Design and synthesis of a new coumarin-based 'turn-on' fluorescent probe selective for Cu+2

Article abstract of DOI:10.1016/j.tetlet.2012.07.082

The novel coumarin-based 'turn-on' fluorescent probe (E)-3-(2,5- dimethoxybenzylideneamino)-7-hydroxy-2H-chromen-2-one (MGM) was designed, synthesized, and characterized. This compound shows high selectivity for Cu ⁺², combined with a large fluorescence enhancement upon binding to Cu²⁺. Benesi-Hildebrand and Job plots demonstrate that the stoichiometry of the Cu²⁺ complex formed is 2:1. Preliminary studies employing epifluorescence microscopy demonstrated that Cu⁺² could be imaged in human neuroblastoma SH-SY5Y cells treated with MGM.

Full text of DOI:10.1016/j.tetlet.2012.07.082

Tetrahedron Letters 53 (2012) 5280–5283
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Design and synthesis of a new coumarin-based ‘turn-on’ fluorescent probe
selective for Cu+2
Olimpo García-Beltrán a,e,f, , Natalia Mena b,e, Leidi C. Friedrich c, José Carlos Netto-Ferreira c,d
,
⇑
Víctor Vargas a, Frank H. Quina c, Marco T. Núñez b,e, Bruce K. Cassels a,e,
⇑
a Department of Chemistry, Faculty of Sciences, University of Chile, Santiago, Chile
b Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile
c Institute of Chemistry and the USP Research Consortium for Photochemical Technology, (NAP-PhotoTech), University of São Paulo, São Paulo, Brazil
d Department of Chemical Engineering, University of São Paulo, São Paulo, Brazil
e Institute for Cell Dynamics and Biotechnology, University of Chile, Santiago, Chile
f Departamento de Ciencias Químicas, Facultad de Ciencias Exactas, Universidad Andrés Bello, Avenida República 275, Piso 3, Santiago, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
The novel coumarin-based ‘turn-on’ fluorescent probe (E)-3-(2,5-dimethoxybenzylideneamino)-7-
hydroxy-2H-chromen-2-one (MGM) was designed, synthesized, and characterized. This compound
shows high selectivity for Cu+2, combined with a large fluorescence enhancement upon binding to
Cu2+. Benesi–Hildebrand and Job plots demonstrate that the stoichiometry of the Cu2+ complex formed
is 2:1. Preliminary studies employing epifluorescence microscopy demonstrated that Cu+2 could be
imaged in human neuroblastoma SH-SY5Y cells treated with MGM.
Received 22 April 2012
Revised 14 July 2012
Accepted 19 July 2012
Available online 31 July 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Cu+2 ion
Turn-on probes
Coumarin Schiff base
Fluorescence sensor
Chemosensor development is an active field of research with
excellent potential in clinical biochemistry, analytical chemistry,
and environmental science.1 Copper is a very important transition
metal in the body, being the third most abundant metal after iron
and zinc,2 but it can also catalyze the formation of reactive oxygen
species (ROS) capable of damaging biomolecules. Research has
connected the cellular toxicity of copper ions with serious diseases,
including prion disease.3,4 In addition, alterations in cellular levels
of copper have been associated with several neurodegenerative
diseases, such as Menkes and Wilson diseases,5–7 Alzheimer’s
and Parkinson’s diseases.8
The molecular recognition of a variety of different metal cations
by MGM was investigated by UV–vis and fluorescence spectros-
copy. All absorption and emission spectral studies were performed
in freshly purified CH3CN at room temperature, while the corre-
sponding metal chlorides were used as the source of the metal
cations. The absorption spectrum of MGM in CH3CN exhibits a
band with a maximum at 375 nm, the intensity of which gradually
decreases as the concentration of Cu+2 is increased (Supplementary
Fig. S3). Concomitantly, a new absorption band builds up at
300 nm, which we originally ascribed to the MGM–Cu+2 complex.
The fluorescence spectrum of MGM (kexc = 375 nm) showed lit-
In this work, the novel fluorescence probe MGM was prepared
via a conventional four-step synthesis from commercial precur-
sors. Vilsmeier–Haack formylation of resorcinol gave 2,4-dihy-
droxybenzaldehyde, which was subsequently condensed with
N-acetylglycine (Knoevenagel) and hydrolyzed in situ to afford
3-amino-7-hydroxycoumarin. The condensation of 3-amino-7-
hydroxycoumarin with 2,5-dimethoxybenzaldehyde then provided
MGM (Scheme 1), which was characterized by 1H NMR and 13C
NMR spectroscopy (Supplementary Figs. S1A and S1B).
tle or no enhancement in the presence of 200 lM cations such as
Fe+2, Fe+3, Ca+2, Co+2, Mg+2, Mn+2, Zn+2, Cd+2, Pb+2, or Hg+2. In sharp
contrast, there was an enormous increase of its fluorescence inten-
sity upon the addition of 200 l
M Cu+2 (Fig. 1a). The specificity of
the enhancement of the fluorescence of this ‘turn-on’ compound
for Cu+2 and a visual indication of the Cu2+-induced fluorescence
enhancement are indicated in Figure 1b and Figure 1c, respectively.
Titration of MGM with Cu+2 resulted in an increase in the
fluorescence intensity and a slight blue shift of the fluorescence
emission at intermediate Cu2+ concentrations (Fig. 2). Upon
375 nm excitation, the emission quantum yield (
determined to be = 0.0003, using quinine sulfate as standard.
Upon addition of 200
M Cu+2 (10 equiv), the quantum yield in-
creased almost 50-fold to = 0.014.
U) of MGM was
U
⇑
Corresponding author. Tel.: +56 2 978 7253; fax: +56 2 271 3888.
E-mail addresses: ojgarciab@ug.uchile.cl (O. García-Beltrán), bcassels@u.uchile.cl
l
U
(B.K. Cassels).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.07.082

