Rapid and sensitive fluorescent probes for monoamine oxidases B to A at low concentrations

Article abstract of DOI:10.1016/j.tetlet.2012.09.074

A new monoamine oxidase (MAO) fluorescent probe that displays response for both monoamine oxidase type-A (MAO-A) and monoamine oxidase type-B (MAO-B) is reported. This sensing platform does not require any additional enzymes or reagents. Furthermore, the

Full text of DOI:10.1016/j.tetlet.2012.09.074

Tetrahedron Letters 53 (2012) 6881–6884
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Tetrahedron Letters
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Rapid and sensitive fluorescent probes for monoamine oxidases B to A at low
concentrations
⇑
Yexiang Zhang, Yufang Xu, Shaoying Tan, Lin Xu, Xuhong Qian
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new monoamine oxidase (MAO) fluorescent probe that displays response for both monoamine oxidase
type-A (MAO-A) and monoamine oxidase type-B (MAO-B) is reported. This sensing platform does not
require any additional enzymes or reagents. Furthermore, the probes have better sensitivity and shorter
response time by comparison with similar probes, and have potentials in differentiation of MAO-A and
MAO-B upon further optimization.
Received 7 August 2012
Revised 13 September 2012
Accepted 18 September 2012
Available online 17 October 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescent probes
Monoamine oxidase type-B
Coumarin derivatives
Low concentrations
Monoamine oxidases (MAOs) are found in the mitochondria of
most mammalian cells in the central nervous system and periphe-
ral organs which catalyze the oxidative deamination of their sub-
strates.1 These enzymes play a vital role in neurotransmitter
amine deactivation, catabolism, and metabolism.2 Abnormal levels
of MAOs in humans are associated with depression, schizophrenia,
substance abuse, attention deficit disorder, migraines, irregular
sexual maturation, and other diseases.3
In humans, there are two forms of MAO which are designated as
monoamine oxidase type-A (MAO-A) and monoamine oxidase
type-B (MAO-B).4 Their differences include the amino acid se-
quences, tissue distribution, three-dimensional structure, inhibitor
selectivity, and substrate preferences.5 For these reasons, they
show different substrate and inhibitor specificities: serotonin is
metabolized by MAO-A, which is inhibited by low concentrations
of clorgyline; while dopamine is metabolized by MAO-B, which
can be inhibited by nanomolar concentrations of selegiline
(l-deprenyl).6 Within the human brain, MAO-A and MAO-B are
expressed to varying degrees depending upon the cell type.7
Because of the medical importance of MAOs, the MAO inhibitors
have been developed as drugs for treating clinical depression, Par-
kinson’s disease, Alzheimer’s disease, and cerebral ischemia and
strokes.8 Especially, the inhibitors specific for MAO-B have been
developed using pyrrole derivatives, 3-arylcoumarins derivatives.9
The fluorescence detection method is preferred for its satisfac-
tory sensitivity, facile operation, and real-time monitoring in envi-
ronmental and biological samples. The fluorescent detection for
MAOs has become a hot topic these years. For example, Sames
et al. have developed a sensor for both MAO-A and MAO-B, based
on a coumarin system, fluorescence of which can be recovered
upon formation of an indole via an aldehyde intermediate.10 Chang
et al. have also developed a probe for MAOs based on a tandem
amine oxidation/elimination mechanism using resorufin as a fluo-
rescent reporter.11 Ahn et al. have developed a fluorescent probe
for MAOs based on two-photon absorbing material.12 Zhu et al.
have done a lot of work for sensing MAO-B lately.13 Other groups
have also developed some probes for MAO by using peptide-PNA
or 19F magnetic resonance imaging.14
However, the sensitivity and response time of known probes,
which are important for future medical applications, were still
needed to be improved. Also, most of them could not distinguish
MAO-A and MAO-B very well. Here, a new fluorescent probe CR1
for MAO, which can distinguish MAO-A and MAO-B to a certain ex-
tent, has been developed. Furthermore, the sensitivity of the probe
for MAOs has been enhanced which is evaluated by the response
time and the concentration test for MAOs.
Matos and his group have developed a selective MAO-B inhibitor
whose result showed that the introduction of a p-methoxy group
increases the inhibitory activity.15 Inspired by this inhibitor and
the known MAO fluorescent probes;10–13 we design a coumarin
probe with a p-methoxy group, and a propyl linker between the
amine substrate and fluorophore (Scheme 1). Based on such a pro-
posal, fluorescence of the probe would be quenched because of the
free amine of the aniline and the rotation of the linker, while the
fluorescence of the coumarin system would be recovered upon
MAO in buffer, since the amine was oxidized by MAO to afford imin-
ium or aldehyde intermediates that would undergo b-elimination,
⇑
Corresponding author. Tel.: +86 21 64251399; fax: +86 21 64252603.
E-mail address: xhqian@ecust.edu.cn (X. Qian).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.09.074

