An improved water-soluble/stereospecific biotransformation of aporphine alkaloids in Stephania epigaea to 4R-hydroxyaporphine alkaloids by Clonostachys rogersoniana

Article abstract of DOI:10.1016/j.procbio.2016.04.016

Aporphine alkaloids were transformed into the corresponding stereospecific 4R-hydroxyaporphine alkaloids through the solid-state fermentation of Stephania epigaea with Clonostachys rogersoniana. This process was validated by both solid- and liquid-state fermentations with aporphine alkaloids as substrates. Cytochrome P450 enzymes were confirmed to participate in the catalysis of this biotransformation. 4R-Hydroxyaporphine alkaloids exhibit the same levels of acetylcholinesterase (AChE) inhibitory and cytotoxic activities as aporphine alkaloids and are considerably more water soluble than aporphine alkaloids, indicating their potential as water-soluble AChE inhibitors and antitumor agents. This paper suggests that C. rogersoniana fermentation can facilitate a novel biotransformation of aporphine alkaloids in S. epigaea to 4R-hydroxyaporphine alkaloids.

Full text of DOI:10.1016/j.procbio.2016.04.016

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Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
An improved water-soluble/stereospecific biotransformation of
aporphine alkaloids in Stephania epigaea to 4R-hydroxyaporphine
alkaloids by Clonostachys rogersoniana
Le Caia,1, Jian-Wei Donga,1, Li-Xing Zhaob, Hao Zhoua, Yun Xinga, Ying Lia, Zhen-Jie Lia,
Wei-He Duana, Xue-Jiao Lia, Zhong-Tao Dinga,∗
a Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University,
Kunming 650091, China
b Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Aporphine alkaloids were transformed into the corresponding stereospecific 4R-hydroxyaporphine
alkaloids through the solid-state fermentation of Stephania epigaea with Clonostachys rogersoniana.
This process was validated by both solid- and liquid-state fermentations with aporphine alkaloids as
substrates. Cytochrome P450 enzymes were confirmed to participate in the catalysis of this biotransfor-
mation. 4R-Hydroxyaporphine alkaloids exhibit the same levels of acetylcholinesterase (AChE) inhibitory
and cytotoxic activities as aporphine alkaloids and are considerably more water soluble than aporphine
alkaloids, indicating their potential as water-soluble AChE inhibitors and antitumor agents. This paper
suggests that C. rogersoniana fermentation can facilitate a novel biotransformation of aporphine alkaloids
in S. epigaea to 4R-hydroxyaporphine alkaloids.
Received 16 January 2016
Received in revised form 16 April 2016
Accepted 19 April 2016
Available online xxx
Keywords:
Aporphine alkaloid
4R-Hydroxyaporphine alkaloid
Stephania epigaea
© 2016 Elsevier Ltd. All rights reserved.
Biotransformation
Clonostachys rogersoniana
Water solubility
1. Introduction
the fermentation of Bletilla striata [10], Bletilla formosana [11], and
Asparagus filicinus [12] facilitates biotransformations of their com-
Stephania epigaea Lo is a herbaceous liana primarily found in
the Yunnan and Sichuan provinces of China [1]. Previous phyto-
chemical studies have shown that aporphine [2], morphine [3],
and bisbenzylisoquinoline alkaloids [4,5] are the major chemi-
cal constituents of this plant, some of which exhibit significant
acetylcholinesterase (AChE) inhibitory [2] and cytotoxic activities
[6]. Aporphine alkaloids, an important group of plant secondary
metabolites, have long been used in traditional medicine to treat
various diseases [6]. (R)-Dicentrine (1a) (Fig. 1), a major apor-
phine alkaloid in S. epigaea, was found to exhibit significant AChE
inhibitory [2] and cytotoxic activities [7,8].
Herbal fermentation processing, which has been practiced for
more than 4000 years in China, is used to produce secondary
metabolites from domestic plants in bulk by utilizing the metabolic
mechanisms of microorganisms. In recent years, some interesting
biotransformation reactions have been developed for herbal fer-
mentation processing [9]. Our previous studies have shown that
ponents and enhances their bioactivities.
This paper reports on an efficient biotransformation of
aporphine alkaloids to the corresponding stereospecific 4R-
hydroxyaporphine alkaloids during the solid-state fermentation
processing of S. epigaea with Clonostachys rogersoniana. Liquid-state
fermentation was also found to facilitate this transformation with
very high yields. Cytochrome P450 enzymes were confirmed to
participate in the catalysis of this biotransformation. It is worth
noting that 4R-hydroxyaporphine alkaloids exhibit the same levels
of AChE inhibitory and cytotoxic activities as aporphine alkaloids
and are considerably more water soluble than aporphine alka-
loids, indicating their potential as water-soluble AChE inhibitors
and antitumor agents.
2. Materials and methods
2.1. Chemicals and instruments
Optical rotations were measured using a Jasco P-1020 digital
polarimeter (Jasco, Tokyo, Japan). Electronic circular dichro-
ism (ECD) spectra were recorded using a Chirascan circular
dichroism spectrometer (Applied Photophysics Ltd., UK). Nuclear
∗
Corresponding author.
