Novel strategy of constructing fluorescent probe for MAO-B via cascade reaction and its application in imaging MAO-B in human astrocyte

Article abstract of DOI:10.1016/j.cclet.2018.05.018

To detect monoamine oxidase B (MAO-B), the level of which is one of the most important indicators of neurodegenerative diseases such as Parkinson's disease, a new type of cascade reaction based on the formation of coumarin was applied in this research. After the reaction with MAO-B, the protecting group of hydroxyl group in probes (DEAN-MA and DEAB-MA) was removed, and the fluorescence intensity significantly increased (λ_em = 456 nm) as the structure of coumarin was formed subsequently. The probes showed excellent sensitivity and selectivity to MAO-B. The detection limit of DEAN-MA and DEAB-MA were 0.6 ng/mL and 0.2 ng/mL, respectively. We succeeded in detecting MAO-B in vitro and imaging it in human astrocyte (U87).

Full text of DOI:10.1016/j.cclet.2018.05.018

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CCLET 4550 No. of Pages 4
Chinese Chemical Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
Novel strategy of constructing fluorescent probe for MAO-B via cascade
reaction and its application in imaging MAO-B in human astrocyte
Huihuan Qin, Lingling Li, Kun Li*, Xiaoqi Yu*
Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 20 March 2018
Received in revised form 8 May 2018
Accepted 8 May 2018
Available online xxx
To detect monoamine oxidase B (MAO-B), the level of which is one of the most important indicators of
neurodegenerative diseases such as Parkinson's disease, a new type of cascade reaction based on the
formation of coumarin was applied in this research. After the reaction with MAO-B, the protecting group
of hydroxyl group in probes (DEAN-MA and DEAB-MA) was removed, and the fluorescence intensity
significantly increased (lem = 456 nm) as the structure of coumarin was formed subsequently. The probes
Keywords:
showed excellent sensitivity and selectivity to MAO-B. The detection limit of DEAN-MA and DEAB-MA
were 0.6 ng/mL and 0.2 ng/mL, respectively. We succeeded in detecting MAO-B in vitro and imaging it in
human astrocyte (U87).
Monoamine oxidase-B
Fluorescent probe
Covalent-assembly
Coumarin
©
2018 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Cell imaging
Monoamine oxidase B (MAO-B) belongs to the flavin mono-
amine oxidase family, which catalyzes the oxidation of biogenic
and xenobiotic amines and plays an important role in the
catabolism of neuroactive and vasoactive amines in the central
nervous system and peripheral tissues. MAO dysfunction is
thought to be responsible for several psychiatric and neurological
disorders. Especially, the level of MAO-B is related to Alzheimer’s
disease and Parkinson’s disease [1,2]. Therefore, detecting MAO-B
in human nerve cells is of great significance. Various technologies
have been developed for the detection of MAO-B. Compared with
technologies like positron emission tomography (PET) [3,4],
magnetic resonance imaging (MRI) [5,6], and enzyme-linked
immunosorbent assay (ELISA) that exploits bio-reducible agents,
fluorescence imaging as a powerful non-invasive method with
high sensitivity is regarded particularly suitable for monoamine
oxidase detection [7–14].
developing “off-on” fluorescent probe from a zero background is
highly desirable. Probe via “covalent-assembly” strategy (raised by
Yang group) could provide an approach to get “off-on” or
ratiometric fluorescent probe, since a new fluorophore can be
formed via covalent reaction of two fragments [15]. The
fundamental feature of “covalent-assembly” probe is that it
warrants both a colorimetric change and a turn-on fluorescent
signal from
a zero background. Moreover, the conjugative
covalent-assembly of the backbone may cause a “red shift” of
the fluorescence spectra, since a new fluorescent conjugated
structure is developed.
In fact, several “covalent-assembly” probes have been success-
fully applied in the detection of small molecules including sulfur-
containing species (SCS) and reactive oxygen species (ROS) [16–
24]. However, such strategy is hardly applied in the detection of
MAO [12,13]. Sames group [13] reported a coumarin switch for
In recent years, many fluorescent probes have been reported for
MAO-B, most of which explored the fluorescence-enhancement
strategy by removing the reducible group and releasing the
fluorescence signal of the fluorophore based on MAO-B’s redox
reaction [10] (Scheme 1a). However, one shortage of this design
strategy is that the original fluorescence intensity of the probes is
hard to form a zero background before reacting with MAO, which
would influence the sensitivity of the detection. Therefore,
MAO-B, based on a sequence of enzymatic oxidation and
intramolecular cyclization (Scheme 1b). In this work, a different
design strategy was applied in the “covalent-assembly” to detect
MAO-B. Instead of expanding the conjugate planes of the
fluorophore, we newly built a fluorescent skeleton. As indicated
in Scheme 1c, after reacting with MAO-B, the re-exposed oxygen
anion will attack the nearest carbonyl carbon atom, leading to the
formation of the fluorophore and the off-on fluorescence effect.
As a kind of important fluorophore, coumarin has large
conjugate system, strong molecular electron transfer ability, good
thermodynamic and photochemical stability. As a result, a large
number of coumarin based fluorescent probes have been reported
*
Corresponding authors.
E-mail addresses: kli@scu.edu.cn (K. Li), xqyu@scu.edu.cn (Y. Xiaoqi).
https://doi.org/10.1016/j.cclet.2018.05.018
001-8417/© 2018 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
1
Please cite this article in press as: H. Qin, et al., Novel strategy of constructing fluorescent probe for MAO-B via cascade reaction and its
application in imaging MAO-B in human astrocyte, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.05.018

