Synthesis and characterization of 2-(2′-hydroxy-5′- chlorophenyl)-6-chloro- 4(3H)-quinazolinone-based fluorogenic probes for cellular imaging of monoamine oxidases

Article abstract of DOI:10.1002/asia.201000025

Monoamine oxidases (MAOs) catalyze the oxidative deamination of neuro-transmitters and biogenic amines. A new class of activity-based fluorescent probes based on the alkylation of 2-(2'-hydroxyphenyl)-4(3 H)-quinazolinone (HPQ) with primary, secondary, or

Full text of DOI:10.1002/asia.201000025

DOI: 10.1002/asia.201000025
Synthesis and Characterization of 2-(2’-hydroxy-5’-chlorophenyl)-6-chloro-
4ACHTUNGTRENNUNG(3H)-Quinazolinone-Based Fluorogenic Probes for Cellular Imaging of
Monoamine Oxidases
Junxin Aw, Qing Shao, Yanmei Yang, Tingting Jiang, Chungyen Ang, and
Bengang Xing*[a]
Monoamine oxidases (MAOs) are essential FAD-depen-
dent enzymes which can efficiently catalyze the oxidative
deamination of neurotransmitters and biogenic amines.[1–2]
There are two isoforms, MAO A and MAO B, that are
abundant in the liver, gastrointestinal tract, blood platelets,
and central nervous systems.[3] These enzymes play an im-
portant role in metabolism and neural development by regu-
lating the homeostasis of amine neurotransmitters and pe-
ripheral dietary amines. Any excess or deficiency of these
enzymes will lead to various neurological and psychiatric
disorders[4] such as depression, Parkinsonꢀs and Alzheimersꢀs
diseases, or even the growth inhibition and progression of
tumor.[5–7] Thus, the development of suitable MAO sub-
strates which can be used for selective and sensitive moni-
toring of enzyme activity in a complex biological system is
of great significance.
Recently, fluorescence techniques have attracted consider-
able attention as simple, effective, and powerful tools[8,9] for
real-time monitoring of protein and enzyme activities in vi-
tro and in vivo.[10,11] Fluorescent detection is more advanta-
geous compared to colorimetric or radioisotope assay owing
to its high sensitivity, relative safety, low cost, and easy han-
dling.[12,13] To date, several standard chromogenic, radio-
chemical, and fluorogenic substrates have been successfully
employed to identify MAO activity in vitro.[14–20] However,
simple and effective fluorescent probes which can provide a
direct and sensitive readout of the MAO activity in living
cells are still highly required since most of the existing meth-
ods are less sensitive,[17–19] require a secondary activating
enzyme to release the signal for detection,[15] or do not pro-
vide live cell fluorescence imaging for the enzyme func-
tions.[14–20]
Herein, we present the rational design and synthesis of a
new class of activity-based fluorescent probes for real-time
imaging of MAO function in living cells. The general con-
cept for MAO imaging relies on the fact that MAO enzymes
catalyze the FAD-dependent oxidation of primary, secon-
dary, and tertiary amines to iminium intermediates, which
are further non-enzymatically hydrolyzed to the correspond-
ing aldehydes to facilitate the release of a fluorescent prod-
uct through a b-elimination process (Scheme 1). In this
study, we chose 2-(2’-hydroxy-5’-chlorophenyl)-6-chloro-4-
AHCTUNGTREG(NNNU 3H)-quinazolinone (HPQ) derivatives as fluorescent re-
porters. The HPQ fluorophores are generally insoluble in
water and highly fluorescent in the solid state owing to the
[a] J. Aw, Q. Shao, Y. Yang, T. Jiang, C. Ang, Prof. B. Xing
Division of Chemistry & Biological Chemistry
School of Physical & Mathematical Sciences
Nanyang Technological University
21 Nanyang Link, Singapore 637371 (Singapore)
Fax : (+65)67911961
E-mail: bengang@ntu.edu.sg
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201000025.
Scheme 1. Fluorescence detection of MAO activity by oxidative deamina-
tion of HPQ derivatives and subsequent b-elimination.
