Regio-selective Reduction of Oxysophoridine by Microorganism Isolated from Soil

Article abstract of DOI:10.1002/jccs.201500501

Oxysophoridine (OSR), an alkaloid extracted from a traditional Chinese medicine, attracted more and more attention because of remarkable biological activities. Biocatalysis of OSR by 86 stains including soil microbes and endophytic fungi were investigated. Incubation of OSR with soil bacteria T003 identified as Escherichia coli could lead to only one reductive product with highly regio-selectivity. The structural elucidations of transformation product were achieved mainly by the NMR spectroscopic methods and references. Effect of pH, substrate concentration, conversion time and strain concentration was studied, the optimization of reaction was analyzed by LC-MS. The production of sophoridine (SR) from OSR was highest at pH 7.0, 36 h of incubation and in presence of 625 μg/mL substrate concentration, 50 μL/mL strain concentration respectively. Biocatalysis was considered the alternatives to preparation of sophoridine with green and sustainable synthetic processes except for less time-consuming and more environmentally friendly.

Full text of DOI:10.1002/jccs.201500501

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Regio-selective Reduction of Oxysophoridine by Microorganism Isolated from Soil
a
Shaobin Fu, * Yi Li, Qingfeng Meng, Changwu Yue, Yongzhen Sun and Gang Wei
a
b
c
d
e
a
Department of Pharmacy, Zunyi Medical College, Zunyi, 563000, China
Department of Public Health, Zunyi Medical College, Zunyi, 563000, China
b
c
Guizhou Key Laboratory of Microbial Resources & Drug Development, Zunyi Medical University, Zunyi, 563003,
China
d
Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China
Materials Science and Engineering, Commonwealth Scientific and Industrial Research Organization (CSIRO), 2070,
Australia
e
(Received: Dec. 6, 2015; Accepted: Mar. 18, 2016; Published Online: Apr. 23, 2016; DOI: 10.1002/jccs.201500501)
Oxysophoridine (OSR), an alkaloid extracted from a traditional Chinese medicine, attracted more and
more attention because of remarkable biological activities. Biocatalysis of OSR by 86 stains including soil
microbes and endophytic fungi were investigated. Incubation of OSR with soil bacteria T003 identified as
Escherichia coli could lead to only one reductive product with highly regio-selectivity. The structural elu-
cidations of transformation product were achieved mainly by the NMR spectroscopic methods and refer-
ences. Effect of pH, substrate concentration, conversion time and strain concentration was studied, the op-
timization of reaction was analyzed by LC-MS. The production of sophoridine (SR) from OSR was high-
est at pH 7.0, 36 h of incubation and in presence of 625 mg/mL substrate concentration, 50 mL/mL strain
concentration respectively. Biocatalysis was considered the alternatives to preparation of sophoridine
with green and sustainable synthetic processes except for less time-consuming and more environmentally
friendly.
Keywords: Biocatalysis; Region-stereoselectivity; Soil microbes; Natural product; Condition
optimization.
INTRODUCTION
Oxysophoridine (Sophoridine N-oxide,OSR) was
have demonstrated their catalytic capacities at non-acti-
vated positions of the skeleton that were difficult to achieve
5
by classical chemical means. However, there was no re-
isolated a natural alkaloid extracted from a traditional Chi-
nese medicine Sophora lopecuroides L. It is reported that
9
OSR displayed significant activities, such as anti-tumor,
port on the structural modifications of OSR by microbes.
Herein, the present was to study the structural modification
of OSR by microbial transformation to obtain OSR deriva-
tives.
1
anti-arrhythmia, anti-inflammation, central inhibi-
2
7
4
,10,11,13,15
3,8
antinociception. Besides, Wang et al.
tory,
found that OSR had a neuroprotective effect against cere-
6
bral ischemia/reperfusion injury in mice. OSR may reduce
RESULTS AND DISCUSSION
streptozotocin-induced diabetic rat aorta lesions and have a
1
protective effect on the ultrastructure of the aorta.