E-mail address: ztding@ynu.edu.cn (Z.-T. Ding).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.procbio.2016.04.016
1359-5113/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: L. Cai, et al., An improved water-soluble/stereospecific biotransformation of apor-
phine alkaloids in Stephania epigaea to 4R-hydroxyaporphine alkaloids by Clonostachys rogersoniana, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.04.016

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20 mL of seed culture medium (potato dextrose broth (PDB); 1 L of
R
3
4
water, 200 g of potato, and 20 g of dextrose) and placed on a rotary
shaker shaking at 120 r/min at 28 ◦C for 3 days. Subsequently, 10 g
of S. epigaea was added to a 100-mL Erlenmeyer flask to serve as
the fermentation culture medium. After this fermentation culture
medium was infiltrated with 8 mL water and sterilized at 121 ◦C
for 30 min, the seed cultures were added and the medium was
incubated at 28 ◦C for 30 days. Then, the herbs, before and after fer-
mentation, were immersed in methanol for 24 h and extracted with
an ultrasonic cleaner thrice (30 min each). The methanol extract of
the non-fermented S. epigaea was further subjected to silica gel
CC (chloroform–methanol, (50:1, 30:1)) and Sephadex LH-20CC
(methanol) to yield 1a (166 mg). Further silica gel CC purification
of the methanol extract of the C. rogersoniana-fermented S. epigaea
was achieved by eluting with chloroform–methanol (50:1, 20:1)
and Sephadex LH-20 (methanol) to afford 1b (160.5 mg).
2
O
5
3a
1b
6a
6
1
N
O
CH3
1a
H
11a
7
11
10
7a
8
H3CO
9
OCH3
1a R = H
(R)-dicentrine
1b R = OH (4R,6aR)-4-hydroxydicentrine
Fig. 1. The structures of 1a and 1b.
magnetic resonance (NMR) spectra were recorded on a Bruker
Avance 400 MHz spectrometer (Bruker, Karlsruhe, Germany) using
tetramethylsilane (TMS) as the internal reference. High-resolution
electrospray ionization mass spectrometry (HRESIMS) and elec-
trospray ionization mass spectrometry (ESIMS) analyses were
conducted using an Agilent G3250AA (Agilent, Santa Clara, CA,
USA) and an Auto Spec Premier P776 spectrometers (Waters, Mil-
ford, MA, USA). Silica gel (200–300 mesh, Qingdao Marine Chemical
Group Co., Qingdao, China) was used for column chromatography
(CC). The fractions were monitored by thin-layer chromatography
(TLC) and visualized by spraying with a modified Dragendorff’s
reagent.
2.4. Solid-state fermentation of aporphine alkaloids
C. rogersoniana grown in a shaking culture at 28 ◦C for 3 days in
250-mL conical flasks containing 50 mL of sterile PDB was used as
the seed medium. The potato containing 20 mg of aporphine alka-
loid, without excess water, was used as the solid-state fermentation
medium. After sterilization, the mature strains in the seed medium
were added to the fermentation medium and incubated at 28 ◦C
for 30 days. Then, the medium was immersed in methanol for 24 h
and extracted thrice with an ultrasonic cleaner (30 min each). The
methanol extract was further subjected to repeated silica gel CC
and Sephadex LH-20 to afford the product.
(R)-(+)-␣-Methoxy-␣-(trifluoromethyl)phenylacetic
acid
(>98%,
ee:
99.5%)
and
(S)-(−)-␣-methoxy-␣-
(trifluoromethyl)phenylacetic acid (>98%, ee: 99.5%) were
purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
N,N’-Diisopropylcarbodiimide
and
4-dimethylaminopyridine
2.5. Liquid-state fermentation of aporphine alkaloids
were obtained from Adamas Reagent Co., Ltd. (Shanghai, China).
Methanol and acetonitrile (high-performance liquid chromatog-
raphy (HPLC) grade) were purchased from Fisher Scientific Co.,
Ltd. (Fair Lawn, NJ, USA). 2-Methyl-1,2-di-3-pyridyl-1-propanone
(metyrapone, 96%), CDCl3 (99.9 atom% D, contains 0.03% TMS) and
MeOD (99.9 atom% D, contains 0.03% TMS) were obtained from
Sigma-Aldrich (Shanghai, China). 1-Aminobenzotriazole (ABT,
98%), piperonyl butoxide (PBO, 90%), and n-tetradecane (98%)
were purchased from J&K Scientific Ltd. (Beijing, China). The water
(resistivity ≥18.25 Mꢀ/cm) used was purified using a water-
purifying system (Chengdu, China). Dicentrine (≥96%), romeline
(≥95%), romerine (≥95%), N-methyllaurotetanine (≥95%), and
dehydrodicentrine (≥95%) were previously isolated from S. epigaea
by our group [2]. All other chemicals used were of analytical grade.
C. rogersoniana was grown in a shaking culture at 28 ◦C for 3
days in 250-mL conical flasks that contained 50 mL of sterile PDB.
