Fluorenyl-Schiff-base ligands and their dicopper(II) complexes. Synthesis, structural and spectroscopic characterization and DNA binding assays

Article abstract of DOI:10.1016/j.poly.2018.01.003

Schiff-base ligands L1 and L2 containing fluorene as fluorophore were synthesized thorough condensation of salicylaldehyde and 2,6-diformyl 4-methylphenol with 6-(9-fluorenyl)-1,4,8,11-tetraazaundecane-5,7-dione, respectively. A comprehensive spectroscopic analysis was performed by elemental analysis, ESI-MS, ¹H and ¹³C NMR, FTIR, EPR, UV–Vis and emission spectroscopies. Ligand L1 binds copper(II) to form [Cu₂(L1)₂]·2H₂O that crystallizes in orthorhombic system and space group P2₁/n. Assignments to the UV–Vis and EPR spectra allowed access to Jahn–Teller stabilization energies (E_JT) at 6345 and 5435 cm^?1 for complexes [Cu₂(L1)₂]·2H₂O and [Cu₂(L2)](ClO₄)₄·4H₂O, respectively, and to their corresponding spin orbit coupling constants λ = ?428 and ?398 cm^?1. The EPR spectrum of [Cu₂(L2)](ClO₄)₄·4H₂O in the solid state showed a half-field Δm_S = ±2 transition, supporting the dicopper composition. The fluorenyl copper(II) derivatives demonstrated a moderate binding to ct-DNA than the fluorenyl-ligand molecule following the increasing order of (K_b): L1 < [Cu₂(L1)₂]·2H₂O < [Cu₂(L2)](ClO₄)₄·4H₂O.

Full text of DOI:10.1016/j.poly.2018.01.003

Accepted Manuscript
Fluorenyl-Schiff-base ligands and their dicopper(II) complexes. Synthesis,
structural and spectroscopic characterization and DNA binding assays
Fabiane dos Santos Carlos, Marcelo Carpes Nunes, Bernardo Almeida Iglesias,
Priscilla J. Zambiazi, Manfredo Hörner, Guilherme A. de Moraes, Guilherme
Sippel Machado, Ronny Rocha Ribeiro, Fábio Souza Nunes
PII:
S0277-5387(18)30003-2
https://doi.org/10.1016/j.poly.2018.01.003
POLY 12995
DOI:
Reference:
Polyhedron
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 August 2017
17 November 2017
2 January 2018
Please cite this article as: F.d.S. Carlos, M.C. Nunes, B.A. Iglesias, P.J. Zambiazi, M. Hörner, G.A. de Moraes, G.S.
Machado, R.R. Ribeiro, F.S. Nunes, Fluorenyl-Schiff-base ligands and their dicopper(II) complexes. Synthesis,
structural and spectroscopic characterization and DNA binding assays, Polyhedron (2018), doi: https://doi.org/
10.1016/j.poly.2018.01.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
Fluorenyl-Schiff-base ligands and their dicopper(II) complexes. Synthesis,
structural and spectroscopic characterization and DNA binding assays
Fabiane dos Santos Carlos,a Marcelo Carpes Nunes,a Bernardo Almeida Iglesias,b
Priscilla J. Zambiazi,b Manfredo Hörner,b Guilherme A. de Moraes,b Guilherme Sippel
Machado,c Ronny Rocha Ribeiroa and Fábio Souza Nunesa*
aDepartamento de Química, Universidade Federal do Paraná, Cx. Postal 19081,
81531980 Curitiba, PR, Brazil
bDepartamento de Química, Universidade Federal de Santa Maria, 97105-900, Santa
Maria, RS, Brazil
cCentro de Estudos do Mar, Universidade Federal do Paraná, Pontal do Paraná, Cx.
Postal 61, 83255-976, PR, Brazil
* Correspondence to: Fábio S. Nunes (fsnunes@ufpr.br)
FAX 55-41-3361-3186