Screening of OSR by biocatalysis
0
86 strains isolated were used to screening of OSR and
18 strains belong to two kinds of reactions showed activity
of transforming OSR into other structures while the endo-
phytic fungi had not such capacity. Two types of reactions
were found, one reaction can produce two products with
smaller polarity and the other with the only product was de-
tected. T003 which was identified as Escherichia coli on
16S rDNA was selected to prepare the transformation pro-
Derivatives of natural product were synthysied by
chemical methods while biotransformation because of its
mild conditions, environment-friendly, sustainable at-
tracted more and more attention in recent years. Microor-
ganisms with multi-enzyme systems were known to have
the ability to perform the regio- and stereo-selective bio-
catalysis to organic compounds. Thus, microbial catalysis
technique is increasingly being used as one of the feasible
approaches by far for structurally modifying natural and
1
duct because of its high region-selectivity.
Preparation of transform product of OSR
2
synthetic compounds. A good number of microorganisms
Strain T003 with only one product was selected to
*
Corresponding author. Tel: +86-0851-28642513; Fax: +86-0851-28609493; Email: fushb@126.com
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Article
Fu et al.
prepare the only derivative (Figure 1). The transformation
product was obtained by silica gel column chromatogra-
phy, Sephadex LH-20 and recrystallization.Product: White
1
crystalline solid (methanol). H-NMR (CDCl
3
) (Figure 2):
3
2
.21~3.45 (3H, m, 11-H, 17-2H), 2.78 (2H, m, 10,2-H),
.32 (3H, m, 14-2H, 6-H), 2.12 (2H, m, 10, 2-H), 2.07 (2H,
m, 3-Ha, 5-H), 1.92 (2H, m, 12,4-H), 1.85~1.01 (10H, m).
1
3
C-NMR (CDCl
6.0 (C-2), 55.8 (C-11), 50.4 (C-10), 47.7 (C-17), 41.0
C-7), 32.6 (C-14), 30.9 (C-5), 30.3 (C-12), 28.2 (C-4),
3.8 (C-3), 21.9 (C-8), 21.6 (C-9), 19.0 (C-13). Compared
3
) (Figure 3): 170.1 (C-15), 63.4 (C-6),
5
(
2
1
13
with H-NMR and C-NMR, spectral data with those re-
1
4
ported in the literature, its structure was confirmed as SR.
Optimization of reaction conditions
It could be found that pH 7, 8 and 9 were suitable for
both the growth of the strain and catalytic activity of en-
zyme, and the highest SR production occurred when pH
value was at 7 (Figure 4). Apparently, OSR of 625 mg/ml
was optimum for SR production; above this concentration,
production of SR decreased, SR production was considered
unnormal which suddenly dropped to 0, however, from the
OSR reduction, the optimal substrate concentration could
also be inferred (Figure 5). Every test tube was harvested
Fig. 1. Selectively transformation of OSR by strain
T003.
1
Fig. 2. H-NMR of transformation product of T003.
332
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
New Preparation Method of Oxysophoridine by Biocatalysis
1
Fig. 3. C-NMR of transformation product of T003.
3
Fig. 4. Effect of pH on transformation.
Fig. 5. Effect of substrate concentration on transfor-
mation.
ever 12 h. The results demonstrated that SR reached to
maximum at 36 h (Figure 6). The production of H reached
to the maximal levelof 856 mg/mL. Strain concentration
could also affect the SR production from OSR. According
to Figure 7, with the increase of strain concentration, the
conversion activity gradually increased. SR reached the
maximum production (50 mL/mL) and then decreased.
corded on an Agilent NMR Spectrometer operating at 400 MHz in
3
CDCl with TMS as an internal standard. TLC analyses were car-
ried out on pre-coated silica gel GF254 plates (0.25 mm thick,
Qingdao Oceanic Chemicals, China). LC-MS (AB SCIEX) was
carried out on C-18 column with methanol – water = 85:15 and
with the condition of CUR: 40.00, CAD; Medium; IS:-4500.00;
TEM: 550.00; GS1: 55.00; GS2: 55.00; DP: -140.00; EP: -10.00;
CE: -40.00; CXP: -15.00. Column chromatography was carried out
on silica gel (200–300 mesh, Qingdao Oceanic Chemicals, China).
