A novel fluorescent chemosensor for Cu2+ ion based on a new hexadentate ligand receptor: X-ray single crystal of the perchlorate salt of the ligand, ion selectivity assays and TD-DFT study

Article abstract of DOI:10.1016/j.ica.2020.120061

New hexadentate chelates, H₂L¹ and L¹, were synthesized. The Schiff base imine ligand, L¹, was prepared by reaction of 2,2′-[ethane-1,2-diylbis(oxy)]dibenzaldehyde and propanolamine, and characterized by infrare

Full text of DOI:10.1016/j.ica.2020.120061

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Research paper
A novel fluorescent chemosensor for Cu2+ ion based on a new hexadentate li‐
gand receptor: X-ray single crystal of the perchlorate salt of the ligand, ion se‐
lectivity assays and TD-DFT study
Reza Golbedaghi, Licínia L.G. Justino, Marzyeh Bahrampour, Rui Fausto
PII:
DOI:
Reference:
S0020-1693(20)31261-5
https://doi.org/10.1016/j.ica.2020.120061
ICA 120061
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Inorganica Chimica Acta
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1 October 2020
2 October 2020
Please cite this article as: R. Golbedaghi, L.L.G. Justino, M. Bahrampour, R. Fausto, A novel fluorescent
chemosensor for Cu2+ ion based on a new hexadentate ligand receptor: X-ray single crystal of the perchlorate salt
of the ligand, ion selectivity assays and TD-DFT study, Inorganica Chimica Acta (2020), doi: https://doi.org/
10.1016/j.ica.2020.120061
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A novel fluorescent chemosensor for Cu2+ ion based on a new hexadentate
ligand receptor: X-ray single crystal of the perchlorate salt of the ligand,
ion selectivity assays and TD-DFT study
Reza Golbedaghi,a,b,* Licínia L.G. Justino,b,* Marzyeh Bahrampoura and Rui Faustob
a Chemistry Department, Payame Noor University, 19395-4697 Tehran, Iran
b University of Coimbra, CQC, Department of Chemistry, P-3004535 Coimbra, Portugal
Abstract
New hexadentate chelates, H2L1 and L1 were synthesized. The Schiff base imine ligand, L1, was
prepared by reaction of 2,2'-[ethane-1,2-diylbis(oxy)]dibenzaldehyde and propanolamine, and
1
characterized by infrared (IR), H-NMR and 13C-NMR spectroscopies. The amine ligand, H2L1, was
prepared in the form of perchlorate salt ([H4L1](ClO4)2,) by reduction of L1 with NaBH4, and was
characterized by IR (in KBr pellet), 1H-NMR, 13C-NMR and 2D NMR correlation spectroscopy (COSY)
(in solution). The X-ray crystal structure of [H4L1](ClO4)2 was also determined and discussed.
Fluorescence studies showed that H2L1 is a highly selective novel fluorescence chemosensor for the Cu2+
cation. The sensor shows selectivity for Cu2+ over 12 other metal cations. In presence of Cu2+ (5
equivalents in the M range) the fluorescence emission of the sensor, at 309 nm, is quenched by more
than 98 %. TD-DFT calculations show that ligand to metal charge transfer transitions in the complex
formed between Cu2+ and the amine ligand contribute to the quenching of the fluorescence. An explanation
based on TD-DFT results is also proposed for the weak fluorescence quenching in the presence of Zn2+.
Keywords: Hexadentate Schiff base; X-ray structure; Fluorescence; Cu2+ chemosensor; TD-DFT
 Corresponding Authors, Fax: +98(852)4246001, E-mail: golbedaghi82@gmail.com (R. Golbedaghi); E-mail:
liciniaj@ci.uc.pt (Licínia L. G. Justino)

1. Introduction
Schiff bases are compounds in which an aldehyde or ketone carbonyl functional group has been substituted
by an imine or azomethine group. These compounds are extensively used for industrial applications and
also show a vast range of biological activities. Schiff bases were first reported by Hugo Schiff, in 1864
[1]. Since then, a large diversity of methods for their synthesis have been reported. The classical synthesis
of these compounds involves the condensation of a primary amine with a carbonyl compound [2] under
specific conditions (e.g., azeotropic distillation), and is described by the following chemical equation:
RNH2 + R-COH
R-N=CH-R + H2O.
The chemical and physical properties of Schiff bases and their reduced amine forms have been
investigated extensively in different contexts, such as synthesis, identification and qualitative
determination of aldehydes or ketones, purification of carbonyl or amino compounds, or protection of
these compounds in complex or sensitive reactions [3,4]. In the present work, we explore the use of a
hexadentate Schiff base in its reduced salt form as a chemosensor for the Cu2+ cation.
Development of new fluorescent chemosensors is a challenging and important field of research in
supramolecular chemistry [5-7]. In particular, ion sensing is of great relevance for the understanding of
biochemical processes taking place in cellular media [8]. Among the essential transition metals in
biological systems, the most abundant ones are Fe, Zn and Cu [9,10].
Other heavy metal ions, such as Hg2+ and Cd2+, constitute a serious risk to living organisms due to
their high toxicity. Systems based on coumarin have been used in the development of fluorescent
chemosensors for the detection of anions (e.g., CN-, F-), metal ions (such as Cu2+/Cu+, Zn2+, Fe3+/Fe2+,
Hg2+, Cd2+, Al3+, alkali metal ions, etc.), reactive sulfur species, biological thiols, reactive oxygen and
nitrogen species, etc. These studies have recently been reviewed [11]. Near-infrared fluorescence sensors
2

