Zn2+ and Cu2+ complexes of a fluorescent scorpiand-type oxadiazole azamacrocyclic ligand: Crystal structures, solution studies and optical properties

Article abstract of DOI:10.1039/c9dt04764f

A ligand comprised of a macrocyclic pyridinophane core having a pendant arm containing a secondary amine group linked through a methylene spacer to a pyridyl-oxadiazole-phenyl (PyPD) fluorescent system has been prepared (L). The crystal structures of [ZnL](ClO₄)₂ and [CuL](ClO₄)₂ show that M²⁺ is coordinated to all the nitrogen atoms of the macrocyclic core, the secondary amine of the pendant arm and the nitrogen atom of the pyridine group of the fluorescent moiety, the latter bond being clearly weaker than the one with the pyridine of the macrocycle. Solution studies showed the formation of a highly stable Cu²⁺ complex with 1 : 1 stoichiometry, whereas with Zn²⁺ least stable complexes were formed and, given the right conditions, a [Zn₃L₂]⁶⁺ species was also detected, but it was not possible to isolate this species in the solid state. Following Zn²⁺ coordination, a strong chelation-induced enhancement of fluorescence was observed, a behaviour that was not observed with any of the other metal cations tested.

Full text of DOI:10.1039/c9dt04764f

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Trans., 2020, DOI: 10.1039/C9DT04764F.
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DOI: 10.1039/C9DT04764F
ARTICLE
Zn2+ and Cu2+ complexes of a Fluorescent Scorpiand-type
Oxadiazole Azamacrocycle Ligand: Crystal Structures, Solution
Study and Optical Properties
Received 00th January 20xx,
Accepted 00th January 20xx
Gianluca Ambrosi,a M. Paz Clares,b Isabel Pont,b Mauro Formica,a Vieri Fusi,*a Angela Ricci,a Paola
Paoli,c Patrizia Rossi,c Enrique García-España*b and Mario Inclán.b
DOI: 10.1039/x0xx00000x
A ligand constituted by a macrocyclic pyridinophane core having a pendant arm containing a secondary amine group linked
through a methylene spacer to a pyridyl-oxadiazole-phenyl (PyPD) fluorescent system has been prepared (L). The crystal
structures of [ZnL](ClO4)2 and [CuL](ClO4)2 show that the M2+ is coordinated by all the nitrogen atoms of the macrocyclic core,
the secondary amine of the pendant arm and the nitrogen atom of the pyridine group of the fluorescent moiety, being this
latter bond clearly weaker than the one with the pyridine of the macrocycle. Solution studies showed the formation of a
highly stable Cu2+ complex of 1:1 stoichiometry, whereas with Zn2+ least stable complexes were formed and, given the right
conditions, also a [Zn3L2]6+ specie was detected that was not possible to isolate in the solid state. Following Zn2+ coordination,
a strong chelation enhancement of the fluorescence was observed, a behaviour that was not registered with any of the
other metal cations tested.
selectively determine the zinc content in biological media, even
in the presence of copper.7
With this aim, we have developed a new polyaza macrocyclic
Introduction
Zinc is a ubiquitous and multifunctional metal in human
metabolism, being involved in the catalytic function of almost
300 enzymes. It is present in all major biochemical pathways
and it is essential for DNA metabolism and recognition by
numerous proteins.1 However, in recent years, the focus on
zinc research has shifted towards its neurobiological roles and,
particularly, its implication in the pathogenesis of Alzheimer’s
disease (AD) and other neurodegenerative disorders.2
Concentrations of zinc in the brain are about ten times the
levels found in serum, and are highest in the same regions of
the brain that are affected by AD, pointing towards a role of zinc
homeostasis in the development of this pathology.3
One of the major drawbacks of currently available sensors for
zinc is the lack of selectivity of the response in the presence of
other metal cations and, particularly, a strong interference in
the presence of competing copper cations.4 This is a relevant
issue since copper is also present in the brain in high
concentrations and is considered to play a role in the
development of AD.5 Although this problem has been recently
overcome with the use of a ratiometric sensor,6 it is still of great
importance to develop highly sensitive molecular probes to
ligand (Chart 1) bearing a covalently attached 2-(5-phenyl-
[1,3,4]oxadiazol-2-yl)-pyridine (PyPD) unit. This photoactive
moiety has been chosen because it has interesting coordination
properties towards metal ions, besides being strongly emissive.
Due to this interesting photochemical behaviour, PyPD has
been inserted in materials for different applications;8 for
example, selected metal-complexes are at the base of light-
emitting electrochemical cells8a as well as they exhibited
electro-luminescence properites.8b In this work, the PyPD unit
was linked through a methylene spacer to the nitrogen of the
pendant arm of the scorpiand ligand 7,9 obtaining the
chemosensor L.
