2,4,5-Triaryl imidazole probes for the selective chromo-fluorogenic detection of Cu(II). Prospective use of the Cu(II) complexes for the optical recognition of biothiols

Article abstract of DOI:10.1016/j.poly.2019.05.055

The sensing behaviour toward metal cations and biothiols of two 2,4,5-triarylimidazole probes (3a and 3b) is tested in acetonitrile and in acetonitrile–water. In acetonitrile the two probes present charge-transfer absorption bands in the 320–350 nm interv

Full text of DOI:10.1016/j.poly.2019.05.055

Accepted Manuscript
2,4,5-triaryl imidazole probes for the selective chromo-fluorogenic detection of
Cu(II). Prospective use of the Cu(II) complexes for the optical recognition of
biothiols
Hazem Essam Okda, Sameh El Sayed, Ismael Otri, R. Cristina M. Ferreira,
Susana P.G. Costa, M. Manuela M. Raposo, Ramón Martínez-Má ñez, Félix
Sancenón
PII:
S0277-5387(19)30393-6
https://doi.org/10.1016/j.poly.2019.05.055
POLY 13980
DOI:
Reference:
Polyhedron
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 April 2019
28 May 2019
28 May 2019
Please cite this article as: H. Essam Okda, S. El Sayed, I. Otri, R.C.M. Ferreira, S.P.G. Costa, M.M.M. Raposo, R.
Martínez-Má ñez, F. Sancenón, 2,4,5-triaryl imidazole probes for the selective chromo-fluorogenic detection of
Cu(II). Prospective use of the Cu(II) complexes for the optical recognition of biothiols, Polyhedron (2019), doi:
https://doi.org/10.1016/j.poly.2019.05.055
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2,4,5-triaryl imidazole probes for the selective chromo-
fluorogenic detection of Cu(II). Prospective use of the Cu(II)
complexes for the optical recognition of biothiols
Hazem Essam Okda,a,b,c Sameh El Sayed,a,b,c Ismael Otri,a,b,c R. Cristina M. Ferreira,d Susana
P. G. Costa,d M. Manuela M. Raposo,*d Ramón Martínez-Máñez,*a,b,c and Félix Sancenóna,b,c
a Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo
Tecnológico (IDM), Universitat Politècnica de València, Universitat de València. Spain. E-mail:
rmaez@qim.upv.es
b Departamento de Química, Universitat Politècnica de València. Camino de Vera s/n, 46022,
València, Spain.
c CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN).
d Centro de Química, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal.
E-mail: mfox@quimica.uminho.pt
Abstract
The sensing behaviour toward metal cations and biothiols of two 2,4,5-triarylimidazole probes
(3a and 3b) is tested in acetonitrile and in acetonitrile-water. In acetonitrile the two probes
present charge-transfer absorption bands in the 320-350 nm interval. Among all cations tested
only Cu(II) is able to induce bathochromic shifts of the absorption band in the two probes, which
is reflected in marked colour changes. Colour modulations are ascribed to the formation of 1:1
Cu(II)-probe complexes in which the cation interacts with the imidazole acceptor heterocycle.
Besides, the two probes present intense emission bands (at 404 and 437 nm for 3a and 3b
respectively) in acetonitrile that are quenched selectively by Cu(II). Probe 3a is soluble in
acetonitrile-water 1:1 (v/v) and Cu(II) also induces bathochromic shifts of the absorption bands.
Moreover, the emission bands of probe 3a in this mixed aqueous solutions is quenched in the
1

