A family of hydroxypyrone ligands designed and synthesized as iron chelators

Article abstract of DOI:10.1016/j.jinorgbio.2013.06.009

The use of chelating agents for iron and aluminum in different clinical applications has found increasing attention in the last thirty years. Desferal, deferiprone and deferasirox, chelating agents nowadays in use, are based on hydroxamic groups, hydroxyl-substituted pyridinones or aromatic ring systems. With the aim of designing new chelators, we synthesized a series of kojic acid derivatives composed by two kojic units joined by linkers variously substituted. The huge advantages of these molecules are that they are easy and cheap to produce. Preliminary works on complex formation equilibria of the first group of ligands with iron and aluminium highlighted extremely good pMe values and gave evidence of the ability to scavenge iron from inside cells. On these bases a second set of bis-kojic ligands, whose linkers between the kojic chelating moieties are differentiated both in terms of type and size, has been designed, synthesized and characterized. The structural characterization of these new ligands is presented, and the protonation and iron^III complex formation equilibria studied by potentiometry, UV-Visible spectrophotometry, electrospray ionization mass (ESI-MS) and ¹H NMR spectroscopy will be described and discussed.

Full text of DOI:10.1016/j.jinorgbio.2013.06.009

Journal of Inorganic Biochemistry 127 (2013) 220–231
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
A family of hydroxypyrone ligands designed and synthesized as
iron chelators
a
a
a
Leonardo Toso a, Guido Crisponi a, , Valeria M. Nurchi , Miriam Crespo-Alonso , Joanna I. Lachowicz ,
M. Amelia Santos b, Sergio M. Marques b, Juan Niclós-Gutiérrez c, Josefa M. González-Pérez c,
Alicia Domínguez-Martín c, Duane Choquesillo-Lazarte d, Zbigniew Szewczuk e
⁎
a
Dipartimento di Scienze Chimiche e Geologiche, Università di Cagliari, Cittadella Universitaria, 09042 Monserrato Cagliari, Italy
Centro Quimica Estrutural, Instituto Superior Tecnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
Department of Inorganic Chemistry, Faculty of Pharmacy, Campus Cartuja, University of Granada, E-18071 Granada, Spain
Laboratorio de Estudios Cristalográficos, IACT, CSIC-Universidad de Granada, Av. de las Palmeras 4, E-18100 Armilla, Granada, Spain
b
c
d
e
Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50-383 Wroclaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The use of chelating agents for iron and aluminum in different clinical applications has found increasing
attention in the last thirty years. Desferal, deferiprone and deferasirox, chelating agents nowadays in use,
are based on hydroxamic groups, hydroxyl-substituted pyridinones or aromatic ring systems. With the aim
of designing new chelators, we synthesized a series of kojic acid derivatives composed by two kojic units
joined by linkers variously substituted. The huge advantages of these molecules are that they are easy and
cheap to produce. Preliminary works on complex formation equilibria of the first group of ligands with
iron and aluminium highlighted extremely good pMe values and gave evidence of the ability to scavenge
iron from inside cells. On these bases a second set of bis–kojic ligands, whose linkers between the kojic
chelating moieties are differentiated both in terms of type and size, has been designed, synthesized and
characterized. The structural characterization of these new ligands is presented, and the protonation and
ironIII complex formation equilibria studied by potentiometry, UV–Visible spectrophotometry, electrospray
ionization mass (ESI-MS) and 1H NMR spectroscopy will be described and discussed.
Received 26 December 2012
Received in revised form 15 June 2013
Accepted 17 June 2013
Available online 26 June 2013
Keywords:
Kojic acid
FeIII
Solution equilibria
X-Ray structure
Synthesis
Hydroxypyrones
© 2013 Elsevier Inc. All rights reserved.
1. Introduction
In a previous work, evidence was given on the formation of MeL,
MeL2, and MeL3 complexes of AlIII and FeIII with kojic acid, confirmed
In the last two decades there has been an increasing interest in the
use of chelation therapy for various health diseases depending on iron
and aluminium overload [1–6]. Drugs in current use, such as desferal,
deferiprone and deferasirox, include 1–3 chelating groups, typically
hydroxamic groups, hydroxyl-substituted pyridinones or aromatic
ring systems. In the above mentioned reviews the numerous drawbacks
limiting in some instance the use of these drugs are presented and
discussed, and the need of new effective iron chelators is stressed.
With the aim of designing new ligands that form complexes with high
stability, selectivity, lipophilicity and bioavailability, which satisfy both
the chemical requirements and biological constraints for an effective
therapeutic agent, we synthesized some derivatives of kojic acid,
and studied their complex formation equilibria with FeIII and AlIII, as
well as those with the parent ligand kojic acid, 5-hydroxy-2-
(hydroxymethyl)-4H-pyran-4-one, (Scheme 1).
by the X-ray structure of the FeL3 complex, and of diverse protonated
species of Me2L2 and MeL2 complexes with L1 [7]. On the basis of the
extremely good pFe value (23.1) characterizing L1, and of its ability to
scavenge iron from inside cells [8], we extended the investigation to
the related compounds in which vanillin and ortho-vanillin (L2 and
L3) substituents were inserted on the linker joining the two kojic
units. Similar complexes to those formed with L2 were found. In the
binuclear Me2L2 complexes, each metal ion is coordinated by two
COC(OH)-chelating moieties, one from each coordinating molecules:
actually, the length of the linker between the two kojic units prevents
metal ion coordination by both kojic units on the same molecule. The
found pFe values (18.9 for L2 and 22.2 for L3), lower than that of
desferal (26.6) and comparable with that of deferiprone (20.7), were
very encouraging [9,10]. These ligands are easy and cheap to produce,
as the starting materials, kojic acid and vanillin, are not expensive.
Hence, they are worthy of further tests on their toxicity and their
capacity to remove iron and/or aluminium from intra-cellular sites in
living organisms. Aimed at improving the interaction between the
kojic moieties and the metal ions, we have designed and synthesized
a new set of bis–kojic ligands whose linkers (between the two kojic
⁎
Corresponding author at: Dipartimento di Scienze Chimiche e Geologiche, Cittadella
Universitaria, 09042 Monserrato, Cagliari, Italy. Tel.: +39 070 675 4476; fax: +39 070 675
4478.
E-mail address: crisponi@unica.it (G. Crisponi).
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.06.009