2
Abstract
Schiff-base ligands L1 and L2 containing fluorene as fluorophore were
synthesized thorough condensation of salicylaldehyde and 2,6-diformyl 4-methylphenol
with
respectively.
A
comprehensive spectroscopic analysis was performed by elemental analysis, ESI-MS,
13
1H- and C-NMR, FTIR, EPR, UV-Vis and emission spectroscopies. Ligand L1 binds
copper(II) to form [Cu2(L1)2]∙2H2O that crystallizes in orthorhombic system and space
group P21/n. Assignments to the UV-vis and EPR spectra allowed access to Jahn-Teller
stabilization energies (EJT) at 6345 and 5435 cm-1 for complexes [Cu2(L1)2].2H2O and
.
[Cu2(L2)](ClO4)4 4H2O, respectively, and to their corresponding spin orbit coupling
constants λ = -428 and -398 cm-1. The EPR spectrum of [Cu2(L2)](ClO4)4∙4H2O in the
solid state showed a half-field mS = 2 transition, supporting the dicopper
composition. The fluorenyl copper(II) derivatives demonstrated a moderate binding to
ct-DNA than the fluorenyl-ligand molecule following the increasing order of (Kb): L1 <
.
[Cu2(L1)2].2H2O < [Cu2(L2)](ClO4)4 4H2O.
Keywords: Schiff-base ligands; Binuclear copper complex;
salicylaldehyde; 2,6-Diformyl 4-methylphenol
1. Introduction
Copper is an essential element, but it is also a pollutant and can be toxic when in
high concentrations in the environment [1]. Copper concentration in the environment
varies and comes from natural occurrence, mining and industrial activity. Literature

3
presents several fluorescent sensors for copper ion, with varying degrees of sensitivity,
selectivity and difficulty of preparation [2-19].
Condensation of 2,6-diformyl-4-methylphenol and diamines produces
Robson-type ligands that have been investigated over the years because their
coordination compounds show interesting magnetic, redox and structural properties [20-
25].
Continuing our general interest in the synthesis of metal complexes of
polyfunctional ligands we have recently reported the structural, magnetic and
spectroscopic properties of a bis(semicarbazone) series prepared by Schiff condensation
of 2,6-diformyl 4-methylphenol with semicarbazide hydrochloride in 1:2 molar ratio
[21].
In this work we report on the preparation, characterization and properties of two
coordination compounds that contain luminescent ligands (Scheme 1). Ligands 1 and 2
were prepared by connecting
a
diamidodiamine
(
to two equivalents of salicylaldehyde and 2,6-diformyl-4-
methylphenol, respectively. Both ligands contain fluorene, which is a fluorophore with
recognized photophysical properties [26-30].
2. Experimental
2.1. Preparations

4
The chemicals used in this work were purchased from Sigma-Aldrich and used
as supplied. 6-(9-fluorenil)-1,4,8,11-tetraazaundecano-5,7-dione and 2,6-diformyl-4-
methyl-phenol were prepared as described elsewhere [26,34].
Ligand L1 (Scheme 1) and [Cu2(L1)2]∙2H2O were synthesized according to
Luo et al. [35]. Elemental analysis calculated(found) for L1, C34H32N4O4, 560.64 g
1
mol-1: C% 72.85 (72.84); H% 5.66 (5.75); N% 10.26 (10.00). H-NMR ( in ppm):
7.835, d, H1; 7.885, t, H2; 7.196, t, H3; 7.412, d, H4; 4.662, d, H5; 2.851, d, H6; 3.497-
3.676, m, H8 and H9; 8.390, s, H10; 6.868, m, H11 and H12; 7.321, m, H13, H14 and
H15.
ESI-MS (negative mode) at m/z: Calculated(observed) for [L1-H]-:
559.63(559.53). Yield for [Cu2(L1)2]∙2H2O was 0.071 g, 28%. Elemental analysis
calculated(found) for C68H64N8O10Cu2,1280 g mol-1: C% 63.99 (63.79); H% 4.74
(4.64); N% 9.00 (8.78).
Ligand L2 was prepared by template method. Mg(NO3)2∙6H2O (1.00 g, 3.9
mmol) and 0.69 g (1.95 mmol) 6-(9-fluorenil)-1,4,8,11-tetraazaundecano-5,7-dione
were dissolved in 30 mL of methanol and mixed with a solution of 0.32 g (1.95 mmol)
of 2,6-diformyl-4-methylformol in 10 mL of methanol. The system was kept under
reflux for 24h, resulting in an orange solution. One third of the volume was removed
under vacuum and 20 mL of diethyl ether was added. The orange product was collected
by filtration, washed with ether and dried under vacuum. Yield was 0.73 g (30%).
Elemental analysis calculated(found) for C58H56Mg2N12O18, 1257,75 g mol-1: C% 55.54
(55.48); H% 4.42 (4.33); N% 13.17 (13.39). 1H-NMR ( in ppm): 7.195-8.534, m, H1;
4.655, d, H2; 3.311-3.352, m, H4; 3.012, d, H3; 2.775-2.857, m, H5; 2.156-2.350, m,
H6. ESI-MS (positive mode) at m/z: Calculated(observed) for [L2+H]+: 961.44(961.44).