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COMMUNICATION
intramolecular hydrogen bonding between the imine nitro-
gen and the phenolic hydrogen atoms. Compared to the
common fluorescent dyes, these molecules display extreme
photostability with a large stokes shift (> 100 nm), which
allow them to be easily focused and distinguished from most
cell and tissue autofluorescence.[21–24] Modification of the 2’-
hydroxyl group in HPQ molecule efficiently eliminates its
long wavelength fluorescence, providing an ideal molecular
switch with which to amplify the fluorescence signals for
enzyme detection. Such mechanism-based fluorogenic
probes have exhibited high specificity in the detection of
particular enzymes including alkaline phosphatase,[21] b-glu-
curonidase,[22] and acyl hydrolase.[24] In this investigation,
HPQ fluorophores are alkylated with aminopropyl groups
for MAO enzymatic reactions. Upon MAO treatment, the
amine oxidation and subsequent b-elimination result in the
release of acrolein and a green fluorescent precipitate,
HPQ, thus allowing a direct and effective identification of
enzyme activity in real time.
orescent measurements. Typically, these probes and MAO
enzymes were incubated in 100 mm of Tris-HCl buffer
(pH 7.90) at 378C for 2 h. All the MAO substrates 1, 2, and
3 were very stable in aqueous solutions and as expected,
were almost nonfluorescent before MAO enzymatic oxida-
tion owing to alkylation of the 2’-hydroxyl group in the
HPQ fluorophore. However, upon treatment with MAO A
and B, HPQ molecules were released and significant fluores-
cence enhancement around the wavelength 530 nm was ob-
served in all substrates (Figure 1, and Figure S1 in the Sup-
porting Information).
Scheme 2 shows the synthesis of MAO-HPQ fluorescent
substrates. Our strategy for the preparation of substrates
was divided into two sections: firstly, HPQ fluorophore was
Figure 1. Fluorescence measurement of MAO enzymatic reactions. Emis-
sion spectrum of MAO-HPQ 1 (200 mm) upon treatment with MAO A
(a) and MAO B (b) in Tris-HCl buffer (100 mm, pH 7.90). Enzyme con-
centration: 10 mgmLÀ1. Excitation wavelength: 360 nm; emission wave-
length: 530 nm.
Scheme 2. Synthetic route for fluorogenic probes MAO-HPQ 1–3.
The formation of HPQ fluorophore in the enzymatic reac-
tions was further confirmed by HPLC analysis. In the pres-
ence of each MAO isozyme, the retention time of enzymatic
product was the same as that of HPQ (retention time:
21.8 min), approving the enzyme-mediated reactions to re-
lease HPQ fluorophores from all the substrates (Figure 2,
and Figure S2 in Supporting Information).
Fluorescent enhancement and HPLC results confirmed
that all the primary, secondary, and tertiary MAO-HPQ flu-
orescent substrates underwent an oxidative deamination cat-
alyzed by MAO enzymes, followed by b-elimination, which
resulted in the release of HPQ fluorescent precipitates. The
ratios of fluorescence intensity in the presence and absence
of MAO enzymes were used to estimate MAO activities.
synthesized by refluxing 2-amino-5-chlorobenzamide and 5-
chlorosalicylaldehyde in ethanol in the presence of a catalyt-
ic amount of TsOH·H2O followed by in situ oxidation with
dichloro-dicyanoquinone (DDQ). The fluorophore was then
alkylated with N,N-dimethyl-3-chloropropylamine 3a using
cesium carbonate as base to afford MAO-HPQ 3 in 60%
yield. These alkylation conditions were also applied for the
synthesis of N-Boc-protected precursors of other two
probes, 1b and 2b (yield: 75% and 71%), respectively,
which were further deprotected using TFA and triisopropyl-
silane to give MAO-HPQ 1 and 2 quantitatively.
The activity of these three probes (MAO-HPQ 1–3) to-
wards both MAO isozymes were investigated by in vitro flu-
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Chem. Asian J. 2010, 5, 1317 – 1321

Quinazolinone-Based Fluorogenic Probes for Cellular Imaging of Monoamine Oxidases
Figure 2. HPLC analysis of enzymatic reaction of MAO-HPQ 1 with
MAO isozymes. Absorbance wavelength: 360 nm
The maximum fluorescence enhancement in the primary,
secondary, and tertiary amine MAO substrates was 307-fold,
15-fold, and 5-fold for MAO A and 300-fold, 20-fold, and 7-
fold for MAO B, respectively (Figure 3), indicating that the
different amine substrates exhibited different activities
toward MAO enzymes. Of the three MAO-HPQ derivatives,
substrate 1 exhibited an intensive fluorescence signal and
the highest signal-to-background ratio at 530 nm, affording a
convenient means with which to measure MAO activity.