are also relevant in the context of applications to in vivo bioimaging, due to the low background
interference, high image sensitivity and minimal damage to tissues [12].
Fluorescent sensors for Cu2+ ion have been developed and used to clarify the physiological role of
this metal ion [13-17], which is known to be involved in several cellular processes, such as functional and
structural strengthening of proteins and gene expression, and also in processes taking place in human
nervous system [18,19]. Both high and low copper concentrations can disrupt normal activity of living
systems and lead to diseases, such as Alzheimer's [20], Menkes-Wilson disease [21] and prion-based
infections [22]. In spite of the considerable attention that has been given to the design of sensitive and
selective fluorescent sensors for the detection of Cu2+ ions [23-25], the search for better Cu2+
chemosensors is still an active field of research. The Cu2+ ion is a strong quencher of fluorescence both
via energy and electron transfer mechanisms. However, many copper sensors developed hitherto exhibit
a fluorescence “turn-off” response that frequently leads to limited resolution [26-30].
Naphthol derived [31] and oligothiophene-phenylamine derived [32] Schiff base ligands have been
reported, respectively, as selective fluorescent probes for the detection of Zn2+ [31] and Hg2+ and Cu2+
[32]. The Mn(II)/(III) complexes of a hexadentate bisaldehyde-type Schiff base have also been
characterized and the ligand was used for the determination of Fe(III) [33].
As described in details below, in the present investigation a novel hexadentate Schiff base (L1) and
its reduced amine derivative form (in its neutral H2L1, and cationic [H4L1]2+ forms) were synthesized and
characterized. The capability of H2L1 to act as a highly sensitive fluorescent chemosensor for Cu2+ ion is
demonstrated.
3

2. Experimental
2.1. General information
2,2'-[ethane-1,2-diylbis(oxy)]dibenzaldehyde was prepared according to the literature method [34].
Salicylaldehyde and NaBH4 were obtained from Merck. Infrared (IR) and nuclear magnetic resonance
(NMR) spectra were measured on a Perkin Elmer FT-IRGX and a Bruker DPX 300 spectrometer,
respectively. The fluorescence spectra were recorded on a Sinco FS2 spectrofluorimeter; excitation and
emission band widths were set as 5.0 and 10.0 nm, respectively.
2.2 X-ray crystal structure determination
Single crystal X-ray data for compound [H4L1](ClO4) were collected at 298 K on a Bruker Stoe IPDS-II
diffractometer diffractometer using graphite monochromated MoK radiation ( = 0.71073 Å). The linear
absorption coefficients, scattering factors for the atoms, and anomalous dispersion corrections were taken
from the International Tables for X-ray Crystallography [35]. The data integration and reduction were
carried out with the SAINT software [36]. An empirical absorption correction was applied to the collected
reflections using SADABS [37], and the space group was determined using XPREP [38]. The structure
was solved by the direct method using SHELXTL-2017 [39] and refined on F2 by full-matrix least-squares
using the SHELXL-2017 program package [40]. All non-hydrogen atoms were refined anisotropically.
The hydrogen atoms attached to carbon atoms were positioned geometrically and treated as riding atoms
using SIR-97 default parameters.
2.3. Syntheses (see Scheme 1)
2.3.1. Preparation of the precursor aldehyde (2,2'-[ethane-1,2-diylbis(oxy)]dibenzaldehyde)
1,2-dibromoethane (0.1 mol) and NaOH (0.2 mol) were mixed and heated under reflux in 500 mL aqueous
ethanol (300 mL H2O and 200 mL EtOH). Salicylaldehyde (0.2 mol) in 30 mL EtOH was added, and the
4