Scorpiand-type ligands are macrocycles appended with a side
chain which contains additional, potentially coordinating, donor
atoms.10 They have drawn attention for their unique
coordination properties: (i) the macrocycle can bind the metal
cation with high kinetic and thermodynamic stability; and (ii)
the flexible pendant arm can further coordinate the metal ion
in an axial positions, according to a labile binding mode.11 This
special coordination behaviour, with the metal weakly bound to
one of the positions, along with the possibility to functionalize
the side chain with chromophoric moieties, makes them
excellent candidates for the development of optical
chemosensors.12
a. Department of Pure and Applied Sciences, University of Urbino “Carlo Bo”, Via
della Stazione 4, I-61029 Urbino, Italy. Tel/Fax: +39-0722-304882. e-mail:
vieri.fusi@uniurb.it
Here we report the synthesis, the binding properties and
fluorescence sensing of the new ligand L towards both Zn2+ and
Cu2+ ions in aqueous solution.
b. Institute of Molecular Sciences, University of Valencia, C/ Catedrático José Beltrán
2, 46980 Paterna (Valencia), Spain. E-mail:Enrique.garcia-es@uv.es
c. Department of Industrial Engineering, University of Firenze, Via S. Marta 3, I-
50139 Firenze, Italy
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx00000x
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ARTICLE
Journal Name
solutions and determination of the equivalent point by the
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DOI: 10.1039/C9DT04764F
Gran’s method which gives the standard potential E0 and the
ionic product of water [pKw = 13.73(1)].16 The computer
program HYPERQUAD was used to calculate the protonation
and stability constants from the titration curves.17 The pH range
investigated was 2.5–10.5. The concentration of the ligand in
each experiment was close to 1.0 x 10-3 mol dm-3. In the case of
the metal complexes, experiments with different M:L ratios
were performed (i.e. 1:1, 3:2, 2:1). A minimum of two different
titration curves were acquired for each experiment and were
treated first as a single set. Finally, they were merged together
and treated simultaneously, leaving all values of the constants
free to be refined, to obtain the final stability constants, without
registering significant variations in their values.
N
N
O
N
NH
N
N
L
NH
HN
UV-vis and Fluorescence titrations
Chart 1 Schematic drawing of L
A 10 µM solution of L (HEPES buffer, pH = 7.4) was titrated with
different metal cations. The absorbance and fluorescence
spectra was registered upon the addition of 0, 0.25, 0.5, 0.75, 1,
1.5, 2, 5 and 10 equivalents of each metal cation. Each solution
was left to incubate for 30 minutes at room temperature, to
achieve full equilibria.
Experimental section
General Methods
UV absorption spectra were recorded at 298.1 ± 0.1 K on a
Varian Cary-100 spectrophotometer equipped with
a
Synthesis
temperature control unit. Fluorescence emission spectra were
recorded on a Varian Cary-Eclypse spectrofluorometer. The
fluorescence quantum yields (Фf) were determined by
comparing the integrated fluorescence spectra of the sample
with 2,2’-biphenol in acetonitrile (Фf = 0.29).13 IR spectra were
recorded on a Shimadzu FTIR-8300 spectrometer. Melting
points were determined on a Büchi melting point B 540
apparatus and are uncorrected. ESI-MS spectra were recorded
on a Thermo Quest LCQ Duo LC/MS/MS spectrometer. The NMR
spectra were recorded on a Bruker Avance 400 spectrometer,
operating at 400.13 and 100.61 MHz for 1H and 13C,
respectively, equipped with a PABBO Z-gradients direct probe
All chemicals were purchased in the highest quality
commercially available. The solvents were RP grade, unless
otherwise indicated. Compound L was obtained following the
synthetic procedure reported in Scheme 1. 6-(2-aminoethyl)-
3,6,9-triaza-1-(2,6)-pyridinecyclodecaphane (7) was prepared
as reported.9
2-(6-methyl-2-pyridyl)-5-phenyl[1,3,4]oxadiazole
(4).
6-
Methyl-2-pyridinecarboxylic acid 1 (7 g, 0.05 mol), thionyl
chloride (35 mL, 0.5 mol) and DMF (2,5 mL) were refluxed for 90
min in dry dichloromethane (165 mL). The mixture containing
the acyl chloride 2 was evaporated and dried under vacuum.
The resultant solid was dissolved in dry 1,4-dioxane (100 mL)
and the benzhydrazide 3 (7 g, 0.05 mol) was added. The reaction
mixture was heated under reflux for about 12 h, until evolution
of HCl ceased then, POCl3 (8,3 mL) was added and the reaction
mixture was heated for further 3 h. The reaction mixture,
brought to room temperature, was carefully poured into 165
mL of ice-cold water and then neutralized with Na2CO3 to give a
precipitate. The solid was filtered, washed with water and dried
at 60 °C in a vacuum. The crude product was purified by column
chromatography (silica gel, CHCl3), affording 4 as a white solid
(6 g; 50 %). MS m/z (ESI): 238.1 (M+H+); 1H NMR (CDCl3): δ = 8.23
(2H, m), 8.10 (1H, d, J=7.8 Hz) 7.79 (1H, t, J=7.8 Hz), 7.55 (3H,
m), 7.34 (1H, d, J=7.6 Hz), 2.71 (3H, s); 13C NMR (CDCl3): δ =
165.3, 164.0, 159.6, 142.9, 137.3, 131.9, 129.0, 127.2, 125.6,
123.6, 120.4, 24.6 ppm; Anal. for C14H11N3O: Calcd C 70.87, H
4.67, N 17.71; Found C 70.7, H 4.6, N 17.6.
1
and a variable temperature unit. H and 13C NMR spectra are
referenced to residual solvent signals. The assignment of the
NMR resonances was supported by 135-DEPT and 2D
experiments. Two-dimensional experiments (COSY, NOESY and
HMQC) were recorded using standard Bruker pulse sequences.
The 2D-NOESY experiments were recorded using a mixing time
(d8) of 0.35 s. All reagents and solvents used were of analytical
grade.
