Biotransformation of triptolide by Cunninghamella blakesleana

Article abstract of DOI:10.1016/S0040-4020(03)00605-7

Biotransformation of triptolide 1 by Cunninghamella blakesleana (AS 3.970) was carried out. Seven biotransformation products were obtained and four of them were characterized as new compounds. On the basis of their NMR and mass spectral data, their struct

Full text of DOI:10.1016/S0040-4020(03)00605-7

Tetrahedron 59 (2003) 4209–4213
Biotransformation of triptolide by Cunninghamella blakesleana
Lili Ning,a,b Jixun Zhan,a Guiqin Qu,a Lei Zhong,a Hongzhu Guo,a Kaishun Bib and Dean Guoa
,
*
aThe State Key Lab of Nat and Biomimetic Drugs, School of Pharmaceutical Sciences and Modern Research Center for Traditional
Chinese Medicine, Peking University, Beijing 100083, People’s Republic of China
bShenyang Pharmaceutical University, Shenyang, Liaoning 110016, People’s Republic of China
Received 11 December 2002; revised 27 February 2003; accepted 31 March 2003
Abstract—Biotransformation of triptolide 1 by Cunninghamella blakesleana (AS 3.970) was carried out. Seven biotransformation products
were obtained and four of them were characterized as new compounds. On the basis of their NMR and mass spectral data, their
structures were characterized as 5a-hydroxytriptolide 2, 1b-hydroxytriptolide 3, triptodiolide 4, 16-hydroxytriptolide 5, triptolidenol 6,
19a-hydroxytriptolide 7 and 19b-hydroxytriptolide 8. All the new transformed products (2, 3, 7 and 8) were found to exhibit potent in vitro
cytotoxicity against some human tumor cell lines. q 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
isolated by chromatographic methods after an additional 5
days’ incubation. Their structures were characterized as
5a-hydroxytriptolide 2, 1b-hydroxytriptolide 3, tripto-
diolide 4, 16-hydroxytriptolide 5, triptolidenol 6, 19a-
hydroxytriptolide 7 and 19b-hydroxytriptolide 8, respect-
ively, on the basis of their IR, 1D NMR, 2D NMR and mass
spectroscopic data. Among the products isolated, com-
pounds 2, 3, 7 and 8 have not been reported before. The
biotransformation reaction is illustrated in Scheme 1.
Tripterygium wilfordii Hook.f, Lei Gong Teng in Chinese,
was used in traditional Chinese medicine for the treatment
of various diseases including rheumatoid arthritis, nephritis,
systemic lupus erythematous and skin disorders, as well as
in male-fertility control.1,2 Triptolide, a diterpenoid triep-
oxide, isolated from the Chinese herbal plant, T. wilfordii
Hook.f by Kupcham in 19723 has been shown to be effective
in the treatment of autoimmune diseases4 and to have potent
antileukemic and antitumor activities.5,6
Product 2 was obtained as colorless crystals and had the
molecular formula of C20H25O7 (m/z 377.1595 [MþH]þ,
calcd 377.1600) based on its HRMS, suggesting that a
hydroxyl group has been introduced into the substrate
molecule. The IR absorbance peaks at 3470, 1762, 1677,
1079, 1029 cm21 indicated the presence of hydroxyl and
ketone groups. 1H and 13C NMR spectra of 2 were similar to
those of 1 except for the signals corresponding to C-5, C-10
and H-5. The signal of H-5 (d 2.67, br.d) disappeared in the
1H NMR spectrum of 2. In the HMQC spectrum, a new
signal (d 70.12 ppm) of a quaternary carbon supports the
fact that a tertiary carbon was hydroxylated. On the basis of
1H–1H COSY and HMBC analysis, the hydroxylated
tertiary carbon was assigned to C5. Additionally, the proton
signal at d 5.33 ppm (5-OH) showed correlations with the
signal at d 1.75 ppm (H-1a), d 2.17 ppm (H-6a) and d
4.86 ppm (H-19) in the NOESY spectrum. Therefore,
compound 2 was identified as 5a-hydroxytriptolide, which
is a new compound. All the 13C and 1H NMR data of 2 were
As a biologically active agent, the application of triptolide
was limited due to its strong toxicity. To find more effective
compounds with less toxicity, structural modifications of
triptolide and its analogues by chemical synthetic methods
have been carried out extensively in recent years.7–10
Biotransformation is now becoming a useful tool for
structural modifications of bioactive natural products.
