Fluorescent probes for detecting monoamine oxidase activity and cell imaging

Article abstract of DOI:10.1039/c3ob42326c

A series of new fluorogenic probes for monoamine oxidases (MAOs) were reported based on an oxidation and β-elimination mechanism. The limits of detection of the probes for MAO-A and -B were determined to be 3.5 and 6.0 μg mL^-1 respectively. The

Full text of DOI:10.1039/c3ob42326c

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Fluorescent probes for detecting monoamine
oxidase activity and cell imaging†
Xuefeng Li,a Huatang Zhang,b Yusheng Xie,b Yi Hu,c Hongyan Sun*b and Qing Zhu*a
Cite this: Org. Biomol. Chem., 2014,
12, 2033
Received 21st November 2013,
Accepted 25th January 2014
DOI: 10.1039/c3ob42326c
www.rsc.org/obc
A series of new fluorogenic probes for monoamine oxidases
Among the various approaches, fluorescence spectroscopy
(MAOs) were reported based on an oxidation and β-elimination methods for detecting MAOs have attracted much attention
mechanism. The limits of detection of the probes for MAO-A and due to their high sensitivity and simplicity.5 The previously
-B were determined to be 3.5 and 6.0 µg mL−1 respectively. These developed methods all reveal good specificity toward MAO-B,
probes displayed strong activity towards MAOs, especially MAO-B. whereas few of them have reported the limit of detection
Cellular imaging studies were also successfully conducted with (LOD) of the assays.6 Zhu et al., for example, synthesized two
MCF-7 cells.
simple coumarin probes, which exhibited good sensitivity
towards MAOs.7 In another study, two resorufin probes were
designed based on an amine oxidation/β-elimination mechan-
ism. Significant fluorescence enhancements can be observed
upon reaction with MAOs.8 However, coumarin’s excitation
wavelength, which is near the ultraviolet region, restricts its
usage in cellular imaging studies. The high price of resorufin
also prevents the probe from being widely adopted, and more-
over, no cell imaging studies were conducted for this probe. It
is therefore imperative to develop novel fluorescent probes for
detecting MAOs in cells. The improved fluorescent properties
and low cost will allow the probe to be widely adopted in bio-
logical applications.
In this study, we designed and synthesized a series of novel
activity-based fluorescent probes to detect MAOs. Our design
of the probes is based on the fluorescein scaffold, which
belongs to the family of xanthene dyes and has become a
prevalent fluorophore in fluorescence spectroscopy studies due
to its excellent spectral properties.9 Fluorescein-based probes
have been widely used to detect activities of various classes of
enzymes.10 We noticed that, when the two phenolic OH groups
of a fluorescein derivative are alkylated, the derivative is non-
fluorescent, and this property provides a molecular off–on
switch for enzymatic activity-based detection.11 Therefore, in
our design, one of the hydroxyl groups is linked to an amino-
propoxy group, which is the substrate of MAOs, and the other
is connected to different functional groups to attain high
selectivity and excellent cell-permeability as reported in our
previous paper.12
Monoamine oxidases (MAOs) are flavin adenine dinucleotide
(FAD)-containing enzymes that catalyze the α-carbon oxidation
of amine neurotransmitters in human brains.1 MAOs are
encoded by two distinct genes and expressed as two isozymes,
MAO-A and MAO-B. Despite their substantial similarities,
these two isozymes differ in many aspects, including substrate
specificities, tissue distribution and sensitivity to inhibitors.2
For example, MAO-A mainly metabolizes serotonin and norepi-
nephrine, and MAO-B preferentially breaks down benzylamine
and phenylethylamine. Notwithstanding, tyramine and dopa-
mine can be metabolized to their corresponding aldehydes by
both isoforms.3 Recent studies have shown that MAOs are
closely linked to the increasing concentration of amines in
neurons and are responsible for various types of adverse
health conditions, such as depression, senility, Alzheimer’s
disease, Parkinson’s disease and schizophrenia.4 Due to
MAOs’ important roles in these diseases, strong interest has
been generated in the research community to develop various
types of assays to detect MAO activity.
aInstitute of Bioengineering, Zhejiang University of Technology, Chaowang Road 18,
Hangzhou 310014, China. E-mail: zhuq@zjut.edu.cn; Fax: +86-571-88320781;
Tel: +86-571-88320781
bDepartment of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee
Avenue, Kowloon, Hong Kong, China. E-mail: hongysun@cityu.edu.hk
cCAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety,
CAS Key Lab of Nuclear Radiation and Nuclear Energy Technology, Center for
Multidisciplinary Research, Institute of High Energy Physics, Chinese Academy of
Sciences (CAS), Beijing 100049, China
†Electronic supplementary information (ESI) available: Experimental procedures
and spectral analyses. See DOI: 10.1039/c3ob42326c
Our design is shown in Scheme 1; the 3-aminopropoxy
group, a substrate of MAOs, is installed on one phenolic OH of
fluorescein. Under the catalysis of MAOs, the propylamino
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Scheme 1 Design principle of the fluorescent probes for MAO.
