Synthesis of fluorescent drug molecules for competitive binding assay based on molecularly imprinted polymers

Article abstract of DOI:10.1039/c9ra00422j

Fluorescent immunosorbent assay (FIA) is very promising for sensitive and selective analysis in bio-medical applications. Here, we proposed an assay, using fluorescent engineering of analytes and the corresponding molecularly imprinted polymers (MIPs) as a plastic antibody. Three drug molecules (metronidazole, zidovudine and lamivudine) were condensed with 9-aminoacridine, using succinic anhydride as a spacer. The target products were characterized with ¹H-NMR, IR and mass spectrometry. UV-vis absorption and fluorescent properties of the fluorophore-labeled drug molecules were investigated. Feasibility of the fluorescent biomimetic immunosorbent assay based on MIPs was demonstrated in the solution. This work will provide sound foundation for the future application in real sample.

Full text of DOI:10.1039/c9ra00422j

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Synthesis of fluorescent drug molecules for
competitive binding assay based on molecularly
Cite this: RSC Adv., 2019, 9, 6779
imprinted polymers†
a
a
Muyasier Wubulikasimu,a Turghun Muhammad,
Mukhtar Imerhasan,
*
*
Nurmemet Hudaberdi,b Wenwu Yang,a Jianzhang Zhao
and Xiaojun Peng
c
c
Fluorescent immunosorbent assay (FIA) is very promising for sensitive and selective analysis in bio-
medical applications. Here, we proposed an assay, using fluorescent engineering of analytes and the
corresponding molecularly imprinted polymers (MIPs) as a plastic antibody. Three drug molecules
(metronidazole, zidovudine and lamivudine) were condensed with 9-aminoacridine, using succinic
anhydride as
a
spacer. The target products were characterized with 1H-NMR, IR and mass
Received 17th January 2019
Accepted 13th February 2019
spectrometry. UV-vis absorption and fluorescent properties of the fluorophore-labeled drug
molecules were investigated. Feasibility of the fluorescent biomimetic immunosorbent assay based on
MIPs was demonstrated in the solution. This work will provide sound foundation for the future
application in real sample.
DOI: 10.1039/c9ra00422j
rsc.li/rsc-advances
immunodeciency virus type 1 (HIV-1).5 Zidovudine has been
approved for the treatment of HIV-1, and even helping with
prevention of maternal-fetal HIV-1 transmission.6 Lamivudine
is a potent and effective anti-retroviral drug, which is prescribed
in the human immune virus (HIV) therapy. It is a water-soluble
nucleoside analog and plays a vital role to inhibit reverse
transcriptase.7
Currently, these drugs are oen determined and analysed by
HPLC and HPLC-MS. However, their uorescence analysis is
rarely reported in relevant eld. Herein, it is important to
develop a uorescent biomimetic immunosorbent assay which
is simple, selective and accurate.
Fluorescence reagents play an extremely important role and
have always been the focus of research in the eld of chemistry
and biological analysis.8 Among them, 9-aminoacridine (9-AA) is
the parent compound of a family of pharmacologically active
model substances9 and is also a uorescent dye of nitrogen
heterocyclic bases. 9-AA has the active amino group which can be
used to label other molecules. The molecules containing 9-AA
moiety have been shown to have high biological activity such as
antibacterial, mutagenic and antitumor effect.10,11 Owing to good
uorescence, physicochemical properties, biological activities,
and as well as carrying with active amino group, 9-AA has been
widely utilized in different areas such as pharmacology, toxi-
cology, organic synthesis, and biochemistry.12,13 However, utili-
zation this molecule for uorescence labelling and further use in
immune assay is not found in literature reports.