solution was heated under reflux for 4 days. After cooling, the resulting yellow crystals were filtered,
washed and recrystallized (yield: 87%, purity > 98%) [34,41].
2.3.2. Preparation of ligand L1
The precursor aldehyde (0.27 g, 1 mmol) and propanolamine (0.150 g, 2 mmol) were mixed and heated
at 40 ºC for 30 min in EtOH (30 mL). The solution was filtered and the filtrate was reduced to ca. 10 mL.
The resulting yellow crystalline product was filtered off, washed with cold ethanol, and dried. Yield: 90%.
Anal. Calc. for C22H28N2O4: C, 68.71; H, 7.29; N, 7.32. Found: C, 68.36; H, 7.17; N, 7.26. IR (KBr, cm-
1): 1637 (imine), 1H-NMR (CDCl3, 300 MHz): δ 1.80 (m, 4H), 3.60 (t, 4H), 3.67 (t, 4H), 4.32 (m, 4H),
6.90-7.86 (m, 8H), 8.58 (s, 2H). 13C-NMR (CDCl3, 75 MHz): δ 33.5 (C-10), {59.5, 61.4, 67.2 (C-1, C-9,
C11)}, {112.6, 121.4, 124.7, 127.4, 132.0 (aromatic rings)}, {157.9, 157.2 (C-2, C-8)}.
2.3.3. Preparation of H2L1 and crystalline [H4L1](ClO4)2
The Schiff base ligand L1 was reduced with NaBH4, in ethanol solution at room temperature, to its amine
H2L1 form. After stirring the solution during 3 h, a white precipitate was formed. The precipitate was then
filtered, washed with cold ethanol and dried. Anal. Calc. for C22H32N2O4: C, 68.01; H, 8.30; N, 7.21.
Found: C, 67.62; H, 8.18; N, 7.19. IR (KBr, cm-1): 3302 (νN–H). The precipitate (H2L1) was subsequently
dissolved (0.39 g, 1 mmol) in an equimolar acetonitrile/methanol solution. NaClO4 (0.25 g, 2 mmol)
dissolved in methanol (5 mL) was then added and the solution heated at 40 ºC for 30 min in presence of
HCl. The solution was filtered and the filtrate was reduced to ca. 10 mL. The resulting white crystalline
product was filtered off, washed with cold ethanol, and dried. The afforded material, [H4L1](ClO4)2, was
recrystallized from an equimolar acetonitrile/methanol solution. Yield: 82%. Anal. Calc. for
C22H34Cl2N2O12: C, 44.82; H, 5.77 N, 4.75. Found: C, 44.76; H, 5.67; N, 4.68. IR (KBr, cm-1): 1497
(νC=C), 3299 (νN–H). 1H-NMR (CDCl3, 300 MHz): δ 1.65 (m, 4H), 2.79 (t, 4H), 3.74 (t, 4H), 3.80 (m,
4H), 4.40 (s, 4H), 6.94-7.26 (m, 8H). 13C-NMR (CDCl3, 75 MHz): δ 30.7 (C-10), {48.6, 49.0, 63.8, 66.7
5

(C-1, C-8, C9, C11)}, 111.5-156.7 (aromatic rings), DEPT 135º: {30.7, 48.6, 49.0, 63.7, 66.7 (CH2
aliphatic)}, {111.5, 121.0, 128.5, 130.2 (CH aromatics)}.
Crystals for single crystal X-ray structure determination were obtained by slow diffusion of diethyl ether
vapour into a methanol-ethanol solution of the compound.
2.4. Spectroscopic response experiments
Stock solutions (1 mM) of Ni2+, Co2+, Al3+, Mn2+, Mg2+, Zn2+, Hg2+, Pb2+, Cd2+, Ag+, Cr3+, and Cu2+ in
H2O/dimethyl formamide (DMF) (1:1, v/v) were prepared from the corresponding nitrate salts. Stock
solutions of L1 and H2L1 (0.2 mM) in DMF were also prepared. The test solutions were then obtained by
adding 0.1 mL of the probe’s stock solution and an appropriate aliquot of each metal stock solution in a
test tube, and subsequently diluting the obtained mixture with H2O-DMF (1:1, v/v) to give the final
concentrations of the probe (10 M) and metal ions (in the 0-50 M range).
2.5. Computational Details
The structure of the Cu(II)/H2L1 complex was investigated at the density functional theory (DFT) level
with the CAM-B3LYP [42] exchange correlation functional using the GAUSSIAN09 [43] code. The
LANL2DZ [44,45] valence and effective core potential (ECP) functions were used for copper and the
double-ζ plus polarization 6–31G(d,p) [46,47] basis set was used for the remaining atoms. Cu(II) ion has
an open-shell electronic configuration (d9), and therefore the calculations were carried out using the
unrestricted (U-CAM-B3LYP) formalism. A conformational study was carried out to determine the most
stable conformers of the H2L1 ligand in water. Starting from the modified X-ray structure of the [H4L1]2+
fragment, conformers were generated by using the molecular mechanics Dreiding force field [48] as
implemented in MarvinSketch [49] and the lower energy 22 conformers were then optimized at the CAM-
B3LYP/6–31G(d,p) level of theory. The lowest energy conformer has anti conformation and Ci symmetry.
6

