is one of the most important anticancer drugs, and is used for
treatment of ovarian, breast, and various other cancers world-
wide.1 From the pharmacological point of view, the pathway
of the biological degradation of taxol is interesting and impor-
tant. However, little attention has been paid to the biological
degradation of taxol by cultured cells, because it is hardly dis-
solved in aqueous solution. Use of an ester-linked taxol–sugar
conjugate, which is higher water-soluble than taxol and would
be hydrolyzed by hydrolytic enzymes in the living cells, is a
new approach to biodegradation research of taxol. Additionally,
taxol–sugar conjugates have attracted much attention clinically,
because of their potential to be useful prodrugs.2 The biodegra-
dation pathway of taxol–sugar conjugates is also important.
Herein, we report the biodegradation of taxol with cultured cells
of Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942,
Marchantia polymorpha, Nicotiana tabacum, and Glycine
max using a water-soluble taxol derivative, 7-acetyltaxol-200-yl
glucoside, as the substrate.
In contrast, biotransformation of 1 by cultured cells of liver-
wort M. polymorpha or higher plants, N. tabacum and G. max,
was examined. Biotransformation was performed under illumi-
nation (4000 lux, M. polymorpha) or in the dark (N. tabacum
Table 1. Biotransformation of 7-acetyltaxol-200-yl glucoside (1)
by cultured cells
Biotransformation of 7-acetyltaxol-200-yl glucoside (1)3 was
examined by incubating 1 with each cell cultures.4 Yield of the
products was calculated on the basis of the peak area from HPLC
using the calibration curves prepared by the HPLC analyses of
each compounds. The structures of the products were identified
based on their HRFABMS, 1H and 13C NMR, H–H COSY,
C–H COSY, and HMBC data (Figure 1).
Cell line
Products/%
2
3
4
5
6
7
8
Synechocystis sp. PCC 6803
Synechococcus sp. PCC 7942 31
7
0
2 33 16 27 10
24 23
0
17 10
25
16
4
8
15
0
0
2
0
0
0
M. polymorpha
N. tabacum
G. max
8
0
0
2
Biotransformation of 1 with cultured Synechocystis sp. PCC
6803 cells gave taxol (2, 7%), 7-epi-taxol (4, 2%),5 the cytotox-
icity of which is only slightly less than that of taxol,6 baccatin III
0
6
11 10
0
0
0
Copyright Ó 2008 The Chemical Society of Japan

Chemistry Letters Vol.37, No.3 (2008)
363
and G. max) in freshly prepared medium (MSK-2 medium for M.
polymorpha and MS medium for N. tabacum and G. max). After
the 7 day-incubation of 1 with cultured M. polymorpha cells, five
products 2 (17%), 3 (10%), 4 (8%), 5 (2%), and 6 (8%) were iso-
lated. Feeding experiment of 10-deacetyltaxol (3) or baccatin III
(5) revealed that 10-deacetylbaccatin III (6) was predominantly
formed from 5 rather than 3. On the other hand, cultured
N. tabacum cells converted 1 into three products 2 (25%), 5
(11%), and 6 (10%). Biotransformation of 1 with G. max
gave only two products 2 (16%) and 3 (6%). No epimerization
occurred through the transformation with both N. tabacum
and G. max. In the case of the biotransformation by these
three cell cultures, a large amount of unreacted substrate 1 was
recovered.
The results of this experiment revealed, for the first time, di-
verse biodegradation pathways of taxol in cultured cells by using
a water-soluble taxol derivative, 7-acetyltaxol-200-yl glucoside,
as the substrate. Two cyanobacteria, Synechocystis sp. PCC
6803 and Synechococcus sp. PCC 7942, were able to catalyze
hydrolysis at 13-position of taxol, deacetylation at 10-position
of baccatin III and 7-epi-baccatin III, and epimerization at
7-position of taxol and baccatin III. Liverwort M. polymorpha
could catalyze hydrolysis at 13-position of taxol, deacetylation
at 10-position of taxol and baccatin III, and epimerization at
7-position of taxol. On the other hand, higher plants, N. tabacum
and G. max, could not catalyze epimerization of taxol skeleton.