presence of Cu(II). The potential use of the 1:1 complex formed between 3a and Cu(II) for the
chromo-fluorogenic detection of biothiols (GSH, Cys and Hcy) in aqueous environments is also
tested. At this respect, addition of GSH, Cys and Hcy to acetonitrile-water 1:1 v/v solutions of
3a-Cu(II) complex induces a hypsochromic shift of the visible band (reflected in a bleaching of
the solutions) with a marked emission increase at 470 nm.
Keywords
Imidazole-based probes; Cu(II) detection; biothiols recognition; Cu(II) imaging; GSH imaging
1. Introduction
In the last years, the development of chromo-fluorogenic chemosensors for transition metal
cations has attracted the attention of researchers around the world.[1] These chromo-
fluorogenic chemosensors are generally formed by two subunits, namely the binding site and
the signalling group, that can be covalently linked or forming a supramolecular assembly.[2] In
the first case, interaction of transition metal cations with the binding sites induce
rearrangements in the -conjugated system of the signalling unit which are reflected in colour
and/or emission changes.[3] In the second approach, interaction of the transition metal cation
with the binding site induced the displacement of the signalling unit from the initial complex to
the solution.[4]
Among transition metal cations, Cu(II) is one of the most abundant essential element in
the human body and plays vital roles in several physiological processes. For instance, it has
been reported that Cu(II) stimulates the proliferation of endothelial cells and is necessary for the
secretion of several angiogenic factors by tumour cells.[5,6] Aside from its biological and
environmental importance, copper is widely used in metallurgical, pharmaceutical and
agrochemical industries.[7] As a result of the extensive applications of Cu(II) in life science and
industry, it has become one of the first hazard environmental pollutants.[8] Despite the
important role played by Cu(II) in several biological processes, abnormal levels of this cation
can cause serious health problems on humans due to its ability to displace other vital metal ions
in some enzyme-catalysed reactions.[9] In addition, high concentrations of Cu(II) in cells was
documented to cause toxicity and different neurodegenerative diseases such as Menkes,
Wilson's and Alzheimer.[10] Therefore, simple and rapid sensing tools to monitor Cu(II) levels in
2

biological and environmental media are of importance. Besides, the World Health Organization
(WHO) has recommended the maximum allowable level of Cu(II) in drinking water at 2.0 ppm
(~30 μM).[11,12]
Currently, several complex analytical techniques are used to detect metal ions such as
electrochemical measurements, atomic absorption spectrometry and inductively coupled plasma
mass spectroscopy.[13,14] However, these techniques are time-consuming, needs sample pre-
treatment, used expensive equipment’s, required trained personnel and cannot be used in situ.
On the other hand, biothiols (such as the amino acids cysteine (Cys) and homocysteine
(Hcy) and the tripeptide glutathione (GSH)) play vital roles in cellular processes related with the
damage of cellular components by reactive oxygen species (ROS).[15,16] Levels of biothiols in
cells are controlled by the equilibrium between thiols and disulfides. Besides, the relative levels
of biothiols can be used as biomarkers for aging, neurodegenerative pathologies and many
other diseases such as cancer, AIDS and cystic fibrosis among others.[17,18] Thus, taking into
account the above mentioned facts, determination of biothiols especially in biological samples is
an important issue. Actually, different analytical methods are available to determination of
biothiol levels including ca. HPLC, capillary electrophoresis, and mass spectrometry.[19-21] As
an alternative to these classical techniques, in the past two decades, a number of chromo-
fluorogenic probes for biothiol detection have been reported.[22-24] Most of the described
examples are based on the chemodosimeter approach and are synthesized taking into account
the nucleophilic character of thiol moieties in these biomolecules.[25-31] Also, the displacement
approach has been extensively used to design biothiol selective chromo-fluorogenic probes. For
this purpose, the emission quenching features of transition metal ions such as Cu(II) has been
widely used. The basis of these probes is the generation of non-emissive Cu(II) complexes with
fluorophores equipped with coordinating subunits. In the presence of biothiols, there is a
demetallation process, restoring the full emission of the fluorophore.[32-37] However, many of
the described biothiols chemosensors did not work in pure water or in mixed aqueous
environments. Besides, the selectivity achieved in most cases is low because probes are
unable to distinguish between Cys, Hcy and GSH. In fact, the synthesis and characterization of
probes for the selective recognition of individual biothiol in aqueous environments is of interest.
O
O
O
2a
O
H
N
O
O
3
N
N
NH4OAc/AcOH
N
H
1
O
3a
NH4OAc/AcOH
N
O
N