EMF Measurements
The potentiometric titrations were carried out at 298.1 ± 0.1 K
using 0.15 M NaClO4 as the supporting electrolyte. The
experimental procedure (burette, potentiometer, cell, stirrer,
microcomputer, etc.) has been fully described elsewhere.14 The
acquisition of the EMF data was carried out with the computer
program PASAT.15 The reference electrode was an Ag/AgCl
electrode in saturated KCl solution. The glass electrode was
calibrated as a hydrogen-ion concentration probe by titration of
previously standardized amounts of HCl with CO2 free NaOH
2-(6-dibromomethyl-2-pyridyl)-5-phenyl[1,3,4]oxadiazole (5).
4 (6 g, 0.025 mol), N-bromosuccinimide (9,8 g 0.055 mol), and
2 | J. Name., 2012, 00, 1-3
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2,2’-azobis(2-methylpropionitrile) (125 mg, 0.75 mmol) were with 0.1 M NaOH and saturated with solid NaClO4. After a few
View Article Online
DOI: 10.1039/C9DT04764F
dissolved in 240 mL of CCl4 under nitrogen. The reaction was minutes a colourless microcrystalline solid precipitated in the
stirred under reflux for 10 h and then cooled, filtered and the case of Zn2+ (27 mg, 72 %) while a green-blue microcrystalline
solvent removed under reduced pressure. The crude product solid precipitated in the case of Cu2+.
was purified by chromatography on silica gel (eluent CH2Cl2) to [ZnL](ClO4)2: MS m/z (ESI): 274.0 (ZnL2+); Anal. Calcd. for
afford 5 as a white solid (4 g, 40 %). MS m/z (ESI): 393.9 (M+H+); C27H32Cl2N8O9Zn: C 43.30; H 4.31; N 14.96. Found: C 43.2; H 4.4;
1H NMR (CDCl3): δ = 8.23 (3H, m), 8.05 (2H, m) 7.56 (3H, m), 6.80 N 15.0.
(1H, s); 13C NMR (CDCl3): δ = 172.0, 138.8, 133.8, 132.2, 130.2, [CuL](ClO4)2: Anal. Calcd. for C27H32Cl2N8O9Cu: C 43.41; H 4.32;
129.3, 129.1, 128.5, 127.4, 123.6, 123.5, 122.1 ppm; Anal. for N 15.00. Found: C 43.4; H 4.3; N 15.0.
C14H9Br2N3O: Calcd C 42.56, H 2.30, N 10.64; Found C 42.3, H Crystals suitable for X-ray analysis were obtained by slow
2.3, N 10.5.
evaporation of an aqueous solution containing the complex.
CAUTION! Perchlorate salts of organic compounds are
2-(6-formyl-2-pyridyl)-5-phenyl[1,3,4]oxadiazole (6). 5 (3.9 g, potentially explosive; these compounds must be prepared and
0,01 mol) and 40 % dimethylamine aqueous solution (50 mL) handled with great care.
were kept at 60 °C overnight. The cooled resulting solution was
extracted with CH2Cl2 (3 x 50 mL); the organic layer was washed
with a saturated solution of NaHCO3 (4x25 mL). The collected
organic layers were dried over anhydrous Na2SO4, concentrated
and purified by chromatography on silica gel (eluent CH2Cl2)
affording 6 as a white solid (1.7 g, 68 %). MS m/z (ESI): 252.1
X-ray Crystallography
Intensity data for compounds [ZnL](ClO4)2 and [CuL](ClO4)2 were
collected at 100 K and 110 K, respectively, on an Oxford
Diffraction Xcalibur diffractometer using the Cu-Kα radiation
(λ = 1.54184 Å). Data collection and reduction were performed
with the program CrysAlisPro;18 absorption correction was
performed with the SCALE3 ABSPACK scaling algorithm in
CrysAlisPro. The structures were solved by using the SIR-200419
package and subsequently refined on the F2 values by the full-
matrix least-squares program SHELXL-2014.20 In both cases
anisotropic thermal parameters were used for all the non-
hydrogen atoms. The hydrogen atoms were found in the Fourier
difference map and their positions were freely refined (with the
exception of the hydrogen atom bonded to one nitrogen atom
N(5) in [ZnL](ClO4)2 and N(4) in [CuL](ClO4)2, and to the carbon
atoms C(2) and C(13) in [ZnL](ClO4)2 were set to a fixed distance)
which while their isotropic thermal parameters were set
accordingly to those of the atoms to which they are bound.
Geometrical calculations were performed with PARST9721 and
molecular plots were produced by the ORTEP3,22 Mercury 3.7,23
and Discovery Visualizer 4.024 programs. Crystallographic data
and refinement parameters for [ZnL](ClO4)2 and [CuL](ClO4)2 are
available in the ESI (Tables S1). CCDC 1529591-1952561
contains the supplementary crystallographic data for this paper.
These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
1
(M+H+); H NMR (CDCl3): δ = 10.24 (1H, s), 8.54 (1H, m), 8.26
(2H, m), 8.13 (2H, m), 7.60 (3H, m); 13C NMR (CDCl3): δ = 192.5,
165.9, 163.1, 153.0, 144.2, 138.5, 132.3, 129.2, 127.4, 127.0,
123.4, 123.2 ppm; Anal. for C14H9N3O2: Calcd C 66.93, H 3.61, N
16.72; Found C 67.0, H 3.7, N 16.5.