Hydrolysis occurred at 13-position of taxol (N. tabacum),
and 10-position of taxol (G. max) or baccatin III (N. tabacum).
Further studies on the enzymes participating in the hydrolysis
and epimerization of these taxoid compounds are now in
progress.
13C NMR (100 MHz, CD3OD): ꢀ 11.3 (C-19), 14.7 (C-18),
20.7 (CH3 in 10Ac), 22.2 (C-16), 23.1 (CH3 in 4Ac), 26.7
(C-17), 34.2 (C-6), 36.3 (C-14), 44.5 (C-3, C-15), 57.1 (C-
30), 57.7 (C-8), 62.4 (C-6000), 65.9 (C-200), 71.4 (C-7, C-13),
72.1 (C-4000), 73.6 (C-5000), 74.1 (C-2000), 74.8 (C-20), 75.1
(C-3000), 75.7 (C-2), 76.6 (C-10), 77.2 (C-20), 78.8 (C-1),
81.8 (C-4), 85.0 (C-5), 100.5, 100.6 (C-1000), 128.6, 129.1,
129.7, 131.2, 132.5, 134.1, 134.5, 135.4, 139.8 (C-11,
Ar-C in NBz, Ar-C in OBz, Ar-C in Ph), 142.1 (C-12),
167.6 (C=O in OBz), 170.0 (C=O in NBz), 170.9 (C-100),
171.3 (C=O in 4Ac), 171.9 (C=O in 10Ac), 174.3 (C-10),
203.1 (C-9).
4
Typical biotransformation procedures were as follows. Prior
to this experiment, 5 g (fresh weight) of mature cultured cells
of cyanobacterium, Synechocystis sp. PCC 6803, was indi-
vidually transplanted to 300-mL conical flasks containing
100 mL of freshly prepared BG-11 medium. The cultures
were grown for 2 weeks on a rotary shaker (120 rpm) under
illumination (4000 lux). A total of 0.03 mmol of a water-
soluble taxol derivative, 1, was added to three 300-mL con-
ical flasks (0.01 mmol/flask) containing the cultured cyano-
bacterium cells. The flasks were incubated at 25 ꢁC for fur-
ther 7 days on a rotary shaker (120 rpm) under illumination
(4000 lux). After incubation, the cells and medium were sep-
arated by centrifugation at 10000 g for 5 min. The cells were
extracted (ꢂ3) by homogenization in MeOH, and the extract
was concentrated. The residue was partitioned between H2O
and CH2Cl2. The medium was extracted (ꢂ3) with CH2Cl2.
The CH2Cl2 fractions were combined, concentrated, and
purified by HPLC [column: YMC-Pack R&D ODS column
(150 ꢂ 30 mm); solvent: MeOH–H2O (13:7, v/v); detection:
UV (227 nm); flow rate: 1.0 mL/min] to give products.
Spectral data for product 4: HRFABMS: m=z 876.3207
References and Notes
1
5
1
M. C. Wani, H. L. Taylor, M. E. Wall, P. Coggon, A. T.
McPhail, J. Am. Chem. Soc. 1971, 93, 2325.