Scheme 1. One pot synthesis of imidazole probes 3a and 3b.
Taking into account our interest in the development of chromo-fluorogenic probes for
biomolecules[38-45] we report herein the synthesis, characterization and binding studies toward
Cu(II) and biothiols (GSH, Hcy and Cys), of two probes containing 2,4,5-trisubstitued imidazole
moieties (3a and 3b in Scheme 1). The prepared probes contain electron donor (furan) and
electron acceptor (phenanthroline) rings of different strength covalently linked with an imidazole
heterocycle. 2,4,5-triaryl(heteroaryl)-imidazole based chromophores have received increasing
attention due to their distinctive optical properties, applications in medicinal chemistry and
materials sciences, and as nonlinear optical materials (SHG chromophores, two-photon
absorbing molecules) and thermally stable luminescent materials for several applications (such
as OLEDs and fluorescent probes).
2. Experimental section
Materials and methods. All melting points were measured on a Stuart SMP3 melting
point apparatus. TLC analyses were carried out on 0.25 mm thick pre-coated silica
plates (Merck Fertigplatten Kieselgel 60F254) and spots were visualised under UV light.
Chromatography on silica gel was carried out on Merck Kieselgel (230-240 mesh). IR
spectra were determined on a BOMEM MB 104 spectrophotometer using KBr discs.
NMR spectra were obtained on a Bruker Avance III 400 at an operating frequency of 400
MHz for 1H and 100.6 MHz for 13a using the solvent peak as internal reference at 25 ºC.
All chemical shifts are given in ppm using δH Me4Si = 0 ppm as reference. Assignments
were supported by spin decoupling-double resonance and bidimensional heteronuclear
correlation techniques. UV/Vis titration profiles were carried out with JASCO V-650
spectrophotometer (Easton, MD, USA). Fluorescence measurements were recorded with
4

a
JASCO FP-8500 spectrophotometer. Commercially available reagents 4-
(dimethylamino) benzaldehyde (1), 1,2-di(furan-2-yl)ethane-1,2-dione (2a), 1,10-
phenanthroline-5,6-dione (2b) and ammonium acetate were purchased from Sigma-
Aldrich and Acros and used as received. TLC analyses were carried out on 0.25 mm
thick pre-coated silica plates (Merck Fertigplatten Kieselgel 60F254) and spots were
visualized under UV light. Chromatography on silica gel was carried out on Merck
Kieselgel (230-240 mesh).
Synthesis of 4-(4,5-di(furan-2-yl)-1H-imidazol-2-yl)-N,N-dimethylbenzenamine (3a). 4-
(dimethylamino) benzaldehyde (1, 0.15 g, 1 mmol), 1,2-di(furan-2-yl)ethane-1,2-dione (2a, 0.19
g, 1 mmol) and NH4OAc (1.54 g, 20 mmol) were dissolved in glacial acetic acid (5 mL), followed
by stirring and heating at reflux for 8 h. The reaction mixture was then cooled to room
temperature, ethyl acetate was added (15 mL) and the mixture was washed with water (3 x 10
mL). After drying the organic phase with anhydrous MgSO4, the solution was filtered and the
solvent was evaporated to dryness. The resulting crude product was purified by column
chromatography (silica gel, DCM/MeOH 100:1 v/v); yield (64 mg, 60%) as Grey solid. Mp =
240.2-240.9 ºC.
1H NMR (400 MHz, DMSO-d6):  = 2.95 (s, 6H), 6.55 (s, 1H), 6.63 (br s, 1H), 6.71 (d, J =
2.0 Hz, 1H), 6.77 (dd, J = 8.8 and 2.0 Hz, 2H), 6.89 (d, J = 2.8 Hz, 1H), 7.68 (br s, 1H),
7.78 (br s, 1H), 7.90 (dd, J = 8.8 and 2.0 Hz, 2H), 12.47 (s, 1H, NH) ppm. 13C NMR
(100.6 MHz, DMSO-d6):  = 39.89, 106.56, 107.81, 111.33, 111.80, 117.50, 118.27,
126.68, 129.15, 141.61, 142.22, 144.79, 147.23, 149.59, 150.53 ppm. IR (Nujol): ν =
3570, 1665, 1612, 1530, 1494, 1222, 1202, 1169, 1122, 1074, 1014, 986, 943, 911, 885,
815 cm-1. HRMS-EI m/z: calcd for C19H17N3O2+H+: 320.1399; measured: 320.1394.
Synthesis of 4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-N,N-dimethylbenzenamine (3b).
4-(dimethylamino) benzaldehyde (1, 0.15 g, 1 mmol), 1,10-phenanthroline-5,6-dione (2b, 0.2 g,
1 mmol) and NH4OAc (1.54 g, 20 mmol) were dissolved in glacial acetic acid (5 mL), followed by
stirring and heating at reflux for 8 h. The reaction mixture was then cooled to room temperature,
ethyl acetate was added (15 mL) and the mixture was washed with water (3 x 10 mL). After
drying the organic phase with anhydrous MgSO4, the solution was filtered and the solvent was
5