EXPERIMENTAL
General experimental procedure: NMR spectra were re-
J. Chin. Chem. Soc. 2016, 63, 331-335
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Fu et al.
were incubated for another 3 days. For endophytic fungi, the same
quantity of substrate was added after incubation for 48 hours and
the cultures were incubated for another 5 days. Then the cultures
were then extracted three times with EtOAc. The extracts were
evaporated under reduced pressure and were analyzed by TLC.
Culture with microorganisms but without substrate and culture
without microorganism but with substrate were both designated
as the controls and were incubated in the same conditions as de-
scribed above.
Biocatalysis of OSR by T003 and purification of the
product: The preparative scale biocatalysis of OSR by soil mi-
crobes was carried out in 8.2 L medium in sixteen 1 L flask and
Fig. 6. Effect of conversion time on transformation.
9
00 mg substrate in total was added. Incubation conditions were
the same as mentioned above. But after being extracted with
EtOAc, the product can be detected in the fermentation liquid.
The aqueous phase of the fermentation liquid was vaporated un-
der reduced pressure again. About 4.04 g crude extract was ob-
tained.
The crude extracts were subjected to silica gel column
chromatography that was adjusted pH > 7 with diethylamine and
eluted with mixtures of chloroform/methol from 40:1 to 0:1. Frac-
tion A, B, C and D were obtained. Fraction B was further purified
by Sephadex LH-20 with chloroform /methol, gel column chro-
matography with chloroform/methol several times and recry-
stallization, and then the only transformation product (96.8 mg)
were obtained.
Fig. 7. Effect of strain concentration on transforma-
tion.
Optimization of reaction conditions: Optimization of pH
(
from 5 to 9), substrate concentration (1250 mg/ml, 625 mg/ml,
Visualization of the TLC plates was performed by Bismuth potas-
sium iodide spray reagent that is specific chromogenic agent to
alkloids. General solvents and reagents were purchased from
Chengdu kelong Chemical Industry Company, Chengdu, China.
Substrate: Substrate OSR (with purity > 98%) was pur-
chased from Beijing Gold- moutain Chromatography Science and
Technology Co. Ltd.
312.5 mg/ml, 156.25 mg/ml and 78.125 mg/ml), transformation
time (12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h) and strain concentra-
tion (12.5 ml/ml, 25 ml/ml, 37.5 ml/ml, 50 ml/ml, 65.5, 62.5 ml/ml)
was investigated, and the optimization of reaction was analyzed
by LC-MS.
ACKNOWLEDGEMENTS
Microorganisms: Microbial strains consist of soil micro-
organisms isolated from soil in the campus of Zunyi Medical Uni-
versity and endophytes isolated from plant in Guizhou province.
This research was financially supported by The
National Natural Sciences Foundation of China (No.
21462057), Program of Ministry of Education “Chunhui
Plan” (No. Z2014091), Guizhou Science and Technology
Department (QKHLH-2014-7555), Program for Guizhou
Key Laboratory of Microbial Resources & Drug Develop-
ment (No. GZMRD-2014-003) and Ntional Training Pro-
gram of Innovation and Entrepreneurship for Undergradu-
ates (201410661013).
1
Soil microbes were isolated by soil dilution method.
Screening procedures: The preliminary screening experi-
ment of soil microbes was carried out in LB media and endo-
phytes in PDA media. Fermentations were carried out according
to a standard two-stage protocol. The cultures were grown in 100
mL Erlenmeyer flasks containing 40 mL of medium. The flasks
were incubated at 37 °C for soil microbes and 28 °C for endo-
phytes and 140 rpm on a shaker. And then 2 mg substrate (in etha-
nol) was added after 6 hours for soil microbes and the cultures
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