This conformer shows OH…N intramolecular hydrogen bridges. The second most stable conformer is only
0.25 kJ/mol above the lowest energy conformer and has syn conformation. It also contains OH…N and
NH…O intramolecular hydrogen bridges. The nature of the stationary points found was checked by
vibrational analysis, and only those which are true minima (with no imaginary frequencies) were
considered. Single point DFT calculations were carried out to obtain the energy difference between the
syn conformation adopted by the ligand in the complex and the anti conformation of the free ligand. To
do this, the metal ion was formally removed from the optimized geometry of the complex and the terminal
deprotonated oxygen atoms were saturated so as to have the same chemical formula as the free ligand.
Since a very large number of intramolecular interactions (in particular, H-bridges) becomes possible
involving the terminal OH and the nitrogen atoms, and the intramolecular network established depends
on the orientation given to the added hydrogen atoms, we have used the ReadOpt option of GAUSSIAN09
to optimize the coordinates of these two hydrogen atoms, while maintaining the remaining coordinates of
this syn conformation frozen. We have then compared the energy of this syn conformation with the energy
of the most stable anti conformation of the free ligand H2L1. In these calculations, the tight convergence
criterion (scf=tight) was used. The single point calculations were repeated including the effect of the
solvent water through the PCM model [50,51]. Respectively, the 40 and 10 lowest-energy vertical
electronic excited states were calculated for the optimized geometries of the complex and of the ligand by
2
〈 〉
time-dependent DFT (TD-DFT) [52] using the same functional and basis sets. The values of 푆 obtained
from the U-DFT calculations for the complex were compared with the expected values for the spin state
to confirm that spin contamination was negligible in all cases. The same basis sets and functional were
used in the DFT and TD-DFT (restricted formalism) calculations for the Zn(II) complex.
7

1
3
6
2
7
O
O
4
5
8
O
O
HC
N
CH
2
Propanolamine
EtOH/40 0
(L1)
N
C
9
HOC
COH
10
HO
OH
11
E
t
O
H
/
r
t
O
O
O
O
H2C
HN
H2C
CH2
NH
CH2
2 NaClO4 /HCl
CH3CN/MeOH
+
+H2N
NH2
HO
(H2L1)
HO
OH
OH
([H4L1]2+
)
Scheme 1. Syntheses of L1 and H2L1 and [H4L1]2+ (the cation was produced as perchlorate salt).
3. Results and Discussion
3.1. Syntheses and characterization of the compounds
The Schiff base compound L1 was synthesized as described above, and then reduced to the amine H2L1
using NaBH4. The H2L1 compound was subsequently converted to its perchlorate salt, [H4L1](ClO4)2.
The compounds were found to be quite stable in air and they can be stored in a desiccator for long
periods of time without decomposing. Both the imine and the amine derivative, in its perchlorate salt form
1
([H4L1](ClO4)2), were characterized by, IR, elemental analysis and both H- and 13C-NMR. The
perchlorate salt of the amine species was further investigated by Distortionless Enhancement by
Polarization Transfer (DEPT) 135º and 2D NMR correlation spectroscopy (COSY) experiments, and
single crystal X-ray diffraction (IR and NMR data are provided in Figures S1-S8, in the Supporting
8

Information; X-ray data is available from the Cambrigde Crystalographic Data Centre, CCDC #1424449;
http://www.ccdc.cam.ac.uk/conts/retrieving.html).
Condensation of the primary amino group of the propanolamine precursor during the synthesis of
L1 is confirmed by the lack of bands in the characteristic N–H stretching region (3150–3450 cm-1) in the
infrared spectrum of the reaction product, and also by the presence of a strong imine C=N stretching band
at 1637 cm-1 (Figure S1). On the other hand, in the IR spectrum of [H4L1](ClO4)2 (Figure S2), the band
corresponding to the C=N stretching mode of the imine group is absent, while bands due to the stretching
vibrations of the amine N–H bonds are easily observed superimposed with the broad feature due to the
OH stretching modes (particularly characteristic is the band observed at 3299 cm-1). These observations
confirm the successful reduction of L1 to H2L1, followed by transformation of the later into the
corresponding perchlorate salt.
All obtained NMR data (1H-NMR, 13C-NMR, DEPT 135º and COSY), as well as the X-ray crystal
structure of [H4L1](ClO4)2, are fully consistent with the proposed structures for the compounds (Figures
S3-S8; see also assignments of 1H-NMR and 13C-NMR spectra in the Experimental section). The COSY
data obtained for the amine compound (Figure S8) show the characteristic splitting of the diastereotopic
protons in the aliphatic region, as expected for this species.
Suitable crystals of [H4L1](ClO4)2 were obtained by slow diffusion of diethyl ether vapour into a
methanol-ethanol solution of the compound. A summary of the crystal data, and data collection and
refinement details are given in Table 1. An ORTEP diagram of the molecular structure of [H4L1]2+ unit in
the crystal is shown in Figure 1 and the crystal packing is presented in Figure 2a.
Table 1. Crystal data and structure refinement parameters for [H4L1](ClO4)2 crystal.
Empirical formula
Formula weight
Temperature (K)
C22H34Cl2N2O12
589.41
293
9