1 Introduction
Fluorescence labeling with uorescent protein or organic
molecules is one of the most common methodologies used for
bioanalytical purposes.1 Recently, the development and inno-
vation of uorescent labeling has greatly facilitated the drug
analysis. Metronidazole, zidovudine, lamivudine are common
and fundamental drugs which are playing important roles in
treatment of several types of diseases. And safety use and
research about these drugs have been attracted worldwide
attention in medical and chemical communities. Metronidazole
is a nitroimidazole antibiotic that was introduced in 1950s. It
has been widely used for infections caused by protozoa (e.g.,
trichomoniasis, giardiasis, amoebiasis) and anaerobic bacteria
(e.g., Clostridium spp., Bacteroides spp.).2 Nowadays metronida-
zole is still commonly used because of its potent activity,
attractive pharmacokinetic and pharmacodynamic properties,
favorable side-effect prole, and low cost.3 It has excellent
bioavailability (>90%) and penetrates cerebrospinal uid and
hepatic abscesses. Zidovudine (AZT) belongs to the class of
nucleoside reverse-transcriptase inhibitors (NRTIs),4 and it is
a
pyrimidine synthetic analogue active against human
aCollege of Chemistry & Chemical Engineering, Xinjiang University, Xinjiang Key
Laboratory of Oil and Gas Fine Chemicals, Urumqi 830046, P. R. China. E-mail:
imerhasan@xju.edu.cn; turghunm@sina.com
bYili Chuanning Biotechnology Co., Ltd., Yining 835000, P. R. China
cState Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian
116024, P. R. China
Now, labelling of proteins and drug molecules have been
carried out using click-chemistry, condensation reaction, ester/
amide bond formation, and thiol-ene reactions.14 It is well-
known that the labelling reagent shouldn't bring obvious
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra00422j
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change about the properties of analyte such as chemical and competitors, to develop competitive binding assay for the three
biological activities. To address this issue, a spacer is oen used drugs using corresponding MIPs (plastic antibodies).
to connect the target molecule with a uorescence reagent.
Carrying with reactive groups, the spacer can facilitate labelling
reactions. As a spacer, succinic anhydride (SA) is oen used to
2 Results and discussion
2.1 Preparation
connect amines or alcohols by ester or amide bond.15,16 SA
produces a four-carbon chain between the two molecules
through two step reactions.17–19 SA is a good choice in the
labelling of drug molecules.
Structures of target compounds are depicted in Scheme 1(A).
The target compounds MNZ-F, AZT-F, 3TC-F were obtained by
conjugation of drug molecules with uorescent labeling
reagent 9-AA via spacer succinic anhydride. Synthetic routes of
drug-linker-uorophore was shown in Scheme 1(B). Nucleo-
philic addition reaction between the active group of succinic
anhydrides and –OH or –NH2 group of drug molecules (MNZ,
AZT, 3TC-ES) gives the intermediate (D-SA). Then, the inter-
mediate was used as a building block for the synthesis of the
target compounds. N-Hydroxysuccinimide was used to
produce activated intermediates (D-SA-HAS). Then the inter-
mediate was further labeled with 9-AA by nucleophilic
substitution to yield the target compounds. Molecular struc-
tures of MNZ-F, AZT-F and 3TC-F were conrmed with 1H
NMR, IR and MS spectra. The yields of MNZ-F, AZT-F and 3TC-
F were 29%, 27% and 14%, respectively. Conjugating surface
and steric hindrance of 3TC-F are larger than that of MNZ-F,
It has been proved on many occasions that molecularly
imprinted polymers (MIPs) have the potential to become cost-
efficient and robust alternatives to natural antibodies as a recog-
nition element in bioanalytical assays.20 MIPs, as a biomimetic
synthetic receptor, have been widely used in all respects, due to
specic recognition, high specicity, simple preparation, low cost
and high temperature tolerance.21,22 Hence, MIPs were also
commonly used in immunoassay.23,24 The immune adsorption
experiment is simple and fast, meanwhile, uorescence properties
can improve the sensitivity of drug analysis.
In this study, we selected 9-AA as a uorescent reagent and
succinic anhydride as a spacer to label drug molecules (metroni-
dazole, zidovudine and lamivudine) in Scheme 1. The synthesized
molecules (MNZ-F, AZT-F, 3TC-F) were further characterized and
veried. Finally, the labeled drug molecules were used as
Scheme 1 (A) Fluorescent labelled drug molecules, MNZ-F, AZT-F and 3TC-F; (B) synthetic route of the fluorescence labelling process (a)
pyridine or anhydrous acetone, rt, stir 24 h; (b) anhydrous acetone or anhydrous dimethylformamide (DMF), dimethylaminopyridine (DMAP), N,N-
dicyclohexylcarbodiimide (DCC), rt, stir 36 h; (c) anhydrous DMF, DMAP, rt, stir 36 h.