evaporated to dryness. The resulting crude product was purified by column chromatography
(silica gel, DCM/MeOH 98:2 v/v); yield (8 mg, 7%) as yellow oil.
1H NMR (400 MHz, DMSO-d6):  = 3.02 (s, 6H), 6.90 (d, J = 9.2 Hz, 2H), 7.81-7.84 (m,
2H,), 8.11 (dd, J = 8.8 and 2.0 Hz, 2H), 8.92 (dd, J = 8.0 and 1.6 Hz, 2H), 9.00 (dd, J =
4.0 and 2.0 Hz, 2H), 13.40 (s, 1H, NH) ppm. 13C NMR (100.6 MHz, DMSO-d6):  = 40.13,
111.97, 117.29, 123.33, 127.45, 127.62, 129.77, 142.94, 147.34, 147.40, 151.21, 151.88
ppm. IR (liquid film): ν = 3414, 2925, 1657, 1611, 1502, 1438, 1372, 1351, 1195, 1168,
1125, 1067, 1024, 946, 814, 739 cm-1. HRMS-EI m/z: calcd for C21H17N5+H+: 340.1562;
measured: 340.1560.
UV-visible and emission measurements. Stock solutions of the cations (i.e., Cu(II),
Pb(II), Mg(II), Ge(II), Ca(II), Zn(II), Co(II), Ni(II), Ba(II), Cd(II), Hg(II), Fe(III), In(III), As(III),
Al(III), Cr(III), Ga(III), K(I), Li(I) and Na(I) as perchlorate salts) were prepared at (1.0 × 10-
3
mol L-1) in acetonitrile. The concentrations of probes used in spectroscopy
measurements were ca. (5.0 × 10-5 mol L-1) and (1.0 × 10-5 mol L-1). We took care that
the maximum addition of cations solutions did not exceed 10% of the volume of the
receptor to avoid significant changes in the total solution concentration. In the
experiments that required the addition of excess of ions (20 equiv.), corrections of the
volume and concentration were made. The UV/Vis and fluorometric titrations were
carried out at room temperature (25 ºC).
3. Results and discussion
Synthesis and characterization of the probes. Heteroaromatic commercially available diones
2a and 2b containing furyl and phenanthroline rings (see Scheme 1) were used as precursors
for the synthesis of 3a and 3b. The synthesis of probes 3a and 3b was previously
described.[45,46] The final probes were prepared through a condensation reaction between 2a
and 2b diones and 4-(N,N-dimethylamino) benzaldehyde (1) in presence of ammonium acetate
and glacial acetic acid at reflux under Debus-Radziszewski imidazole synthesis conditions (see
also Scheme 1).[47] Probes were synthesized in fair to moderate yields (Table 1) as a
consequence of several factors such as the difficulty in the purification step due to the polarity of
the compounds as well as their decomposition during the long reaction times under hard
1
conditions (acetic acid reflux). The final probes 3a and 3b were characterized by H and 13C-
6