The substrate (10.0 mg, 5.0 mg each) dissolved in MeOH (0.5 mL)
was evenly distributed between two flasks which contained the
aforementioned 50-mL sterile PDB and 3-days cultured strains, and
incubation was continued for 7 days. The broth was extracted with
EtOAc thrice to afford the extract.
2.6. Preparation of (S)- and (R)-MTPA esters of 1b
To a stirred solution of 1b (10.0 mg, 0.028 mmol) in CDCl3
(2 mL), (S)-(−)-MTPA-OH (5 ␮L, 0.029 mmol) and (R)-(+)-MTPA-OH
(5 ␮L, 0.029 mmol) were added, respectively. Then, 10.0 mg of N,Nꢀ-
diisopropylcarbodiimide and 10.0 mg of 4-dimethylaminopyridine
were added. The reaction mixture was stirred at room temper-
ature for 30 min and concentrated under vacuum at 45 ◦C. The
residues were purified by silica gel CC (chloroform–methanol,
20:1) to afford (S)-MTPA ester (10.0 mg, 62.2%) and (R)-MTPA ester
(10.5 mg, 65.3%), respectively.
2.2. Materials and microorganisms
The roots of S. epigaea were collected from Kunming, Yunnan,
China, in December 2014 and identified by Prof. Shugang Lu at the
School of Life Sciences, Yunnan University. A voucher specimen
(2014-dbr-01) was deposited at the Key Laboratory of Medicinal
Chemistry for Natural Resource, Ministry of Education, Yunnan Uni-
versity, Kunming, Yunnan, China.
2.7. AChE inhibitory activity
C. rogersoniana 828H2 was obtained from the Yunnan Institute
of Microbiology, Yunnan Province, China. The nucleotide sequence
data reported in this paper were deposited in GenBank (accession
number KT625993).
AChE inhibition was performed spectrophotometrically using
the Ellman method [13] with slight modifications [2]. The com-
pounds were dissolved in 10% dimethyl sulfoxide (DMSO), and
the reaction mixture (total volume of 200 ␮L) containing 110 ␮L
of phosphate buffer (PB, 0.1 M, pH 8.0), 10 ␮L of the test com-
pound (50 ␮M, final concentration), and 40 ␮L of AChE (0.02 U/mL,
final concentration) was incubated for 20 min (30 ◦C). Then, the
reaction was initiated by adding 40 ␮L of a solution containing
5,5ꢀ-dithiobis(2-nitrobenzoic acid) (DTNB, 0.625 mM) and acetylth-
iocholine iodide (ATCI, 0.625 mM) to assay the AChE inhibitory
activity. The hydrolysis of acetylthiocholine was monitored at
412 nm every 30 s for 30 min using a Tecan Infinite® M1000 Pro
2.3. Herbal fermentation procedure
Potato dextrose agar (PDA) (1 L of water, 200 g of potato, 20 g
of dextrose, and 15 g of agar) slant culture medium was inocu-
lated with the aforementioned plant pathogenic fungi and held
in a constant-temperature incubator at 28 ◦C for 5 d. Then, the
mature slants were added to a conical flask (150 mL) that contained
Please cite this article in press as: L. Cai, et al., An improved water-soluble/stereospecific biotransformation of apor-
phine alkaloids in Stephania epigaea to 4R-hydroxyaporphine alkaloids by Clonostachys rogersoniana, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.04.016

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Table 1
3
microplate reader. Tacrine and huperzine A (Hup A) were used
as the positive controls with final concentrations of 0.333 and
0.05 ␮M, respectively. All of the reactions were performed in trip-
licate. The percentage inhibition (I%) was calculated as follows:
The NMR spectroscopic data for 1a and 1b in CDCl3.a
position
1a
1b
ıC
ıH mult. (J in Hz)
ıC
ıH mult. (J in Hz)
1
146.4, C
116.4, C
126.3, C
141.6, C
106.6, CH
126.4, C
29.0, CH2
147.4, C
116.3, C
130.9, C
142.9, C
104.8, CH
126.7, C
66.5, CH
1a
1b
2
3
3a
4
I% = [(AE − AB) − (AS − AB)]/(AE − AB) × 100
where AE is the absorbance of the enzyme without the test com-
pound, AS is the absorbance of the enzyme with the test compound,
and AB is the background absorbance.
6.42 s
6.94 s
3.08 m
2.58 m
4.91 br s
The IC50 values of the potential AChE inhibitory compounds
were calculated from the inhibition curves obtained by plotting the
percentage inhibition at various concentrations of the inhibitor.