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Fig. 1 (A) Fluorescence excitation (EX) and emission (EM) spectra of MNZ-F in methanol solution; (B) fluorescence emission spectra of MNZ-F in
various solvents. (lex ¼ 366 nm, sensitivity of 2, slit width of 10/10 nm).
AZT-F, so yield of 3TC-F is lower than that of other two also reported in the case of acridine pyrene-3-carboxaldehyde27
products.
and 7-methoxy-4-methylcoumarins.28,29 Moreover, AZT-F dis-
played the highest uorescence intensity in water, but 9-AA and
MNZ-F showed the highest in methanol.
The results showed that the uorescent labelled drugs have
good uorescence properties. The molecule AZT-F has higher
relative uorescence quantum yield (0.564) as compared to that
of MNZ-F (0.370) and 3TC-F (0.251). Relative uorescence
quantum yield was given in Table 1. Calibration curve param-
eters were listed in Table 2. The data showed that there was
a good linear relationship between the concentration of the
labeled drugs and the relative uorescence intensity.
2.2 Fluorescence characteristics
Firstly, optically matched solutions of 9-AA, MNZ-F and AZT-F
were prepared. Excitation spectra (EX) and emission spectra
(EM) of MNZ-F was shown in Fig. 1(A). The results showed that
9-AA has two strong peaks in the range of 300–600 nm in the
excitation spectrum (Fig. S1A†). The maximum excitation
wavelengths of 9-AA in methanol are at 405 nm and 425 nm,
respectively. However, compared to labeling reagent, MNZ-F
and AZT-F (Fig. S2A†) showed only a strong peak in the range
of 300–600 nm. Excitation peak position of MNZ-F and AZT-F
were blue-shied to 400 nm and 366 nm. The maximum
emission wavelength of 9-AA in methanol are 425 nm and
460 nm, while, emission peak position of MNZ-F and AZT-F
were blue-shied to 440 nm, 430 nm, respectively. The
behavior differences in photoluminescence might be caused by
the thiazole ring and pyrrolidine ring in the uorescent labeling
drugs.
2.3 Competitive binding assay
Principle of the uorescent competitive binding assay is shown
in Scheme 2. The imprinted polymers were utilized in
a competitive assay to detect MNZ and AZT. The assay principle
is based on the competition of the uorescently labeled
compound and unlabeled analyte in solution for the binding
Generally, the polarity of a solvent inuences the emission
spectra of uorophores in quantum yields and spectral shis.25
A uorescent probe is usually strongly uorescent in hydro-
phobic environment but weakly uorescent in hydrophilic
environment (or in more polar solvents).25,26 Interestingly, the
opposite behavior was observed on MNZ-F, 9-AA and AZT-F,
shown in Fig. 1(B), S1B and S2B,† where uorescence emis-
sion is stronger in water than in ethanol. This phenomenon was
Table 2 Calibration curve parameters
Labeled
drug
Linear range
(mmol)
Regression equation
y ¼ 92.62 + 2401.77x
y ¼ 48.81 + 4400.03x
R2
Solvent
MNZ-F
0.006–0.18
0.9394 Methane
chloride
0.9975 Acetonitrile
0.9887 Acetonitrile
AZT-F
3TC-F
0.01–0.20
y ¼ ꢁ20.00 + 5405.00x 0.01–0.20
Table 1 Relative fluorescence quantum yields
Refractive
index h
Relative quantum
yield
Compound
Solvent
Gradient (ꢀ10–5
)
Quinine sulfate
9-AA
MNZ-F
AZT-F
3TC-F
0.1 M H2SO4
Ethanol
Water
Water
Water
1.333
1.362
1.333
1.333
1.333
6.56
4.56
4.44
6.76
3.01
0.547
0.397
0.370
0.564
0.251
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increasing of MNZ concentration. The results of AZT competi-
tive assay are shown in the ESI (Fig. S3†). A good linearity (R2 ¼
0.9818) was found between uorescence intensity and MNZ
concentration, shown in Fig. 2(B). Further work needs to be
done to conrm feasibility of the assay in the real sample matrix
from the point of optimum amount of MIPs, concentration of
the label, solvent and nding out IC50
.
