An activity-based fluorogenic probe for sensitive and selective monoamine oxidase-B detection

Article abstract of DOI:10.1039/c2cc33089j

We report the design and synthesis of a new class of fluorogenic probes based on monoamine oxidase-triggered oxidative C-O bond cleavage. The selectivity of probe P1 towards MAO-B was 22-fold higher than that towards MAO-A.

Full text of DOI:10.1039/c2cc33089j

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An activity-based fluorogenic probe for sensitive and selective
monoamine oxidase-B detectionw
Shaobo Long, Lin Chen, Yumi Xiang, Minggui Song, Yuguo Zheng and Qing Zhu*
Received 29th April 2012, Accepted 21st May 2012
DOI: 10.1039/c2cc33089j
We report the design and synthesis of a new class of fluorogenic
probes based on monoamine oxidase-triggered oxidative C–O
bond cleavage. The selectivity of probe P1 towards MAO-B was
22-fold higher than that towards MAO-A.
approach is urgently needed. In this report, we designed and
synthesised a novel class of activity-based fluorescent probes
that specifically target MAO-B.
Unlike previously developed fluorescent probes based on
oxidation/b-elimination, our general strategy was based
on a MAO-B-catalysed a-carbon oxidation mechanism.15
4-Phenoxy-1-methyl-1,2,3,6-tetrahydropyridine, a specific MAO-B
substrate, undergoes a-carbon oxidation to afford dihydro-
pyridinium, which can be rapidly hydrolysed to yield amino
enone and phenol. Therefore, N-alkylated tetrahydropyridines
were designed as an enzyme recognition head group, which
was then conjugated to coumarin as fluorescent reporter owing
to its favourable photophysical properties and biological com-
patibility. Due to the electron-withdrawing effect of the
recognition group, the probes were virtually non-fluorescent
and provided a molecular switch for activity detection. After
treatment with MAO-B, the tetrahydropyridine derivatives
yielded the intermediate dihydropyridinium via a-carbon
oxidation and readily underwent rapid hydrolysis in water.
Consequently, amino enone derivatives and blue fluorescent
4-methylcoumarin were produced (Fig. 1). Our approach
offers the distinct advantages of directly and specifically
monitoring MAO-B activity without the need for an additional
secondary activating enzyme.
Monoamine oxidases (MAOs) are flavoenzymes that catalyze
the oxidative deamination of a number of biogenic and
xenobiotic amines to their corresponding aldehydes via the
consumption of oxygen and production of hydrogen peroxide.1
These enzymes play important roles in the central nervous
system by maintaining the homeostasis of neurotransmitters
such as dopamine and serotonin, as well as in the peripheral
nervous system by metabolising dietary amines and pharma-
ceuticals.2,3 Two isoforms of MAO were identified and desig-
nated as MAO-A and MAO-B, which are coded by similar but
distinct genes. These two isoforms possess different substrate
preferences and inhibitor specificities.4–6 MAO-B is the
predominant isoform of MAO found in the human brain,
and unusually high MAO-B activity in the brain is observed
in the neurological degeneration processes of Alzheimer’s
disease, Huntington’s disease, some forms of Parkinson’s
disease, and normal aging.7–9
Due to the vital roles of MAOs in neurological health and
diseases, there is great interest in the development of sensitive
and selective detecting methods for monitoring MAO activity.
Consequently, a number of interesting fluorimetric and colori-
metric approaches for assaying MAO activity in vitro have
been described.10 For example, several assays detect and
monitor MAOs via the oxidation of propylamine by MAO
and subsequent b-elimination reaction to liberate free coumarin
or luciferin.11–14 However, these detection methods are limited
by the required addition of enzymes or other activating
reagents (such as NaIO4 or BSA) that can potentially affect
the physical properties of the MAOs and thus increase the
background signal. These probes target both MAO-A and
MAO-B for the non-specific oxidation of propylamine by
MAOs. To understand the functions of MAO-B in Alzheimer’s
and Huntington’s diseases better, a sensitive and specific detection
To examine the feasibility of our strategy and investigate the
effect of the N-substituted group and reporters on the sensitivity
and selectivity, four probes were chemically synthesised from
coumarin (Scheme 1). 7-Hydroxy-4-methyl coumarin (1),
which was obtained from resorcinol (see ESIw), was reacted
with pyridine salts with methyl iodide, allyl bromide or benzyl
groups 2–4 to give intermediates 5–7 in 75, 61 and 61% yields,
Institute of Bioengineering, Zhejiang University of Technology,
Chaowang Road 18, China. E-mail: zhuq@zjut.edu.cn;
Fax: +86-571-88320781; Tel: +86-571-88320781
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc33089j
Fig. 1 Design principle of MAO-B fluorogenic probes.
c
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exhibited more than 22-fold enhancement compared with
MAO-A; thus, probe P1 is a highly selective fluorogenic probe
for the detection of MAO-B.