6882
Y. Zhang et al. / Tetrahedron Letters 53 (2012) 6881–6884
Scheme 1. The design of a new MAO fluorescent probe CR1.
and formed fluorescent-hydroxyl coumarin as product. In addition,
the p-methoxy group was introduced to enhance the sensitivity to
MAO-B. We anticipate that the intensity of the fluorescence will be
enhanced more upon MAO-B than A at low concentrations, and fur-
thermore, the fluorescence intensity of the probe will increase with
increasing concentration of MAO-A and MAO-B.
Scheme 2a outlines the synthesis of probes CR1 and CR2 and
the principle of its design. The hydroxyl coumarin was easily syn-
thesized according to the literature using the Pechmann reaction.16
Reactions of N-methyl-3-chloropropylamine or 3-chloropropyl-
amine in the presence of (Boc)2O and potassium carbonate fur-
nished the amines in a total yield of 90% and 92%, respectively.
Deprotection of Boc after the reaction of the protected amine and
hydroxyl coumarin, the probes CR1 and CR2 were obtained in a to-
tal yield of 61% and 74%, respectively. The probe was characterized
by 1H NMR, 13C NMR, and mass spectrometry. The principle of
sensing MAO is presented at Scheme 2b. At low concentrations,
the probe CR1 is oxidized by MAO-B but not MAO-A to form imin-
ium or aldehyde intermediates that would undergo b-elimination,
and fluorescent-hydroxyl coumarin is formed as product which
enhances the intensity of fluorescence. At higher concentration,
the selectivity of CR1 is lost and turn-on fluorescence is observed
for both MAO-A and B.
Spectroscopy determination of CR1 and CR2 was carried out in
HEPES buffer (100 mM pH 7.4 with 5% glycerol and 1% DMSO at
37 °C). The spectrum showed that CR1 and CR2 featured one obvi-
ous absorption band at 345 nm and emission band at 465 nm
which were characteristic of the coumarin core. The effect of pH
on fluorescence properties of the probes CR1 and CR2 was deter-
mined. As shown in Figure 1, the spectra of emission remained
Scheme 2. (a) The synthesis route of probes CR1 and CR2; (b) the principle of the sensing process.

Y. Zhang et al. / Tetrahedron Letters 53 (2012) 6881–6884
6883
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0
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20
0
(b)
(a)
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12
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4
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12
pH
pH
Figure 1. (a) The pH dependence of the fluorescence intensity of CR1 in H2O. (b) The pH dependence of the fluorescence intensity of CR2 in H2O.
150
100
50
300
250
200
150
100
50
CR1
CR1+ MAO A
CR1+ MAO B
(a)
CR1
CR1+ MAO A
CR1+ MAO B
(b)
0
0
350
400
450
500
550
600
650
350
400
450
500
550
600
650
Wavelength (nm)
Wavelength (nm)
Figure 2. (a) Emission spectra of CR1 (1
lmol/L), CR1+MAO-A (5
lg/ml), and CR1+MAO-B (5
lg/ml). Reactions were performed at 37 °C in enzyme assay buffer (100 mM
HEPES, pH 7.4 with 5% glycerol and 1% DMSO). (b) Emission spectra of CR1 (2
lmol/L), CR1+MAO-A (5
lg/ml), and CR1+MAO-B (5
lg/ml). Reactions were performed at 37 °C
in enzyme assay buffer (100 mM HEPES, pH 7.4 with 5% glycerol and 1% DMSO).
1000
800
600
400
200
0
700
CR2
CR1
CR2+ MAO A
CR2+ MAO B
(a)
(b)
CR1+ MAO A
600
CR1+ MAO B
500
400
300
200
100
0
350
400
450
500
550
600
650
350
400
450
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550
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650
Wavelength (nm)
Wavelength (nm)
Figure 3. (a) Emission spectra of CR1 (1
l
mol/L), CR1+MAO-A (50
lg/ml), and CR1+MAO-B (50
lg/ml). Reactions were performed at 37 °C in enzyme assay buffer (100 mM
HEPES, pH 7.4 with 5% glycerol and 1% DMSO). (b) Emission spectra of CR2 (1
l
mol/L), CR2+MAO-A (50
l
g/ml), and CR2+MAO-B (50 g/ml). Reactions were performed at
l
37 °C in enzyme assay buffer (100 mM HEPES, pH 7.4 with 5% glycerol and 1% DMSO).
stable with the pH from 2 to 10. This indicates that the probes
could be used for sensing MAO in a wide range of pH. Then, we
determined the quantum yield of the two compounds. As we
expected before, the probes CR1 and CR2 have very weak fluores-
cence (U = 0.11) compared with the hydroxyl coumarin (U = 0.49),
which meant that the fluorescent intensity of the probe should in-
crease upon the addition of MAOs.
Then, we wanted to find out whether there was different re-
sponse after adding MAO-A and MAO-B at low concentrations.
First, we tested the probe CR1 with MAO-A and MAO-B. We found
that the fluorescence intensity of CR1 both increased 29% and 31%
with MAO-B at the concentrations of 1
almost no increase with MAO-A (Fig. 2).
lmol/L and 2 lmol/L but
Later, we tested the fluorescence intensity of the probe CR1
response to MAO-A and MAO-B at the higher concentration. As
shown in Figure 3a, the fluorescence intensity of probe CR1 was in-
creased from 140 to 500 and 600, respectively. The large responses
for the fluorescent probe were due to the formation of fluorescent-
hydroxyl coumarin after b-elimination of the probe. Here, the fluo-
rescent intensity of product, fluorescent-hydroxyl coumarin (CH)