6-{2-[N-(6-(5-phenyl[1,3,4]oxadiazole-2-yl)-2-
pyridyl)methylamino]ethyl}-3,6,9-triaza-1-(2,6)-
pyridinecyclodecaphane (L∙3HClO4∙H2O). 7 (236 mg, 0.95
mmol) and 6 (238 mg, 0.95 mmol) were dissolved in dry ethanol
(30 mL); the solution was stirred at room temperature for 2 h,
then sodium borohydride (359 mg, 9.5 mmol) was added and
the mixture kept under stirring for another 2 hours. The solvent
was removed under reduced pressure and water (10 mL) was
carefully added. The solution was extracted with CH2Cl2 (6x20
mL). The organic layers were dried over anhydrous Na2SO4 and
solvent evaporated under reduced pressure. The residue was
dissolved in ethanol (30 mL) and treated with
a 4:1
ethanol/perchloric acid solution, yielding a white solid. The
product was then recrystallized from methanol/ethanol
affording L as L·3HClO4·H2O salt (487 mg, 62 %). MS m/z (ESI):
1
243.0 (H2L2+); H-NMR (D2O, 25 °C): δ = 8.09 (1H, d, J=7.2 Hz),
8.00 (3H, m), 7.82 (1H, t, J=7.8 Hz), 7.52 (4H, m) 7.32 (2H, d,
J=7.8 Hz), 4.50 (4H, s), 4.47 (2H, s), 3.35 (2H, t, J=7.1 Hz), 3.16
(4H, t, J=5.3 Hz), 3.06 (2H, t, J=7.1 Hz), 2.81 (4H, t, J=5.3 Hz); 13
C
NMR (D2O, 25 °C): δ = 172.5, 166.0, 163.8, 151.8, 151.7, 148.9,
141.8, 139.8, 133.0, 129.4, 127.1, 126.0, 123.2, 122.3, 50.8,
50.7, 50.4, 49.5, 45.8, 43.1 ppm; Anal. for C27H37Cl3N8O14 Calcd
C 40.34, H 4.64, N 13.94; Found C 40.5, H 4.7, N 13.8.
[ML](ClO4)2. A sample of Zn(ClO4)2.6H2O (37 mg, 0.1 mmol) or
Cu(ClO4)2.6H2O (37 mg, 0.1 mmol) in water (5 mL) was added
to an aqueous solution (5 mL) containing L·3HClO4.H2O (40 mg,
0.05 mmol). The pH of the resulting solution was adjusted to 7
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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COOH
COCl
SOCl2
N
N
CH3
(1)
CH3
(2)
N
N
CONHNH2
i) Dioxane
ii) POCl3
2
O
N
H3C
(3)
(4)
N
N
N
N
NBS, AIBN
CCl4
Me2NH, 40%
water solution
O
4
O
N
N
C
N
Br2HC
O
H
(5)
(6)
N
NH2
O
N
N
N
NH
i) EtOH
6
NH
HN
ii) NaBH4
N
L
NH
HN
N
(7)
Scheme 1 Synthesis of L
bromosuccinimide gave rise to the dibromide (5) that was
converted in the aldehyde derivative 6 using dimethylamine.
The desired compound L was obtained by coupling of the
polyamine 7 with 1 equivalent of 2-(6-formyl-2-pyridyl)-5-
phenyl[1,3,4]oxadiazole (6) in ethanol and subsequent
reduction with NaBH4. The ligand was precipitated by treatment
with an ethanol/perchloric acid 4:1 solution, and then further
purified as its hydroperchlorate salt by recrystallization from
methanol/ethanol.
Results and discussion
Synthesis
The synthetic pathway used to obtain L is depicted in Scheme 1.
The heteroaromatic scaffold 2-(6-formyl-2-pyridyl)-5-
phenyl[1,3,4]oxadiazole (6) showing the aldehyde function
suitable to selectively react with the primary amine function of
macrocycle 7, was synthesized starting from the precursors 2
and 3 following a similar procedure to that reported in ref. 21.
The acyl chloride
pyridinecarboxylic acid
2
was prepared from 6-methyl-2-
using thionyl chloride in
1
dichloromethane; it was freshly used without further
purification. The coupling to obtain 4 occurred in two steps; the
first provides the formation of the dihydrazide intermediate
which was not isolated but, using POCl3, it was cyclized at the
dihydrazide fragment to afford the oxadiazole ring in a good
yield. The bromination of 4, carried out by an excess of N-
4 | J. Name., 2012, 00, 1-3
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ARTICLE
Description of the [ZnL](ClO4)2 and [CuL](ClO4)2 structures
H(2N)...O(12), 2.21(4) Å, 155(4)°; N(4)-H(4N)...O22, 2.15(4) Å,
171(4)° in [ZnL]2+ and N(2)-H(2N)...O(12), 2.17(3) Å, 158(3)°;
N(4)-H(4N)...O22, 2.12(2) Å, 174(2)° in [CuL]2+).
The crystal structures of the two metal compounds are
isomorphous and isostructural. In both cases the asymmetric
unit consists of one [ML]2+ (M=Zn2+ or Cu2+) cation and two
perchlorate counterions. The overall shapes of the two metal
complexes are essentially identical (Figure 1). The metal cation
is hexa-coordinated by six nitrogen atoms, four coming from the
macrocycle and two from the dangling arm. The coordination
geometry of each metal ion is distorted octahedral (Figures 2
and S1), as indicated by the value of the N-M-N angles
comprised between adjacent donors which vary from 77.7(1) to
106.4(1)° in [ZnL]2+ and from 77.65(8) to 104.38(8)° in [CuL]2+,
respectively (Table 1). In the copper complex the M-N distances
involving the two axial donors N(3) (the tertiary amine of the
macrocyclic unit) and N(6) (belonging to the pyridine moiety of
the dangling arm) are significantly longer than the other four.