5
Scheme 1. Synthesis of L1 (top) and [Mg2(L2)](NO3)4 (bottom).
[Cu2(L2](ClO4)4∙4H2O. Copper perchlorate hexahydrate (0.088 g, 0,24 mmol)
dissolved in 3 mL of methanol was mixed to [Mg2(L2)](NO3)4 (0.300 g, 0.24 mmol) in
3 mL of methanol. After reflux for 2h, the pH was set to 9 using triethylamine, and
reflux was maintained for two more hours. After filtration, 2/3 of the volume was
removed, leading to a brown powder, which was collected, washed with cold ethanol
and dried under vacuum. Yield was 0.328 g, 88%. Elemental analysis calculated(found)
for C58H60N8O26Cl4Cu2,1558 g mol-1: C% 44.35 (44.83); H% 4.06 (4.14); N% 7.46
(7.21).

6
2.2. Apparatus
Elemental analyses were done in a Perkin-Elmer 2400 analyzer. Mass spectra
were measured in a high resolution ESI-MS on a microTOF QII mass spectrometer
1
13
(Bruker Daltonics, Billerica, MA) from dimethylsulfoxide solutions. H and C NMR
spectra were recorded in a Bruker Avance HD spectrometer at 400 MHz, DMSO was
used as solvent and TMS as the internal reference. The chemical shifts are expressed in
δ (ppm).
Electronic spectra in the UV-Vis range (190-820 nm) were obtained on a diode
array Hewlett-Packard 8452A or Varian Cary 50 spectrophotometer in dimethylsufoxide
solutions using a 1.0 cm path length quartz cell.
Fluorescence measurements and fluorescence quantum yields were recorded in a
1.0 cm optical path length of a quartz cuvette using a Varian Cary Eclipse or Shimadzu
RF5301-PC spectrofluorimeter with a concentration of 1x10-5 mol.L-1 to minimize the
re-absorption. The equipment was set at 1.5 nm slit width and 600 nm.min-1 scan rate
for both excitation and emission spectra. The solutions were always purged with
nitrogen prior to the measurements to avoid quenching by dioxygen molecules.
Re-absorption of the fluorescence was minimized using absorbance values smaller than
0.100 for a standard 1.0 cm optical path length quartz cuvette.
Green crystals were isolated after the slow diffusion of layers of water and
methanol into a dimethylsulfoxide solution of [Cu2(L1)2]. A single crystal fixed on a
glass fiber was used for the X-ray data collection. Data were collected with a Bruker
APEX II CCD areadetector diffractometer and graphitemonochromatized Mo-K
radiation using COSMO program [36]. Cell refinement, data reduction and the
absorption correction were performed using SAINT and SADABS programs, respectively
[36]. The structure of complex 1 were solved by direct methods [37] and refined on F2