L. Toso et al. / Journal of Inorganic Biochemistry 127 (2013) 220–231
221
Scheme 1. Chemical structures and acronyms of studied ligands.
chelating moieties) are differentiated both in terms of type and size
(Scheme 2).
Biofutura Pharma product. All the products were used without any
further purification.
In this paper we will report the synthesis of the new derivatives,
namely L4, L5, L6, and L7 containing an amine group in the linker and
also L8, which is a L1 analog but with insertion of a methyl group in
the linker. The acid-base properties, together with the equilibrium
constants for the formation of FeIII complexes as well as the structure
characterization of L6, L7 and L8 by X-Ray diffraction will be presented
herein.
A previous described method was used in the preparation of 0.1 M
carbonate free KOH solution [11]. Ligand solutions were acidified
with stoichiometric equivalents of HCl. FeIII solution was prepared by
dissolving the required amount of FeCl3 in pure double distilled water
to which a stoichiometric amount of HCl was previously added to pre-
vent hydrolysis. This solution was standardized by spectrophotometric
titration with desferal.
2. Experimental
2.2. Synthesis
2.1. Reagents
All the ligands were prepared from 6-alkylation of kojic acid (KA)
with an aldehyde and a primary amine or an inorganic base, depending
on desired presence or absence of amine group on that alkyl substituent.
The particular operations are reported in the following for each single
ligand. For each ligand preparation, a KA (1.00 g, 7.1 mmol) solution
in ethanol (25 mL) was used. All the reactions were followed by TLC,
using a CH2Cl2:MeOH (12:1) mixture as eluent. The synthetized products
Kojic acid, benzylamine hydrochloride, piperazine, methylamine
hydrochloride, NaOH, KOH, FeCl3, methanol, ethanol and 2-isopropanol
were purchased from Aldrich, and N-benzylmethylamine was an Alfa
Aesar product. Formaldehyde was purchased from Merck, HCl from
Fluka, and KCl and ZnO from Carlo Erba (Milan, Italy). Desferal was a

222
L. Toso et al. / Journal of Inorganic Biochemistry 127 (2013) 220–231
Scheme 2. Chemical structures and acronyms of studied ligands.
were fully characterized by different standard instrumental techniques.
NMR spectra were recorded on a Bruker AVANCE III spectrometer at
300 MHz and 400 MHz for 1H NMR and 13C NMR respectively. Chemical
shifts (δ) are reported in ppm related to tetramethylsilane (TMS). Infra-
red spectra were recorded by using KBr pellets on a Jasco FT-IR 410
spectrometer. The electrospray ionization mass spectra (ESI-MS) were
obtained on a 500 MS LC Ion Trap (Varian Inc., Palo Alto, CA, USA)
mass spectrometer equipped with an ESI ion source, operated in the
positive and negative ion modes. The elemental analyses were
performed on a Fisons EA1108 CHNS-O within the limit of 0.4%.
Although some of the compounds gave unsatisfactory analysis values,
for which found values differ by more than 0.5% from Calc. values,
crystal structures of all of these compounds were determined by X-ray
diffraction (XRD) technique or their structures were supported by spec-
troscopic (FT-IR, 13C-NMR and ESI-MS) analyses.
ethanolic solution. The solution was left under stirring for 3.5 h in a
water bath at 40/50 °C. Solvent was removed under vacuum. Red impu-
rities were removed from the white precipitate by several double
distilled cold water washings, filtrations and dryings under vacuum.
Recrystallization yielded the pure final product (0.84 g, 57%).
Mp = 126–128 °C. Elemental analysis found (calc. for C21H21NO8): C,
59.43 (60.72); H, 5.58 (5.10); N, 3.42 (3.37). FT-IR (KBr, 4000–
400 cm−1): two types of νas(CH2) 2960 and 2925, two types of
νs(CH2) 2888 and 2851, ν(C_O) 1644, δ(CH2) 1454, ν(C\N)tertiary amine
1360, ν(C\O\C)cyclic 1223, π(C\H)ar (kojic) 858, π(C\H)ar (benzyl)
747 and 701. 1H-NMR (300 MHz, MeOD): δ 7.203(5H,Ph), 6.295(2H,S),
4.306(4H, s), 3.792(4H, s), 3.751(2H, s). 13C-NMR (300 MHz, MeOD):
δ 139.10(C), 144.86(C), 149.24(C), 169.68(C), 176.27(C), 109.97(CH),
128.59(CH), 129.32(CH), 130.16(CH), 52.26(CH2), 60.44(CH2),
61.21(CH2). ESI-MS(–): MH−(414.2), [M + Cl]−(450.2).
2.2.1. 6,6′-(Methylazanediyl)bis(methylene)bis(5-hydroxy-2-
(hydroxymethyl)-4H-pyran-4-one), L4
2.2.3. 6,6′-(Piperazine-1,4-diylbis(methylene))bis(5-hydroxy-2-
(hydroxymethyl)-4H-pyran-4-one), L6
Methylamine hydrochloride (0.260 g, 3.8 mmol), previously neutral-
ized with NaOH (3.8 mmol), was added to a solution containing formal-
dehyde (580 μL, 7.1 mmol) in ethanol (2 mL). The mixture was stirred
for 30 min at room temperature, then added drop wise to the kojic
ethanolic solution described above. The solution was left under stirring
for 1.5 h in a water bath at 40/50 °C. Solvent was removed under vacuum.
Addition of CH3CN yielded a white precipitate that was filtered off,
washed with cold CH3CN, and dried under vacuum for 6 h. Recrystalli-
zation afforded the pure product as a white solid (1.09 g, 92% yield).
Mp = 150–152 °C. Elemental analysis found (calc. for C15H17NO8): C,
52.76 (53.10); H, 5.48 (5.05);
Piperazine (0.305 g, 3.5 mmol) was added to a solution of formalde-
hyde (580 μL, 7.1 mmol) in ethanol (2 mL). The mixture was stirred for
30 min at room temperature and then added drop wise to a kojic
ethanolic solution. The solution was left under stirring for 5 h during
which a fine white precipitate appeared. Upon refrigeration for 2 h,
the reaction mixture was filtered off, washed with cold ethanol
and dried under vacuum for 6 h. Recrystallization afforded the final
product 1.35 g, 97%). Mp N 350 °C. Elemental analysis found (calc. for
C18H22N2O8): C, 54.18 (54.82); H, 6.07 (5.62); N, 7.17 (7.10). FT-IR
(KBr, 4000–400 cm−1): two types of νas(CH2) 2951 and 2917, two
types of νs(CH2) 2880 and 2839, ν(C_O) 1653, δ(CH2) 1460,
ν(C\O\C)cyclic 1244, π(C\H)ar (kojic) 867, π(C\H)ar (piperazine)
824.1H-NMR (300 MHz, MeOD + KOD): δ 6.120(2H,S), 4.179(4H, s),
3.530(4H, s), 2.490(8H, s). 13C-NMR (400 MHz, MeOD + KOD): δ
148.782 (C), 154.936 (C), 165.674 (C), 183.262 (C), 109.646 (CH),
53.455 (CH2), 55.127 (CH2), 61.801 (CH2). ESI-MS(–): MH−(393.3).
N, 4.20 (4.13). FT-IR (KBr, 4000–400 cm−1): νas(CH3) 2963,
νas(CH2) 2923, νd(CH3) 2888, νs(CH2) 2853, ν(C_O) 1665, δ(CH2)
1460, ν(C\O\C)cyclic 1218, π(C\H)ar (kojic) 854.1H-NMR (400 MHz,
MeOD): δ 6.380(2H, s), 4.350(4H, s), 3.763(4H, s), 2.403(3H, s). 13C-
NMR (400 MHz, MeOD): δ 145.30 (C), 148.03 (C), 169.86 (C), 179.95
(C), 110.09 (CH), 43.11 (CH2), 54.37 (CH2), 61.23 (CH2). ESI-MS(–):
MH−(338.2), [M + Cl]−(374.2).
2.2.3.1. Synthesis of L6 crystal. L6 (15 mg) was dissolved in distilled
water (12 mL), aided by drop wise addition of HCl 0.01 M. Afterwards,
isopropanol (4 mL) was added and the solution was left stirring for
30 min and then filtered into a crystallization device to remove possible
impurities. The solution was left stand at room temperature in order
to evaporate. Evaporation of the solvents was controlled with the aid
of a plastic film. After three weeks, parallelepiped colorless crystals
appeared suitable for XRD.
2.2.2. 6,6′-(Benzylazanediyl)bis(methylene)bis(5-hydroxy-2-
(hydroxymethyl)-4H-pyran-4-one), L5
Benzylamine hydrochloride (0.51 g, 3.5 mmol), previously neutral-
ized with NaOH (3.5 mmol), was added to a solution of formaldehyde
(633 μL, 7.8 mmol) in ethanol (2 mL). The mixture was stirred for
30 min at room temperature and then added drop wise to the kojic