Interestingly, the long bond distance with the pyridine nitrogen
atom of the tail can be ascribed to the steric hindrance
originated by the bulky aromatic system. As a matter of fact, in
the Cu2+ and Mn2+ complexes of a related ligand having just the
pyridine ring in the tail (reference codes FUTPIV and ZUHPAV,
respectively),26 which show a very similar overall shape with
respect to [ML]2+ (Figure S2 in ESI), the M-pyridine nitrogen
distances lie among the shortest ones in the coordination
spheres. The axial positions in the Cu2+ complex (FUTPIV) are
defined in this case by the secondary amines closest to the
pyridine ring in the macrocyclic core.27
The phenyl and oxadiazole moieties in [ML]2+ almost share a
plane in agreement with the structural data deposited in the
Cambridge Structural Database (CSD, version 5.37)19 about this
molecular fragment, but they are almost perpendicular to the
adjacent pyridine ring (while the three aromatic rings are
usually coplanar (from CSD data referring to only-organic
compounds). Most probably, both the steric interactions
between close bulky groups, i.e. the PD tail and the pyridine
belonging to the macrocycle, which disfavour a coplanar
arrangement of the three PyPD rings, and the stabilizing π-
stacking interactions between the oxadiazole and the
macrocyclic pyridine ring (they are parallel displaced, the ring
centroids are, in both molecular structures at 3.5Å) might
explain such an arrangement for the phenyl-oxadiazole moiety
(Figures 2 and S1). Finally, from Figure 2b it appears that also
the zinc ion can hardly be accessed by an incoming ligand
without the removal of any metal-ligand bond (the same applies
for the copper complex).
Table 1 Bond distances (Å) and angles (°) about the coordination
sphere of the [ZnL]2+ and [CuL]2+ cations.
M-N
Distances (Å)
[ZnL]2+
[CuL]2+
1.950(2)
2.147(2)
2.279(2)
2.182(2)
2.016(2)
2.362(2)
M-N(1)
M-N(2)
M-N(3)
M-N(4)
M-N(5)
M-N(6)
2.068(3)
2.206(30)
2.222(3)
2.249(3)
2.113(3)
2.255(3)
N-M-N
Angles (°)
[ZnL]2+
78.1(1)
94.9(1)
78.1(1)
175.6(1)
106.4(1)
81.8(1)
148.8(1)
99.6(1)
100.1(1)
80.4(1)
81.0(1)
158.6(1)
102.7(1)
105.7(1)
77.7(1)
[CuL]2+
80.73(9)
96.66(8)
80.56(8)
177.53(9)
104.38(8)
82.16(8)
152.81(9)
97.58(9)
99.67(8)
80.60(8)
81.29(9)
158.92(7)
100.40(9)
103.98(8)
77.65(8)
N(1)-M-N(2)
N(1)-M-N(3)
N(1)-M-N(4)
N(1)-M-N(5)
N(1)-M-N(6)
N(2)-M-N(3)
N(2)-M-N(4)
N(2)-M-N(5)
N(2) -M-N(6)
N(3)-M-N(4)
N(3)-M-N(5)
N(3)-M-N(6)
N(4)-M-N(5)
N(4)-M-N(6)
N(5)-M-N(6)
Fig. 1 Superimposition of the [ZnL]2+ and [CuL]2+ cations as found in the X-ray
structures of [ZnL](ClO4)2 (all atoms ball and stick) and ([CuL](ClO4)2 (stick, green).
Structures are superimposed by the metal cation and the six donor atoms.
Regarding the crystal packing, in both crystal structures each
perchlorate anion is kept in place by H-bonds involving as
donors the secondary nitrogen atoms of the macrocycle (N(2)-
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290 nm (ε = 16100 cm-1mol-1dm3), both ascribedViteow AtrhticelePOynPlinDe
DOI: 10.1039/C9DT04764F
chromophores (Figure S3a) with the first containing also the
absorbance of the pyridine unit inserted in the macrocyclic
skeleton. The UV-Vis spectra showed only minimal changes in
the 2-12 pH range investigated, suggesting that the pyridine
moieties of the macrocycle and of the PyPD fluorophore are not
directly involved in the protonation steps but, at most, in the
stabilization of the protonated species via H-bonding. On the
contrary, a different behaviour was observed in the emission
spectra where the intensity of the emission is highly pH-
dependent. Fluorescence emission spectra were recorded upon
exciting at 264 or 290 nm in aqueous solution in the pH range
2-12; the emission intensity does not change when exciting at
the different wavelengths, supporting that both bands are due
to the PyPD moiety. The spectra (λex = 275 nm) are reported in
Figure S3b while, the trend of the fluorescence emission
intensity (■) versus pH (λem = 364 nm) is shown in Figure 3
together with the distribution diagram of the species.
Fig. 2 (a) ORTEP-3 view of [ZnL]2+ as found in the solid state structure of [ZnL](ClO4)2.
Ellipsoids are drawn at 15% probability. (b) Space-filling model of the [ZnL]2+ cation as
found in the solid state structure.