NMR, and IR. The data obtained are in full agreement with the proposed formulations (see
Experimental section).
Table 1. Yields and 1H NMR data of imidazole probes 3a and 3b at 400 MHz in DMSO-D6.
δH NH
Probe
Yield (%)
(ppm)
12.45
13.40
3a
3b
60
7
One of the most characteristic 1H-NMR signals of probes 3a and 3b is that
corresponding to the N-H proton in the imidazole ring (see Table 1). As could be seen, a clear
correlation between the electronic nature of the rings (furan and phenanthroline) directly linked
with imidazole and the shift of the N-H proton of this heterocycle was observed. At this respect,
probe 3b bearing the electron deficient phenanthroline system exhibited the highest chemical
shift for the nitrogen proton of the imidazole ring (13.40 ppm). On the other hand, probe 3a
functionalized with electron rich furan heterocycle exhibit lower chemical shifts for the nitrogen
proton of the imidazole ring (12.45 ppm) when compared to 3b.
UV/Vis and emission spectroscopic behaviour of probes in the presence of selected
metal cations in acetonitrile. In a first step, UV-visible changes of both probes in acetonitrile
were studied. Acetonitrile solutions of 3a and 3b (1.0 x 10-5 mol L-1) showed intense absorption
bands in the UV zone (321 nm for 3a and 341 nm for 3b). The observed absorption bands are
ascribed to charge-transfer transitions between the N,N-dimethylamino donor moiety and
imidazole acceptor heterocycle. Besides, the wavelength of the charge-transfer band was
affected by the aromatic units directly connected with the central imidazole heterocycle. At this
respect, the presence of electron deficient phenanthroline unit in probe 3b induced the larger
charge-transfer character and the associated absorption appeared at the higher wavelength
(341 nm). On the other hand, in probe 3a, the imidazole core was linked to an electron donating
heterocycle (furan) and, as a consequence, absorption bands are shifted to lower wavelengths
(321 nm).
Besides, both probes were highly emissive in acetonitrile upon excitation at the
maximum of the absorbance of the visible band. At this respect, excitation at 321 nm of
acetonitrile solutions of probe 3a (1.0 x 10-5 mol L-1) induced the appearance of a marked
7

emission band centred at 404 nm. Nearly the same results were obtained for probe 3b with an
intense emission centred at 437 nm. Besides, the quantum yield of the two probes (determined
using pyrene as standard, see Supporting Information for details) was 0.41 and 0.31 for 3a and
3b respectively.
Figure 1. UV/Vis titration profile of probe 3a in acetonitrile (1.0 x 10-5 mol L-1) upon addition of increasing
quantities of Cu(II) cation (0-10 eq.). Inset: Absorbance at 563 nm vs equivalents of Cu(II) added.
Then, UV-visible changes in acetonitrile solutions of both probes (1.0 x 10-5 mol L-1) in
the presence of increasing amounts (from 0.1 to 10 eq.) of selected metal cations (Cu(II), Pb(II),
Mg(II), Ge(II), Ca(II), Zn(II), Co(II), Ni(II), Ba(II), Cd(II), Hg(II), Fe(III), In(III), As(III), Al(III), Cr(III),
Ga(III), K(I), Li(I) and Na(I)) were tested. The two probes showed nearly the same behaviour
and in all cases only Cu(II) induced the progressive appearance of a new redshifted absorption
band. Figure 1 shows the set of spectra obtained upon addition of increasing quantities of Cu(II)
cation to an acetonitrile solution of probe 3a. As could be seen in Figure 1, addition of Cu(II)
induced a progressive bathochromic shift together with a marked decrease of the band centred
at 321 nm with the concomitant appearance of a redshifted absorption at 563 nm. Besides, a
marked colour change from colourless to violet was observed.
Nearly the same behaviour was observed with probe 3b in the presence of Cu(II) cation.
Addition of Cu(II) to an acetonitrile solution of 3b induced the appearance of a new absorbance
at 466 nm with a marked colour change from colourless to yellow. Changes upon addition of
Cu(II) to acetonitrile solutions of 3a and 3b was ascribed to the formation of complexes in which
the cation interacts with the acceptor part of the probes (i.e. the imidazole heterocycle).
8