L. Toso et al. / Journal of Inorganic Biochemistry 127 (2013) 220–231
223
the pure final product (4.61 g, 68%). Mp = 207-208 °C. Elemental anal-
ysis found (calc. for C14H14O8): C, 54.20 (55.70); H, 5.03 (4.55). FT-IR
(KBr, 4000–400 cm−1) νas(CH3) 2983, νas(CH2) 2940, νd(CH3) 2879,
νs(CH2) 2850, ν(C_O) 1653, δ(CH2) 1463, ν(C\O\C)cyclic 1265,
π(C\H)ar (kojic) 857. 1H-NMR (300 MHz, D2O + KOD, pH = 6):
6.560(2H, s), 4.530 (4H, s), 1.660 (3H, d).
2.2.5.1. Synthesis of L8 crystal. L8 (10 mg) was dissolved in EtOH
(10 mL) under moderate heating (35 °C) and stirred until a clear color-
less solution was obtained. Afterwards, the solution was filtered into a
crystallization device and covered with a plastic film to control the
evaporation of the solvent. In one week, small parallelepiped colorless
crystals appeared. In order to improve the size of the crystals, successive
filtrations of the mother liquors were performed until suitable crystals
for XRD purposes were collected.
Fig. 1. Centro-symmetrical H2L62+ ion in the crystal of ligand L6. Intra-molecular
H-bonding interactions are depicted in dotted lines.
2.2.4. 2-((Benzyl(methyl)amino)methyl)-3-hydroxy-6-(hydroxymethyl)-
4H-pyran-4-one, L7
2.3. Potentiometric–Spectrophotometric measurements
N-Benzylmethylamine (967 μL, 7.8 mmol) was added to a solution
of formaldehyde (633 μL, 7.8 mmol) in ethanol (2 mL). The mixture
was stirred for 30 min at room temperature and then added drop
wise to the kojic ethanolic solution described above. The solution was
left under stirring for 5 h. Solvent was removed under vacuum until a
white precipitate was formed. Upon refrigeration for 2 h it was filtered
off washed with cold ethanol and dried under vacuum for 6 h. Recrystal-
lization afforded the final pure product (1.69 g, 87%). Mp = 137–139 °C.
Elemental analysis found (calc. for C15H17NO4.): C, 65.12 (65.44); H, 6.14
(6.22); N, 5.11 (5.09). FT-IR (KBr, 4000–400 cm−1) νas(CH3) 2989, two
types of νas(CH2) 2951 and 2938, νd(CH3) 2884, νs(CH2) 2831, ν(C_O)
1662, δ(CH3) 1496, δ(CH2) 1465, ν(C\N)tertiary amine 1360, ν(C\O\C)-
Protonation and complex-formation equilibrium studies were car-
ried out under the same conditions described in a previous publication
[7]. The operating ligand concentrations ranged from 3 × 10−4 to
3 × 10−3 M according to the examined ligand. The studies of complex
formation were carried using constant ligand concentration, and 1:1,
1:2, and 1:3 metal/ligand molar ratios. Combined potentiometric–
spectrophotometric measurements were performed for protonation
equilibria in the 200–400 nm spectral range and in the 400–800 nm
spectral range for FeIII complexes, using 0.2 and 1 cm path lengths.
Protonation and complex formation data were analyzed using the
Hyperquad program [13].
1244, π(C\H)ar (kojic) 853, π(C\H)ar (benzyl) 747 and 701.
cyclic
1H-NMR (300 MHz, D2O + KOD, pH = 12): δ 7.351(5H,Ph), 6.331(1H,
2.4. ESI-MS analysis of complexes
S), 4.417(2H, s), 3.684(2H, s), 3.601(2H, s), 2.249(3H, s).
ESI-MS spectra were carried out on a Bruker microTOF-Q spec-
trometer (Bruker Daltonik, Bremen, Germany) equipped with an ESI
source. Samples were dissolved in water and methanol (molar ratio
2.2.4.1. Synthesis of L7 crystal. L7 (30 mg) was dissolved in a H2O/MeOH
(1:1) mixture (16 mL). After 30 min stirring, a clear colorless solution
was obtained. The solution was filtered into a crystallization device
and covered with a plastic film to control the evaporation of the solvents.
In one week, parallelepiped colorless crystals were collected for XRD
analysis.
1:1) and the final pH was ~7. The ligand concentration was ~10−5
M
and the ligand to metal molar ratio was 1:10. The experiment parame-
ters were as follows: scan range: m/z 100–1600; drying gas: nitrogen;
and temperature: 200 °C, ion source voltage 4500 V, collision energy
10 eV. The instrument was operating in the positive ion mode and cali-
brated externally with Tunemix™ mixture (Bruker Daltonik, Germany).
Analyte solutions were inserted at a flow rate of 3 μl/min. Compass Data
Analysis (Bruker Daltonik, Germany) program was used to determine
the formulae of the complexes. The distance between peaks allowed
calculating the charge of the analyzed ions. In the MS/MS experiments,
the ion corresponding to signal at m/z 392 was selected on the quadru-
pole and subsequently fragmented in the collision cell. Argon was used
as a collision gas. The obtained fragments were directed to the mass
2.2.5. 2-2′-Ethanediylbis(3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-
one), L8
Synthesis of L8 was carried out according to literature methods
[12]. 6.2 g of kojic acid and an equivalent amount of aldehyde were
dissolved in (100 mL) ethanol. The reaction, catalyzed by of concen-
trated ammonium hydroxide (1 mL), was heated for three hours at
60 °C. Then the solvent was left to evaporate and the beje precipitate
was washed with cold ethyl alcohol. Recrystallization from ethanol yield
Fig. 2. View in the ab plane of the H-bonded 3D network of the crystal of L6. A) Chloride ions and water molecules are hosted in the corresponding channels throughout the c axis.
B) Chloride ions and water molecules are removed and the total accessible voids are plotted in green space fill.