5
54.4, CH2
3.04 m
2.41 m
61.5, CH2
3.28 m
2.31 m
6a
7
62.2, CH
34.0, CH2
3.01 br s
2.97 dd (11.6, 5.8)
2.45 dd (11.6, 5.8)
62.5, CH
33.8, CH2
3.15 br d (14.0)
3.01 dd (14.0, 4.0)
2.61 dd (14.0, 4.0)
2.8. Cytotoxic activity
7a
8
9
10
11
11a
OCH2O
128.2, C
111.1, CH
147.5, C
148.0, C
110.3, CH
123.4, C
123.4, C
111.4, CH
147.8, C
148.5, C
110.6, CH
128.2, C
Cytotoxicity was assayed using the trypan blue method [14]
with a human leukemia cell line (HL-60) and using the MTT assay
[15] with a liver hepatocellular carcinoma cell line (SMMC-7721),
lung carcinoma cell line (A-549), breast carcinoma cell line (MCF-
7), and colon carcinoma cell line (SW480). The cell lines were
purchased from America Type Culture Collection (ATCC; Rockville,
MD, USA) and cultured in RPMI-1640 medium (Gibco, New York,
NY, USA) supplemented with 100 U/mL of penicillin, 100 ␮g/mL
of streptomycin, 1 mM glutamine and 10% heat-inactivated fetal
bovine serum (Gibco). cis-Dichlorodiammineplatinum (DDP) and
taxol were selected as the positive controls. Each sample was
assayed in triplicate.
6.72 s
7.60 s
6.71 s
7.61 s
100.4, CH2
5.82 s
5.98 s
101.0, CH2
6.04 s
5.90 s
a
1H NMR recorded at 400 MHz; 13C NMR recorded at 100 MHz.
2.11. Effects of the inhibitors metyrapone, ABT, and PBO and the
inducer n-tetradecane
The inhibitors metyrapone, ABT, and PBO (40 ␮g/mL, final
concentration) and the inducer n-tetradecane (2%, v/v, final con-
centration) were added to the 24-h spore suspension. After an
additional 48 h of culturing, 1 mL of the substrate (5 mg/mL) was
added. The spore suspension without the inhibitors and inducer
was selected as the positive control. The contents of the product
and reactant were determined by HPLC after 6 h, 12 h, 1 day, 2 days,
3 days, 4 days, and 7 days, respectively. Each sample was repeated
in triplicate.
2.9. Determining reaction yield by HPLC analysis
All samples and standards were filtered through a 0.45-␮m fil-
ter prior to injection into a Waters HPLC system equipped with a
Waters 600 quaternary pump, a Waters 2487 dual-ꢁ absorbance
detector, an Agilent Zorbax SB-C18 (250 × 4.6 mm i.d., 5 ␮m) col-
umn (SN: USCL054040), and an Empower data-processing station.
A gradient elution system consisting of solvents A (0.02% diethy-
lamine) and B (50% acetonitrile–methanol) was used for the
analysis, and the gradient program was as follows: 0–5 min, 20%
solvent B; 5–10 min, 20–40% solvent B; 10–25 min, 40–70% sol-
vent B; 25–30 min, 70% solvent B; 30–40 min, 70–100% solvent B;
40–45 min, 100% solvent B; and 45–50 min, 100–20% solvent B. The
peaks were confirmed by ultraviolet absorption at a wavelength of
280 nm and by the retention times. The flow rate was 0.8 mL/min,
the column temperature was set to 25 ◦C, and the injection volume
was 20 ␮L. Each sample was analyzed in triplicate. The contents
of products were determined according to the corresponding cali-
bration curves; further conversions were calculated on the basis of
these contents.
2.12. Theoretical electronic circular dichroism (ECD) calculation
The theoretical calculations of compounds 1a and 1b were per-
formed using the Gaussian program by the Yunnan Electronic
Computing Center. Two geometries of 1a and four geometries of
1b (Fig. S21) were previously optimized using the density func-
tional theory (DFT) method at the B3LYP/6-31G(d,p) level, and the
excitation energies and rotational strengths were calculated using
time-dependent density functional theory (TDDFT) at the B3LYP/6-
31 G(d,p) level [18]. The ECD spectra were simulated from the
electronic excitation energies and velocity rotational strengths.
2.13. Spectroscopic data
2.10. Water solubility analysis
(R)-Dicentrine (1a): white amorphous powder; HRESIMS m/z
340.1544 (Calcd. for C20H22NO4 340.1548); 1H and 13C NMR spec-
troscopic data, see Table 1.
Water solubility was measured using a previously reported
method [16,17]. A total of 10.0 mg of compound was added to
1.5-mL Eppendorf tubes containing 0.5 mL of HPLC-grade water.
The tubes were placed in an ultrasonic cleaner at 25 ◦C for 20 min.
The excess solid was separated by centrifugation (10,000 r/min) for
5 min, and the supernatant was passed through a 0.45 ␮m filter. The
concentrations of the samples were determined using a Waters
HPLC system on Agilent Zorbax SB-C18 (250 × 4.6 mm i.d., 5 ␮m)
with a flow rate of 0.8 mL/min. The separation conditions were the
same as those reported in Section 2.9. Each sample was analyzed in
triplicate. The contents were calculated according to the standard
curves.
(4R,6aR)-4-Hydroxydicentrine (1b): white amorphous powder;
25
␣
−39.6 (c 0.020, CHCl3); ESIMS m/z 356 [M+H]+; HRESIMS
[
]
D
m/z 356.1503 (Calcd. for C20H22NO5 356.1492); 1H and 13C NMR
spectroscopic data, see Table 1.