3 Experimental section
3.1 General methods
All of the chemicals of reagent grade quality were commercially
available and used as received. 9-Aminoacridine (9-AA) was
synthesized according to ref. 31. Metronidazole, zidovudine and
lamivudine were purchased from Hangzhou Dayang General
Co. Ltd. (Hangzhou, China). Acetic anhydride was purchased
from Kaifeng Chemical Reagent Factory (Kaifeng, China).
Dimethylaminopyridine, N,N-dicyclohexylcarbodiimide, dime-
thylformamide and pyridine were purchased from Sinopharm
Chemical Reagent Co. Ltd. (Shanghai, China). All uorescence
spectral measurements were conducted by a uorescence
spectrophotometer (970 CRT Shanghai, China) with a tunable
excitation light source. UV-vis analysis was performed a UV-
1800 spectrophotometer (Shimadzu, Japan). 1H NMR spectra
were measured using a ZAB-HS-400 NMR (Varian, America)
using trimethylsilane as interior label. Fourier transform
infrared spectroscopy was performed using KBr pellets on
Scheme 2 Principle of the fluorescent biomimetic immunosorbent
assay. MIPs: molecularly imprinted polymers (plastic antibody).
sites of the MIPs. If there is less analyte in the sample (le row
of Scheme 2), the uorescent label will have more chance to
occupy the limited binding sites. In the consequence, the
supernatant solution shows weaker uorescence. Oppositely,
there more analyte present in the sample (right row of Scheme
2), less label will bind with MIPs and stronger uorescent
shown in the supernatant. As labeled compound, MNZ labeled
with 9-AA was used. Aer removing the polymers by centrifu-
gation, supernatant was measured by uorescence
spectrophotometer.
a EQUINOX 55 (Bruker, Germany) between 400 and 4000 cmꢁ1
.
Mass spectrum were detected in EMS mode. For all experiments
double distilled water was used.
The application of the competitive uorescent assay was
investigated with a xed concentration of the MNZ imprinted
polymer and using MNZ (0–0.36 mM) as an analyte, in the
presence of 0.018 mM MNZ-F in chloroform. The results are
shown in the Fig. 2(A). MNZ competed with MNZ-F for binding
to the imprinted polymers. And the MNZ imprinted polymers
were reported elsewhere by our group.30 Aer centrifugation,
the uorescent intensity of the solution increased with the
3.2 Measurement of quantum yield
We determined the uorescence quantum yield of the products
obtained according to the reported method.32 Quinine sulfate
was used as a reference standard uorophore and using 0.1 M
H2SO4 as solvent, the emission and absorption spectra of all
compounds were listed in Table 1. The relative uorescence
quantum yield was calculated by eqn (1):
Fig. 2 (A) Fluorescence spectra of competitive binding assay of 0.018 mM MNZ-F in the presence of 0–0.36 mM MNZ (lex ¼ 395 nm, sensitivity
of 2, slit width of 20/10 nm) in chloroform solution; (B) relationship between fluorescence intensity and concentration of MNZ.
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ꢀ
ꢁꢀ
ꢁ
2
temperature for 36 h. Under stirring, reaction mixture was poured
into the cool distillated water (the ask was kept cold with an ice
bath), gradually formed deposit. Aer 1 h, the suspension was
ltrated with vacuum, washed with water and dried at 90 ꢂC. Crude
product was obtained as yellow powder and it was puried by
column chromatography over silica gel (30 cm ꢀ 2 cm, eluting
with v (CHCl3) : v (C2H5OH) ¼ 40 : 3). Solvent was removed under
reduced pressure and dried in vacuum. Product was obtained as
a yellow powder 7 mg, yield: 29%. 1H NMR (400 MHz, DMSO-d6) d:
10.79 (s, 1H), 8.16 (t, J ¼ 8.0 Hz, 8.0 Hz, 4H), 8.04 (s, 1H), 7.89–7.85
(m, 2H), 7.64–7.61 (m, 2H), 4.60 (t, J ¼ 8.0 Hz, 8.0 Hz, 2H), 4.43 (t, J
¼ 8.0 Hz, 4.0 Hz, 2H), 2.94 (t, J ¼ 8.0 Hz, 4.0 Hz, 2H), 2.68 (t, J ¼ 8.0
Hz, 8.0 Hz, 2H), 2.46 (s, 3H) ppm. IR (KBr): nmax 3412, 1740, 1263,
Gradx
hx
Fx ¼ FST
(1)
GradST
hST
where F is uorescence quantum yield, the subscript x repre-
sents the sample to be tested, subscript ST denotes reference,
Grad is the slope of the curve derived from the integral area of
uorescence intensity and the absorbance corresponding to
different absorbance (provided in the Fig. S4†), h is the refrac-
tive index of the solvent, the refractive index of 0.1 M H2SO4 and
water is 1.333.