group will be oxidized to the corresponding imine, which will purified and characterized by 1H (or 13C)-NMR and mass
be subsequently hydrolyzed to yield the aldehyde. The result- spectrometry.
ing aldehyde derivative is unstable and will undergo β-elimi-
Next we carried out MAO assays with fluorescein probes
nation quickly, leading to the breakage of the ether bond. The 1–4. Preliminary experiments showed that all the probes per se
intermediate is quickly air-oxidized to afford fluorescein, and displayed virtually no fluorescence, and the fluorescence inten-
fluorescence can therefore be turned on in the presence of sity increased to a steady state around 40 min after the
MAOs.13
addition of MAOs. We optimized the assay conditions as the
Based on the above hypothesis, we designed and syn- following: a fluorescent probe (200 μM) and an enzyme solu-
thesized four different fluorescent probes. In the specific tion (16 μg mL−1) were mixed with 200 μL boric acid buffer
probe design, one phenolic OH group of fluorescein was sub- (pH = 8.4) in 96-well plates at 37 °C for 40 minutes. All fluo-
stituted with the 3-aminopropoxy group. The other group was rescence spectra were collected at λex/λem = 470/535 nm with a
replaced with various functional groups to improve the speci- microplate reader. In general, no change in fluorescence was
ficity and hydrophobicity of the probes. Scheme 2 outlined the observed in the absence of MAO, whereas the fluorescence
synthetic route of probes 1–4. First, fluorescein 1 was con- intensity increased immediately upon addition of MAO
verted to methyl fluorescein 2 in 72.3% yield. Fluorescein 2 enzymes (Fig. 1a). The results indicated that all four probes
was then reacted with different electrophilic reagents, namely can be used to monitor the activity of MAOs. It is also noted
iodomethane, allyl bromide, propargyl bromide and benzyl
bromide, to obtain compounds 3–6 respectively. Subsequent
reduction in a solution of NaBH4 in MeOH gave intermediates
7–10 with good yields. N-Boc-3-brominepropylamine was then
added to the corresponding intermediates, and compounds
11–14 were obtained. Finally the Boc group was removed by
ZnCl2 and the desired probes were obtained with moderate
yields after the hydrolysis in 2 M NaOH. The compounds were
Fig. 1 (a) Fluorescence intensity of probes 1–4 before and after reac-
tions with MAO-B or MAO-A. The bars represent the fluorescence inten-
sity of the probes (200 µM) after 40 minutes at λex/λem = 470/535 nm in
100 mM aqueous borate buffer (pH = 8.4). The data were derived from
the means of three independent experiments, and the error bars rep-
resent the standard deviation. (b) Fluorescence intensity of probe 1
before and after reaction with MAO-B (solid line), MAO-A (dot line) and
the corresponding inhibitor and inactive enzyme. The spectra were
recorded in 100 mM borate buffer (pH = 8.4) at λex = 470 nm. (c) Data
showing the activity of different concentrations of probe 1 (0–300 µM)
after reaction with MAOs (16 µg mL−1) at 37 °C in enzyme assay buffer
Scheme 2 Synthesis of fluorogenic probes for monoamine oxidase: (a)
H2SO4–MeOH, r.t.; (b) iodomethane, allyl bromide, propargyl bromide
or benzyl bromide, K2CO3 in DMF, r.t.; (c) NaBH4 in MeOH, 0 °C; (d)
Br(CH2)3NHBoc, K2CO3 in DMF, r.t.; (e) ZnCl2–CH2Cl2, r.t.
(100 mM borate buffer, pH = 8.4). The fluorescence intensity was
monitored at λex/λem 470/535 nm. (d) Fluorescence of probe
(200 µM) treated with different concentrations of MAO-A, [enzyme] =
0–0.3 mg mL−1 after 40 minutes in pH = 8.4 borate buffer.