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Y. Zhang et al. / Tetrahedron Letters 53 (2012) 6881–6884
was also determined lonely (see Supplementary data). The inten-
sity of CH was 650, which was almost the same with the probe
after addition of MAO.
and Manufacturing Program (Grant 2009ZX09103-102), and the
Shanghai Leading Academic Discipline Project (B507).
Interestingly, the result of fluorescent test for CR2 was very dif-
ferent from what we expected before. The structure of CR2 is al-
most the same with CR1 but a substitution of methyl, according
to our assumption, the response of CR2 upon addition of MAO is
similar to CR1. (Fig. 3b) But to our surprise, the fluorescence inten-
sity was almost of no change with MAO-A and MAO-B. The reason
maybe the affinity of CR2 with MAOs was much weaker than CR1.
As we mentioned before, although the selectivity is still needed
to be improved, the sensitivity of this probe was increased more
compared with the other known probes. The concentration of this
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.09.
074.
References and notes
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probe in the test was 1
than the most probes, even the lowest concentration of the known
probe (5 mol/L). On the other hand, the response time of sensing
lmol/L and 2 lmol/L which were lower
l
for MAO was tested which is another indicator of the sensitivity of
the probes. The result showed that after addition of the same con-
centration of MAO A (50
lg/mL), the fluorescence intensity of the
probe CR1 (5 mol/L) peaked at 1 min, which was much shorter
l
than the known probe (7.6
l
mol/L, 10–60 min).11 (See Supplemen-
tary data)
In conclusion, we have reported a novel and sensitive mono-
amine oxidase probe. Interestingly, this was the first time to report
a probe whose intensity could be increased by MAO-B but not
MAO-A at a lower concentration. The detailed reason maybe the
addition of the bulky group into the fluorophore which changes
the affinity with the MAO. Furthermore, the probe was a turn-on
probe for both MAO-A and MAO-B at high concentration. On the
other hand, the sensitivity of the probes was much enhanced than
that of the known probe. According to our research and the known
probes,10–13 a molecule with a propyl linker between the amine
substrate and fluorophore can be used as a selective, sensitive fluo-
rescent probe for MAO. So, this might be a good platform to build a
MAO-A and MAO-B distinguishable fluorescent probe.
10. Chen, G.; Yee, D. J.; Gubernator, N. G.; Sames, D. J. Am. Chem. Soc. 2005, 127,
4544.
11. Alber, A. E.; Rawls, K. A.; Chang, C. J. Chem. Commun. 2007, 4647.
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Ahn, K. H. Chem. Commun. 2012, 48, 6833.
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Acknowledgments
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We are grateful for the financial support from the National Basic
Research Program of China (973 Program, 2010CB126100), the
China 111 Project (Grant B07023), the Key New Drug Creation