Wavelength
Crystal system
Space group
K
Monoclinic
P 21/c
Unit cell dimensions
a (Å )
5.533 (11)
b (Å)
18.318 (4)
c (Å)
13.798 (3)
α
90.000
β
99.240 (3)
γ
90.000
1380.4 (5)
V (Å3)
Z, Z´
2, 1/2
1.699
702
Dcalc (Mg/m3)
F(000)
θ range for data collection (°)
Index ranges
2.68–25.99°
-6 <= h <= 6, -22 <= k <= 0,
-17 <= l <= 17
Reflections collected
Independent reflections
Ri
2712
1923
0.016
Absorption correction
Max. and min. transmission
Refinement method
Empirical
0.854 and 0.774
Full matrix least-squares on
F2
restraints
1
R[F2 > 2σ(F2)]
wR(F2)
0.0913
0.3046
The X-ray crystal structure shows that the two aromatic rings of the [H4L1]2+ moiety in the
[H4L1](ClO4)2 crystal are coplanar, and the aliphatic chains are nearly perpendicular to the plane of the
rings, pointing to opposite directions (Figure 2b). The conformation about the central
C1–C1_3 bond, as well as that of the terminal hydroxyl group is anti (OCCO and HOCC dihedrals = 180º),
while those about the C8–N1, N1–C9, C9–C10 and C10–C11 bonds are gauche, anti, anti and gauche’,
respectively (gauche and gauche´ corresponding to conformation-defining dihedral angles of ~60 and –
60º, respectively). The distance between the terminal OH groups (O/O) is 14.24 Å and the longest C/C
distance between carbon atoms of the aromatic rings is 14.60 Å. The crystal unit cell contains two [H4L1]2+
units and four perchlorate anions (Z = 2), and an asymmetric unit with Z´ equal to ½, i.e., one perchlorate
anion and half of the [H4L1]2+ unit are enough to totally determine the crystal structure upon application
of the symmetry figures of the crystal. The crystal packing results in short contacts between all oxygen
10

atoms of the perchlorate anion and hydrogen atoms from the [H4L1]2+ cation, two of these corresponding
to weak H-bonds of type O…HN (2.597, 2.713 Å), one to a O…HO H-bond (2.233 Å), and the remaining
to non-classic O…HC(aromatic/aliphatic) interactions (with lengths in the 2.626-2.713 Å range). The H-bond
network displays also a H-bond interaction between the nitrogen atoms and hydroxyl oxygen atoms of
neighbouring molecules (2.905 Å).
Fig. 1. ORTEP graph of the [H4L1]2+ moiety in the [H4L1](ClO4)2 crystal. The asymmetric unit is constituted by the
left-half of the [H4L1]2+ cation and one perchlorate anion (shown in the figure); the second half of the [H4L1]2+ cation
is shown for clarity.
a)
b)
Fig. 2. a) Packing diagram for [H4L1](ClO4)2 crystal, showing the unit cell axes; b) view of the same fragment of
the crystal unit cell shown in Figure 1 but oriented to stress the relative orientations of the aromatic rings and of the
aliphatic chain of the [H4L1]2+ fragment, as well as the O…HN H-bond established between the perchlorate anion
and the nitrogen tom of the cation (right).
11

3.2. Fluorescence response of the amine ligand to Cu2+and other cations
The fluorescence response of both the imine and amine compounds in presence of various metal ions
(Ni2+, Co2+, Al3+, Mn2+, Mg2+, Zn2+, Hg2+, Pb2+, Cd2+, Ag+, Cr3+ and Cu2+) was investigated in H2O-DMF
(1:1 v/v) solution. The results obtained with the imine compound were discourageous. On the other hand,
for the amine ligand, its intense fluorescence emission with maximum at 309 nm (upon excitation at 274
nm) was found to be quenched at different levels in presence of the different metal ions. The addition (of
5.0 equivalents) of Ni2+, Co2+, Al3+, Mn2+, Mg2+ and Zn2+ cations to the solution of the amine ligand caused
partial reduction of the fluorescence emission (20-30 %), whereas the addition of Hg2+, Pb2+, Cd2+, Ag+
and Cr3+ cations lead to a stronger quenching of the fluorescence (35-55 %). Noteworthy, the addition of
Cu2+ caused an almost total quenching of the fluorescence (over 98 %) (Figure 3a).
Ligand
680
580
480
380
280
180
80
680
580
480
380
280
180
80
a)
b)
0
0
-20
-20
284
334
384
284
334
384
Wavelength (nm)
Wavelength (nm)
Fig. 3. a) Fluorescence emission spectra of the H2L1 ligand (λex. = 274 nm) obtained in the presence of 5.0
equivalents of different metal ions: Ni2+, Co2+, Al3+, Mn2+, Mg2+, Zn2+, Hg2+, Pb2+, Cd2+, Ag+, Cr3+ or Cu2+, in H2O-
DMF (1:1 v/v). The excitation and emission band widths were 5.0 and 10.0 nm, respectively; b) series of
fluorescence emission spectra of H2L1 (λex. = 274 nm) in H2O-DMF (1:1, v/v; [H2L1] = 10.0 μM) in presence of
different concentrations of Cu2+ (0.0-50.0 μM; step of 5 μM). The arrow indicates the direction of increasing Cu2+
concentration (see also Figure 4).
12