O. García-Beltrán et al. / Tetrahedron Letters 53 (2012) 5280–5283
5281
O
O
NH2
N
O
O
OH
a
d
b, c
HO
HO
O
O
HO
OH
HO
MGM
O
1
3
2
Scheme 1. Synthetic route to MGM. Reagents and conditions: a) POCl3, DMF, acetonitrile, 0–5 °C, 2 h; b) acetylglycine, acetic anhydride, anhydrous sodium acetate, reflux
4 h; c) 2:1 HCl/H2O reflux, 2 h; d) 2,5-dimethoxybenzaldehyde, EtOH, reflux, 4 h.
Figure 1. (a) Fluorescence spectra of MGM (20 lM) alone and in the presence of several different metal salts (200 l
M Fe+2, Fe+3, Ca+2, Cu+2, Co+2, Mg+2, Mn+2, Zn+2, Cd+2, Pb+2
or Hg+2) in 90% ACN-H2O mixture. (b) Selectivity of the fluorescence enhancement for MGM with Cu+2 ion in 90% acetonitrile-H2O mixture. Red bars indicate the fluorometric
responses of MGM with 10 equiv of Fe+2, Fe+3, Ca+2, Co+2, Mg+2, Mn+2, Zn+2, Cd+2, Pb+2, and Hg+2 and black bars represent the fluorescence response after the addition of the
same ions and 10 equiv of Cu+2. (c) Photographs of the fluorescence of acetonitrile solutions of MGM (20 M) in the absence (left) and presence (right) of Cu+2 (200
l lM). All
the metal ion solutions were prepared by dissolving their corresponding chloride salts in water.
A Benesi–Hildebrand graph of the fluorescence data (Supple-
mentary Fig. S3) was non-linear, indicating that the stoichiometry
of the Cu+2 complex formed is different from 1:1. Consistent with
this, application of the Method of Continuous Variation resulted
in a Job plot (Supplementary Fig. S5) with a maximum at a mole
fraction of Cu2+ close to 0.33, indicating a preferred 2:1 stoichiom-
etry for the complex.
To determine if the observed changes in absorption and fluores-
cence are due to chelation of Cu2+ by MGM the 1H NMR spectra of
the probe in the absence and presence of Cu+2 were recorded. As
shown in Figure 3, upon addition of Cu+2, the Hb proton signal
(the proton at C4 of the coumarin) at 7.81 ppm, shifts upfield to
7.2 ppm, accompanied by more subtle changes in the rest of the
aromatic region of the spectrum. More disturbingly, the resonance
of the imine proton Ha, at d 9.31 disappears and a typical aldehyde
signal arises at d 10.25. This was interpreted as indicating that
MGM undergoes hydrolysis in the presence of Cu+2, releasing 2,5-
dimethoxybenzaldehyde and 3-amino-7-hydroxycoumarin.
In fact, we were able to show that 2,5-dimethoxybenzaldehyde,
which itself is unusually fluorescent for a benzaldehyde derivative,9
becomes much more strongly fluorescent upon the addition of Cu+2
(Supplementary Fig. S6; Supporting Information), giving the same
emission spectrum observed when MGM was treated with Cu+2
.
Moreover, the fluorescence of 3-amino-7-hydroxy-coumarin is
quenched by Cu+2 (Supplementary Fig. S7), and therefore does not
interfere with the emission of the 2,5-dimethoxybenzaldehyde-
Cu+2 complex (Supplementary Fig. S8). Recent work presented sim-
ilar imino systems, which are not fluorescent.10 However, in the