(S)-MTPA ester of 1b: white amorphous powder; ESIMS m/z
572 [M+H]+; HRESIMS m/z 572.1894 (Calcd. for C30H29F3NO7
572.1891); 1H NMR (CDCl3, 400 MHz): ıH 7.62 (1H, s), 7.57 (2H, m),
7.43 (3H, m), 6.76 (1H, s), 6.55 (1H, s, H-3), 6.29 (1H, br d, J = 6.4 Hz),
6.11 (1H, s), 5.96 (1H, s), 3.91 (6H, s), 3.58 (3H, s), 3.40 (1H, dd,
J = 10.0, 6.4 Hz, H-5a), 3.17 (1H, dd, J = 14.0 Hz, H-6a), 3.07 (1H, dd,
Please cite this article in press as: L. Cai, et al., An improved water-soluble/stereospecific biotransformation of apor-
phine alkaloids in Stephania epigaea to 4R-hydroxyaporphine alkaloids by Clonostachys rogersoniana, Process Biochem (2016),
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J = 14.0, 4.4 Hz), 2.63 (1H, dd, J = 14.0, 4.4 Hz), 2.55 (3H, s, NCH3),
2.39 (1H, t, J = 10.0 Hz, H-5b).
(R)-MTPA ester of 1b: white amorphous powder; ESIMS m/z
572 [M+H]+; HRESIMS m/z 572.1895 (Calcd. for C30H29F3NO7
572.1891); 1H NMR (CDCl3, 400 MHz): ıH 7.61 (1H, s), 7.59 (2H,
m), 7.44 (3H, m), 6.76 (1H, s), 6.27 (1H, s, H-3), 6.26 (1H, m), 6.06
(1H, s), 5.92 (1H, s), 3.91 (3H, s), 3.88 (3H, s), 3.58 (3H, s) 3.50 (1H,
dd, J = 10.0, 6.4 Hz, H-5a), 3.19 (1H, dd, J = 14.0 Hz, H-6a), 3.07 (1H,
dd, J = 14.0, 4.4 Hz), 2.63 (1H, dd, J = 14.0, 4.4 Hz), 2.58 (3H, s, NCH3),
2.49 (1H, t, J = 10.0 Hz, H-5b).
(4R,6aR)-4-Hydroxyromerine (2b): white amorphous powder;
25
␣
[
−9.89 (c 0.020, MeOH); ESIMS m/z 296 [M+H]+; HRESIMS
]
D
m/z 296.1284 (Calcd. for C18H18NO3 296.1281); 1H NMR (CDCl3,
400 MHz) ıH 7.03 (1H, s, H-3), 4.95 (1H, br s, H-4), 3.31 (1H, dd,
J = 10.0, 5.6 Hz, H-5a), 2.36 (1H, t, J = 10.0 Hz, H-5b), 3.24 (1H, br
d, J = 12.4 Hz, H-6a), 3.17 (1H, br d, J = 12.4 Hz, H-7a), 2.68 (1H, t,
J = 12.4 Hz, H-7b), 7.30-7.34 (3H, m, H-8, 9, 10), 8.07 (1H, s, H-11),
6.11 (1H, s, (OCH2O)a), 5.96 (1H, s, (OCH2O)b), 2.58 (3H, s, NCH3);
13C NMR (CDCl3, 100 MHz) ıC 147.5 (s, C-1), 116.1 (s, C-1a), 130.8
(s, C-1b), 143.8 (s, C-2), 105.5 (d, C-3), 127.8 (s, C-3a), 66.7 (d, C-4),
61.6 (t, C-5), 62.3 (d, C-6a), 34.3 (t, C-7), 135.3 (s, C-7a), 127.8 (d,
C-8), 127.3 (d, C-9), 127.1 (d, C-10), 128.4 (d, C-11), 140.0 (s, C-11a),
101.1 (t, OCH2O), 43.3 (q, NCH3).
Fig. 2. HPLC chromatograms of methanol extracts from non-fermented (A) and C.
rogersoniana-fermented (B) S. epigaea.
(4R,6aR)-4-Hydroxy-N-methyllaurotetanine (3b): white amor-
25
D
phous powder; ␣
−16.3 (c 0.020, MeOH); ESIMS m/z 358
[
]
[M+H]+; HRESIMS m/z 358.1655 (Calcd. for C20H24NO5 358.1649);
1H NMR (CDCl3, 400 MHz) ıH 6.87 (1H, s, H-3), 4.51 (1H, br s, H-4),
3.14 (1H, br d, J = 10.0 Hz, H-5a), 2.71 (1H, br d, J = 10.0 Hz, H-5b),
3.03 (1H, br d, J = 10.0 Hz, H-6a), 2.98 (1H, dd, J = 10.0, 4.4 Hz, H-7a),
2.60 (1H, m, H-7b), 6.82 (1H, s, H-8), 8.06 (1H, s, H-11), 3.67 (3H,
s, OCH3-1), 3.92 (3H, s, OCH3-2), 3.90 (3H, s, OCH3-10), 2.58 (3H, s,
NCH3); 13C NMR (CDCl3, 100 MHz) ıC 145.6 (s, C-1), 126.1 (s, C-1a),
131.8 (s, C-1b), 152.8 (s, C-2), 111.1 (d, C-3), 122.6 (s, C-3a), 67.0
(d, C-4), 60.7 (t, C-5), 62.8 (d, C-6a), 33.9 (t, C-7), 130.5 (s, C-7a),
114.1 (d, C-8), 145.2 (s, C-9), 145.9 (s, C-10), 111.3 (d, C-11), 123.9
(s, C-11a), 60.2 (q, OCH3-1), 55.9 (s, OCH3-2), 56.2 (s, OCH3-10), 43.8
(q, NCH3).