Solution studies
Acid-base behavior. Table 2 collects the protonation constants
of the newly prepared ligand L, as well as those of its precursor
7, reported previously.9 L shows three stepwise protonation
constants, which correspond to the protonation of the three
secondary amines of the molecule. The protonation of the 2-
phenyl-5-pyridil-[1,3,4]oxadiazole group, as well as those of the
tertiary amine and the pyridine nitrogen have not been
detected in the pH range of study (2.5-11.0). The global basicity
of L is lower than that of the precursor compound 7 due to the
higher basicity of the primary nitrogen in aqueous solution.
These values are, on the other hand, close to those previously
reported for analogous derivatives with other functionalities in
the pendant arm of the macrocycle.20a,28,29
Table 2 Logarithms of the stepwise and cumulative protonation
constants of L and 7, determined in 0.15 M NaClO4 at 298.1 ± 0.1 K.
Fig. 3 Distribution diagram of the species (−) and trend of the emission (■) at λem = 364
nm, I = 0.15 M NaClO4, T = 298.1± 0.1 K.
Reaction[a]
L + H ⇄ HL
HL + H ⇄ H2L
H2L + H ⇄ H3L
logβ[b]
7
L
10.20(6)[c]
9.18(3)
7.84(4)
27.32(8)
9.84(1)[d]
8.61(2)
6.73(2)
25.18(2)
At pH = 12, in the presence of the non-protonated L species, the
fluorescence is completely quenched. However, when going
from alkaline to acidic pH values, the emission intensity starts
to increase at pH 8 (λem = 364 nm) following the formation of
the H3L3+ species in solution and it reaches the maximum
intensity below pH 4 where the H3L3+ species is fully formed.
Considering that the UV-Vis spectra did not suggest the
involvement of PyPD in protonation, this behaviour can be
rationalized in terms of quenching of the excited state due to
photoinduced intramolecular electron transfer (PET) from the
HOMO of the donor benzyl nitrogen atom to the excited
fluorophore moiety30 occurring if the amine function in the
benzylic position to PyPD shows a lone pair. This means that, for
pH < 4 the aliphatic amine in such position is protonated, i.e. the
nitrogen lone-pair is engaged in the protonation, and the PET
effect is therefore forbidden; on the contrary, in the H2L2+
species, the quenching of the emission suggests that such amine
is deprotonated.
[a]Charges have been omitted. [b]Calculated as log β = ∑j log Kj. [c]Values in
parenthesis are standard deviations in the last significant figure. [d]Taken
from ref. 9
Figure 3 shows the species distribution diagram of L. The three
detected species appear in the 4-10 pH range being H2L2+ the
predominant species at physiological pH values.
UV-Vis Absorption and Fluorescence of L. Absorption and
emission electronic spectra were recorded in aqueous solution
at variable pH to obtain information about the effect of the
protonation on absorption and emission properties of L.
The UV-Vis absorption electronic spectrum of L at pH = 12,
where the neutral species L is present in solution, shows two
main bands with λmax at 264 (ε = 12200 cm-1mol-1dm3) and
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Interaction with Zn2+ and Cu2+. L is a polytopic ligand potentially
able to form metal complexes with stoichiometry higher than
1:1. Moreover, it has been reported that the pyridyl-oxadiazole
moiety can behave as a chelating agent, forming Zn2+ complexes
with 1:1 and 1:2 Zn2+:L stoichiometries.31 To infer the
stoichiometry of the complexes formed in solution and
calculate their stability constants, potentiometric titrations
were performed in water at different metal/ligand mole ratios,
adjusting each one separately and then merging all the data into
a single model. In the case of Zn2+, titrations were rather slow
requiring long equilibration times between consecutive base
additions to reach the equilibrium. The results show the
formation of 1:1 and 3:2 Zn2+:L complexes; for 1:1 stoichiometry
only the [ZnL]2+ species was detected while three species of 3:2
M:L stoichiometry were found: [Zn3L2]6+, [Zn3L2(OH)2]4+ and
[Zn3L2(OH)3]3+. On the other hand, in the case of Cu2+ the
equilibrium was reached rapidly, and the data treatment
revealed the formation of a highly stable (β ˃ 22) [CuL]2+
complex, which predominates throughout the whole pH range
of study (pH ≈ 2.5 – 10.5). The values obtained for the formation
of these species are summarized in Table 3.
View Article Online
DOI: 10.1039/C9DT04764F
Table 3 Stability constants in logarithmic units for the Zn2+ and Cu2+
complexes of L determined in 0.15 M NaClO4 at 298.1 ± 0.1 K.
Fig. 4 Species distribution diagrams for the Zn2+-L system at different mole ratios ([Zn2+
= 1x10-3 M). Charges omitted.
]
Reaction[a]
Zn2+
Cu2+
L + M ⇄ ML
2L + 3M ⇄ M3L2
2L + 3M + 2H2O ⇄ M3L2(OH)2 + 2H
2L + 3M + 3H2O ⇄ M3L2(OH)3 + 3H
16.50(5)[b]
40.98(4)
24.59(5)
14.5(1)
22.65(5)
The stoichiometry of the complexes at low concentration was
also determined by the continuous variation method, or Job’s
plot,32 measuring the area of the fluorescence emission spectra
of both the free ligand and the ligand in the presence of Zn2+ at
–
–
–
[a]Charges have been omitted. [b]Values in parenthesis are standard deviations in
the last significant figure.
different molar ratios, but keeping
a constant total
concentration.33 The experiments were done using HEPES
buffer at pH 7.4 and a total concentration of 1x10-4 M. As seen
in Figure 5, the maximum of the plot is centred at a 0.5 molar
ratio, which suggests a 1:1 stoichiometry of the complex. The
shape of the Job’s plot indicates that the complex formed is very
stable.34
The distribution diagrams built for different Zn2+:L molar ratios
(Figure 4) show how for a 1:1 relation ([Zn2+] = 1x10-3 M) the
[ZnL]2+ complex predominates from intermediate to basic pH
values, while between pH 4-5 the main species is the [Zn3L2]6+
complex. For 3:2 Zn2+:L mole ratios and the same Zn2+
concentration the 3:2 species prevail throughout the whole pH
range.