7
with anisotropic temperature parameters for all non H atoms [38]. The crystallographic
parameters and details of data collection and refinement are given in Table 1. Further
details about structure refinement can be found in the Supplementary Section.
2.3. DNA-binding experiments
Fluorenyl ligand and copper(II) compounds interactions with calf-thymus DNA
(ct-DNA) were performed by spectral measurements at room temperature in phosphate
buffer (PBS) at pH 7.4, using DMSO stock solution of derivatives (10-4 mol.L-1 range).
The DNA pair base concentrations of low molecular weight DNA from calf thymus
DNA was determined by spectroscopy, using the molar extinction coefficients 6.600
L.mol-1.cm-1 at  = 260 nm, respectively.
Compound solutions in dimethylsulfoxide with PBS buffer were titrated with
increasing concentrations of ct-DNA (0-100 mol.L-1). The absorption spectra of
derivatives were acquired in the wavelength range of 300-800 nm. The intrinsic binding
constants (Kb) of fluorenyl compounds were calculated according to the decay of the
absorption bands of compounds using the following Equation (1) through a plot of
[DNA]/(a - f) versus [DNA]:
[DNA]/(a - f) = [DNA]/(b - f) + 1/Kb(b - f)
(1)
where [DNA] is the concentration of ct-DNA in the base pairs, a is the extinction
coefficient (Aobs/[compound]), b and f are the extinction coefficients of free and fully
bound forms, respectively. In plots of [DNA]/(a - f) versus [DNA], Kb is given by the
slope/interception ratio.

8
In the emission fluorescence analysis with ct-DNA, compounds were dissolved in
DMSO and competitive studies were performed through the gradual addition of the
stock solution of the derivatives to the quartz cuvette (1.0 cm path length) containing
ethidium bromide (EB, 2.0 x 10-7 mol.L-1) and DNA (1.0 x 10-5 mol.L-1) in a PBS buffer
solution. The concentration of compounds ranged from 0 to 100 mol.L-1. Samples
were excited at exc = 510 nm and emission spectra were recorded at the range of 550-
800 nm, 3 min after each addition of the complex solution in order to allow incubation
to occur. To distinguish the type of quenching mechanism and the extent of quenching,
the Stern–Volmer type Equation (2) can be used [39]:
F0/F = 1 + kq0[Q] = 1 + KSV[Q]
(2)
where, F0 is the original fluorescence intensity; F is the quenched intensity of the
fluorophore in the presence of the compound (quencher); Q is the quencher, Ksv is the
Stern–Volmer quenching constant; kq is the apparent bimolecular quenching rate
constant and equal to Ksv/0.

9
3. Results and discussion
Structural description of [Cu2(L1)2].2H2O
Suitable crystals for X-ray analysis were obtained after diffusion of water and
methanol into the dimethylsulfoxide solution of [Cu2(L1)2].2H2O. Figure 1 shows the
representation of the molecular structure of the [Cu2(L1)2] unit in [Cu2(L1)2].2H2O.
Crystal structure analysis reveals that two units of L1 bind two copper(II) ions in a
2N2O fashion, through phenolate and imine groups. The flexible ligand is wrapped
around the metal center, which is tetracoordinated and experiences a distorted square
planar geometry. Main angles are O1-Cu1-N1, O1-Cu1N11, N1-Cu1-O11 and N11-
Cu1-O11 at 92.9(4), 90.3(3), 91.8(4) and 94.2(4)o, respectively. The crystallographic
parameters and details of data collection and refinement are given in Table 1. Selected
bond distances and angles are listed in Table 2. Li and co-workers [35] reported on the
crystal structure of the monohydrate complex [Cu2(L1)2].H2O that crystallizes in the
monoclinic system and space group P21/n. Therefore, it is different from the dihydrate
compound ([Cu2(L1)2].2H2O) herein described, which shows an overall composition of
C68H64Cu2N8O10 , a molecular mass of 1280 g.mol-1 and, forms orthorhombic crystals in
the space group Pccn.

10
Figure 1. Projection of the molecular structure of the [Cu2(L1)2] unit in [Cu2(L1)2].2H2O
with the atom labeling scheme. [40]. Atoms represented with arbitrary radii. Water
molecules as crystallization solvents were omitted for clarity.

