A new fluorescent probe for sensing of biothiols and screening of acetylcholinesterase inhibitors

Article abstract of DOI:10.1039/d0ob00020e

A new N₂O-type BODIPY probe (LF-Bop) has been proposed for the selective and sensitive detection of biologically relevant small molecular thiols. This detection is based on the Michael addition reaction between the thiol and nitrostyrene groups in the probe, which decreases the quenching effect from the nitro group, thus resulting in the recovery of the deep-red fluorescence from the BODIPY structure. The results show that LF-Bop is able to detect all tested free thiols through a fluorescence turn-on assay. The lowest limit of detection (LOD) for glutathione was found to be down to nanomolar levels (220 nM). Based on this probe, we have developed a new fluorescence assay for the screening of acetylcholinesterase inhibitors. In total, 11 natural and synthetic alkaloids have been evaluated. Both experimental measurements and theoretical molecular docking results reveal that both natural berberine and its synthetic derivative dihydroberberine are potential inhibitors of acetylcholinesterase.

Full text of DOI:10.1039/d0ob00020e

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DOI: 10.1039/D0OB00020E
ARTICLE
A new fluorescence probe for sensing of biothiols and screening
of acetylcholinesterase inhibitors
Shengjun Wu,a Yuge Li,b Tao Deng,a,* Xiaojuan Wang,a Shiyou Hu,a Guiyuan Peng,b Xin-an Huang,a
Yanwu Ling c,* and Fang Liu a,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A new N2O-type bodipy probe (LF-Bop) has been proposed for selective and sensitive detection of bio-relevant small
molecular thiols. The detection is based on the Michael addition between thiol and nitrostyrene group in the probe, which
decreases the quenching effect from nitro group thus recovering the deep-red fluorescence from bodipy structure. The
result shows that LF-Bop is able to detect all tested free thiols through a fluorescence turn-on manner. The lowest limit of
detection (LOD) toward glutathione is found down to nanomolar levels (220 nM). Based on this probe, we have developed
a new fluorescence assay for the screening of acetylcholinesterase inhibitors. In total, 11 natural and synthetic alkaloids
have been evaluated. Both experimental measurement and theoretical molecular docking reveal that natural berberine
and its synthetic derivative dihydroberberine are both potential inhibitors of acetylcholinesterase.
the strongly electron-deficient 2, 4-dinitrophenyl sulfonyl group is
another important strategy for thiol-response probe design. The
detection is based on the displacement of the nitro quenching
groups induced by thiol’s nucleophilic attacking.24-27 Although great
efforts have been made in this field, searching for much advanced
fluorescence probes for sensing of bio-relevant thiols with
promising sensitivity and selectivity is still desired.
Besides thiol detection and imaging, the probes with thiol-
response functions are also useful in revealing the activities of thiol-
manipulating enzymes. For example, glutathione reductase (GR) can
covert GSSG back to GSH, which is important for living organisms to
maintain their reductive microenvironment. Recently, thiol
Introduction,
Small molecular thiols are important to living organisms, for
example, endogenous thiols such as L-cysteine (L-Cys) and
glutathione (GSH) play critical roles in regulating cellular redox
balance. The disruption of thiol mediated microenvironment
balance will lead to the risks of various diseases.1, Besides,
2
exogenous thiols such as N-acetylcysteine (NAC) are widely applied
as antioxidants for the treatment of human health care issues, due
to the reductive function of thiol groups.3, 4 The importance of thiols
has been attracting great efforts in the development of detection
strategies. Fluorescence assays are most attractive among all
techniques, because of their excellent sensitivity and simplicity.5-8
There have been numerous fluorescence chemical probes for thiol
quantification and imaging based on different reaction
mechanisms.9-14 Michael addition has been extensively used as the
basis for probe construction, with the consideration of the intrinsic
strong nucleophilicity of thiols. Under this technique aspect, a
number of maleimide conjugated fluorescence probes have been
reported for thiol sensing.15-20 The fluorescence of those probes is
quenched by photoinduced electron transfer (PET), since maleimide
detection probes have been developed to monitor GR activities
29
through fluorescent manners.28,
Acetylcholinesterase (AChE) is
another important enzyme in human body, particularly in the
nervous system.30 It’s found that the reaction between AChE and
acetylthiocholine can generate free thiocholine, which offers the
opportunities for AChE activities evaluation using thiol-response
probes.31-33
serves as a perfect PET acceptor.21, Thiol addition can greatly
suppress PET, thus restoring the fluorescence.21, 23 Conjugation with
22
a. Institute of Tropical Medicine and Artemisinin Research Center, Guangzhou
University of Chinese Medicine, Guangzhou 510405, Guangdong, PR. China
E-mail: dengtao@gzucm.edu.cn, fangliu@gzucm.edu.cn
b. The Second Clinical College of Guangzhou University of Chinese Medicine,
Guangzhou 510120, Guangdong, PR. China I
Scheme 1. Schematic show of LF-Bop based fluorescence assay for thiol
detection and AChE inhibitors screening
c. Department of human anatomy, Youjiang Medical University for
Nationalities, Baise 533000, Guangxi, PR. China
Herein, we present a new fluorescence probe for thiol detection.