However, structural modifications of triptolide by biological
methods have not been performed. In the present paper, we
report for the first time the successful biotransformation of
triptolide by Cunninghamella blakesleana (AS 3.970) and
structural elucidation of the biotransformation products.
2. Results and discussion
Triptolide was successfully bioconverted by C. blakesleana
(AS 3.970). The substrate was added into a 2-day-old
cultivation of the microorganisms and seven products were
1
assigned using extensive NMR techniques (DEPT, H–H
COSY, HMQC, HMBC and NOESY). The 1D and 2D NMR
spectral data of 2 were summarized in Table 1.
Keywords: biotransformation; triptolide; Cunninghamella blakesleana.
*
Product 3 was obtained as colorless crystals and its Time of
Flying Mass Spectrum (TOFMS) gave the quasi-molecular
Corresponding author. Tel.: þ86-10-62091516; fax: þ86-10-62092700;
e-mail: gda@bjmu.edu.cn
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00605-7

4210
L. Ning et al. / Tetrahedron 59 (2003) 4209–4213
Scheme 1. Biotransformation of triptolide by Cunninghamella blakesleana.
ion peak at m/z 394 [MþNH4]þ and m/z 399 [MþNa]þ. The
IR spectrum of product 3 revealed the presence of hydroxyl
and ketone groups. High resolution MS [MþH]þ at
377.1598 was consistent with the formula C20H25O7
(calcd 377.1600). By comparing its 13C NMR spectrum
with that of 1, the signal of C-1 had shifted downfield from
29.76 to 71.09 ppm, which suggested 3 to be a hydroxylated
product of 1 at C-1 position. This deduction was also
supported by a long-range coupling between H-20 and C-1
on the basis of its HMBC spectrum. In the NOESY
spectrum, the correlations of 1-OH with H-2b, H-11 and
H-20 strongly supported the orientation of the 1-OH as
b-configuration. This conclusion was supported by the
correlations of H-1 with H-2a and H-5a in the NOESY
spectrum. On the basis of the above analysis, the structure of
3 was elucidated to be the 1b-hydroxy derivative of
compound 1, which is also a new compound. All the 13C
and 1H NMR data of 3 were assigned according to extensive
and NOESY). The 1D and 2D NMR spectral data of 3 were
summarized in Table 2.
Product 4 was obtained as colorless crystals and its TOFMS
gave the quasi-molecular ion peak m/z at 394 [MþNH4]þ.
After comparison of the spectroscopic data with those
reported in the literature,3 compound 4 was identified as
triptodiolide, which is a known structure.
Product 5 was obtained as colorless crystals and its TOFMS
gave the quasi-molecular ion peak m/z at 394 [MþNH4]þ.
Its NMR spectral and chemical data were in good agreement
with those of 16-hydroxytriptolide reported by Ma et al.11
Product 6 was obtained as colorless crystals and its TOFMS
gave the quasi-molecular peak at m/z 394 [MþNH4]þ. It
was a known compound identified as triptolidenol based on
its spectral data, which are in good agreement with those
reported in the literature.12
1
NMR techniques (DEPT, H–H COSY, HMQC, HMBC
Table 1. NMR data and correlated relations in 2D NMR of 2 (DMSO-d6, J in Hz)
1H
13C
HMBC (C!H)
NOESY
1
2
1.74 (ddd, 6, 5.5, 12)
1.03 (dd, 5.5, 12.5)
2.07 (br.d)
23.6
17.3
H-20
H-1b, 11, 5-OH
H-1a, 2b, 11
H-2b
H-1ab
1.94–2.00 (m)
H-1b, 20
3
4
5
6
124.6
163.3
70.1
H-1b, 19
H-19, 5-OH
H-1b, 2a, 6ab, 7, 20, 5-OH
H-7, 5-OH
2.15–2.18 (m)
2.11 (dd, 13, 15)
3.33 (d, 5)
30.8
H-7, 19, 20, 5-OH
H-7, 19, 20,
H-6ab
7
8
9
59.2
61.9
63.1
39.4
56.4
54.5
64.4
71.4
27.8
17.1
18.1
173.8
69.1
16.8
H-6a, 14
H-6a, 7, 14
H-7, 12, 14, 20
H-1ab, 2a, 6b, 20, 5-OH