In a next step, the emission of probes 3a and 3b in acetonitrile (1.0 x 10-5 mol L-1) was
also studied upon addition of increasing amounts of selected cations. A marked selective
response was obtained for Cu(II) that was the unique cation able to induce a remarkable
emission quenching for both probes. Figure 2 shows the set of emission spectra obtained upon
addition of increasing amounts of Cu(II) to probe 3a. Addition of Cu(II) induced a progressive
quenching of the emission band of 3a at 404 nm (excitation at ca. 380 nm) together with a
hypsochromic shift of ca. 10 nm. A similar quenching and hypsochromic shifts of the emission
bands was observed upon addition of Cu(II) to acetonitrile solutions of probe 3b (see
Supporting Information). The obtained results are in accordance with the marked and well-
known quenching behaviour of Cu(II) cation.
Figure 2. Fluorescence titration profile of probe 3a in acetonitrile (1.0 x 10-5 mol L-1) upon addition of
increasing quantities of Cu(II) cation (0-10 equiv.).
From the UV-visible and emission titration profiles obtained for both probes, the limits of
detection (LOD) for Cu(II) in acetonitrile were determined (see Table 2). LODs were assessed
using the 3.3*(σ/s) equation, where σ is the standard error of the predicted Y-value vs. each X
(i.e. Cu(II) concentration), and s is the slope for the linear relationship. Both probes presented
similar Cu(II) LODs in the 0.9-2.0 M range.
Table 2. Limits of detection of Cu(II) measured for 3a and 3b probes in acetonitrile.
Emission
[μM]
Probe
UV/Vis [μM]
3a
0.9
1.0
9

3b
1.2
2.0
Determination of binding stoichiometries and stability constants of probe 3a and 3b with
Cu(II) in acetonitrile. In order to complete the characterization of the complex formed between
probe 3a and 3b and Cu(II) cation we carried out UV-visible and emission studies to obtain
Job’s plots to assess the binding stoichiometry and to determine the stability constants.
Figure 3. Job’s plot for the binding of 3a with Cu(II). Absorbance at 555 nm was plotted as a function of
the molar ratio [Cu(II)]/([3a] + [Cu(II)]). The total concentration of Cu(II) and probe 3a was (2.0 × 10−5 mol
L-1).
Job’s plots, constructed using UV-visible measurements in acetonitrile, showed that 3a
and 3b formed 1:1 complexes with Cu(II). As an example, Figure 3 showed the Job’s plot
obtained for probe 3a. Besides, from the UV-visible and fluorescence titration profiles obtained
for probes 3a and 3b in the presence of Cu(II) cation, the stability constant for the formation of
the corresponding 1:1 complexes were determined. The obtained values are shown in Table 3.
Table 3. Logarithms of binding constants measured for the interaction of probes 3a and 3b with Cu(II) in
acetonitrile.
Log K
Probe
UV/Vis
5.1 ± 0.1
5.8 ± 0.9
Emission
6.4 ± 0.3
5.8 ± 0.1
3a
3b
10