224
L. Toso et al. / Journal of Inorganic Biochemistry 127 (2013) 220–231
Fig. 3. Overlapped view of the two non-equivalent molecules within the asymmetric unit in the crystal of ligand L7. Molecule 1 is depicted in darker line whereas molecule 2 is
depicted in lighter line. Numbering scheme is also provided. Intra-molecular H-bonding interactions are omitted for clarity.
analyzer and registered as an MS/MS spectrum. The collision energy was
10 eV.
In addition, one red parallelepiped crystal obtained from the synthe-
sis of one Fe/L6 complex was mounted on a MiTeGen Micromount™
and this sample was used for data collection. Data was collected at the
ESRF synchrotron ID14-1 beamline (λ = 0.9340 Å, 100 K). Previous at-
tempts to determine the structure of these iron compound using con-
ventional X-ray diffraction facilities were unsuccessful provided the
poor diffraction power of the crystal samples. Only a tentative model
for the iron(III) compound Fe2(L6)3 can be proposed due to the limited
resolution achieved in the experiment. Constraints were used to model
the aromatic rings and the piperazine moiety of L6 ligand. The diffuse
scattering contribution from solvent and data noise was removed
using the SQUEEZE procedure in PLATON [17].
2.5. Crystal structure determination
Measured crystals L6, L7 and L8 were prepared under inert condi-
tions immersed in perfluoropolyether as protecting oil for manipulation.
Suitable crystals were mounted on MiTeGen Micromounts™ and these
samples were used for data collection. Data were collected with Bruker
SMART APEX [L6 (293 K), L7 (296 K) and L8 (100 K)] diffractometer.
The data were processed with APEX2 [14] program and corrected for
absorption using SADABS [15]. The structures were solved by direct
methods, which revealed the position of all non-hydrogen atoms.
These atoms were refined on F2 by a full-matrix least-squares procedure
using anisotropic displacement parameters [16]. All hydrogen atoms
were located in difference Fourier maps and included as fixed contribu-
tions riding on attached atoms with isotropic thermal displacement
parameters 1.2 times those of the respective atom. Geometric calcula-
tions were carried out with PLATON [17] and drawings were produced
with PLATON and MERCURY [18]. Additional crystal data and more
information about the X-ray structural analyses are shown in
Supplementary material S1 to S3. Crystallographic data for the structural
analysis have been deposited with the Cambridge Crystallographic Data
Centre, CCDC 914446–914448 from L6 to L8, respectively. Copies of this
information may be obtained free of charge on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: 44 1223 336 033; e-mail:
deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
3. Results and discussion
3.1. Crystal structure of ligands L6, L7, L8
3.1.1. Crystal structure of the ligand L6: (H2L6)Cl2·2H2O
The crystal of ligand L6 (monoclinic system, space group P21/c) con-
sists of one dichlorhydrate acid molecule and two water molecules.
Note that an inversion center is located in the middle of the acid mole-
cule, thus L6 is centro-symmetric (Fig. 1).
The molecule shows the most stable trans-e, e-conformation. The
kojic acid moieties are stabilized by two intra-molecular H-bonds
involving the OH phenol-like groups as donors and the O keto–kojic
groups as acceptors [O14\H⋯O12 (2.742(2) Å, 113.3°)]. This latter fea-
ture has also been observed in closely related kojic-like compounds [9].
Fig. 4. Fragment of one zig-zag 1D chain in the crystal of ligand L7. Note that the OH phenol-like group is involved in a bifurcated interaction acting as H-donors for one intra- and
one inter-molecular interactions. Likewise, the O keto–kojic group also acts as double H-acceptor from one intra- and one inter-molecular interactions.