11
Table 1. Crystal data and structure refinement for [Cu2(L1)2].2H2O.
Empirical formula
Formula weight, g.mol-1
C68H64Cu2N8O10
1280.35
T (K)
296(2)
Radiation, λ / (Å)
1.54178
Crystal system, space group
Unit cell dimensions, a, b, c /Å
Orthorombic, Pccn
a = 27.6139(10)
b = 19.5971(7)
c = 27.5767(10)
14923.2(9)
Volume /Å3
Z, Calculated Density /g.cm3
Absorption coefficient /mm1
F(000)
8, 1.140
1.154
5328
Crystal size (mm3)
0.305 x 0.294 x 0.144
2.765 to 60.189
-30 h  30
-22 k  22
-31  l  30
199757
Theta range /°
Index ranges
Reflections collected
Independent reflections
Completeness to theta max
Refinement method
11076 [Rint =0.0756 ]
99.3%
2
Full-matrix leastsquares on F
Data / restraints / parameters
11057 /42/781
2
1.513
Goodness-of-fit on F
R1 = 0.1425,
wR2 = 0.3893
R1 = 0.1978,
wR2 = 0.4138
Final R indices [I >2(I)]
R indices (all data)
Largest diff. peak and hole /e.Å3
-3 *
1.915 and -0.743 e.Å
* Highest peak 1.91 at 0.2874 0.7607 0.1616 [ 3.28 Å from H86 ].
*Deepest hole -0.74 at 0.4307 0.0990 0.0466 [ 0.20 Å from O15 ].

12
Table 2. Selected bond lengths / Å and angles / ° for [Cu2(L1)2].2H2O.
O(1)-Cu(1)
O(11)-Cu(1) 1.880(8) O(1)-Cu(1)-N(11)
N(1)-Cu(1) 1.964(10) O(11)-Cu(1)-N(1)
N(11)-Cu(1) 1.963(8) O(11)-Cu(1)-N(11)
O(4)-Cu(2) 1.888(6) O(11)-Cu(1)-O(1)
O(14)-Cu(2) 1.883(7) N(11)-Cu(1)-N(1)
N(4)-Cu(2) 2.007(8) O(14)-Cu(2)-O(4)
N(14)-Cu(2) 1.968(9) O(14)-Cu(2)-N(14)
1.880(7) O(1)-Cu(1)-N(1)
92.9(4)
90.3(3)
91.8(4)
94.2(4)
155.2(3)
158.2(3)
154.5(3)
93.5(3)
symmetry transformations used to generate equivalent atoms: () -1+x, y, z.
Despite the fact that compound [Cu2(L1)2].2H2O presents two water molecules
as crystallization solvates, curiously, the crystal structure of the complex shows
predominantly intramolecular hydrogen bonding, while the bifurcated intermolecular
hydrogen bondings discussed below, do not include the crystallization solvates. The
crystal structure of [Cu2(L1)2].2H2O in the centrosymmetric Pccn space group, reveals
an infinite one-dimensional 1D-chain with the base vector [010]. [Cu2(L1)2] molecules
are related by a two-fold screw axis along the [010] crystallographic direction with
screw component [0,1/2,0]. The 1D chain results from hydrogen bonds with a bifurcated
acceptor geometry (D1H1, D2H2)∙∙∙A (D = donor atom, A = acceptor atom), (N12-
H12, C75H75)∙∙∙O3, symmetry code () 1-x, -1/2+y, 1/2-z (Figure 2). Tab. 3 lists the
geometrical parameters of the bifurcated hydrogen bonding, which presents a classical
hydrogen bonding component N12-H12∙∙∙O3, and, a non-classical hydrogen bonding
component C75H75∙∙∙O3, respectively connected to the same acceptor O3 atom. Fig.
2 depicts a section of the supramolecular arrangement of the [Cu2(L1)2] molecules in
the unit cell of [Cu2(L1)2].2H2O as discussed above, symmetry codes ():1-x, -1/2+y,
1/2-z ; (): x, -1+y, z ; (): 1-x, 1/2+y, 1/2-z).

13
Table 3. Hydrogen-bonding geometric parameters (Å, °)
DH∙∙∙A
DH
H∙∙∙A
D∙∙∙A
DH∙∙∙A
N12H12∙∙∙O3
C75H75∙∙∙O3
0.86
0.98
2.080
2.560
2.914(9)
163.15
149.49
3.413(10)
(D = donor atom, A = acceptor atom)
Symmetry code: () 1-x, -1/2+y, 1/2-z

14
Figure 2. Section of the supramolecular arrangement of the [Cu2(L1)2] molecules
related by the along the [010] crystallographic direction in the unit cell of
[Cu2(L1)2].2H2O symmetry codes ():1-x, -1/2+y, 1/2-z ; (): x, -1+y, z ; (): 1-x, 1/2+y,
1/2-z). Radii of the represented atoms are arbitrary [40].