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Fig. 1. (a) The fluorescence response to MAO-B (10
fluorescence response of probe DEAN-MA (10 mol/L) to different concentration of
MAO-B. (b) The fluorescence response of probe DEAB-MA (10 mol/L) to MAO-B
10 g/mL) and the image of fluorescence response of probe DEAN-MA (10 mol/L)
to different concentration of MAO-B. Spectra collected 1 h after MAO-B addition.
ex: 360 nm (DEAB-MA), 375 nm (DEAN-MA). Solvent: HEPES buffer (100 mmol/L,
mg/mL) and the image of
m
m
Scheme 1. (a) The reaction process of probes with MAO-B in most previous
research. (b) Previously reported “covalent assembly” MAO-B probe. (c) The
(
m
m
“
covalent assembly” strategy applied in this work.
l
pH 7.4, 1% DMSO and 5% glycerol included) [14].
for anions, metal ions and small molecules [25–28]. As an
important reactant of coumarin in synthesis, salicylaldehyde
compound has a hydroxyl moiety which is the key reaction group.
If the hydroxyl group was protected, the formation of coumarin
will be restricted because of the lack of nucleophilic group. Once
the protecting group was removed, coumarin skeleton would be
formed. In the light of “covalent-assembly” strategy, we want to
develop a novel approach for MAO-B detection via this cascade
reaction. In this work, two salicylaldehyde derivatives decorated
with the 3-aminopropoxy group were designed for the detection of
MAO-B. With the sequence of enzymatic oxidation by MAO-B and
intramolecular cyclization, fluorescence signal of coumarin would
be turned on. Furthermore, the development of inhibitors of MAO-
B is an important item in the treatment of neurodegenerative
diseases [29,30]. We therefore detected the inhibition of pargyline
Fig. 2. The signal-to-background ratio of probe DEAN-MA (a) and DEAB-MA (b) (10
m
mol/L) to several distractors. Spectra was collected 1 h after substance addition.
The concentration of enzymes except MAOs was 1 mg/mL, and the concentration of
and amino acids was 1 mmol/L. Insert: 1. horse radish peroxidase, 2. glucose-
oxidase, 3. H O , 4. sparsely cotton like thermophilic lipase, 5. L-GSH, 6. L-cysteine,
2 2
h O
(
N-benzyl-N-methylprop-2-yn-1-amine) to MAO-B using our
2
2
probes. It was proved that both DEAN-MA and DEAB-MA were
capable of screening MAO-B inhibitors. Importantly, it has been
suggested that the expression level of MAO-B in nerve cells is
higher than non-nerve cells [31], we therefore applied our probes
to the imaging of MAO-B in both HL-7702 cells (human liver cells)
and U87 cells (human astrocyte).
7. L-serine, 8. pancrelipase, 9. trypsin, 10. L-high cysteine, 11. D-glutamic acid, 12.
acyl transferase, 13. MAO-A (10 g/mL), 14. MAO-B (10 g/mL).
m
m
some oxidoreductase and other related enzymes. As depicted in
Fig. 2, probe DEAN-MA exhibited high selectivity to MAO-B over
the species tested, which may be ascribed to the specific oxidation
of the substrate by the enzyme. Probe DEAB-MA also showed high
signal to background ratio after reacting with MAO-B, however,
Firstly, the enzyme activity of MAO-B was tested to be 4.8 U/mg
via UVÀvis absorption spectra (Fig. S1 in Supporting information)
interferences from L-GSH and L-cysteine were also hard to omit.
[
32]. Then the reaction conditions for probe DEAN-MA and DEAB-