L. Toso et al. / Journal of Inorganic Biochemistry 127 (2013) 220–231
225
Fig. 5. Asymmetric unit in the crystal of ligand L8. Intra-molecular H-bonding interactions
are plotted. Only one of the disordered positions of the hydroxyl-methyl arm is depicted
for clarity.
Fig. 7. Speciation curves of ligand L7, with the overlapping trends of the heights of UV
bands at 276 nm (△) and 322 nm (◊), obtained by spectral decomposition of the spectra
in Fig. 6 with the Specpeak program (19).
molecules are not equivalent mainly deals with the different torsion
angles defined by the benzyl moiety [molecule 1: bN12–C14–C15–C16
85.78° or molecule 2: bN32–C34–C35–C36 108.05°] (see Fig. 3).
As already described for compound L6, each L7 molecule exhibits
one intra-molecular H-bond that, again, involve the OH phenol-like
group as donor and the O keto–kojic group as acceptor [molecule 1:
O1\H⋯O4 (2.757(2) Å, 112.8°) or molecule 2: O21\H⋯O24 (2.764(2)
Å, 112.6°)]. Moreover, the independent two molecules within the asym-
metric unit are associated by one inter-molecular H-bonding interac-
tion [O28\H⋯N12 (2.901(2) Å, 169.0°)] building pairs of molecules.
In the crystal, the aforementioned pairs of molecules are further
associated by rather strong inter-molecular H-bonds building zig-zag 1D
chains [O1\H⋯O24 (2.700(2) Å, 153.2°) and O21\H⋯O4 (2.654(2) Å,
152.6°)] (Fig. 4).
Table 1
Protonation constants of the ligands at 25 °C, 0.1 M KCl ionic strength, obtained from
potentiometric titration curves using the Hyperquad program [13].
L4
L5
L6
L7
L8
logK1
logK2
logK3
logK4
9.19(3)
7.51(3)
4.38(5)
–
9.01(2)
7.61(2)
3.35(2)
–
8.52(4)
7.81(2)
5.49(5)
1.97(5)
8.49(1)
6.02(2)
9.49(3)
6.69(5)
–
–
–
–
The crystal of L6 is a 3D network built thanks to the involvement of
H2O and H2L62+ and Cl− ions in H-bonding interactions (see Table
S1.3). In particular, chloride ions and water molecules are located in
cavities that extend along the c axis and kept inside due to hydrogen
bonding interactions involving all the donors from L6 acid molecule.
The total accessible void calculated by PLATON [17] is found to be
18.8% of the crystal volume (Fig. 2).
Adjacent chains connect to each other by additional H-bonds (see
Table S2.3) leading to corrugated ribbons. Finally, hydrophobic inter-
actions connect close ribbons to accomplish the 3D architecture of the
crystal.
3.1.3. Crystal structure of the ligand L8
3.1.2. Crystal structure of the ligand L7: (L7)2
The crystal of ligand L8 crystallizes in the triclinic system, space
group P-1. The asymmetric unit consists of just one organic molecule
(Fig. 5).
Ligand L7 crystallizes in the monoclinic system, space group P21/c.
The asymmetric unit of the crystal comprises two crystallographically
independent molecules of ligand L7. The reason why the two former
One of the hydroxyl-methyl arms in L8 is delocalized in three
different positions [O27A 0.393(12), O27B 0.325(14), O27C 0.283(6)].
Fig. 6. Spectra collected during the potentiometric titration of ligand L7. Blue (darker
lines) spectra are collected in the pH range relative to the first deprotonation, pink (lighter
lines) spectra in the pH range relative to the second deprotonation.
Fig. 8. Chemical shifts of CH3 (blue squares), CH2(B) (green triangles) and CH2(K) (red
crosses) of L7 ligand as a function of pH.

226
L. Toso et al. / Journal of Inorganic Biochemistry 127 (2013) 220–231
Table 2
Table 4
Chemical shifts of H3, CH2(K), CH2(B) and CH3 protons in the species LH+2 , LH and L− of
ligand L7, calculated with the HyperNMR program [20].
Complex formation constants of the five ligands with FeIII at 25 °C, 0.1 M KCl ionic
strength, obtained from potentiometric–spectrophotometric data using the Hyperquad
program [13]. Charges are omitted for simplicity.
Species
H3
CH2(K)
CH2(B)
CH3
L4
L5
L6
L7
L8
LH+2
LH
L-
6.51
6.41
6.35
4.49
4.03
3.71
4.49
4.00
3.72
2.98
2.60
2.27
FeLH3
FeLH2
FeLH
–
–
25.25(3)
–
–
–
22.21(3)
–
21.9(2)
–
–
–
–
–
18.4(3)
Fe2L2
–
–
–
–
41.11(4)
Fe2L2H-1
Fe2L3H4
Fe2L3H3
Fe2L3H2
Fe2L3H
Fe2L3
–
–
–
–
38.97(2)
–
Table 3
69.1(1)
66.10(4)
61.6(2)
56.19(1)
–
70.3(3)
66.2(1)
61.2(2)
–
–
Chemical shifts of in the species LH3+, LH2, LH− and L2− of ligands L4 and L5, calculated
with the HyperNMR program [20].
67.2(2)
64.1(2)
59.9(1)
53.5(1)
–
–
–
–
–
–
L4
L5
49.75(1)
49.4(2)
–
55.08(4)
Species
H3
CH2(N)
CH3
H3
CH2(N)
CH(Bn)
FeL2H2
FeL2H
FeL2
FeL2H−1
FeL3
–
–
–
–
–
–
–
–
–
–
–
–
33.0(1)
28.2(2)
22.6(2)
15.7(1)
27.1(1)
16.7
–
–
–
–
LH+3
LH2
LH−
L2−
6.57
6.49
6.39
6.39
4.61
3.87
3.93
3.76
3.11
2.55
2.58
2.31
6.50
6.41
6.32
6.29
4.65
3.98
3.95
3.90
4.61
3.92
3.90
3.73
pFe
18.1
19.3
17.7
20.0
The mean planes of the two kojic moieties define a dihedral angle of
85.02°. This value, and therefore the orientation of the kojic moieties
within the organic molecule, is similar to that described for a related
bis–kojic derivatives of ortho-vanillin [9]. In accordance to the above-
described L6 and L7 ligands, in L8 can also be observed in the corre-
sponding intra-molecular interactions that involve the OH phenol-like
group and the O keto–kojic group [O26\H⋯O25 (2.774(2) Å, 111.63°)
and O36\H⋯O35 (2.756(2) Å, 112.35°)].
The crystal packing of L8 resembles to that of compound L7. Thus,
zig-zag 1D chains are built by symmetry related molecules kinked by
inter-molecular H-bonding interactions [O(26)\H⋯O25 (2.653(2) Å,
147.5°) and O36\H⋯O35 (2.754(2) Å, 154.6°)]. Adjacent chains are
further connected by H-bonds (see Table S3.3) leading to corrugated
ribbons. Again, the 3D framework is reached by hydrophobic interac-
tions between neighboring ribbons.
The ligands L4 and L5 are characterized by three protonation con-
stants, corresponding to the two kojic phenolates and to the nitrogen
atom in the linker; L6 by four protonation constants relative to the
two kojic units and to the two nitrogen atoms in the piperazine ring;
L7 by two protonation constants attributable to the kojic phenolate
and the nitrogen atom; L8 by the two protonation constants relative
to the two kojic phenolates. The parent molecule kojic acid is character-
ized by a logK value of 7.70, and the protonation constant of a nitrogen
atom in a tertiary amine can be roughly estimated from literature
data that range from a value of logK = 8.05 for triethanolamine to
10.70 for triethylamine. As previously reported [7], the L− and LH
species of kojic acid are characterized by UV bands at 315 nm (ε =
5900 M−1 cm−1) and 270 nm (ε = 8700 M−1 cm−1), respectively,
with sharp isosbestic points at 242 nm and 290 nm.
In the following the protonation sequences of the five ligands, based
on the spectrophotometric and 1H NMR results, are proposed. This dis-
cussion starts with the simpler L7 compound, presenting only a kojic
phenol group and a protonable nitrogen atom on the linker. Analysis
of Fig. 6 (spectra a), in which the spectra collected during the potentio-
metric titration are presented, shows the band of the fully protonated
form LH+2 quite similar to that of the protonated species LH of kojic
acid, differing only in the wavelength of the maximum, now shifted
from 270 to 276 nm (ε = 8850 M−1 cm−1). The intensity of this
3.2. Protonation equilibria
The protonation equilibria of the five ligands have been studied by
potentiometry, UV spectrophotometry and 1H NMR spectroscopy. The
protonation constants evaluated from the potentiometric titration
curves at 25 °C and 0.1 M ionic strength for the five ligands are reported
in Table 1.
Scheme 3. Deprotonation sequence of ligand L4.