H-12
H-11, 14
H-11, 14, 15, 16, 17, 14-OH
14-OH
H-14, 16, 17
H-15, 17
H-15, 16
10
11
12
13
14
15
16
17
18
19
20
5-OH
14-OH
3.72 (d, 3)
3.52 (d, 3)
H-1ab, 12
H-11, 15, 16, 17
3.36 (d, 7)
2.15 (sept, 7)
0.74 (d, 7)
0.88 (d, 7)
H-15, 16, 17, 14-OH
H-12, 14, 15, 17
H-12, 14, 15, 17
H-15, 16
4.86 (s)
0.98 (s)
5.33 (s)
4.61 (d, 7)
H-6a, 5-OH
H-6ab, 2b
H-6a, 12, 19
H-7, 14, 15

L. Ning et al. / Tetrahedron 59 (2003) 4209–4213
Table 2. NMR data and correlated relations in 2D NMR of 3 (DMSO-d6, J in Hz)
4211
1H
13C
HMBC (C!H)
H-2a, 20, 1-OH
1-OH
NOESY
1
2
3.74 (quin, 4.5)
2.21 (br.d)
1.80–1.84 (m)
71.1
26.2
H-2a, 5, 11, 1-OH
H-2a, 20
H-2b, 12
3
4
5
6
121.9
161.5
39.8
H-2a
H-2ab, 19
H-6ab, 7, 20
H-7
2.64 (br.d)
H-1a, 5, 7
H-5, 6b, 7
H-6a, 20
H-6a
2.13–2.19 (m)
1.85 (dd, 14, 15)
3.34 (d, 5.5)
22.3
7
8
9
59.6
61.1
64.7
40.0
54.9
54.0
66.1
71.4
27.4
16.7
17.6
172.6
70.2
8.8
H-6ab, 14
H-6ab, 7, 14
H-11, 14-OH
H-2ab, 5, 6ab, 20, 1-OH
H-12
10
11
12
13
14
15
16
17
18
19
20
1-OH
14-OH
4.21 (d, 3)
3.59 (d, 3)
H-1, 5, 12, 14, 1-OH
H-11, 15, 16, 17
H-11, 14
H-11, 12, 14, 15, 16, 17, 14-OH
H-7, 14, 15, 16, 17
H-14, 16, 17
H-15, 17
H-15, 16
3.32 (d, 8)
H-15, 16, 17, 14-OH
H-12, 14, 16, 17, 14-OH
H-12, 14, 15, 17
2.12 (sept, 7)
0.76 (d, 7)
0.88 (d, 6.5)
H-12, 14, 15, 16
4.76 (q, 18)
1.01 (s)
4.01 (d, 4)
4.61 (d, 8)
H-5, 6ab
H-2b, 6b, 1-OH
H-1, 2b, 11, 2-OH
H-14, 15
1
Products 7 and 8 were obtained as a pair of epimers at a ratio
of about 3:1 on the basis of their 1H NMR spectral data. We
had tried to separate the epimers by a number of methods
but finally failed. They were colorless crystals and their
TOFMS showed the quasi-molecular peak at m/z 394
[MþNH4]þ and m/z 399 [MþNa]þ. Products 7 and 8 did not
produce a mauve spot as products 3–5 did on TLC by
spraying with the Kedde solution but produced a brown spot
with 10% H2SO4. This result suggested the loss of
metheylene group adjacent to a,b-unsaturated lactone
which was necessary for the Kedde reaction. Hence one
can deduce that the metheylene of C-19 might have been
substituted in products 7 and 8. IR spectra showed strong
hydroxyl group absorption at 3432 and 3274 cm21. High-
resolution mass measurement on the [MþHþ] at m/z
377.1596 was consistent with the molecular formula
C20H24O7 (Calcd 377.1600), suggesting that a hydroxyl
group had been introduced into the substrate molecule at
C-19. The 13C NMR spectra of 7 and 8 showed new peaks at
97.2 and 98.1 ppm, respectively. DEPT analyses showed
that the number of secondary carbons changed from 4 to 3,
and the number of tertiary carbons increased from 6 to 7. All
these data suggested that 7 and 8 were hydroxylated
products of triptolide at C-19. All the H and 13C NMR
data were assigned according to 1H–1H COSY, HSQC and
HMBC spectra. The relative stereochemistry was confirmed
by NOESY experiment (Scheme 2). The strong correlation
between 2.49 ppm (H-5) and 7.79 ppm (19-OH) of 7
suggested the a-configuration of 19-hydroxytriptolide in
7. Therefore, 19-OH in 8 should be of b-configuration. The
signal at 6.00 ppm (H-19) of 7 correlated with H-6a and
H-6b supported the orientation of the 19-OH as a-con-
figuration in 7. The signal at 6.05 ppm (H-19) of 8 was not
observed to have correlations with H-6a and H-6b. On the
basis of the above analysis, the structures of 7 and 8 were
identified as 19a-hydroxytriptolide and 19b-hydroxytripto-
lide, respectively, which are a pair of new compounds. The
1H and 13C NMR spectral data of compounds 7 and 8 were
summarized in Table 3.