Therefore, only DEAN-MA was applied in the living cell imaging
experiments. Furthermore, both DEAN-MA and DEAB-MA showed
selectivity towards MAO-B to MAO-A under the same concentra-
tion, which indicates that probes are more suitable for detecting
MAO-B than MAO-A.
Fig. 3 shows the fluorescence change of probes with MAO-B at
various concentrations. As is seen, the fluorescence intensity of the
probes increased gradually with increasing concentration of MAO-
B. A good linearity between the fluorescence intensity and the
MA with MAO-B were studied, including the effects of pH and the
concentration of probes. The enzymatic reaction was carried out in
the pH range of 5.3 À 9.0. And It was discovered that the
fluorescence barely changed under acidic conditions (pH ꢀ 7.0),
while a significant enhancement of the probe could be observed in
biological conditions (pH around 7.4) after incubation with MAO-B
for 1 h. This indicated that DEAN-MA and DEAB-MA should be able
to serve as sensitive MAO-B probes under physiological conditions
(
Fig. S2 in Supporting information). Following that, the optimum
concentration of MAO-B range of 0–10 mg/mL with an equation of
concentration of the probes to react with MAO-B (10 g/mL) were
m
2
I
456 = 13.82209 Â [MAO-B] +38.11 (R = 0.960) was observed. The
studied. As indicated in Fig. S3 (Supporting information), the
fluorescence intensity reached a plateau when the concentration
detection limit (k = 3) of DEAN-MA and DEAB-MA was respectively
determined to be 0.6 ng/mL and 0.2 ng/mL [33], which was better
or comparable to previously published MAO probes.
of the probes reached 10
mmol/L. The following experiments were
therefore all carried out using 10
the probes.
mmol/L as the concentrations of
The probe can also be used for the screening of potential MAO-B
inhibitors. As shown in Fig. 4, with increasing concentrations of
pargyline (N-benzyl-N-methylprop-2-yn-1-amine), a typical in-
hibitor of MAO-B, the emission intensity gradually decreased. The
IC50 value of pargyline obtained by using the logistic fit of the
As seen from Fig. 1, fluorescence studies revealed that probe
DEAB-MA and DEAN-AM had very weak fluorescence in HEPES
buffer. Reaction of probe DEAN-MA and DEAB-AM with MAO-B
leaded to the change of fluorescence signal at 456 nm from “off”
state to “on” state. These results indicated that both fluorescence
probes DEAN-MA and DEAB-MA were MAO-B reactive.
experimental data was 9.3
mmol/L for DEAN-MA and 2.6 mmol/L
for DEAB-MA. This result suggests that DEAN-MA and DEAB-MA
have the potential of being applied to the screening of MAO-B
inhibitors.
We then studied the specificity and selectivity of the probes
towards MAO-B. We examined various potential interfering
The reaction mechanism was proved by ESI-MS of the reacting
product and the fluorescence spectra. The peak at m/z 255.0655
species in parallel under the same conditions, including H
serine, glutamic acid, cysteine, glutathione, GSH, high-cysteine,
2 2
O ,
Please cite this article in press as: H. Qin, et al., Novel strategy of constructing fluorescent probe for MAO-B via cascade reaction and its
application in imaging MAO-B in human astrocyte, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.05.018