[M + Na]þ; H NMR (CDCl3): ꢀ 1.16 (3H, s, H-16), 1.21
(3H, s, H-17), 1.68 (3H, s, H-19), 1.81 (3H, s, H-18), 2.17
(3H, s, CH3 in 10Ac), 2.25 (1H, dd, J ¼ 15:4, 9.0 Hz,
H-14a), 2.29 (1H, m, H-6a), 2.35 (1H, m, H-6b), 2.41 (1H,
dd, J ¼ 15:4, 9.0 Hz, H-14b), 2.51 (3H, s, CH3 in 4Ac),
3.70 (1H, d, J ¼ 12:0 Hz, H-7), 3.90 (1H, d, J ¼ 7:2 Hz,
H-3), 4.39 (2H, m, H-20), 4.81 (1H, d, J ¼ 3:0 Hz, H-20),
4.91 (1H, d, J ¼ 9:2 Hz, H-5), 5.76 (1H, d, J ¼ 7:2 Hz,
H-2), 5.82 (1H, d, J ¼ 9:2 Hz, H-30), 6.25 (1H, t,
J ¼ 9:2 Hz, H-13), 6.80 (1H, s, H-10), 7.35–7.58 (10H, m,
m-H in NBz, p-H in NBz, m-H in OBz, o-H in Ph, m-H in
Ph, p-H in Ph), 7.62 (1H, t, J ¼ 7:6 Hz, p-H in OBz), 7.75
(2H, d, J ¼ 8:2 Hz, o-H in NBz), 8.17 (2H, d, J ¼ 8:2 Hz,
o-H in OBz); 13C NMR (CDCl3): ꢀ 14.7 (C-18), 16.0
(C-19), 20.8 (CH3 in 10Ac), 21.4 (C-16), 22.5 (CH3 in
4Ac), 26.0 (C-17), 35.5 (C-14), 36.2 (C-6), 40.3 (C-3),
42.7 (C-15), 55.0 (C-30), 57.7 (C-8), 72.5 (C-13), 73.2
(C-20), 75.5 (C-2), 75.7 (C-7), 77.5 (C-20), 78.0 (C-10),
79.0 (C-1), 82.0 (C-4), 83.0 (C-5), 126.9, 128.5, 129.0,
129.2, 133.5, 133.7, 138.1, 139.6 (C-11, C-12, Ar-C in
NBz, Ar-C in OBz, Ar-C in Ph), 167.0 (C=O in NBz),
167.3 (C=O in OBz), 170.0 (C=O in 10Ac), 172.3 (C=O
in 4Ac), 173.0 (C-10), 207.5 (C-9).
2
Y. Luo, G. D. Prestwich, Bioconjugate Chem. 1999, 10, 755;
Y. Luo, M. R. Ziebell, G. D. Prestwich, Biomacromolecules
2000, 1, 208.
3
Coupling of 20-TES ester of taxol with 2,3,4,6-tetra-O-
benzylglucosyloxyacetic acid (1.2 equiv.) [EDCI, DMAP,
CH2Cl2, rt] provided 7-acetyl-20-TES-taxol-200-yl 2,3,4,6-
tetra-O-benzylglucoside (99%), which was deprotected with
Pd black in wet acetic acid to give 1 (98%). Spectral data for
1: HRFABMS: m=z 1096.3136 [M + Na]þ; 1H NMR
(400 MHz, CD3OD): ꢀ 1.10 (3H, s, H-16), 1.16 (3H, s, H-
17), 1.78 (3H, s, H-19), 1.82 (1H, m, H-6ꢁ), 1.87 (3H, s,
H-18), 2.02 (1H, dd, J ¼ 15:2, 9.0 Hz, H-14a), 2.15 (3H, s,
CH3 in 10Ac), 2.21 (1H, dd, J ¼ 15:2, 9.0 Hz, H-14b),
2.37 (3H, s, CH3 in 4Ac), 2.59 (1H, m, H-6ꢂ), 3.30–3.80
(8H, m, H-200, 2000, 3000, 4000, 5000, 6000), 3.90 (1H, d,
J ¼ 7:2 Hz, H-3), 4.19 (3H, m, H-7, 20), 4.75 (1H, d,
J ¼ 5:2 Hz, H-20), 4.92–4.95 (1H, m, H-1000), 5.01 (1H, d,
J ¼ 9:2 Hz, H-5), 5.60 (2H, m, H-2, 30), 6.15 (1H, t,
J ¼ 9:0 Hz, H-13), 6.21 (1H, s, H-10), 7.28 (1H, t,
J ¼ 7:6 Hz, p-H in Ph), 7.39–7.59 (9H, m, m-H in NBz,
p-H in NBz, m-H in OBz, o-H in Ph, m-H in Ph), 7.66
(1H, t, J ¼ 7:6 Hz, p-H in OBz), 7.85 (2H, d, J ¼ 8:2 Hz,
o-H in NBz), 8.11 (2H, d, J ¼ 8:2 Hz, o-H in OBz);
6
C. H. O. Huang, D. G. I. Kingston, N. F. Magri, G.
Samaranayake, J. Nat. Prod. 1986, 49, 665.
Published on the web (Advance View) March 5, 2008; doi:10.1246/cl.2008.362