To obtain the kinetic parameters for probe P1, it was
assayed over a wide range of concentrations (0 to 200 mM)
with MAO-B. Km, Vmax, and the fluorescence intensity were
determined, and a representative enzyme kinetic plot is shown
in Fig. 3A. These kinetic experiments yielded the following
Michaelis–Menten constant values for probe P1: Km
=
59.63 mM and Vmax = 1.42 nmol mg minÀ1. The initial rate
verified that it was a good substrate for MAO-B, indicating
that the reporter motif did not interfere with its reactivity. The
distinct turn-on response to probe P1 resulted from the bright
coumarin dye as well as the relatively fast kinetics of its reactions
with MAO-B and rapid hydrolysis of dihydropyridinium.
Fig. 3B displays a representative fluorescence response of probe
P1 upon the addition of MAO-B, whereas almost no fluores-
cence was detected in the control experiment (no MAO-B).
To gain better insight into the recognition of the most potent
probe P1 with respect to human MAO-A and MAO-B,
a molecular docking study was carried out using the Discovery
studio 2.1 software. The crystallographic structures of
MAO-A [Protein Data Bank (PDB) code 2Z5X] and MAO-B
Fig. 2 MAOs selectivity of probes P1–P3 in 100 mM borate buffer
(pH = 8.4). The bars represent the fluorescence intensity at 460 nm
after 2 h of reaction of 200 mM probes with MAO A or B/diamine
oxidase/inactivating enzyme/borate buffer (control). The excitation
wavelength was 360 nm. Data represent the means of three separate
experiments and error bars represents standard deviation.
Scheme 1 Synthesis of the fluorogenic monoamine oxidase probes.
Reagents and conditions: (a) CH3COCH3/Et3N; (b) NaBH4/MeOH, 0 1C.
respectively. Reduction with sodium borohydride in methanol
provided probes P1–P3 in good yields. All intermediates and
1
probes were characterised by H NMR and MS.
We next assessed the reactivities of probes P1–P3 with
MAO-A and MAO-B. To approximate the in vivo environ-
ment, we employed mitochondrial preparations rather than
purified enzymes. MAO-A and MAO-B were prepared from
human placenta and liver, respectively. To demonstrate the
MAO activity-dependent characteristics, the responses of the
probes towards various proteins, including MAOs, diamine
oxidase, and inactivated MAO-B, were examined (Fig. 1). The
probes and suspension of mitochondria or other enzymes were
incubated in borate buffer (pH = 8.4), and the fluorescence
spectra were collected 2 h thereafter at lex/lem of 360/460 nm.
As shown in Fig. 1, all probes did not respond to inactivated
MAO-B and other enzymes, indicating an activity-based
mechanism. Only the addition of MAO-B triggered a marked
increase in fluorescence intensity, whereas MAO-A did not
elicit a good response, as expected (Fig. 1). The predicted
formation of coumarin was further confirmed by HPLC-MS.
The fluorescence intensity of probe P1 towards MAO-B
Fig. 3 The fluorescent assays for MAO activity with probe P1. (A)
Detection of enzyme kinetics parameters for probe P1. Data show the
activity over a range of concentrations of P1 (0–200 mM) in reaction
with MAO-B (40 mg mLÀ1) at 37 1C in enzyme assay buffer (100 mM
borate buffer, pH = 8.4). The fluorescence intensity was monitored at
460 nm (lex = 360 nm). (B) Fluorescence emission spectra of probe P1
(200 mM) treated with MAO-B (40 mg mLÀ1) at 37 1C in enzyme assay
buffer (100 mM borate, pH = 8.4) after 0, 10, 20, 30, 40, 50 and
60 min. The excitation wavelength was 360 nm.
c
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This work was supported by Zhejiang Natural Science Fund
(No. Y2080303).
Notes and references
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Fig. 4 Binding mode of probe P1 to the human MAO-A (A) and
MAO-B (B) active site. Carbons are in gray, nitrogen in blue, oxygens
in red, and hydrogens in white.
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(PDB code 2BK3) were retrieved from the PDB. Based on the
docking study information, the high MAO-B selectivity of
probe P1 can be explained as follows. First, the most favour-
able binding modes of probe P1 with MAO-A/B (Fig. 4)
showed that no hydrogen bond was formed between probe
P1 and MAO-A. In contrast, a pivotal pair of hydrogen
bonds was found between the nitrogen of probe P1 and the
hydroxyl group of Tyr60 in the active site of MAO-B. Second,
energy evaluation revealed that the binding free energy of
probe P1 with MAO-A was 41000 kcal molÀ1 (calculated by
MM-PBSA), which was much higher than that with MAO-B
(À10.2 kcal molÀ1).
In summary, we took advantage of a simple and effective
a-carbon oxidation–hydrolysis mechanism to design and
synthesize a novel class of fluorogenic probes that can be used
to measure the enzyme activities of MAO-B. The important
advantages of the probes include the direct monitoring of
MAO activity in aqueous solution without the need for
additional proteins or activating reagents, as well as good
selectivity and sensitivity towards MAO-B.
c
7166 Chem. Commun., 2012, 48, 7164–7166
This journal is The Royal Society of Chemistry 2012