Figure 4. Enzymatic kinetics of MAO activity with MAO-HPQ 1. Data
show the relative reaction rates over a range of concentrations of sub-
strate. These values were obtained from an average of at least six inde-
pendent experiments for each concentration and were collected over a
time frame of 60 min. The reactions were performed at 378C in Tris-HCl
Buffer at pH 7.90. These readings were collected at a final enzyme con-
centration of 5 mgmLÀ1. Excitation wavelength: 360 nm; emission wave-
length: 530 nm.
Encouraged by the favorable fluorescent properties of
MAO-HPQ 1, we finally investigated the applicability of
this probe to image MAO activity in living cells. In this
study, the PC12 cell line was chosen as our main target
owing to its high expression of endogenous MAO. As a neg-
ative control, C6 glioma cell was used as there is no MAO
expression in this cell line. In a typical experiment, both
PC12 cells and C6 glioma cells were cultured and incubated
with 100 mm of MAO-HPQ 1 in Dulbeccoꢀs Modified Eagle
Medium at 37 8C for 1 h to obtain an effective live cell fluo-
rescent imaging. The cellular imaging measurements were
acquired using a confocal fluorescence microscope with ex-
citation filter 360/40 nm and emission filter 535/40 nm.
After 1 hour incubation of MAO-HPQ 1, a bright fluores-
cence signal was observed inside the PC12 cells (Figure 5b),
confirming the good cell-membrane permeability of this flu-
orescent probe and its enzymatic oxidation for the release
of the HPQ fluorophores. In contrast, there was little HPQ
fluorescence signal observed in C6 glioma cells (Figure 5a),
indicating that there was no obvious amino oxidation occur-
ing in the cells which do not express MAO enzymes. The
cellular imaging results demonstrated the fact that MAO-
HPQ 1 was able to report the MAO activity in MAO-ex-
pressed cells.
Figure 3. Fluorescence enhancement of MAO-HPQ substrates: 1–3
(200 mm) in 100 mm of Tris-HCl buffer, pH 7.90 incubated with enzymes
(10 mgmLÀ1) for 2 h.
Further analysis of the enzyme kinetics of MAO-HPQ 1
with both MAO A and B was also carried out in Tris-HCl
buffer at 378C. Figure 4 shows a representative enzyme ki-
netics plot of MAO-HPQ 1 for MAO A and B oxidation.
Measurement of the fluorescent signal at different substrate
concentrations provided the Michaelis–Menten kinetics con-
stants. These observed kinetic parameters were determined
to be: Km =146.1Æ7.21 mm,
K
cat =9.76Æ0.49 minÀ1 for
MAO A and Km =106.8Æ5.06 mm,
K
cat =8.47Æ0.42 minÀ1
for MAO B. The enzyme catalytic efficiency (Kcat/Km) for
MAO A and B are 6.68ꢂ104 mÀ1 minÀ1 and 7.93ꢂ
104 mÀ1 minÀ1, respectively.
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B. Xing et al.
COMMUNICATION
Experimental Section
Experimental details, including the synthesis of substrates, enzymatic re-
action conditions, kinetics studies, and imaging assays, can be found in
the Supporting Information.
A quantitative analysis of the enzyme kinetics of MAO-HPQ 1 with both
MAO A and B was carried out in Tris-HCl buffer at 378C. To a series of
different MAO-HPQ 1 solutions (concentration range: 0–250 mm) were
added a solution of MAO A or MAO B enzyme with final concentration
5 mgmLÀ1. Tris-HCl buffer was added to adjust the final volume to
100 mL. The rate of enzymatic oxidation was monitored with fluorescence
enhancement at 530 nm. The values of enzyme kinetic parameters (Km
and Kcat) were determined from the standard Lineweaver–Burk plot, the
double-reciprocal plot of the reaction rate versus MAO-HPQ 1 concen-
tration.
Figure 5. Representative fluorescence images of C6 glioma cells and
PC12 cells with MAO-HPQ 1 (100 mm) and inhibitors (100 mm) incubated
separately at 378C with 5% CO2 for 60 min. Excitation filter: 360/40 nm;
emission filter: 535/40 nm. a) C6 glioma cells incubated with MAO-HPQ
1; b) PC12 cells incubated with MAO-HPQ 1; c) clorgyline pretreated
PC12 cells incubated with MAO-HPQ 1; d) pargyline pretreated PC12
cells incubated with MAO-HPQ 1.