The probe LF-Bop is made of an N2O-type bodipy dye and an α,β-
unsaturated nitro group as fluorescence quencher (Scheme 1). The
strong electron withdrawing effect of nitro group in the conjugation
E-mail: lyw@ymcn.edu.cn
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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system is thought to induce fluorescence quenching probably reaction rate between LF-Bop and GSH. In contrast,ViLeFw-BAroticpleaOlonlninee
through intramolecular charge transfer (ICT) process. Upon Michael without GSH only exhibits background lDevOeI:l 10fl.u10o3r9e/sDc0eOncBe0.00G2S0HE
addition by thiols, the conjugation of nitrostyrene is disrupted, thus addition does not affect the UV-vis spectrum of LF-Bop too much,
leading to electron transferring effect decrease and fluorescence as the main fluorogenic structure is maintained during the reaction
recovery. The detection of various endogenous and exogenous bio- (Fig. S2). LF-Bop behaves similarly in terms of NAC detection as
relevant thiols, including 2-mercaptoethanol (BME), H2S, L-Cys, D- illustrated in Fig. S2. The most important photo-physical parameters
Cys, GSH and NAC, have been evaluated by monitoring the deep- of LF-Bop including molar extinction coefficient, stokes shift and
red fluorescence (peaked at 637 nm) changes. More importantly, quantum yield were then measured in the presence or in the
with the good performance of LF-Bop on thiol detection, we absence of thiols, the result was presented in Table S1.
developed an efficient fluorescence assay for AChE inhibitors
screening.
B(OH)2
OH
Pd(PPh3)4
Na2CO3 (aq)
POBr3
DCM, 45 o
DMF
Toluene, 75 oC, N2
C
O
Br
N
N
H
N
1
yield 84%
H
B(OH)2
N
OHC
POCl3
DCM
NaOH (aq)
EtOH, 85 o
CHO
N
H
C
OH
2
yield 75%
NO2
CHO
CH3NO2
O
O
N
pyrrolidine
toluene
B
B
N
N
N
Fig. 1. A) Kinetic monitoring fluorescence intensity I637 of LF-Bop solution (5
μM) in the presence of GSH (200 μM) ; B) Fluoresces spectra of LF-Bop
solution (5 μM) after reaction (10 min) with GSH at series concentrations (0-
400 μM); C) I637 against the concentrations of GSH; D) Linear correlation
when GSH between 0-50 μM. All measurements were performed in PBS
(pH7.4):DMSO (1:1) solution, LF-Bop was excited at 581 nm.
LF-Bop
yield 54%
BOBPY-CHO (3)
yield 70%
Scheme 2. Synthetic route of probe LF-Bop
Results and discussion
The axial N2O-type bodipy was designed as the fluorescence
indicator mostly because of its promising red fluorescence emitting
property, and meanwhile, the axial-substitution can prevent their
possible aggregations through steric protection. Benzaldehyde
capped bodipy-CHO (compound 3 in Scheme 2) was synthesized
according to previously reported methods (Scheme S1).34, 35 Bodipy-
CHO was then reacted with nitromethane to form the final product
LF-Bop using pyrrolidine as the catalyst (Scheme S2). The important
intermediates and final product have been well characterized by
NMR and LC-MS. Thiol detection property was firstly examined by
adding GSH into LF-Bop solutions in a 96-well plate followed by
fluorescence monitoring using a plate reader. As found in Fig. 1 and
Fig. S1, GSH addition leads to fluorescence increases from LF-Bop
solutions. The fluorescence intensity of the reaction solutions varies
between different solvent ratios (PBS:DMSO). The reaction product
is weak fluorescent in pure aqueous solutions and becomes strong
fluorescent in organic solvents. Similar hydrophobicity sensitive
properties have been reported on some other kinds of bodipy
probes.36, 37 The ratio of PBS (pH 7.4) to DMSO was finally fixed at
1:1 for the rest tests after screening. Fig. 1A shows that the
fluorescence from LF-Bop/GSH solution reaches to an
intensity plateau within 10 minutes, which indicates a reasonable
Fig. 2. LF-Bop selectively responds to thiols against other biorelevant amino
acids as indicated by the increase of fluorescence (I637, excited at 581 nm).