Fig. 3. Key 1H–1H COSY (
1b.
), HMBC (
) and NOESY (
) correlations of
results suggest the presence of a hydroxyl group substituted at C-4
or C-7 in compound 1b. Heteronuclear multiple-bond correlations
(HMBCs) (Fig. 3) from H-4 (ıH 4.93 (1H, br d, J = 4.4 Hz)) to C-3a
(ıC 126.7 s), C-1b (ıC 130.9 s), and C-5 (ıC 61.5 t) confirmed that
the hydroxyl group was substituted at C-4. Therefore, the planar
structure of 1b was identified as 4-hydroxydicentrine.
3. Results and discussion
3.1. Screening of biotransformation
The absolute configuration of the newly installed chiral center
at C-4 was determined using the advanced Mosher’s method [19].
The treatment of 1b with (R)- and (S)-MTPA-OH (␣-methyloxy-
trifluoromethylphenylacetic acid) yielded (R)- and (S)-MTPA ester
derivatives, respectively. The calculation of the 1H NMR ꢂıS-R
values for the mono-MTPA esters of 1b confirmed an R absolute con-
figuration at C-4 (Fig. 4). The absence of a NOESY correlation (Fig.
S12) from H-4 (ıH 4.93 (1H, br d, J = 4.4 Hz)) to H-6a (ıH 3.19 (1H, br
d, J = 18.0 Hz)), combined with the absolute configuration of 1a, con-
firmed the R configuration at C-6a of 1b. The (4R,6aR)-isomer was
further confirmed by comparing the experimental and calculated
ECD spectra of four alternative isomers (Fig. S21). Thus, the struc-
ture of 1b was established to be (4R,6aR)-4-hydroxydicentrine.
4-Hydroxyaporphine alkaloids are a type of alkaloid found
in a few natural sources, such as plants of Launelia philippiana
[20], Glaucium vitellinum [21], and S. venosa [22], among others.
This paper might provide an approach for finding new natural 4-
hydroxyaporphine alkaloids.
Sixteen plant pathogenic fungi were initially screened for their
ability to catalyze biotransformation reactions using S. epigaea as
the substrate. HPLC and TLC were used to monitor the transforma-
tion process during fermentation. The HPLC (Fig. 2) and TLC (Fig.
S1) experiments revealed that C. rogersoniana fermentation could
facilitate prominent biotransformation.
3.2. Identification of 1a and 1b
The primary metabolized compound 1a in the original material
was isolated as a white amorphous power and identified to be (R)-
dicentrine based on NMR data (Table 1) analysis [2] and comparing
its experimental and calculated ECD spectra (Fig. S5).
The corresponding compound 1b produced after fermentation
was isolated as a white amorphous power, and its molecular for-
mula was deduced to be C20H21NO5 by HRESIMS at m/z 356.1494
[M+H]+ (calcd. for C20H22NO5 356.1492). The NMR spectra of com-
pound 1b (Table 1) presented the characteristic NMR features of an
aporphine alkaloid. Comparison of the NMR data between 1b and
1a revealed that the primary difference is that 1b contains only two
methylenes, which is one less than 1a. Compound 1b also presented
signals for an additional oxygenated methine (ıH 4.93 (1H, br d,
J = 4.4 Hz), ıC 66.5 d). Together with its molecular formula, these
3.3. Solid-state fermentation of aporphine alkaloids
To confirm the biotransformation between (R)-dicentrine
and (4R,6aR)-4-hydroxydicentrine, (R)-dicentrine was used as
substrate in the biotransformation. Potatoes, without (4R,6aR)-4-
hydroxydicentrine or (R)-dicentrine, were used as the fermentation
Please cite this article in press as: L. Cai, et al., An improved water-soluble/stereospecific biotransformation of apor-
phine alkaloids in Stephania epigaea to 4R-hydroxyaporphine alkaloids by Clonostachys rogersoniana, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.04.016

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Fig. 4. (A) The Newman projection of the C4-C*
illustrating Mosher’s esters of 1b; (B) Mosher’s ester analysis of 1b, and ꢂıS-R of 1H NMR for S- and R-MTPA esters of 1b.
(MTPA)
conversions (99.5 2.9% (yield, 5.21 0.15 mg), 97.4 3.8% (yield,
5.15 0.20 mg), and 99.9 3.0% (yield, 5.23 0.16 mg), respec-
tively) after 7 days of liquid fermentation (Fig. 5). The high
conversions and good compatibility demonstrated that fermen-
tation with C. rogersoniana is indeed an efficient, simple, and
straightforward approach for facilitating the regioselective and
stereospecific R-hydroxylation of aporphine alkaloids at C-4.