However, at low concentrations (below 0.1 mM), only the
[ZnL]2+ species is relevant, as is shown in the distribution
diagrams (Figure S4) and as it has been also demonstrated by
mass spectrometry (Figures S5).
Fig. 5 Job plot for complex formation between L and Zn2+. Fluorescence emission spectra
were recorded at 297 nm in a pH 7.4 solution (0.05 M HEPES buffer) at a total
concentration of [L]+[Zn2+]=1x10-4 M.
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In the case of the Cu2+ cation, because of the very high stability
Journal Name
View Article Online
of the 1:1 complex, the [CuL]2+ specie predominates (formation As above reported, the binding of Zn2+ changes the absorption
DOI: 10.1039/C9DT04764F
percentage ˃ 99 %) throughout the whole pH range.
profile with the disappearance of the band at lower energy. The
profile reaches a plateau after the addition of one equivalent of
Fluorescence and UV-Vis of Zn2+/L and Cu2+/L systems. Zn2+ and does not experiment any other change with the
Spectrophotometric and spectrofluorimetric studies were addition of further amounts of Zn2+ (see inset of Figure 6a).
carried out in aqueous and acetonitrile solutions to further The emission band, measured under the same conditions by
evaluate the role of the the PyPD moiety in the coordination of exciting at the isosbestic point (275 nm), undergoes a little red
both Zn2+ and Cu2+ and its photochemical response to these shift, from 364 to 377 nm, and a 4-fold enhancement of the
metal ions. The UV-Vis and fluorescence spectra of L were emission upon Zn2+ addition (see Figure 6b). These effects in the
recorded at variable pH using different Zn2+ or Cu2+ equivalents. absorption and emission spectra can be simultaneously justified
The UV-Vis spectra obtained for the Zn2+/L system show that the by the partial inhibition of the PET effect due to the engagement
band at lower energy (λmax = 290 nm) disappears upon of the aliphatic amine lone-pairs of the side-arm in the metal
formation of the Zn2+-L species; this spectral feature suggests ion coordination as well as by the involvement of the pyridine
that the PyPD moiety is probably involved in the coordination belonging to the PyPD fragment in the binding of Zn2+ (Chelation
of the Zn2+ ion in solution. On the other hand, the fluorescent Enhancement of the Fluorescence (CHEF)), in a similar
emission of the free ligand decreases by increasing the pH. environment to that found in the solid state. The Zn2+ binding
However, in the presence of Zn2+ the quenching is prevented could also interrupt the conjugation among the three rings of
and there is not a complete quenching of the emission at basic PyPD, as shown in the solid state, giving rise to the
pH (see Figure S6). The titrations carried out with 1:1 or 3:2 disappearance of the lower energy band in the absorption
Zn2+:L mole ratios gave similar results (Figure S7) because, as spectra as well as to the change in the emission wavelength.
above commented, in the diluted concentrations used in the The UV-Vis spectra of the Cu2+/L system are similar to those of
measurements ([L] = 5.0·10−6 M) only the mononuclear species the Zn2+/L with respect to the disappearance of the band at λmax
prevails in solution.
= 290 nm while, the band at higher energy (264 nm) increases;
To better understand this effect and considering the biological all of it supporting an analogous involvement of L in the
relevance of Zn2+, emission and absorption titrations of L with stabilization of Cu2+, as found in the solid state. In the emission
Zn2+ were performed in buffered (pH = 7.4) aqueous solution. titration experiments, the coordination of the paramagnetic
At this pH value, the [ZnL]2+ species is fully formed in 1:1 Zn2+/L Cu2+ quencher gives rise, as expected, to the total quenching of
mole ratio. All the spectra were recorded waiting 2 h to allow the fluorescence (see Figure S8).
the stabilization of the species formed (that is up to obtain While the absorption spectra support that the whole
invariance of the spectral profile). Figures 6a and 6b, show the framework of L, including the PyPD moiety, is involved in the
UV-Vis and fluorescence titrations, respectively.
metal ion coordination, the fluorescence experiments show a
higher positive and naked eye detectable response to Zn2+ than
to Cu2+.
To widen the fluorescent behaviour of L in the presence of
metal ions, different metal ions (i.e. Cd2+, Co2+, Hg2+, Mn2+, Ni2+
and Pb2+) were tested in the above experimental conditions.
The result is that the presence of all such metal ions in solution
decrease the emission (Figure 7, orange bars) suggesting that L
is able to bind all of them but, as mentioned above, only Zn2+ is
able to switch on the emission at the physiological pH of 7.4.
Competition experiments were also carried out adding two
equivalents of all metal ions to the buffer 7.4 solution
containing the [ZnL]2+ cation (Figure 7, green bars).