L. Toso et al. / Journal of Inorganic Biochemistry 127 (2013) 220–231
227
band decreases at about one half of its height during the first deproton-
ation (blue spectra), with the contemporary appearance of a new band
at 322 nm, with sharp isosbestic points at 242 nm and 290 nm. During
the second deprotonation (red curves) a further decrease in the inten-
sity of the band at 276 nm and an increase in that at 322 nm take
place. The band at 322 nm too is shifted to higher wavelengths and its
ε value at the maximum is slightly higher than that of the corresponding
band of fully deprotonated kojic acid. The trends of the peak intensity
after decomposition of the spectra in the Gaussian band-components
[19], overlapping the speciation plot, are clearly visible in Fig. 7.
A similar trend is presented by the chemical shifts of protons H3,
CH2(K), and CH2(B) and CH3 reported in Fig. 8. All these chemical
shifts present an upfield shift during the two deprotonations, more
marked for the three proton groups proximal to the nitrogen atom.
The chemical shifts of all the kind of protons in the species LH+2 , LH
and L−, calculated with the HyperNMR program [20], are reported in
Table 2.
Fig. 9. Spectra collected in pH range 0.8–2.4, from solutions containing L4 = 3.44 ×
10−4 M and FeIII = 1.72 × 10−4 M, using 1 cm path length.
The variations of both UV bands and chemical shifts, about of the
same order of magnitude during the first and the second deprotonation,
could lead to the hypothesis of a micro-dissociation scheme, in which
the first proton is lost in comparable measure both by the nitrogen
atom and by the phenolic group. The logK2 value (6.02) corresponding
to the first deprotonation step, ~1.7 units lower than that of kojic acid
(and much lower than that of a tertiary amine nitrogen atom), implies
a particular stabilization of the monoprotonated species such as the
formation of a hydrogen bond between the phenolate O− and the
charged NH+ nitrogen, or between the phenolic group and the
deprotonated nitrogen. Actually, whether the proton is lost by phenolic
group or by nitrogen atom, the remaining proton is equally shared in a
hydrogen bonding between these groups. Such a situation may well
explain the observed experimental trends in which all the UV and
NMR signals feel in an equal effect on both deprotonation steps.
Ligands L4 and L5 were characterized by the UV spectra with a
maximum at about 275 nm for the fully protonated species, with ε
values almost double with respect to those of kojic acid and ligand L7;
during the first deprotonation step, this band slightly decreased in
both ligands, with the appearance of a new band at about 328 nm; in
the following steps a more marked decrease of the 275 nm band and
an increase of the 328 nm band were observed.
Ligand L8 is characterized by two protonation constants almost
equal to those of L1, and a trend of UV spectra and NMR chemical
shifts similar to that previously reported for L1 ligand was observed.
3.3. Iron complexes
The complex formation equilibria of all the five ligands with FeIII
have been studied by combined potentiometric–spectrophotometric
techniques. In fact, being FeIII almost completely complexed when
Characteristic variations of the chemical shifts of the H3, CH2(N),
and CH3 protons in L4, and H3, CH2(N), and CH(Bn) protons in L5
are observed (Table 3), which allow to propose a sequence in the
deprotonations of these ligands similar to that described above for L7.
Hence, the first deprotonation leads to a neutral species in which a pro-
ton is shared through hydrogen bonding between the nitrogen atom
and one of the two kojic phenol groups, inducing the strong upfield
shift of the groups neighboring to the nitrogen atom, and that of H3 in
the kojic ring. The second deprotonation, taking place on the kojic
moiety not involved in the hydrogen bonding, is characterized both by
a strong spectral variation of the UV bands related to the kojic moiety,
and by an upfield shift of the NMR signals of the H3 proton, while
those of the CH2(N), CH3 and CH(Bn) protons remain unaffected; in
the last step, mainly in L4, a further upfield shift is experienced by
hydrogen atoms neighbor to nitrogen for the loss of the shared proton,
and a further strong spectral variation of the UV bands. This sequence
is briefly sketched in Scheme 3.
Concerning the ligand L6 containing the piperazine ring on the linker,
it is characterized by four protonation constants, logK1 = 8.52, logK2
=
7.81, logK3 = 5.49, and logK4 = 1.97. Presumably the general mecha-
nism of deprotonation could be similar to that observed for L4 and L5,
with the involvement of a further basic nitrogen atom. The low L6
water solubility avoided the acquisition of reliable NMR spectra. Further-
more, the small differences between the protonation constants lead to a
continuous variation with pH of the UV absorbance bands, without any
clear inflection. All these facts prevented to speculate upon the depro-
tonation mechanism.
Fig. 10. Trend of the band absorbances at 468 nm (A) and 545 nm (B) reported as a
function of pH, after decomposition of the spectra in Fig. 9.