LF-Bop (5 μM) was used for test, all analytes were used at 200 μM. NaHS
(200 μM) was applied to generate H2S.
Other kinds of bio-relevant thiols including BME, L-Cys, D-Cys and
NAC have also been chosen to check the fluorescence
responsiveness of LF-Bop. As illustrated in Fig. 2, LF-Bop can indeed
detect those tested thiols as indicated by the increased
fluorescence. However, all the tested bio-relevant amino acids at
the same concentrations to GSH (200 μM) cannot lead to
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fluorescence enhancement of LF-Bop. The result implies that resulted to several approved drugs (e.g. tacrinVeie,w dAroticnleepOenlzinile,
although both thiol and amine groups are all strong nucleophiles, rivastigmine and galantamine) for AD treatmDOenI:t1.041.,14023A9/CDh0EOiBn0h0ib0i2t0oEr
the Michael addition acceptor nitrostyrene prefers thiols rather screening is currently still attracting great attentions in drug
than amines. H2S is a small gaseous signalling biothiol, which also discovery. It has been previously proven that AChE persists great
has strong tendency to undergo Michael addition.35, 38 Fig.2 and Fig. hydrolysis activities toward acetylthiocholine with the generation of
S3 illustrate obviously that H2S can react with LF-Bop, thus leading thiocholine,43, 44 which offers the potentials for inhibitor screening
to fluorescence increase. However, it is less reactive than other by using thiol–response fluorescence assays.
−
tested thiols. Bisulfite ion (HSO3 ) can also react with LF-Bop, as it
With these considerations, we then tested the possibilities to use
has been previously proven to have strong reactivity toward LF-Bop for the evaluation of AChE activity. Since enzymes cannot
Michael acceptors.39 The pH effect on thiol detection by LF-Bop was tolerate high concentrations of DMSO, the enzyme reaction was
then checked. Under the tested conditions from pH 6.5 to pH 10.5, performed in pure aqueous solutions (PBS pH7.4) in the presence of
LF-Bop exhibited promising abilities for thiol detection. Acidic pHs LF-Bop (10 µM) and acetylthiocholine iodide (400 µM). The reaction
affected the detection greatly, probably induced by the decreased was performed at 37 oC for 30 min, equal amount of DMSO was
reactivity of Michael addition under acidic conditions (Fig. S4). The added into the reaction mixture before fluorescence measurement.
fluorescence intensity of the probe is susceptible to basic pHs, pH To check the possible influence caused by changing the sequence of
increasing from 10.5 to 12.5 greatly increases the background reagents addition, kinetic measurement and selectivity study have
fluorescence of LF-Bop in the absence of thiols (Fig. S5).
been re-performed. It’s found that changing the sequence of
reagents addition did not affect the detection and selectivity (Fig.
S7). As shown in Fig. 4B, AChE catalyzed reaction indeed leads to
fluorescence enhancement of LF-Bop. The increase of fluorescence
intensity indicates an AChE concentration dependent manner. In
contrast, AChE itself does not result to fluorescence changes greatly
even at a high concentration (400 mU) (Fig. S8). The result
encouraged us to further explore this assay for inhibitor screening.
In a typical test, potential inhibitors at designed concentrations
were co-incubated with AChE in the presence of acetylthiocholine
and LF-Bop. As proposed in Fig 4A, effective inhibition will lead to
decreased thiocholine and fluorescence when compared to the
assay without inhibitor. These fluorescence changes were then
applied for quantitatively analyzing of inhibition effect (Equation 2).
Fig. 3. HPLC and HRMS characterization of the thiol addition reaction on LF-
Bop. LF-Bop (10 µM) was mixed with 400 µM NAC in PBS (pH 7.4)/DMSO
solution for 30 min before HPLC analysis. UV/VIS monitor was set at 254 nm
and 581 nm.