UV/Vis and emission spectroscopic behaviour of probes 3a and 3b in the presence of
selected metal cations in aqueous environments. In order to assess the possible use of both
probes for Cu(II) detection in real samples, the UV-visible and emission response toward Cu(II)
was tested in aqueous environments. Probes 3a and 3b were fully solubilized in water (pH 7.4)-
acetonitrile 1:1 v/v. In this medium, both probes showed charge-transfer absorption bands
centred at 321 and 339 nm for 3a and 3b respectively. The maxima of the charge-transfer band
in water-acetonitrile 1:1 v/v were very similar to those measured in pure acetonitrile, indicating
that conformation and hydration of the probes did not change, to any remarkable extent, the
energy of their electronic levels in the basal state.
Next, the UV-visible behaviour of aqueous solutions of both probes (5.0×10-5 mol L-1) in
the presence of selected metal cations was tested. None of the metal cations tested induced
changes in the absorption bands of probe 3b. In contrast, for probe 3a, only Cu(II) induced the
appearance of a new redshifted absorption at 563 nm as could be seen in Figure 4 (similar to
that found in acetonitrile) together with marked colour changes (Figure 5).
Figure 4. Absorption of probe 3a at 563 nm (water (pH 7.4)-acetonitrile 1:1 v/v, 5.0×10-5 mol L-1) in the
presence of 10 eq. of selected metal cations.
More in detail, addition of increasing quantities of Cu(II) cation to water (pH 7.4)-
acetonitrile 1:1 v/v (5.0×10-5 mol L-1) solutions of probe 3a induced a moderate decrease of the
absorption at 321 nm and the simultaneous appearance of a new band centred at 563 nm (see
Figure 5). These spectral changes were reflected in a clear colour change from colourless to
11

violet (see inset in Figure 5). Besides, from the titration profile at 489 nm, a limit of detection for
Cu(II) of 2.60 M was determined.
Figure 5. UV-Vis titration profile of probe 3a in water (pH 7.4)-acetonitrile 1:1 v/v (5.0×10-5 mol L-1) upon
addition of increasing quantities of Cu(II) cation (0-10 eq.). Inset: colour change when Cu(II) cation was
added to water (pH 7.4)-acetonitrile 1:1 v/v solutions of probe 3a.
Figure 6. Fluorescence titration profile of probe 3a in water (pH 7.4)-acetonitrile 1:1 v/v (5.0 x 10-5 mol L-1)
upon addition of increasing amounts of Cu(II) cation (from 0 to 10 eq.). Inset: Emission intensity at 420 vs
equivalents of Cu(II) added.
On the other hand, 3a was highly emissive in water (pH 7.4)-acetonitrile 1:1 v/v
solutions showing a remarkable emission band at 420 nm (excitation at 321 nm). Moreover,
fluorescence changes in the presence of selected metal cations was tested. For this purpose,
solutions of probe 3a were excited at the isosbestic point observed in the UV-visible titration
profile (ca. 375 nm) and the emission recorded after the addition of different amounts of
selected cations. From all cations tested, only Cu(II) was able to induce a response which
12

consisted of a marked emission quenching of the emission band 420 nm (see Figure 6). The
obtained results clearly resemble those obtained for probe 3a in acetonitrile in the presence of
Cu(II) cation. From the emission titration profile (see also Figure 6), a LOD for Cu(II) of 3.4 M
for 3a was determined. This LOD is higher than that found in acetonitrile, most likely as
consequence of a stronger solvation of Cu(II) cation in water.
Spectroscopic detection of biothiols. In this section we studied the potential use of the 3a·Cu
complex for the recognition of biothiols.[48] Studies were carried out in the presence of selected
amino acids (Ala, Arg, Asp, Cys, Gln, Glu, Gly, Hcy, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,
Trp, Tyr and Val) and small peptides (GSH).
Figure 7. UV-visible changes of 3a·Cu (6.2 x 10-6 mol L-1) in water (pH 7.4)-acetonitrile 1:1 v/v in the
presence of selected amino acids (0.2 eq.) and biothiols (0.2 eq.).
Figure 8. Changes in the emission band of 3a·Cu complex (6.2 x 10-6 mol L-1) in water (pH 7.4)-acetonitrile
1:1 v/v upon addition of biothiols (0.2 eq.) and selected amino acids (0.2 eq.).
13