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3
Fig. 3. The fluorescence intensity of DEAN-MA (a) and DEAB-MA (10
their linear graphs for DEAN-MA (b) and DEAB-MA (d).
m
mol/L) (c) after reacting with MAO-B (the concentration of MAO-B range is 0 À 10
m
g/mL) for 1 h and
safe to say that DEAN-MA possessed relatively low cytotoxicity and
the following fluorescence imaging experiments could be carried
out with 10
mmol/L DEAN-MA.
Fluorescence imaging of HL-7702 cells and U87 cells was shown
in Fig. 5. The fluorescence in U87 cells was higher than that in HL-
7
702 cells, through which we successfully demonstrated that the
level of MAO-B in nerve cells is higher than somatic cells including
liver epithelial cells. This result is in line with the previous report
[28]. We then studied the distribution of the probe inside cells. As
shown in Fig. 6, there was no obvious colocation effect between
probe DEAN-AM and mitochondria tracker red, lysosome tracker
red, or endoplasmic reticulum tracker red in U87 cells. The result
suggested that probe DEAN-AM was distributed in various
organelles after diffusion into cells.
Fig. 4. The fluorescence intensity of DEAN-MA (a) and DEAB-MA (b) (10
mmol/L)
reacting with MAO-B (10 g/mL) for one more hour after deferent concentration of
m
inhibitor incubated with MAO-B 1 h.
+
[
M-Na ] of fluorescent molecule EMCC was found in the reaction
In summary, the tandem reaction-based probes were used to
mixture with DEAB-MA and MAO-B, and the peaks at m/z 269.0802
+
+
detect MAO-B selectively in vitro under the suitable pH value (pH
[
M-H ] and 291.0616 [M-Na ] of EBCC were found in the reaction
ꢁ
7
.4) and temperature (37 C). The 5.8-fold signal enhancement for
mixture with DEAN-MA (Fig. S4 in Supporting information). The
results of fluorescence spectra showed that the emission spectra of
the theoretical product EMCC was the same to the reaction mixture
of DEAB-MA and MAO-B, and DEAN-MA showed the same result.
These results proved the proposed reaction mechanism between
the probes and MAO-B (Scheme 1c, Fig. S5 in Supporting
information).
Following that, we found the DEAN-MA could response to MAO-
B at lex = 405 nm as well (Fig. S6 in Supporting information), so the
cytotoxicity of probe DEAN-MA to U87 and HL-7702 cells were
studied (Fig. S7 in Supporting information). The results showed
that the cells viability was still more than 80% when the
DEAN-AM and 6.4-fold signal enhancement for DEAB-AM were
detected and both of them showed selectivity between MAO-B and
MAO-A. Importantly, the novel design strategy of utilizing targeted
enzyme to build a fluorophore in the reaction process could be of
great importance for the future design of fluorescent probes.
Meanwhile, probe DEAB-MA showed stronger fluorescence
response and less selectivity, which may be caused by the different
substituent on the benzene ring from DEAN-MA. Moreover, the
inhibition assay suggested the potential use of DEAB-MA and
DEAN-MA on the screening of MAO-B inhibitors. Furthermore,
fluorescence imaging results corresponded with previous pub-
lished results about the higher level of MAO-B in nerve cells.
concentration of DEAN-MA reached 10
mmol/L. Therefore, it was
Fig. 5. The fluorescence image of control of U87 cells (a, b, c) and incubated with
DEAN-MA (d, e, f), control of HL-7702 cells (g, h, i) and incubated with DEAN-MA (j,
k, l). a, d, g, j for blue channel; b, e, h, k for overlay; c, f, i, l for bright.
Fig. 6. The fluorescence image of U87 cells incubated with DEAN-MA and mito-
tracker (a, b, c, d), lyso-tracker (e, f, g, h), ER-tracker (i, j, k, l). a, e, i for red channel of
tracker; b, f, j for blue channel of DEAN-MA; c, g, k for overlay; d, h, l for bright.
Please cite this article in press as: H. Qin, et al., Novel strategy of constructing fluorescent probe for MAO-B via cascade reaction and its
application in imaging MAO-B in human astrocyte, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.05.018

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Altogether, these results proved the great value and potential of
DEAB-MA and DEAN-MA in the future study of MAO-B.
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Please cite this article in press as: H. Qin, et al., Novel strategy of constructing fluorescent probe for MAO-B via cascade reaction and its
application in imaging MAO-B in human astrocyte, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.05.018