To look into the reaction, the solution of LF-Bop and NAC was
further
analyzed
by
high
performance liquid chromatography (HPLC) and high resolution
mass (HRMS). As found in Fig. 3, a new product with retention time
at 9.5 min formed when mixing LF-Bop with NAC. The HRMS result
further confirmed that the product was indeed formed from the
addition reaction between LF-Bop and NAC (see ESI). It should be
noted that LF-Bop itself has reasonable long-term stability when
stored in pure DMSO and DMSO/PBS mixtures in the fridge, as
indicated by the HPLC analysis in Fig. S6.
With the good result of thiol detection, we further explored the
possibility to use LF-Bop for AChE’s inhibitors screening. As we
know, AChE is an important enzyme in human body, whose function
is mainly involved in rapid hydrolysis of the neurotransmitter,
acetylcholine (ACh).40 Owing to its critical roles in acetylcholine-
mediated neurotransmission, AChE is an interesting target for drug
development against neurological disorders such as alzheimer's
disease (AD). Up to date, the discovery of AChE inhibitors has
Fig. 4. A) Schematic show of the procedure to use LF-Bop for AChE activity
evaluation; B) LF-Bop (10 μM) fluorescence turn on assay for monitoring
AChE activity under different concentrations (0-50mU) in the presence of
acetylthiocholine iodide (400 μM). The reaction was performed in PBS 7.4
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buffer, before fluorescence measurement, DMSO was added to reach 3.08 μM respectively (Fig. 5). Some other alkaloids can partially
View Article Online
DOI: 10.1039/D0OB00020E
PBS:DMSO=1:1. I637 was recorded under 581 nm excitation.
inhibit AChE at high concentrations, but cannot reach to their IC50
even up to 160 μM. BBR is actually an actively studied inhibitor of
AChE,46 whereas, there is rare information regarding the inhibition
effect of DB to AChE (Scheme S3). Our result shows that DB has
reduced inhibition effect than BBR, which is also supported by
computational molecular docking result. As shown in Fig. 6A and
6B, both BBR and DB can bind with AChE mostly through hydrogen
bonds, pi-alkyl interaction and so son. Total-score values from the
calculation based on the software Sybyl-X2.1.1 are used to evaluate
the molecular docking effect, where larger numbers indicate
greater potentials. As listed in Table S2, Total-scores for BBR and DB
are 11 and 10 respectively, which indicates a relative weaker
potential for DB docking with AChE. The same trend is found in case
of C-scores, where a value much closer to 5 implies better activity.
BBR and DB have similar binding pockets close to the surface of
AChE, both are different from tacrine’s binding pocket (Fig. S10).
The detailed mechanism of AChE inhibition caused by BBR and DB is
still unclear. Nevertheless, although DB exhibits lower inhibition
effect, it still holds great potentials to inhibit AChE in biological
environments since DB is usually found to have better
biocompatibility and higher membrane penetrability than BBR.47, 48
Table 1. Tested compounds for AChE inhibitor screening.
No.
1
Name
Structure
IC50
NH2
HCl
Tacrine
hydrochloride
3.08 μM
N
N
O
O
O
O
Berberine
hydrochloride
O
O
2
7.15 μM
O
O
Cl
Dihydroberbe
rine
3
4
13.69 μM
>160 μM
N
OH
O
N
Atropine
Piperine
O
O
O
5
6
>160 μM
>160 μM
N
O
OH
Higenamine
hydrochloride
HCl
NH
HO
HO
H
H
N
O
Fig. 5. Inhibition evaluation based on LF-Bop/AChE/acetylthiocholine
fluorescence assay. The inhibition ratio was plotted as the function of
concentrations for A) tacrine, B) berberine, C) dihydroberberine. 10 μM LF-
Bop, 40 mU AChE and 400 μM acetylthiocholine in PBS buffer were used for
test. The reaction was performed at 37 oC for 30 min, equal amount of
DMSO was added into the reaction mixture before fluorescence
measurement.
HO
7
Quinine
>160 μM
H
N
H
H
H
N
8
9
Matrine
>160 μM
>160 μM
N
N
O
H
O-
N+
Oxymatrine
O
N
O
O
N
10
11
Theobromine
Piperaquine
>160 μM
>160 μM
NH
N
Cl
N
N
N
Cl
Fig. 6. A) Interaction between AChE and berberine from molecular docking;
N
N
N
B) Interaction between AChE and dihydroberberine from molecular docking.