Microorganisms have been reported to be successfully applied
to the selective oxygenation of unactivated C H bonds in organic
substrates [25]. At present, the major works are on the hydrox-
ylation of steroids [12], terpenes [24] and other complex natural
products, as well as a few alkaloids, e.g., protoberberine [23]. This
paper described a high-conversion biotransformation facilitating
the 4R-hydroxylation of aporphine alkaloids in S. epigaea. Further-
more, biotransformation is frequently used to synthesize some
important analogues as an alternative to chemical synthesis for
further synthesis research or drug discovery [26–28]. This biotrans-
formation, particularly the liquid-state fermentation that is not
highly time consuming, provided an effective approach for synthe-
sizing analogues of aporphine alkaloids.
OH
R2
R1
R2
N
H
N
H
R1
C. rogersoniana
28 oC
R4
R4
R3
1b-3b
R3
1a-3a
R1
R2
OCH2O
OCH2O
R3
R4
1a (b)
2a (b)
OCH3 OCH3
H
H
OCH3
3a (b) OCH3 OCH3 OH
Fig. 5. Biotransformation of aporphine alkaloids to 4R-hydroxyaporphine alkaloids
by C. rogersoniana.
culture media supplemented with pure (R)-dicentrine. As shown in
Fig. 2, the peak of (4R,6aR)-4-hydroxydicentrine with a retention
time of 26.728 min showed distinct HPLC patterns after 30 days of
solid fermentation. The peak of the initial (R)-dicentrine, however,
completely disappeared. This experiment confirmed the microbial
transformation between these two alkaloids (Fig. 5).
3.5. Effects of the inhibitors metyrapone, ABT, and PBO and the
inducer n-tetradecane
Microorganisms specifically act on a set of compounds via
unique reactions [23,24]. Therefore, some other aporphine alka-
loids are assumed to transform to 4R-hydroxyaporphine alkaloids
during the solid-state fermentation processing of S. epigaea
with C. rogersoniana. To confirm that this transformation occurs,
two other aporphine alkaloids, (R)-romerine (2a) and (R)-N-
methyllaurotetanine (3a), which were previously isolated from S.
epigaea [2], were subjected to biotransformation with C. roger-
soniana. Similarly, the peaks of the initial aporphine alkaloids
(2a and 3a) completely disappeared after 30 days of solid-state
fermentation. Two newly produced compounds were isolated
by silica CC and identified as (4R,6aR)-4-hydroxyromerine (2b)
and (4R,6aR)-4-hydroxy-N-methyllaurotetanine (3b). These exper-
iments suggested that most aporphine alkaloids in S. epigaea may
be transformed into the corresponding 4R-hydroxyaporphine alka-
loids during the solid-state fermentation processing of S. epigaea
with C. rogersoniana.
Cytochrome P450 monooxygenases catalyze the oxidation of
a wide variety of endogenous and exogenous organic substrates
by typically inserting an oxygen atom from atmospheric dioxygen
into a carbon–hydrogen bond to produce the corresponding alco-
hol [25,29]. In the present study, 4R-hydroxyaporphine alkaloids
might have been derived from aporphine alkaloids via the action of
cytochrome P450. To determine the enzyme system involved in this
biotransformation, special inhibition and induction experiments
were performed. PBO, a methylenedioxyphenyl (MDP) or benzodi-
oxole compound, is used to estimate the inhibition and induction
of reactions mediated by cytochrome P450 [30,31]. As shown in
Fig. 6A, at a concentration of 40 ␮g/mL, PBO exhibited a significant
inhibitory effect. At a concentration of 40 ␮g/mL, ABT, a triazole
P450-14DM inhibitor that is widely used as an inactivator of many
P450 isozymes [32], exihibited an inhibitory effect on the bio-
transformation of (R)-dicentrine to (4R,6aR)-4-hydroxydicentrine
(Fig. 6B). Metyrapone is a pyridine derivative that strongly inhibits
the activity of both the oxidized and reduced forms of cytochrome
P450 proteins and is thus used as a potent inhibitor of these
enzymes [33]. In the present study, metyrapone was selected as
an inhibitor to evaluate the biotransformation process. The results
shown in Fig. 6C show that the biotransformation could be inhib-
ited by 40 ␮g/mL of metyrapone. n-Tetradecane is an effective
inducer of cytochrome P450 enzymes [34]. As shown in Fig. 6D, this
transformation is induced by 2% (v/v) n-tetradecane because (R)-
dicentrine is converted to (4R,6aR)-4-hydroxydicentrine (82.18%
within 12 h). The results of the inhibition and induction exper-
iments indicate the role of cytochrome P450 enzymes in the
catalysis of the biotransformation.