700
600
500
400
300
200
100
0
Y
0
20
40
60
[Cu2+](μM)
Fig. 4. Fluorescence intensity (intensity of the fluorescence emission band of H2L1 at 309 nm) as a function of the
Cu2+ concentration, in the range 0.0–50.0 μM.
To further evaluate the affinity of the ligand towards Cu2+, a fluorescence titration assay was carried
out. As Figure 3b shows, the addition of successively increasing amounts of Cu2+ (within the 0.0–50.0 μM
range) to the ligand solutions causes a gradual quenching of the emission of the ligand at 309 nm. As
mentioned previously, the addition of 5.0 equivalents of Cu2+ causes the emission to decrease by more
than 98%. Moreover, 2.5 equivalents of the cation are already enough to reduce the emission by more than
50 %. In fact, as can be seen in Figure 4, a good linear correlation is observed between the fluorescence
emission intensity and the Cu2+ concentration.
To help us determine the stoichiometry of the complex formed between Cu2+ and the amine ligand,
the Job plot method was used. The Job function FJob is calculated from Eq. (1) [53]:
FJob= (1-x) F0-F
(1)
where F and F0 are the fluorescence intensities in the presence and in the absence of Cu2+, respectively, x
is the mole fraction of Cu2+, and the sum of the concentrations of Cu2+ and the ligand is kept constant (10
μM). The Job plot (Figure 5) shows a maximum at x = 0.5, indicating that the stoichiometry of the complex
is 1:1.
13

160
140
120
100
80
60
40
20
0
0
0.2
0.4
0.6
0.8
1
X = [Cu2+]/([Cu2+]+[Ligand])
Fig. 5. Job plot for the H2L1/Cu2+ system, indicating that the complex formed between the two species has 1:1
stoichiometry. The sum of the concentrations of Cu2+ and H2L1was maintained equal to 10 μM.
To check the practical applicability of the ligand as a selective fluorescence sensor for Cu2+, the
effect of addition of other metal ions to a solution of the ligand, in the presence of Cu2+ (1.0 equivalent),
on the ligand fluorescence was examined. As shown in Figure 6, the fluorescence quenching in the
presence of Cu2+ is not significantly affected by the competing ions, indicating that the compound can
indeed be used as a highly-selective sensitive fluorescence chemosensor for Cu2+ detection.
700
600
500
400
300
200
100
0
Ag
Al
Cd
Co
Cr
Hg no Ion Mg
Mn
Ni
Pb
Zn
Fig. 6. Fluorescence response of H2L1 (L, in the legend) to various metal ions (M) in aqueous DMF (50:50, v/v).
The green bar corresponds to the fluorescence intensity in absence of metal ion; blue bars correspond to the
fluorescence intensity after the addition of a given metal ion (50 μM) to a 10 μM solution of H2L1; the red bars
show the change of the emission which occurs with the subsequent addition of 50 μM Cu2+ to the solution (λex.
=
274 nm).
14

3.3. DFT and TD-DFT study of the Cu2+/H2L1 complexation and of the fluorescence quenching
mechanism
Based on the knowledge of the 1:1 stoichiometry of the Cu2+/H2L1 complex formed in solution, DFT
calculations were carried out to investigate its geometry. With this ligand, Cu2+ can form six, five-, or
four-coordinated complexes. Several input structures with different coordination numbers were used in
the calculations and both the protonated and deprotonated states of the terminal OH groups in the complex
were also considered. Irrespectively of the protonation state of the ligand after complexation, a six-
coordinated metal centre, with all the functional groups of the ligand coordinated, was found to be the
most stable configuration. These calculations suggest the structure presented in Figure 7 as the most
probable structure for the complex formed in solution. Based on the knowledge of the geometries of
analogous complexes [54,55], we suggest that the coordinated OH groups most likely will be
deprotonated. Table S1 includes selected calculated structural parameters for the metal centre of the
complex. The predicted Cu-N (2.163 and 2.168 Å) and Cu – O(terminal) (1.866 and 1.872 Å) bond lengths
are within the expected values for analogous Cu(II) complexes [54,55]. The Cu-O(ether) bonds are predicted
to be very weak, with bonds lengths 2.616 and 2.751 Å, in the range of those reported for the crystal
structure of an analogous complex [54].
Fig. 7. Two perspectives of the DFT/U-CAM-B3LYP optimized structure proposed for the complex [CuH2L1].
15

In the Cu2+ complex, the ligand adopts a syn conformation. It is interesting to compare the energy
of this conformation with that of the most stable anti conformer of the free ligand. To assess this aspect,
single point DFT calculations were carried out for the syn conformation adopted by the ligand in the
complex, after formal removal of the metal ion and saturation of the deprotonated oxygen atoms (only the
coordinates of the H atoms used to saturate the deprotonated oxygen atoms were optimized, as indicated
in the Computational Details section). These calculations showed that in the gas phase the anti
conformation of the free ligand is 17.07 kJ/mol lower in energy compared with the syn conformation
adopted in the complex (and 44.76 kJ/mol in water solution).
For the complex to form, this energy penalty involving the conformation of the ligand should be
overcome by the stabilization occurring with the bonding between the ligand and the metal. In our previous
study [56], we have indeed reported the unfavorable conformational rearrangement of an analogous ligand
upon complexation. In that study, the X-ray crystal structure of a Cd2+ complex showed the ligand in a
syn conformation, while theoretical DFT calculations revealed that the most stable conformation of the
free ligand was the anti conformation. That previous study, for which we obtained the X-ray structure of
the complex, supports the present conclusion that it is possible for the complex to form, in spite of having
un unfavorable conformational rearrangement of the ligand. For the ligand of the present study, we believe
that the formation of the complex is further facilitated by the fact that there is a syn conformer of the free
ligand which is only 0.25 kJ/mol above the lowest energy anti conformer, and therefore will be present in
solution in large proportion.
To obtain additional information on the fluorescence quenching mechanism occurring due to the
formation of the Cu(II)/H2L1 complex, TD-DFT calculations were carried out for both the free ligand and
the Cu(II)/H2L1 complex. With the exception of some rare cases, fluorescence properties are usually
determined by the lowest excited state with the same multiplicity as the ground state. Figure 8 shows the
16