Tacrine, a well-known inhibitor of AChE was used as the positive
control.45 Altogether, 11 kinds of natural alkaloids and synthetic
molecules have been tested for their inhibition activities, the result
was listed in Table 1 and Fig. S9. Among all, only berberine (BBR),
dihydroberberine (DB) and the positive control tacrine exhibited
excellent inhibition to AChE with IC50 at 7.15 μM, 13.69 μM and
Experimental
Synthesis of Bodipy-CHO
Compound 1, 2 and BOBPY-CHO (3) were synthesized by referring
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previous study.34, 35 Briefly, to synthesize the intermediate 3, 2, 4- Thiol detection
View Article Online
Detection was based on 96-well plate andDtOhIe: 1u0.s1e03o9f/Da0OVaBr0i0o0sk2a0nE
LUX plate reader. In a typical test, LF-Bop (5 µM) was mixed with
the analytes at designed concentrations in PBS (pH 7.4):DMSO (1:1)
solutions. The thiol-addition reaction was performed at room
temperature for 10 min before measurement. The solutions were
excited at 581 nm, the fluorescence spectra or the intensity at 637
nm was recorded. To prepare PBS:DMSO solutions with different
pHs, PBS buffer with pH at 5.5, 6.5, 7.5, 8.5, 9.5, and 10.5 were
separately mixed with DMSO at the vol/vol ratio of 1:1.
dimethyl-3-ethylpyrrole (2 mmol) was dissolved in dichloromethane
(5 mL), POCl3 (1 mmol) was then added at 0 °C, followed by the
dropwise addition of compound 2 (1 mmol) in dichloromethane (5
mL). The reaction mixture was then stirred at room temperature for
4 h. 4-formylphenylboronic acid (10 mmol) in THF was added to the
reaction mixture. Then the reaction was further stirred at room
temperature and monitored by TLC. After the reaction completed,
the solvent was removed and the crude product was purified by
silica gel column chromatography to give BOBPY-CHO (yield 70%).
1H NMR (400 MHz, CDCl3) δ 9.72 (s, 1H), 8.01 (d, J = 8.2 Hz, 1H), 7.88
– 7.86 (m, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.45 (d, J = 8.1 Hz, 2H), 7.41 –
7.33 (m, 3H), 7.30 – 7.17 (m, 4H), 6.92 – 6.88 (m, 1H), 2.37 (s, 3H),
2.30 – 2.28 (m, 2H), 2.19 (s, 3H), 0.97 – 0.93 (m, 3H). 13C NMR (100
MHz, CDCl3) δ 192.83, 156.67, 150.86, 143.42, 135.35, 134.89,
133.81, 133.36, 132.07, 131.96, 130.63, 128.82, 128.68, 126.43,
125.74, 125.68, 123.52, 120.20, 120.09, 119.71, 118.95, 116.09,
17.49, 14.86, 13.06, 9.65. Mass spectrometry (ESI-HRMS, m/z): [M]+
calcd. for [C30H25BN2O2]+ 456.2009; found 456.1982.
The lowest limit of detection (LOD) for GSH is estimated by a well
established method (S/N=3).50 Briefly,
LOD = 3σ/B
Equation 1
where σ is the standard deviation obtained from three individual
measured fluorescence intensity I637 in the absence of thiols. B is
the slope from the linear fitting of the titration curve.
AChE activity evaluation and inhibitor screening
Since enzymes cannot tolerate high concentrations of DMSO,
the enzyme reaction was performed in pure aqueous solutions (PBS
7.4) in the presence of LF-Bop (10 µM) and acetylthiocholine iodide
Synthesis of probe LF-Bop
A mixture of compound 3 (0.7 mmol), nitromethane (5.39 mmol)
and toluene (3 mL) was stirred at room temperature for 5 minutes.
After that, pyrrolidine (0.25 mmol) was added and the mixture was
stirred overnight. After reaction completed, distilled water was
added and extracted with ethyl acetate. The organic layer was
separated and dried with Na2SO4. After concentrating under
vacuum, the crude product was purified by silica gel column
(dichloromethane/ethyl acetate/petroleum ether=1:1:50) to get LF-
Bop (yield 54%). 1H NMR (400 MHz, d6-DMSO) δ 8.33 (d, J = 8.3 Hz,
1H), 8.24 (d, J = 8.1 Hz, 1H), 8.15 (d, J = 7.0 Hz, 2H), 8.00 – 7.88 (m,
2H), 7.64 – 7.60 (m, 1H), 7.52 – 7.43 (m, 4H), 7.29 (d, J = 8.1 Hz, 1H),
7.15 (d, J = 8.0 Hz, 2H), 7.05 – 7.02 (m, 1H), 5.75 (s, 1H), 2.42 (s, 3H),
2.35 (d, J = 7.6 Hz, 2H), 2.29 (s, 3H), 1.01 – 0.97 (m, 3H). 13C NMR
(100 MHz, d6-DMSO) δ 156.48, 150.72, 142.71, 140.18, 137.48,
135.35, 134.81, 134.12, 132.21, 132.10, 130.51, 129.71, 129.22,
128.89, 128.09, 127.24, 126.62, 125.88, 124.05, 121.05, 120.92,
120.33, 118.74, 118.61, 17.29, 15.20, 13.27, 9.77. Mass
spectrometry (ESI-HRMS, m/z): [M+H]+ calcd. for [C31H27BN3O3]+
500.2145; found 500.2155.