The new products obtained from current investigation were
evaluated for their bioactivity against human tumor cell
lines. By MTT assay, products 2, 3, and mixture of 7 and 8
were found to exhibit potent in vitro cytotoxic activities
against several human tumor cell lines. Product 2 has IC50
values of 0.30, 1.50, 1.05, 0.34, 0.47 mM against KB,
Scheme 2. NOESY correlations of biotransformed products 7 and 8.

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L. Ning et al. / Tetrahedron 59 (2003) 4209–4213
Table 3. 1H and 13C NMR spectral data of 7 and 8 (DMSO-d6, J in Hz)
Perkin–Elmer QSTAR mass spectrometer. All the solvents
used for extraction and isolation were of analytical grade.
TLC was performed on silica gel G. (10–40 mM).
Separation and purification were carried out by column
chromatography on silica gel (200–300 mesh). Silica gel
was purchased from Qingdao Marine Chemical Group Co.,
P. R. China. Triptolide was detected on TLC by spraying
with the Kedde reagent or 10% H2SO4.
Position
1H
13C
1H
13C
1
2
1.19 (ddd, 12, 12, 6)
1.29 (dd, 12, 6)
2.12 (br.d)
29.0 1.19 (ddd, 12, 12, 6)
1.29 (dd, 12, 6)
16.6 2.12 (br.d)
1.92–1.99 (m)
126.8
29.1
16.4
1.92–1.99 (m)
3
4
5
6
127.0
159.3
40.3
160.5
2.49 (br.d)
39.5 2.59 (br.d)
22.3 2.21–2.23 (m)
1.92 (dd, 15, 13)
59.7 3.33 (d, 5.5)
61.0
3.2. Substrate
2.19–2.22 (m)
1.86 (dd, 15, 13)
3.37 (d, 5.5)
22.7
7
8
9
10
11
12
13
14
15
16
17
18
19
20
60.1
60.9
64.3
35.4
55.2
54.3
64.7
71.3
27.5
16.8
17.7
170.6
98.1
14.1
Triptolide 1 was purchased from Institute of Medical
1
Sciences of Fujian. Its structure was characterized by H
64.3
35.3
NMR, 13C NMR and MS spectra. The purity of triptolide,
determined by RP-HPLC evaluation using methanol–water
(60:40) is 98%. The substrate was dissolved in acetone and
diluted to 10 mg/mL before use.
3.86 (d, 3)
3.53 (d, 3)
55.2 3.86 (d, 3)
54.3 3.53 (d, 3)
64.8
71.3 3.31 (d, 7.5)
27.5 2.11 (sept, 7)
16.8 0.75 (d, 7)
17.7 0.87 (d, 7)
170.6
3.31 (d, 7.5)
2.11 (sept, 7)
0.75 (d, 7)
3.3. Microorganisms
0.87 (d, 7)
C. blakesleana (AS 3.970) were purchased from China
General Microbiological Culture Collection Center.
6.00 (d, 8)
0.96 (s)
4.72 (d, 7.5)
7.79 (d, 8)
97.3 6.05 (d, 8.5)
13.9 0.94 (s)
4.36 (d, 7.5)
14-OH
19-OH
7.60 (d, 8.5)
3.4. Medium
All culture and biotransformation experiments were per-
formed in potato medium. Potato medium was produced by
the following procedure: 200 mg of minced husked potato
were boiled in water for one hour, then the solution was
filtered and the filtrate were added with water to 1 L after
addition of 20 mg of glucose.