15
1
3.1. Characterization of compounds. Chemical composition, vibrational, H- and
13C-NMR and mass spectra.
Elemental analyses of compounds are in accordance with the assigned chemical
composition of the compounds. Infrared spectra showed vibrations that are
characteristic of group functions expected for these compounds, such as (O-H) at 3296
cm-1, (N-Hamide) between 1666 and 1620 cm-1 and (C=C) at 741-727 cm-1 [41].
[Mg2(L2)](NO3)4 also showed the stretching mode (N-O) of nitrate ions at 1332-1365
cm-1. Other assignments are shown in Table 4.
Table 4. Vibrational modes of ligands L1 and L2 and their complexes.*
Assignment
(O-H),(N-H)
(C-Haromatic
(C-Halifatic
(C-Hmethyl
L1
CuL1
MgL2
CuL2
3296
3296
3331
3317
)
3061, 2932 3045, 3018 3047, 2908 3035, 2902
2863
-
2895
-
2835
2917
2846
2910
1635
1213
1527
740-727
-
)
)
(C=Oamide),δ(N-Hamide),(N=C) 1666, 1636 1646, 1602 1646, 1614
δ(C-Oaromatic
(C=Caromatic
(C=Caromatic, (C-Haromatic
 (N-Onitrate
 (Cl-Operchlorate
(Cu-N),(Cu-O)
)
1261
1315
1205
1430
1529
1521
)
)
755-728
739-712
741-727
)
-
-
-
-
-
1332-1365
)
-
-
1068
603
609
*CuL1 = [Cu2(L1)2]∙2H2O; MgL2 = [Mg2(L2)](NO3)4; CuL2 = [Cu2(L2](ClO4)4∙4H2O

16
1H- and 13C-NMR spectra of L1 and [Mg2(L2)](NO3)4 are seen in Supplementary
Material Section Figures SM1-SM4, and chemical shifts and integration values in
Tables SM1 and SM2. Hydrogen signals in L1 were assigned as H11 and H12 (6.844-
6.888 ppm, m, 4H,), H13, H14 and H15 (7.284-7.359 ppm, m, 6H) from the phenol ring
and H1 (7.833 ppm, d, 2H) and H2 (7.887, t, 2H) are from the fluorene unit. Hydrogen
H7 from the amide group was not observed, due to the rapid isotopic exchange with the
solvent. Signals at 3.3-3.6 ppm and at 2.5 ppm are from water and dimethylsulfoxide
13
molecules, in that order. C-NMR of L1 showed C14-C17 in the high frequency range
of the spectrum and chemical shifts at 125.304-118.420 ppm, while C13 experience the
electron withdrawing effect of both the –OH and imine (-N=C-C13) and appears at  =
144.304 ppm. This assignment was confirmed since the signal is absent in the DEPT-
135 spectrum as seen Figure SM2. Signals from carbons C5, C6 and C9 were also not
seen in the DEPT-135 experiment, due to their quaternary nature.
1H-NMR spectrum of [Mg2(L2)](NO3)4 showed broad collapsed signals like H1
from the aromatic rings at 7.195-8.534 ppm (m, 28H). The integration of this region
suggests a combined signal with amide and imine hydrogen atoms. The six hydrogen
atoms from the methyl group resonate in high frequency at 2.156-2.350 ppm. Methylene
(-CH2-) and methyl (-CH3) hydrogens C10, C11 and C16 resonate with  at 39.815,
37.704 and 38.672 ppm, respectively. Phenol carbon, C17-OH, shows resonance at
168.4811 ppm, close to the value found for C18-OH at 168.069 ppm in L1. Other
assignments are in Table SM2.