o
(400 µM). The reaction was performed at 37 C for 30 min, equal
amount of DMSO was added into the reaction mixture before
fluorescence measurement. For inhibitor screening, the reaction
solution contains LF-Bop (10 µM) and AChE 40 mU.
Inhibition ratio = 1-(Iw-Iblank)/(Iw/o-Iblank
)
Equation 2
Where, Iw stands for the fluorescence intensity I637 in the presence
of inhibitors, Iw/o stands for I637 in the absence of inhibitors, Iblank
means the background I637 from LF-Bop/AChE solution without the
substrate acetylthiocholine iodide.
Computational molecular docking
The crystal structure of AChE (E.C. 3.1.1.7, PDB ID: 1GQR) was found
from
the
open
source
protein
data
bank
(PDB)
https://www.rcsb.org/. The crystal structure was then open with
Sybyl-X2.1.1, the intrinsic ligand, surrounding water molecules and
ions were removed before docking. Molecular geometry was
optimized with MMFF94 force field. Multi-channel surface was set
as the protomol-generation mode, the fully automatic flexible
molecular docking (Surflex) was performed. Result analysis could be
found in the supporting information.
Synthesis of dihydroberberine
Sodium borohydride (54 mg, 1.43 mmol) was dissolved in 5%
sodium hydroxide aqueous solution, the solution was then added to
the mixture of berberine chloride (450 mg, 1.34 mmol) and
potassium carbonate (550 mg, 4.0 mmol) in methanol (18 mL). The
reaction mixture is stirred at rt for 15 min. The yellow solution
became green, the product was collected by filtration and washed
with water and then ethanol/water (30% v/v). The product was
further purified by recrystallization against ethanol.49 1H NMR (400
MHz, CDCl3) δ 7.08 (s, 1H), 6.65 (s, 2H), 6.49 (s, 1H), 5.86 (d, J = 6.5
Hz, 3H), 4.24 (s, 2H), 3.76 (d, J = 2.1 Hz, 6H), 3.06 – 3.03 (m, 2H),
2.80 – 2.77 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 150.42, 147.29,
146.70, 144.51, 141.66, 128.77, 128.55, 124.55, 122.15, 118.83,
111.48, 107.87, 103.80, 101.03, 96.35, 60.75, 55.97, 49.36, 49.06,
29.85. ESI [M+H]+ m/z calcd. for C20H20NO4 337.1, found 338.7
[M+H]+.
Conclusions
In summary, we have synthesized a novel deep-red N2O-type
bodipy (LF-Bop) for rapid and convenient sensing of bio-relevant
thiols. A nitrostyrene group is incorporated into the probe to induce
fluorescence quenching, which also serves as an acceptor for thiol–
Michael-addition. The deep-red emission of LF-Bop peaked at 637
nm makes it promising for analysis in biological environments due
to the limited autofluorescence of biomolecules in deep-red region.
The time to fully recover the fluorescence is less than 10 min,
indicating a fast reaction rate. Moreover, we show the evidence
that nitrostyrene is a promising Michael-addition acceptor for thiol
probe design. On this basis, a fluorescence assay has been
developed for the screening of AChE inhibitors, which provides the
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Organic & Biomolecular Chemistry
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Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
We gratefully acknowledge the financial support by National
Natural Science Foundation of China (grant number 21807018 and
31560294), the projects of the Department of Education of
Guangdong Province (2018KQNCX047) and the training project of
Guangzhou University of Chinese Medicine (2019QNPY06). We also
acknowledge the Lingnan Medical Research Center of GZUCM for
the support on facilities.
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Organic & Biomolecular Chemistry
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ARTICLE
50. A. Shrivastava and V. Gupta, Chron young sci, 2011, 2, 21-25.
View Article Online
DOI: 10.1039/D0OB00020E
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