3.3 Synthesis
3.3.1 Synthesis of compound MNZ-SA. Compound MNZ-SA 757 cmꢁ1. MS (EMS), m/z calcd for C23H21N5O5, 447.1543, found
was synthesized using the reported method.33,34 In a 50 mL 448.0 [M + H]+, 301.0 [M ꢁ MNZ + Na]+, 277.0 [M ꢁ MNZ], 195.0
ask, metronidazole (6 mmol) was dissolved in pyridine (4 mL). [acridine ammonia fragments + 2H]+.
Then succinic anhydride (9 mmol) was dissolved in anhydrous
pyridine (4 mL) and slowly added dropwise into the above
3.3.5 AZT-F
Compound AZT-F. The details can be found in the ESI.† A yellow
reaction mixture, within 8 minutes under stirring and ice bath powder was obtained, 0.29 g, yield: 27%. 1H NMR (400 MHz,
condition. Aer completion of the reaction, the ice bath was DMSO-d6) d: 11.36 (s, 1H), 10.77 (s, 1H), 8.16 (d, J ¼ 8.0 Hz, 4H),
removed. Under N2 atmosphere and avoiding of light, resulting 7.85 (t, J ¼ 8.0 Hz, 8.0 Hz, 2H), 7.62 (t, J ¼ 8.0 Hz, 8.0 Hz, 2H), 7.46
mixture was stirred for 24 h at room temperature. Solvent was (s, 1H), 6.13 (t, J ¼ 8.0 Hz, 4.0 Hz, 1H), 4.34–4.27 (m, 2H), 4.02–3.98
removed under reduced pressure. Crude product was dissolved (m, 1H), 3.02 (t, J ¼ 8.0 Hz, 8.0 Hz, 2H), 2.83 (dd, J ¼ 8.0 Hz, 4.0 Hz,
in acetone (25 mL) and then diethyl ether (50 mL) was added, 2H), 2.44 (dd, J ¼ 8.0 Hz, 8.0 Hz, 1H), 2.33 (dd, J ¼ 12.0 Hz, 8.0 Hz,
the mixture was kept overnight at room temperature. Suspen- 1H), 1.80 (d, J ¼ 4.0 Hz, 1H), 1.75 (s, 3H) ppm. IR (KBr): nmax 3245,
sion was removed by ltration. The ltrate was treated again by 1708, 1272, 751 cmꢁ1. MS (EMS), m/z calcd for C27H25N7O6,
the procedure mentioned above. Filter cake obtained by two 543.1866, found 544.1 [M + H]+, 301.0 [M ꢁ AZT + Na]+, 277.0 [M ꢁ
ltration steps was combined. The product MNZ-SA was ob- AZT], 195.0 [acridine ammonia fragments + 2H]+.
tained as a white crystal, 1.54 g, yield 95%. 1H NMR (400 MHz,
3.3.6 3TC-F
DMSO-d6) d: 12.22 (s, 1H), 8.04 (s, 1H), 4.57 (t, J ¼ 7.2 Hz, 2H),
Compound 3TC-ES-F. The details can be found in the ESI.† A
1
4.37 (t, J ¼ 7.2 Hz, 2H), 2.64 (s, 3H), 2.45–2.42 (m, 4H) ppm. IR yellow powder was obtained, 0.29 g, yield: 14%. H NMR (400
(KBr): nmax 2534, 1332, 1264 cmꢁ1
.