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O. García-Beltrán et al. / Tetrahedron Letters 53 (2012) 5280–5283
Figure 2. Fluorescence spectra of MGM (20
lM) with different concentrations of
Cu+2
.
presence of Cu+2 they are hydrolyzed to regenerate the amine and 3-
formylcoumarin reactants. In these cases complexation of the free
3-formylcoumarin with Cu+2 is responsible for the increase in
fluorescence.
Copper-selective probes are relatively few in number,11 and
those that can be employed in biological systems are even scarcer.
MGM was therefore evaluated for its potential as a probe for intra-
cellular Cu+2 in SH-SY5Y human neuroblastoma cells. The cells
were initially incubated with MGM under physiological conditions
and then subsequently treated with the histidine-Cu+2 complex as
the source of Cu2+. Fluorescence was monitored using a microplate
fluorescence reader and by epifluorescence microscopy (Fig. 4). The
fluorescence was monitored using a microplate fluorescence read-
er and by epifluorescence microscopy (Fig. 4). The fluorescence
was significantly increased (Fig. 4B) following the addition of his-
tidine-Cu+2, with most of the fluorescent species residing near
the cell periphery.
Figure 4. In vitro tests of the potential of MGM as an intracellular probe for Cu2+
SH-SY5Y cells were incubated with the probe (10 M, 20 min), washed, and the
basal fluorescence measured (A). The cells were then incubated with Cu-His
(200 M, 15 min) and fluorescence measured (B) in a microplate fluorescence
.
l
l
reader and by epifluorescence microscopy. The bar graph represents the mean
-
SEM of the data; n = 6, P < 0.01(⁄).
conditions as the experiments carried out with MGM. After wash-
ing the cells no fluorescence was observed and, more importantly,
none was observed after treatment with histidine-Cu+2 complex.
We therefore concluded that 2,5-dimethoxybenzaldehyde does
not enter the cells. On the contrary, MGM must act as a cell-pene-
trating carrier that enters the cells and, once inside, releases 2,5-
dimethoxybenzaldehyde which fluoresces strongly when copper
is present in the intracellular medium.
As 2,5-dimethoxybenzaldehyde might conceivably be released
in the extracellular medium by hydrolysis of MGM, we incubated
SH-SY5Y cells with the aldehyde (10 lM, 20 min) under the same
Figure 3. Changes in the 1H NMR spectrum of MGM (20 mM) upon addition of Cu+2 (10 equiv) in DMSO–d6 at 25 °C. (A) MGM only; (B) MGM + Cu+2 (after 10 min).

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5283
In conclusion, we have prepared and characterized the novel
References and notes
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Acknowledgments
This work was supported by the Millennium Scientific Initiative
(Grant P05-001-F) and by the Instituto Nacional de Ciência e Tecn-
ologia do Meio Ambiente-USP (Brazil). JCNF thanks the Fundação
de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for a Visit-
ing Professor Fellowship. FHQ and LCF acknowledge fellowship
support from the CNPq-Brazil.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.07.
082.