228
L. Toso et al. / Journal of Inorganic Biochemistry 127 (2013) 220–231
Fig. 11. Speciation plots calculated on the basis of stability constants reported in Table 4, using a ligand concentration 1 × 10−3 M and FeIII concentration 5 × 10−4 M.
mixing reagents before the base potentiometric titrations, the estima-
tion of reliable complex formation constants from potentiometric data
alone was prevented. Therefore the complex formation equilibria have
been studied both in strong acidic solutions (pH 0–2) on sets of solu-
tions at increasing concentrations of HCl until the disappearance of the
bands of complexed iron, and in the pH range 2–10. Simultaneous fitting
of potentiometric and spectrophotometric data allowed obtaining the
stability constants for the complexes of ligands L4–L8 with FeIII, reported
in Table 4.
The three ligands L4, L5, and L6 show a similar behavior; the visible
spectra collected in strong acidic solutions at different metal/ligand
ratios (1/1, 1/2, 1/3) display the 1:1 complex which at increasing pH
soon disappears giving a Fe2L3Hx complex stable in the 2–4 pH range.
Actually, the shape of the spectra in this pH range is similar for all the
three solutions at different metal/ligand ratios, and this is indicative
that a unique species is formed. The ratios of the absorbance values in
the three solutions clearly indicate the formation of a Fe2L3 complex.
This has been confirmed by a Job plot performed at pH 2.9, which

L. Toso et al. / Journal of Inorganic Biochemistry 127 (2013) 220–231
229
Fig. 12. ESI-MS spectra of the system L4-FeIII in a ratio 1:10 at pH 7.
results in two straight lines intersecting at the ligand mole fraction 0.6.
The spectra collected in acidic solutions for the 1:2 ligand/metal molar
ratios for ligand L4 are reported in Fig. 9 as an example.
The spectra were decomposed in four Gaussian bands, with the
program Specpeak [19]. The absorption of the bands at 468 nm and
545 nm are reported as a function of pH in Fig. 10.
and it was evidenced only by spectral analysis. Already at these low pH
values a Fe2L2 complex is formed with the two iron atoms joined by the
two ligands. This complex is further stabilized by the loss at pH 2.1 of a
proton from a coordinated water molecule, presumably a μ water join-
ing the two iron atoms. At pH ≈4 a Fe2L3 complex is formed in which
each iron atom is completely surrounded by six oxygen atoms from
three kojic units.
The ESI-MS spectra, collected at pH ~7, with a 10:1 metal excess,
allows revealing further complexes not lighted by the other solution
equilibrium techniques. The ESI-MS spectrum of FeIII complexes with
L4 is illustrated in Fig. 12. The signal at m/z 393.004 reveals a [L42Fe2]2+
complex formation. The peak at m/z 315.991 corresponds to [L42Fe2]2+
complex without one methylene-kojic acid molecule. Formation of this
complex is probably due to cleavage of the N\C bond during ionization
of the [L42Fe2]2+ complex. The signal corresponding to free ligand is
not present.
The ESI-MS spectrum in Fig. 13 displays the L5 complex formation.
The main peaks appear at m/z 392.026, m/z 469.040 and m/z 478.045.
The peak at m/z 469.040 corresponds to complex [L52Fe2]2+, while
peak at m/z 478.045 represents its hydrated form [L52Fe2]2+. Such
type of complex is stable even without one methylene kojic acid mole-
cule, since the peak at m/z 392.026 is very intense. The peak corre-
sponding to the free ligand is not present.
The speciation plots shown in Fig. 11, calculated on the basis of
stability constants reported in Table 4, allow some considerations: at
very acidic pH (~1) a FeLH2 complex with the two ligands L4 and L5 is
formed, in which presumably FeIII is coordinated by the two oxygen
atoms of a kojic unit, being the nitrogen atom and the second kojic
unit till protonated. At increasing pH a Fe2L3H3 complex appears, in
which the two iron atoms are joined by the three ligands, each bonding
the two metal ions through its kojic moieties; the complex Fe2L3H4
formed only by the ligand L4 is presumably an intermediate in which
two ligands join the two metal ions, and the third ligand binds only
one iron atom with a kojic unit, the second one till protonated. Further
pH increase induces progressive deprotonations with pK's ~ 4.5, 5.4
and 6.4 for L4 and 3.1, 4.2 and 6.4 for L5. These deprotonations can be
reasonably attributed to the loss of protons from the nitrogen atoms in
the linker.
A similar behavior is presented by L6, in which a FeLH3 complex is
initially formed, with iron coordinated by a kojic unit, the second kojic
unit and the two piperazine nitrogen till protonated. Subsequently a
Fe2L3H4 complex is formed, in which the two iron atoms are bridged
by the three ligands through both kojic units, with four of the six piper-
azine nitrogen atoms till protonated. Afterwards, the first two protons
are lost with pK values 4.1 and 5.0, and the two last with the same pK
5.9. After several attempts to crystallize the compound Fe2(L6)3 a red
parallelepiped crystal was obtained and read with Syncroton radiation
but the collected data only allowed seeing a rough model of the com-
plex (See Supplementary Information).
MS/MS fragmentation of the complex corresponding to signal at
m/z 392.025 (Fig. 14) results in loss of another methylene-kojic.
Signal at m/z 315.014 agrees with stoichiometry of [L52Fe2]2+ complex
without two methylene kojic acid molecules.
The representative ESI-MS spectrum of FeIII complexes with L6 is
presented in Fig. 15. The high intensity of the peak at m/z 448.052
corresponds to the [L62Fe2]2+ complex, while the low intensity peak
at m/z 457.053 represents [L62Fe2 + H2O]2+ complex.
Ligand L7, which does not contain a second kojic unit, forms
FeL2Hx and FeL3 complexes, similarly to kojic acid. The FeL2H2 starting
complex, protonated on the nitrogen atoms of both ligands, looses
these protons and a third one, presumably from a coordinated water
molecule at pK's 4.8, 5.6 and 6.9.
4. Conclusions
Five new ligands have been synthesized, structurally character-
ized and their protonation scheme has been explained using the
information of UV and 1H NMR spectra. For all the ligands containing
two kojic units, the complex formation equilibria with FeIII allowed
concluding about the formation of variously protonated 2:3 Fe:L com-
plex as the major species in the whole pH range; the unprotonated
Fe2L3 complex is the unique species at pH 7.4 (see speciation plots
in Fig. 11). Ligand L7, containing only a kojic unit, forms FeL2Hx
As far as the ligand L81 is concerned, its complexation mechanism
starts with the formation of a FeLH complex at extremely low pH (0–1)
1
L8, differently from L4-L7 complexes, does not contain nitrogen atoms, and it is
analogous to the previously studied L1 ligand [7].