MHz, CDCl3) d: 8.34 (dd, J ¼ 8.0 Hz, 8.0 Hz, 2H), 8.13 (d, J ¼ 8.0
Hz, 1H), 7.82 (s, 1H), 7.74 (dd, J ¼ 8.0 Hz, 8.0 Hz, 3H), 7.57 (dd, J
3.3.2 AZT-SA
Compound AZT-SA. The details can be found in the ESI.† A ¼ 16.0 Hz, 8.0 Hz, 2H), 7.20 (s, 1H), 6.49 (d, J ¼ 4.0 Hz, 1H),
yellow grease was obtained, 1.35 g, yield: 92%. 1H NMR (400 6.32–6.29 (m, 1H), 5.40 (dd, J ¼ 8.0 Hz, 4.0 Hz, 1H), 4.63 (ddd, J
MHz, CDCl3) d: 12.01 (s, 1H), 8.81 (s, 1H), 7.22 (d, J ¼ 1.6 Hz, ¼ 12.0 Hz, 8.0 Hz, 4.0 Hz, 2H), 3.66 (dt, J ¼ 8.0 Hz, 8.0 Hz, 8.0
1H), 5.78 (d, J ¼ 1.2 Hz, 1H), 4.52 (dd, J ¼ 8.8, 3.2 Hz, 1H), 4.29 Hz, 1H), 3.33 (dd, J ¼ 12.0 Hz, 4.0 Hz, 1H), 3.12 (t, J ¼ 8.0 Hz, 4.0
(dd, J ¼ 7.6, 4.8 Hz, 1H), 4.21–4.18 (m, 1H), 2.69–2.66 (m, 1H), Hz, 2H), 2.32 (t, J ¼ 8.0 Hz, 4.0 Hz, 2H), 2.15 (s, 3H) ppm. IR
2.51–2.48 (m, 1H), 2.01 (s, 3H), 1.48–1.45 (m, 1H) ppm. IR (KBr): (KBr): nmax 3223, 1733, 1694, 1221, 757 cmꢁ1. MS (EMS), m/z
nmax 2536, 1707, 1289 cmꢁ1
.
calcd for C27H25N5O6S, found 543.2 [M ꢁ CH3CO + K]+, 195.1
3.3.3 3TC-ES-SA
[acridine ammonia fragments + 2H]+and 272 [M ꢁ 3TC ꢁ ES +
Compound 3TC-ES-SA. The details can be found in the ESI.† A 5H]+ fragment ion peak.
1
white powder was obtained, 0.49 g, yield: 86%. H NMR (400
MHz, CDCl3) d: 12.17 (s, 1H), 10.10 (s, 1H), 8.19 (d, J ¼ 7.6 Hz,
1H), 7.22 (d, J ¼ 7.2 Hz, 1H), 6.24 (t, J ¼ 4.0, 5.2 Hz, 1H), 5.44 (t, J
3.4 Competitive binding assay
¼ 3.2 Hz, 1H), 4.50–4.40 (m, 2H), 3.61–3.57 (m, 1H), 3.24–3.34
(m, 2H), 2.64 (t, J ¼ 8.0, 6.0 Hz, 2H), 2.08 (s, 3H) ppm. IR (KBr): Competitive binding assay of MNZ-F was discussed in this
nmax 2657, 1738, 1719 cmꢁ1
.
manuscript as an example, and for AZT-F can be found in the
3.3.4 Synthesis of uorescent labeled metronidazole MNZ- ESI.† Molecularly imprinted polymers synthesized according
F. In a 50 mL ask, 9-aminoacridine (3 mmol) was dissolved in to the procedure reported in our previous work.30,35 MIPs
anhydrous DMF (9 mL). Dimethylaminopyridine (0.15 mmol) was particles of MNZ (20 mg) were mixed with 0.018 mM of MNZ-F
added into the mixture. MNZ-SA-HAS which obtained from MNZ- and the MNZ (nal concentration 0–0.36 mM) in the chloro-
SA (provided in the ESI†) was dissolved in anhydrous DMF (9 mL) form solvent up to a nal volume of 4 mL. The mixture was
and slowly added dropwise into the above reaction mixture, within incubated for 12 h at room temperature in polypropylene
15 minutes under stirring and ice bath condition. Aer completion tubes on a shaking table. Aer shaking, the solution was
of the reaction, the ice bath was removed. Under N2 atmosphere centrifugated and ltrated. Finally, the ltrate was measured
and avoiding of light, the resulting mixture was stirred at room by uorescence spectrometer.
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Conflicts of interest
There are no conicts to declare.
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21 X. A. Ton, V. Acha, P. Bonomi, B. T. S. Bui and K. Haupt,
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grants No. 21462043, 21365020 and
21565025) and the State Key Laboratory of Fine Chemicals (KF
1617).
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