BGC823, MCF-7, Hela and HL-60 cells, respectively.
Product 3 showed significant cytotoxicity against KB,
BGC823, MCF-7, Hela and HL-60 cells with IC50 values
of 0.12, 7.65, 0.87, 0.32, 0.07 mM, respectively. Mixture of
products 7 and 8 had cytotoxic activities against KB and
HL60 cells with IC50 values of 46.5 and 52.6 mM,
respectively.
3.5. Biotransformation
Biotransformation is now becoming an increasingly import-
ant tool available to synthetic chemists in the structural
modifications of natural or synthetic organic compounds.
The rich enzymes in microorganisms could be used for
biotransform or synthesize some natural products. In this
study, we report a powerful method for the preparation of a
variety of derivatives from triptolide using C. blakesleana
(AS 3.970) as a biocatalyst. All of the new products were
found to exhibit potent in vitro cytotoxic activities against
human tumor cell lines. Further studies on their bioactivity
are under investigation.
The screening-scale run was performed in 250 mL
Erlenmeyer flasks containing 60 mL of potato medium.
Microorganisms were transferred into the flasks from the
slants. The cultures were cultivated on a rotary shaker at
160 rpm at 258C. For preparative transformation, 1 mL of
substrate solution was added into a 1000 mL Erlenmeyer
flasks containing 300 mL potato medium that had been pre-
cultured for 48 h. In total 500 mg of substrate were added.
The incubation was continued for 5 days under the same
conditions as described above. Culture controls consisted of
fermentation blanks in which microorganisms were grown
without substrate but with the same amount of acetone.
Substrate controls were composed of sterile medium
containing the same amount of substrate and incubated
under the same conditions.
3. Experimental
3.1. General procedures
3.6. Extraction and isolation of biotransformed products
Melting points were measured with an XT4A micro-melting
point apparatus and uncorrected. Optical rotations were
recorded on a Perkin–Elmer 243B polarimeter using MeOH
as solvent with a 1 cm path length. IR spectra were recorded
on an Avatar 360 FT-IR spectrophotometer in KBr pellets.
1D and 2D NMR spectra were run on a Bruker DRX-500
After 5 days of incubation, the culture was filtered and the
filtrate was extracted with the same volume of ethyl acetate
for three times. The organic phase was evaporated to
dryness in vacuo. The residues were dissolved in acetone.
The solution was spotted on silica gel TLC plate which was
developed by petroleum ether (60–908C)–ethyl acetate
(1:5) and visualized by spraying with 10% H2SO4 solution.
TLC chromatography showed that C. blakesleana had the
ability to biotransform triptolide. Transformed products
were more polar than the substrate. No transformation
products were found in the controls.
spectrometer (500 MHz for 1H NMR and 125 MHz for 13
C
NMR) in DMSO-d6 with TMS as an internal standard. The
Chemical shift values (d) were given in parts per million
(ppm), and the coupling constants were in hertz (Hz). High
resolution positive SIMS were performed on a Bruker Apex
II FI-ICR mass spectrometer. TOFMS were measured with a

L. Ning et al. / Tetrahedron 59 (2003) 4209–4213
4213
The residue (2.5 g) was subjected to column chromatography
over silica gel and eluted with petroleum ether–ethyl
acetate gradient (4:1 to pure ethyl acetate). Bio-
transformation of 1 by C. blakesleana resulted in 90 mg
of 2 (18% yield), 9 mg of 3 (1.8% yield), 15 mg of 4 (3%
yield), 12 mg of 5 (2.4% yield), 10 mg of 6 (2% yield) and
100 mg of 7 and 8 (20% yield).
NSF of China (39925040) and Trans-Century Training
Program Foundation for the Talents by the Ministry of
Education for financial support.
References
3.6.1. 5a-Hydroxytriptolide 2. Colorless crystals; mp
250–2528C; [a]2D5¼2132.9 (c 0.12, MeOH); IR (KBr)
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nmax 3470, 2962, 2932, 1762, 1677, 1079, 1029 cm21
;
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1
377.1600, found 377.1594. The H and 13C NMR spectral
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1
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[2MþNH4]þ, 775 [2MþNa]þ; HRMS: calcd for
C20H25O7 [MþH]þ, 377.1600, found 377.1598. The 1H
and 13C NMR spectral data are summarized in Table 3.
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We thank the National Outstanding Youth Foundation by