Water (pH 7.4)-acetonitrile 1:1 v/v solution of complex 3a·Cu (6.2 x 10-6 mol L-1, formed
in situ by the addition of equimolar quantities of probe and Cu(II) cation) shows an intense
absorption band at 563 nm which was the responsible of the strong violet colour observed.
From all amino acids (0.2 eq.) and peptides (0.2 eq.) tested, only in the presence of thiol-
containing molecules (Cys, Hcy and GSH) a bleaching of the solution was observed (see Figure
7). Besides, water (pH 7.4)-acetonitrile 1:1 v/v solutions of the 3a·Cu complex (6.2 x 10-6 mol L-
1) were weakly emissive and only addition of Cys, Hcy and GSH induced a marked emission
enhancement at 420 nm (see Figure 8). The chromo-fluorogenic changes observed upon
addition of biothiols to the aqueous solutions of 3a·Cu complex are ascribed to a demetallation
of the complex, that released the free probe 3a. Finally, from UV-visible and fluorescence
titration profiles obtained for 3a·Cu, the LODs for biothiols were determined (Table 4).
Table 4. Limits of detection of biothiols measured for 3a·Cu in aqueous environments.
UV-visible (M)
Fluorescence (M)
GSH
0.6
Cys
1.3
Hcy
0.8
GSH
0.6
Complex
Cys
0.9
Hcy
0.7
3a·Cu
4. Conclusions
In summary, we show herein the synthesis and chromo-fluorogenic behaviour toward metal
cations and biothiols of easy to prepare imidazole-containing probes 3a and 3b. Acetonitrile
solutions of both probes were characterized by the presence of a charge-transfer absorption
band in the 320-350 range. Both probes were moderately emissive in acetonitrile. From all
cations tested only Cu(II) induced the growth of new redshifted bands in the 450-570 nm
interval. Besides, the emission bands of both receptors were quenched in the presence of Cu(II)
cation. These spectral changes were ascribed to a preferential coordination of Cu(II) cation with
the electron acceptor imidazole heterocycle. Only probe 3a changed its UV-visible and emission
spectra in the presence of Cu(II) in water-acetonitrile mixtures. On the other hand, weakly
emissive complex 3a·Cu was used to detect biothiols (GSH, Cys and Hcy) in water-acetonitrile
1:1 v/v solution. In the presence of biothiols, the visible bands of the complex disappeared and
the emission intensity of the free probe was fully restored. These changes were ascribed to a
demetallation process.
14

Acknowledgments
We thank the Spanish Government (MAT2015-64139-C4-1-R) and Generalitat Valenciana
(PROMETEO 2018/024). H. E. O. thanks Generalitat Valenciana for his Grisolia fellowship.
Thanks are also due to Fundação para a Ciência e Tecnologia (Portugal) for financial support to
the Portuguese NMR network (PTNMR, Bruker Avance III 400-Univ. Minho), FCT and FEDER
(European Fund for Regional Development)-COMPETEQREN-EU for financial support to the
research centre CQ/UM [PEst-C/QUI/UI0686/2013 (FCOMP-01-0124-FEDER-037302], and a
post-doctoral grant to R.M.F. Batista (SFRH/BPD/79333/2011).
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Graphical abstract synopsis:
Chromo-fluorogenic recogntion of Cu(II) cation and bithiols was achieved using two imidazole-
based probes.
Graphical abstract pictogram:
Cu2+
N
O
N
N
H
O
18