3.4. Liquid-state fermentation of aporphine alkaloids
Liquid-state fermentation is typically more efficient and has
potential industrial applications. However, biotransformation does
not always occur in both liquid-state fermentation and solid-
state fermentation [12]. To investigate this transformation in
liquid-state fermentation, the three aforementioned aporphine
alkaloids, namely, (R)-dicentrine (1a), (R)-romerine (2a), and (R)-
N-methyllaurotetanine (3a), were subjected to a biotransformation
reaction under liquid-state fermentation with C. rogersoniana. The
results showed that 1a–3a could be converted to their corre-
sponding 4R-hydroxyaporphine alkaloids (1b–3b) with very high
Please cite this article in press as: L. Cai, et al., An improved water-soluble/stereospecific biotransformation of aporphine
alkaloids in Stephania epigaea to 4R-hydroxyaporphine alkaloids by Clonostachys rogersoniana, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.04.016

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Fig. 6. Effects of PBO (A, 40 ␮g/mL), ABT (B, 40 ␮g/mL), metyrapone (C, 40 ␮g/mL), and n-tetradecane (D, 2% (v/v)) on the biotransformation of (R)-dicentrine to (4R,6aR)-4-
hydroxydicentrine.
Table 2
IC50 values (ꢃM) for AChE inhibitory and cytotoxic activities of 1a–3a and 1b–3b.
Compound
AChE
Cytotoxic activity
HL-60
SMMC-7721
A-549
MCF-7
SW480
1a
1b
2a
2b
3a
3b
2.94 0.12
2.50 0.05
8.32 0.13
10.56 0.15
>50
9.51 0.03
14.28 0.28
13.67 0.43
12.97 0.20
30.12 0.51
34.65 0.29
10.29 0.54
36.35 1.03
35.41 0.92
45.09 0.88
>40
12.38 0.25
12.74 0.65
20.45 1.10
19.09 0.72
40.54 1.39
39.04 2.08
5.70 0.45
13.20 0.65
18.75 0.99
19.30 0.72
25.23 0.66
35.76 0.95
9.07 0.36
19.18 1.09
17.05 0.76
25.65 1.11
30.73 1.54
34.87 1.90
>50
>40
Tacrinea
HupAa
DDPb
Taxolb
0.19 0.006
0.03 0.001
1.16 0.07
<0.008
8.08 0.35
<0.008
7.10 0.40
<0.008
10.45 0.64
<0.008
8.88 0.45
<0.008
a
Tacrine and hupA (huperzine A) are the positive controls used to estimate the AChE inhibitory capacity.
DDP (cis-dichlorodiammineplatinum) and taxol are the positive controls used to estimate cytotoxic activity.
b
3.6. AChE and cytotoxic activities
3.7. Water solubility
The AChE inhibitory and cytotoxic activities of 1a–3a and 1b–3b
were evaluated. As shown in Table 2, 1b–3b exhibited significant
AChE inhibitory and cytotoxic activities, which were of the same
levels as the original aporphine alkaloids 1a–3a. These experiments
revealed that the stereospecific R-hydroxylation at C-4 has no obvi-
ous effect on the AChE inhibitory and cytotoxic activities of these
aporphine alkaloids. This result is in consistent with the results
of the structure–activity relationship studies of other aporphine
alkaloids [35].
The water solubility of a drug is critical to its bioavailability
in pharmacological preparations. When drugs enter the circula-
tory system, their water solubility can influence their subsequent
bioavailability. The predicted log P values of 4R-hydroxyaporphine
alkaloids are lower than those of aporphine alkaloids (Table 3), indi-
cating the higher water solubilities of 1b–3b. The water solubilities
of 1a–3a and 1b–3b were determined by HPLC based on the corre-
lation between the saturated concentrations of each compound in
water and the corresponding area [16]. It is worth noting that the
Please cite this article in press as: L. Cai, et al., An improved water-soluble/stereospecific biotransformation of apor-
phine alkaloids in Stephania epigaea to 4R-hydroxyaporphine alkaloids by Clonostachys rogersoniana, Process Biochem (2016),
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Table 3
Log P values and water solubility results of aporphine alkaloids and 4R-hydroxyaporphine alkaloids.
Cpd.
Predicted log P valuea
Water solubility (mg/L)
Cpd.
Predicted log P valuea
Water solubility (mg/L)
1a
2a
3a
3.99
4.25
3.03
23.4 0.54
138.3 11.4
248.8 15.9
1b
2b
3b
2.73
2.99
1.84
759.5 36.2
2154.1 45.2
778.8 23.7
a
Calculated using ACD program.
water solubilities of 1b, 2b, and 3b are 759.5 36.2, 2154.1 45.2,
and 778.8 23.7 mg/L, respectively, which are unusually higher
than those of 1a, 2a, and 3a, as well as other compounds reported
in the literature [16,17]. Due to these significant improvements
in water solubility and bioactivities, these 4R-hydroxyaporphine
alkaloids have potential as water-soluble AChE inhibitors and anti-
tumor agents, particularly 1b and 2b.
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Acknowledgments
This work was financially supported by grants from the Natu-
ral Science Foundation of China (Nos. 81460536 and 81460648),
a grant from the Program for Changjiang Scholars and Innovative
Research Team in University (IRT13095), and a grant from the New
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