=
1
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that all the probes displayed higher reactivity towards MAO-B, studying the kinetics of the probes in great detail, we went
compared to MAO-A. The signal-to-background ratios of the further to examine whether our probes are suitable for a cell
four probes for MAO-B were 18.5, 10.7, 3.8, and 9.7, and those imaging study, which was not done in the previous report.7 We
for MAO-A were 11.1, 4.3, 1.9 and 4.1, respectively. The LOD chose MCF-7 cells (human breast carcinoma cells) because
were 3.5 and 6.0 µg mL−1 for MAO-A and -B respectively by they are human MAO positive cells. Specifically, MCF-7 cells
setting the signal to noise ratio as 3 : 1,14 which was compar- were seeded at a density of 1.0 × 106 cells per well in a 6-well
able with the commercially available assay kit.6 Among the tissue culture dish overnight. The cells were incubated with
four probes, probe 1 showed the highest reactivity toward probe 1 in a 5% CO2 incubator at 37 °C for 3 hours. The
MAOs. Consequently, it was chosen for further biological medium was then removed, and the cells were washed twice
experiments. Further data analysis showed a strong fluo- with phosphate buffered saline (PBS, pH = 7.4) to completely
rescence signal at an emission band of 535 nm with the remove excess extracellular probes. For control experiments,
addition of the MAOs (Fig. 1b and Fig. S1, ESI†). The fluo- the cells were treated with a MAO-B inhibitor, selegiline, for
rescence intensity of probe 1 with MAO-B was found to be 60 minutes at 37 °C prior to the incubation with probe 1
about twice as high as that of probe 1 with MAO-A. Very little (50 μM) for 2 hours. Subsequently, the cells were imaged using
fluorescence increase was detected when the enzymes were a fluorescence microscope (Fig. 2). Moreover, addition of the
denatured or inhibited with the corresponding inhibitors, MAO-B inhibitor significantly suppressed the fluorescence
further confirming that the fluorescence intensity correlates signals arising from the breakdown of probe 1. Our experi-
with MAO activity. As shown in Fig. 1c, the fluorescence inten- ments with probe 1 and live MCF-7 cells further suggested that
sity increases with increasing concentrations of probe 1. A the probes reported in our study can serve as promising small-
near linear relationship can be observed between fluorescence molecule tools which directly report MAO’s activity in living
intensity and concentration in the low concentration range. cells.
Fig. 1d shows a fluorescence spectrum of probe 1 with the
In conclusion, we have successfully designed and syn-
addition of various concentrations of MAO-A. Taken together, thesized four novel off–on switch fluorescent probes. Com-
these studies demonstrate that probes 1–4 can be used to pared with previously reported fluorescent probes, these new
monitor the activity of MAOs.
probes display stronger activity towards MAOs and possess
Subsequently, we carried out a more detailed kinetic study improved fluorescent properties. Furthermore, cell imaging
by analyzing the reactions of the four probes in an extensive
range of concentrations with MAO-A and MAO-B. The concen-
tration-dependent data of probes 1–4 were collected and fitted
to a Lineweaver–Burk plot. The Michaelis–Menten constant
was then derived from the plot and is summarized in Table 1.
Results revealed that all Km values of MAO-A are slightly higher
than those of MAO-B, indicating that all the probes have
higher reactivity towards MAO-B. The catalytic efficiency (kcat/Km)
of MAOS is higher for probe 1 than for the other three probes,
suggesting that probe 1 has a better reactivity towards MAOs. It
was noted that our probes produced similar Km values as the
results of Chang’s group and they are much lower than dopa-
mine, a physiological substrate of MAOs. This proves that it is
reasonable to replace resorufin with fluorescein to detect MAO
activity. This study further indicated that probe 1 with a high
kcat value would be the preferable probe for assaying MAOs.
Fig. 2 Fluorescence images of MCF-7 cells with probe 1. Fluorescence
images and bright-field of (a) MCF-7 cells, (b) MCF-7 cells with probe 1
A fluorogenic probe is a powerful tool for cell imaging
at 50 μM, and (c) MCF-7 cells pretreated with the inhibitor selegiline and
studies due to its high signal-to-noise ratio. Thus, after then incubated with probe 1 at 50 μM.
Table 1 The kinetic constants of probes 1–4 with MAOs
Km (μM)
kcat (min−1
)
kcat/Km (M−1 min−1
)
Probe
MAO-A
MAO-B
MAO-A
MAO-B
MAO-A
MAO-B
1
2
3
11.4
8.9
20.6
11.4
6.3
7.7
6.8
11.4
10.5
3.4
3.0
1.5
0.6
0.3
—
3.5
1.9
0.7
0.4
—
263.2 × 103
168.5 × 103
29.1 × 103
26.3 × 103
—
454.5 × 103
279.4 × 103
61.4 × 103
38.1 × 103
—
4
MR27
Dopamine
—
93
—
—
—
—
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studies also proved that the probes are suitable for monitoring
the activity of MAOs in living cells.
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This study was supported by the National Science Foun-
dation of China (no. 21272212 and 21390411), Zhejiang
Natural Science Fund (no. LY12B02018), National Basic
Research Program of China (no. 2011CB933101) and City Uni-
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