17
Figure SM1. 1H-RMN spectrum of L1 in d6-dmso

18
a)
b)
Figure SM2. a) DEPT 13C-RMN spectrum of L1 in d6-dmso; b) regular spectrum with
expansion

19
Figure SM3. 1H-RMN spectrum of [Mg2(L2)](NO3)4 in d6-dmso.

20
Figure SM4. a) DEPT 13C-NMR spectrum of [Mg2(L2)](NO3)4 in d6-dmso; b) regular
spectrum with expansion

21
Table SM1. 1H- and 13C-RMN data of ligand L1 in d6-dmso.
1Ha
Integration
13Cb
(ppm)
(ppm)
2.853, d
3.465 – 3.721, m
4.667, d
6
8, 9
5
1.027 (1H) 166.801
7.958 (8H) 131.597
1.00 (1H) 127.489
4.074 (4H) 127.005
2.101 (2H) 140.334
1
2
3
6.844 – 6.888, m
7.191, t
11,12
3
4
5
7.284 – 7.359, m 13, 14, 15 6.362 (6H) 160.496
6
7.411, d
7.833, d
7.887, t
8.390, s
4
1
2.166 (2H) 57.771
2.177 (2H) 45.968
1.804 (2H) 132.251
1.712 (2H) 57.653
7
8
2
9
10
10
masked 11
116.382 12
144.304 13
125.304 14
119.852 15
118.580 16
118.420 17
168.069 18
a numbering refer to Figure SM1; b numbering refer to Figure SM2

22
Table SM2. 1H- and 13C-RMN data of [Mg2(L2)](NO3)4 in d6-dmso.
1Ha Integration
13Cb
(ppm)
(ppm)
2.156 – 2.350, m
2.775 – 2.857, m
3.012, d
6
5
3
4
2
1
5.891 (6H)
8.015 (8H)
2.052 (2H)
8.190 (8H)
2.00 (2H)
127.653
127.007
126.968
125.219
144.430
1
2
3
3.311 – 3.352, m
4.655, d
4
5
7.195 – 8.534, m
27.781 (28H) 140.493
57.32
6
7
46.375
8
144.135
9
masked
10
11
12
13
14
15
16
37.735
119.997
144.261
125.136
140.398
38.720
168.4811 17
a numbering refer to Figure SM3; b numbering refer to Figure SM4

23
High resolution mass spectrum (HRMS-ESI) of L1 in the negative mode
exhibited a peak at m/z = 559.53, in accordance with the loss of H+ and formation of
[L1-H]- (Figures 3 and 5A). The spectrum of [Mg2(L2)](NO3)4 also in the negative
mode showed a peak at m/z = 209.95 assigned to a tetraaza-fragment, which is
coordinated to a single magnesium ion, as seen in Figures 3 and 5B, and a low intensity
peak at m/z = 982.99 due to the less stable species [Mg(L2)-H]- (Figures 3 and 5C). The
spectrum in the positive mode of [Mg2(L2)](NO3)4 showed a relatively intense signal at
m/z = 961.43, attributed to [L2]+ (Figures 3 and 5D). The assignments are in accordance
with the calculated fragmentation patterns, considering the isotopic distribution of the
elements.
The copper(II)-complexes were also characterized by ESI mass spectrometry.
[Cu2(L1)2(H2O)2] exhibited two sets of fragments in the negative mode at 620.15 and
1279.30, assigned to the [Cu(L1)-H+]- and [Cu2(L1)2(H2O)2]-H+]-, respectively (Figures
4, 5E and 5F). The macrocyclic complex [Cu2(L2)]4+ showed a major set of signals
centered at m/z = 543.14, in the positive mode, and assigned to [Cu2(L2)-2H+]2+
(Figures 4 and 5G). We believe that the more symmetric tetraazacoordination is the
most plausible one, as seen for the magnesium ions. However, considering the large size
and high flexibility of the macrocycle ligand, an alternative mode of coordination
through 3N1O could be possible and has support from the EPR measurements that will
be discussed below.

24
Figure 3. HRMS-ESI negative mode of (top) L1 and (middle) and (bottom)
[Mg2(L2)](NO3)4 dissolved in DMSO. Insets show the three most intense peaks of the
calculated fragmentation patterns (see Figure 5) considering the isotopic distribution of
the elements.

25
Figure 4. HRMS-ESI negative mode of (top) [Cu2(L1)2].2H2O and (bottom)
.
[Cu2(L2)](ClO4)4 4H2O dissolved in DMSO. Insets show the three most intense peaks of
the calculated fragmentation patterns (see Figure 5) considering the isotopic distribution
of the elements.

26
Figure 5. Molecular peaks and fragments assignments according to the experimental
and calculated data in Figures 3 and 4.