For live cell imaging of MAO activity, PC12 cell line (American Type
Culture Collection, No.: CR-1721) were seeded at a density of 2ꢂ105 in a
35 mm diameter m-dish plastic bottom and cultured for 2 days with Nerve
Growth Factor (2.5S, 30 ngmLÀ1, Invitrogen, Carlsbad, CA) in Dulbec-
coꢀs Modified Eagle Medium (DMEM 1X, without phenol red, Gibco/In-
vitrogen, Carlsbad, CA) containing 4.5 gLÀ1 d-glucose, 2 mm GlutaMax-1
(Invitrogen). Control C6 glioma cell lines (American Type Culture Col-
lection, No.: CCL-107) were cultured with the same protocol as PC12
cell lines. After 2 days of culture, the live PC12 and C6 glioma cell lines
were washed twice with DMEM. The live cells were then treated sepa-
rately with 100 mm of MAO-HPQ 1 in DMEM (containing 0.5% of
DMSO) and incubated for 1 hour in an incubator at 378C. The cells were
washed twice with Hankꢀs Balanced Salt solution (Sigma). The fluores-
cence imaging was acquired with a Confocal fluorescence microscope
(Nikon, Eclipse TE2000-E) using a super high pressure mercury lamp
(Nikon, TE2-PS100W) with excitation filter: 360/40 nm; emission filter:
535/40 nm.
Moreover, we further extended the imaging investigations
by utilizing two commonly used inhibitors: clorgyline for
MAO A and pargyline for MAO B.[25–27] The PC12 cells
were pretreated separately with 100 mm of clorgyline and
pargyline, and then incubated with 100 mm of MAO-HPQ 1
at 378C for 1 h. The imaging data revealed that there was
no obvious fluorescence in clorgyline pretreated PC12 cells,
indicating the significant inhibition of the enzyme in the
living cells (Figure 5c), whereas the MAO B inhibitor pargy-
line pretreated PC12 cells still displayed strong fluorescence,
which was similar to the imaging result without inhibitor
treatment (Figure 5d), demonstrating that MAO activity re-
mained in the cell. These results implied that PC12 cells
mainly expressed MAO A enzyme and that its activity could
be selectively suppressed by clorgyline rather than pargyline.
This is in accordance with our in vitro enzyme inhibition
tests (Figure S5 in the Supporting Information) and the re-
sults reported previously.[28,29] In the process of cellular
imaging, the fluorescent probe MAO-HPQ 1 indicates less
toxicity (cell viability assay, Figure S4 in the Supporting In-
formation), and thereby can serve as a novel class of fluoro-
genic probes for real-time imaging of MAO activity in living
cells.
In the inhibition investigation, the live PC12 cell lines were washed twice
with DMEM and pretreated separately with 100 mm clorgyline or pargy-
line in DMEM for 1 hour in an incubator at 378C. MAO-HPQ 1 was
then added to the m-dish with a final concentration of 100 mm and incu-
bated for another 1 hour at 378C. The cells were then washed twice with
Hankꢀs Balanced Salt solution. The fluorescence imaging was acquired
with the Confocal fluorescence microscope.
Acknowledgements
The authors acknowledge URC (RG56/06), A*STAR BMRC (07/1/22/
19/534), and SEP (RG139/06) grants at Nanyang Technological Universi-
ty, Singapore for financial support.
Keywords: cellular imaging · enzymes · fluorogenic probes ·
monoamine oxidases · quinazolinones
In summary, this work describes the design and synthesis
of new and effective fluorogenic probes that can be used to
identify MAO activity in buffer solution and in living cells.
These HPQ-based fluorogenic probes exhibit significant flu-
orescent enhancement and reasonable kinetics upon interac-
tion with MAO A and B enzymes, which facilitate their ap-
plication as biosensors for imaging MAO activity in living
cells. This new type of fluorescent biosensor provides the
possibility to monitor the biological processes of MAOs in
living systems in real time. Moreover, the design strategy re-
ported here could also be utilized for the development of
substrates for other enzymatic assays.
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Received: January 11, 2010
Published online: April 15, 2010
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