main contributions to the lowest energy electronic singlet and doublet excitations, respectively for the
ligand and for the Cu(II) complex. The ligand’s lowest excited singlet state involves transitions from the
HOMO-3 and HOMO-2 orbitals, which have a mixture of π and n character, respectively to the LUMO+1
and LUMO orbitals, with π* character. Although these transitions are parity forbidden, this state will be
populated by internal conversion from higher energy excited states and the decay from the S1 excited state
will give rise to the fluorescence emission observed for the ligand. On the other hand, the first doublet
excited state for the Cu(II)/H2L1 complex has a major contribution (66%) from a β-spin HOMO-1 to
2
LUMO excitation, which involves essentially charge transfer from the ligand to the metal (푑푧 orbital),
and therefore has a dominant ligand-to-metal charge transfer (LMCT) character (Figure 8). Additionally,
Table 2 and Figure 9 show that the next low-lying excited states for the complex all have LMCT character.
This type of transition provides a mechanism for the nonradiative deactivation of the excited state in the
complex. This mechanism readily explains the quenching of the fluorescence. However, we cannot rule
out that energy transfer from the excited ligand to the Cu(II) centre may also occur, and partially contribute
to the radiationless deactivation of the excited state.
L+1
Lb
L
H-1
H
H-1b
[CuH2L1]
H2L1
Fig. 8. Dominant contributions to the lowest energy singlet (left) and doublet (right) electronic excited sates for the
H2L1 ligand and the [CuH2L1] complex, respectively, calculated at the DFT/U-CAM-B3LYP level.
17

Table 2. Vertical excitation energies, wavelengths, oscillator strengths and main contributions to the excited states
of the [CuH2L1] complex calculated at the TD-DFT/U-CAM-B3LYP level.
a
Excited Energy
λcalc.
fa
Main contributions (%)
Dominant character
state
(eV)
(nm)
H2L1 Ligand
H-1→L+1 (24%) + H→L (28%)
[CuH2L1] complex
S1
5.38
230
0.003
π → π*+n → π*
D1
D2
D3
D5
D6
1.61
1.94
2.17
3.20
3.51
768
643
571
388
353
0.000
0.006
0.009
0.042
0.072
H-1→Lβ (66%)
LMCT
LMCT
LMCT
LMCT
LMCT
H→Lβ (42%) + H-12→Lβ (14%)
H-10→Lβ (31%) + H-2→Lβ (21%)
H→Lβ (53%) + H-8→Lβ (11%)
H-2→Lβ (66%)
a For the complex, results are shown for the region up to 250 nm and f≥0.006. Additionally, the first singlet excited state of the
ligand and first doublet excited state of the complex, both with lower f, are also listed.
Fig. 9. Diagram showing the main transitions contributing to the excited states of the complex [CuH2L1].
3.4. TD-DFT analysis of the fluorescence quenching results for the Zn2+/H2L1 system
The fluorescence response experiments show that the fluorescence intensity of H2L1 suffers little change
in the presence of various metals, such as Al(III), Co(II), Ni(II), Zn(II), etc. (Figures 3a and 6). We were
18