27
EPR spectroscopy of [Cu2(L1)2]∙2H2O and [Cu2(L2](ClO4)4∙4H2O
Figure
6
shows the EPR spectra of [Cu2(L1)2(H2O)2] and
[Cu2(L2)](ClO4)4∙4H2O in the solid state and in DMSO:MeOH (1:1 v/v). The spectra of
powdered samples of [Cu2(L1)2(H2O)2] (Cu…Cu at 7.435 Å) does not show any
half-field spin transition, which would be expected if the metal ions were magnetically
coupled. The same behavior was reported in EPR investigation of other binuclear
copper complexes, where the metal centers are even closer, with a Cu…Cu distance of
3.33654(11) Å [42]. In contrast, [Cu2(L2)](ClO4)4∙4H2O exhibits the half-field
transition at a resonance field around 1700 G (Figure 6C), in accordance with the
dicopper(II) composition, and suggesting the metal ions are bridged through the
phenolate oxygen, as given in Figure 5G.
The EPR spectra from frozen solutions at 77K are characteristic of magnetically
diluted mono and binuclear species and show the ms = -½ → ms = ½ transition of a 3d9,
S = ½ ion, which is split in four lines by the hyperfine coupling with the nuclear
magnetic spin of copper ion (I = 3/2) [43]. Spectra of solutions were simulated [44] and
the results are collected in Table 5. Parameters were correlated using Peisach and
Blumberg [45] criteria applied to both parallel hyperfine coupling (Az) and
gyromagnetic component (gz) to help establish the mode of coordination of the ligands.
Based on this analysis, it is suggested a 2N2O and a 3N1O coordination, the oxygenated
ligand coming from water, dimethylsulfoxide or a methanol molecule. It is important to
emphasize that this proposal is presented for species in solution (cf. last column of table
3) and does not exclude the 4N-coordination in the solid state. The complexes showed
some degree of rhombicity as given by different values of gx, gy and gz, but the relation
gz > gx,y indicates a major axial symmetry, characteristic of the unpaired electron of
copper(II) occupying the dx2 y2 orbital [45]. The rhombic character of the complexes is
-

28
most likely caused by different bond lengths between Cu(II) and the N- and O-donor
atoms aggravated by the high flexibility of the ligands.
Figure 6. EPR spectra of [Cu2(L1)2].2H2O (A) solid at room temperature and at 77 K;
(B) in DMSO/MeOH (1:1 v/v) solution; (C) of [Cu2(L2)](ClO4)4∙4H2O solid at room
temperature and at 77 K (inset shows the half field transition observed at 77 K); (D) in
DMSO/MeOH (1:1 v/v) (the bottom spectra are the component species of the
simulation, cf Table 3).

29
Table 5. Parameters for the EPR simulations. 77 K frozen solution spectrum simulation
demonstrates a three species composition for sample Cu2(L2), with 3N1O copper
coordination scheme dominant.
Giromagnetic tensor
Hyperfine constant/MHz
Composition
fraction (%)
Donor atoms
gx
gy
gz
Ax
Ay
Az
Cu2(L1)2
Cu2(L2)
2N2O
2.025
2.074
2.231
46.0
53.3
541
100
2N2O
3N1O
2.015
2.078
2.083
2.079
2.300
2.240
> 20
> 20
515
544
21
48
4O
2.052
2.082
2.400
> 20
404
31
Photophysics of the L1 and [Mg2(L2)](NO3)4
The photophysical properties of L1 and [Mg2(L2)]4+ were measured in
dimethylsulfoxide as seen in Figure 7. UV-vis spectra showed strong intraligand →*
and n→* transitions with maximum molar absorptivities of 4.22x103 L.mol-1.cm-1 at
262 nm for ligand L1, and 2.91x103 L.mol-1.cm-1 at 278 nm for [Mg2(L2)]4+. Excitation
at 278 nm produced emission at 307 nm, which is characteristic of the fluorene
chromophore.
Quantum yields (f) of L1 and [Mg2(L2)]4+ were determined as 1.2% and 5.2%,
respectively, by the method of Brouwer and co-workers [44] using tyrosine (fref = 0.13
± 0.1, 23ºC) as a reference and calculated using Equation 2.