The result is that the emission of the [ZnL]2+ species is
substantially not perturbed by the presence of other metal ions
with the exception of Hg2+ in which the system shows a total
quenching of the emission, which can be attributed to the so-
called heavy metal effect exerted by Hg2+.30a However, the
competition experiments suggests the selective fluorescence
response of L to the presence of Zn2+ over all the other
interfering metal ions tested.
Fig. 6 Absorption (a) and emission (b) spectra of the Zn2+/L system in an aqueous buffer
(HEPES, 0.5 M) solution at pH = 7.4, obtained by adding several amounts of Zn2+ up to 2
equivalents with respect to L; [L] = 3 x 10-5 M. Inset: variation of the absorbance at
λ = 293 nm and of the emission at 377 nm as a function of the added Zn2+
.
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cations are coordinated by all the nitrogen aVtoiewmAsrticolefOntlhinee
DOI: 10.1039/C9DT04764F
macrocyclic core, the secondary amine of the pendant arm and
the nitrogen atom of the pyridine group of the fluorescent
moiety, being this latter bond clearly weaker than the one with
the pyridine atom of the macrocycle.
Fig. 7 Bar-plot showing the emission of L (10M, HEPES buffer pH 7.4) with 2 equivalents
of different metal cations (orange bars), and the emission of [ZnL]2+ complex in the
presence of 2 equivalents of different metal cations (green bars).
Fig. 8 Emission spectra of the Zn2+/L system in acetonitrile solution obtained by adding
several amounts of Zn2+ up to 2 equivalents with respect to L; [L] = 1.0 x 10-5 M.
Considering this, the same titration experiments were
performed in acetonitrile because as it is well known, the
dielectric constant of the solvent plays a crucial role in
fluorescent chemosensors activated/deactivated by a PET
effect; the intensity of the emission increases by lowering the
polarity of the solvent.35 This aspect is interesting to evaluate
the possibility to include such system in polymers or
nanoparticles, which are often the base for optical devices
based on chemosensors.36 The titrations gave rise to similar
results as those in aqueous solution (See Figures 8 and S9),
preserving similar absorption and fluorescent profiles as well as
same absorption and emission wavelengths. However, the
addition of Zn2+ yields a much higher emission than either in
aqueous solution or compared to free L. The emission increase
due to the CHEF effect is more than 80-fold with respect to free
L giving rise to an optic response and sensing of Zn2+ in the
medium. In fact, in these experimental conditions, the [ZnL]2+
species becomes highly fluorescent being the calculated
quantum yield Фf = 0.24 at 374 nm. This highlights not only the
properties of L to signal the Zn2+ ion but also that the new
developed fluorophore PyPD is highly fluorescent and has the
ability to coordinate a metal giving rise to a spectrophotometric
response. Also in this solvent, the emission reaches its
maximum after the addition of one equivalent of Zn2+
supporting the formation of a mononuclear [ZnL]2+ species in
this medium too.
Addition of Zn2+ to give the species [ZnL]2+ induces a chelation
enhancement of the fluorescence, a behaviour that was not
observed with Cu2+ and with any of the other metal cations
studied which, on the contrary, induced a quenching of the
fluorescence.
The OFF-ON fluorescent response of L to the presence of Zn2+
ion is quite marked in physiological pH 7.4 aqueous solution and
becomes even more pronounced in acetonitrile solution (more
than 80-fold increase). For the two metals cations studied, the
PyPD fluorophore is involved in the metal ion coordination via
the contained pyridine moiety. The coordination pattern
exhibited by the [ZnL]2+ cation in the solid state is also preserved
in solution suggesting a further possible application of this
species as fluorescent chemosensor for anions in which it acts
both as metallo-receptor and sensor for anions; preliminary
studies in this direction are already yielding interesting results.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by Spanish MICINN and MEC and
FEDER funds from the European Union (grants CTQ2016-78499-
C6-1-R, CTQ2017-90852-REDC, Unidad de Excelencia María de
Maeztu MDM-15-0538), Generalitat Valenciana (PROMETEO II
2015-002), the University of Valencia (Subprograma Atracció de
Talent), the Italian Ministero dell’Istruzione dell’Università e
della Ricerca (MIUR) (project 2015MP34H3 and project
2017EKCS35), and by University of Urbino (Department of Pure
and Applied Sciences – Grant DISPEA_FUSI_PROG2017 and
Conclusions
A ligand constituted by a macrocyclic pyridinophane core having
a pendant arm containing a secondary nitrogen atom linked
through a methylene spacer to a pyridyl-oxadiazole-phenyl
(PyPD) fluorescent system has been prepared (L). The crystal
structures of [ZnL](ClO4)2 and [CuL](ClO4)2 show that the metal
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20 G. M. Sheldrick, Acta Cryst., 2015, C71, 3.
21 M. Nardelli, J. Appl. Cryst., 1995, 28, 659.
“Progetti di Valorizzazione” DiSPeA. CRIST (Centro di
Cristallografia Strutturale of the University of Firenze) is
gratefully acknowledged for X-ray facilities.
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DOI: 10.1039/C9DT04764F
22 L. J. Farrugia, J. Appl. Cryst., 2012, 45, 849.
23 F. Macrae, I. J. Bruno, J. A. Chisholm, P. R. Edgington, P.
McCabe, E. Pidcock, L. Rodriguez-Monge, R. Taylor, J. van de
Streek, P. A. Wood, J. Appl. Cryst., 2008, 41, 466.
24 Dassault Systèmes BIOVIA, Discovery Studio Visualizer,
Release 4.5, San Diego: Dassault Systèmes, 2015.
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