also interested in understanding the reasons for these different behaviours, compared with Cu(II). In this
study, we have chosen one particular case, Zn(II), and we have carried out DFT and TD-DFT studies on
the Zn(II)/H2L1 system to help explain the experimental results. We will assume that Zn(II) will form a
complex with H2L1 and that this complex is analogous to the Cu(II) complex. Based on this assumption,
we have optimized the structure of the Zn(II) complex and carried out TD-DFT calculations to obtain
information on its vertical singlet excitation energies and character of excited states. Selected structural
parameters and TD-DFT results are presented in Table S2 and Table 3 respectively. The TD-DFT results
show that the weak quenching of the fluorescence in the presence of Zn(II) comes from the fact that the
S1 excited state of the Zn(II) complex has the same character and is very similar to the S1 excited state of
the free ligand (its energy is also only slightly lower in the complex) (Figures 8 and 10). This excited state
of the Zn(II) complex is localized on the ligand and corresponds to a π → π*+n → π* excitation, exactly
as in the free ligand. The molecular orbitals involved in the higher energy excitations listed in Table 3 are
shown in Figure S9.
Table 3. Vertical excitation energies, wavelengths, oscillator strengths and main contributions to the excited states
of the [ZnH2L1] complex calculated at the TD-DFT/CAM-B3LYP level.
a
Excited Energy
λcalc.
fa
Main contributions (%)
Dominant character
state
(eV)
5.38
5.30
5.34
(nm)
H2L1 Ligand
H-1→L+1 (24%) + H→L (28%)
[ZnH2L1] complex
H-4→L+1 (34%) + H→L+1 (22%) +
H-5→L+1 (18%)
S1
230
233
232
0.003
0.043
0.038
π → π*+n → π*
π → π*+n → π*
π → π*+n → π*
S1
S2
H-5→L (34%) + H-4→L (21%) +
H-7→L+2 (13%)
S7
S8
6.00
6.04
207
205
0.105
0.042
H→L (23%) + H→L+3 (19%) +
H-1→L+2 (21%) + H-5→L+2 (17%) +
H-7→L (14%)
π → π*+n → π*
π → π*+n → π*
S9
6.14
202
0.026
H-1→L+1 (78%)
n → π*
a Results are shown for the region up to 200 nm and f≥0.010 for the complex. Additionally, the first singlet excited state of the
ligand, with f=0.003, is also listed.
19

L+1
H-4
H-5
H
Fig. 10. Diagram showing the main transitions contributing to the S1 excited state of the model considered for the
[ZnH2L1] complex.
4. Conclusions
In this study, the syntheses and characterization of a new hexadentate Schiff base and its reduced
amine derivative were reported. The amine derivative was found to be a promising highly-selective
sensitive fluorescence chemosensor for the detection of Cu2+.
The structures of the compounds were characterized from their infrared and NMR (13C,1H-NMR,
DEPT 135º, COSY) spectra, and elemental analysis. The single crystal structure for the amine derivative,
in its perchlorate salt form, was also reported and discussed. Fluorescence studies were carried out and
these revealed that the amine compound presents high selectivity and appropriate fluorescence response
to the Cu2+ cation, and therefore can be considered a promising fluorescence chemosensor for the Cu2+
cation. This amine chemosensor shows selectivity for Cu2+ over twelve other metal cations, and it works
. It also exhibits over 98% of fluorescence quenching in presence (of 5
equivalents) of the copper ion. DFT and TD-DFT calculations showed that LMCT transitions in the
complex formed between Cu2+ and the amine ligand provide a pathway for the radiationless deactivation
of the excited state. The theoretical results also provide a possible explanation for the weak fluorescence
20

quenching of the ligand in the presence of Zn(II). These results open up the possibility of using the new
amine chemosensor as a fluorescent probe for applications in a vast range of fields where the analytical
determination of Cu2+ is relevant. We also hope that this study can motivate future research, including the
study of the use of this ligand as a sensor in aqueous medium in the absence of DMF, which could have
impact in environmental sciences and biological applications.
Supplementary data
Figures S1, S2 and S3, showing the room temperature infrared spectra of L1, H2L1 and [H4L1](ClO4)2 in
KBr pellet. Figures S4 to S9, presenting the NMR data for the studied imine and amine compounds. Figure
S10 showing the selected molecular orbitals of the [ZnH2L1] complex. Tables S1 and S2 presenting
selected geometrical parameters for the optimized structures. CCDC 1424449 contains the supplementary
crystallographic data for [H4L1](ClO4)2. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre,
12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44)1223-336-033, or e-mail: deposit@ccdc.cam.ac.uk.
Acknowledgments
R.G. and M.B. are grateful to the Payame Noor University for financial support. R.F., L.L.G.J. and R.G.
thank the financial support from the Portuguese “Fundação para a Ciência e a Tecnologia” (FCT) (Project
PTDC/QEQ-QFI/3284/2014 – POCI-01-0145-FEDER-016617). Additional funding from the Project
“MATIS - Materiais e Tecnologias Industriais Sustentáveis” (reference: CENTRO-01-0145-FEDER-
000014), co-funded by the European Regional Development Fund (FEDER), through the “Programa
Operacional Regional do Centro” (CENTRO2020) is also acknowledged. The Coimbra Chemistry Centre
21

(CQC) is supported by FCT (Project UI0313/QUI/2013) and COMPETE-UE. The authors also thank Prof.
H.D. Burrows for his comments on this manuscript.
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26

Highlights





New hexadentate Schiff base and its reduced amine derivative were synthesized
Amine derivative is a promising highly-selective fluorescence chemosensor for Cu2+
Sensor exhibits fluorescence quenching over 98 % in presence of the copper ion
Sensor works at the M range of concentrations
LMCT transitions in the complex contribute to the quenching of the fluorescence
27

Author Statement:
Reza Golbedaghi: Conceptualization, Methodology, Investigation, Writing- Original draft preparation.
Licínia L.G. Justino: Methodology, Investigation, Writing- Original draft preparation.
Marzyeh Bahrampour: Methodology, Investigation.
Rui Fausto: Supervision; Writing- Reviewing and Editing.
28