230
L. Toso et al. / Journal of Inorganic Biochemistry 127 (2013) 220–231
Fig. 13. ESI-MS spectra of the system L5-FeIII in a ratio 1:10 at pH 7.
complexes and, in a lower amount, a FeL3 complex. The prevailing spe-
cies at pH 7.4 is FeL2H−1, stabilized by the deprotonation of a coordinat-
ed water molecule.
The excellent chelating properties recommend further toxicological and
pharmacological research on these new promising ligands.
The pFe values reported in Table 4 can be used to evaluate and to
compare the chelating ability of the various ligands. These data give ev-
idence of a strong chelating ability of L8 ligand. The remaining ligands
L4–L6, containing nitrogen atoms in the linker, show a minor chelating
efficiency, pFe ranging from 19.3 for L5 to 17.7 for L6. Interaction of iron
with L7 ligand, having only a coordinating kojic unit, is characterized by
a pFe value 16.7, ca. 3 units higher than that of simple kojic acid. This
significant difference should depend on two facts: the stabilization
due to the loss of a proton from a coordinated water molecule, and to
a kind of a hydrophobic cage formed by the groups not directly implied
in coordination of the metal ion. According to the recent work of Evans
[21], L8 ligand, anionic at physiological pH and characterized by a pFe of
20.0, and perhaps L5, partially in the anionic form LH−1 at physiological
pH and with pFe = 19.3, should be able to mobilize FeIII from transferrin.
Acknowledgments
GC and JIL acknowledge Regione Sardegna for the financial support
CRP-27564 to the project “Integrated approach in the design of chelators
for the treatment of metal overload diseases”. MCA is grateful to RAS for
the program Master and Back — Percorsi di rientro, PRR-MAB-A2011-
19107. MAS acknowledges FCT for financial support, project PEst-OE/
QUI/UI0100/2011.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.jinorgbio.2013.06.009.
Fig. 14. CAD spectrum of peak at m/z 392.025 in Fig. 13.

L. Toso et al. / Journal of Inorganic Biochemistry 127 (2013) 220–231
231
Fig. 15. ESI-MS spectra of the system L6-FeIII in a ratio 1:10 at pH 7.
[10] V.M. Nurchi, G. Crisponi, T. Pivetta, M. Donatoni, M. Remelli, J. Inorg. Biochem. 102
(2008) 684–692.
References
[11] V.M. Nurchi, G. Crisponi, M. Crespo-Alonso, J.I. Lachowicz, Z. Szewczuk, G.J.S. Cooper,
Dalton Trans. 42 (2013) 6161–6170.
[1] G. Crisponi, M. Remelli, Coord. Chem. Rev. 252 (2008) 1225–1240.
[2] Y. Ma, T. Zhou, X. Kong, R.C. Hider, Curr. Med. Chem. 19 (2012) 2816–2827.
[3] G. Crisponi, V.M. Nurchi, V. Bertolasi, M. Remelli, G. Faa, Coord. Chem. Rev. 256
(2012) 89–104.
[4] M.A. Santos, S.M. Marques, S. Chaves, Coord. Chem. Rev. 256 (2012) 240–259.
[5] G. Crisponi, V.M. Nurchi, G. Faa, M. Remelli, Monatsh. Chem. 142 (2011)
331–340.
[12] H.N. Barham, G.N. Reed, J. Am. Chem. Soc. 60 (1938) 1541–1545.
[13] P. Gans, A. Sabatini, A. Vacca, Talanta 43 (1996) 1739–1753.
[14] Bruker, APEX2 Software, Bruker AXS Inc., Madison, Wisconsin, USA, 2010. (v2010.3-0).
[15] G.M. Sheldrick, SADABS, Program for Empirical Absorption Correction of Area
Detector Data, University of Gottingen, Germany, 2009.
[16] G.M. Sheldrick, Acta Crystallogr. Sect. A 64 (2007) 112–122.
[17] A.L. Spek, Utrecht University, Utrecht, The Netherlands, 2010.
[18] C.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. Crystallogr. 41
(2008) 466–470.
[6] G. Crisponi, A. Dean, V. Di Marco, J.I. Lachowicz, V.M. Nurchi, M. Remelli, A. Tapparo,
Anal. Bioanal. Chem. 405 (2013) 585–601.
[7] V.M. Nurchi, G. Crisponi, J.I. Lachowicz, S. Murgia, T. Pivetta, M. Remelli, A. Rescigno, J.
Niclós-Gutíerrez, J.M. González-Pérez, A. Domínguez-Martín, A. Castiñeiras, Z.
Szewczuk, J. Inorg. Biochem. 104 (2010) 560–569.
[19] M.C. Aragoni, M. Arca, G. Crisponi, V.M. Nurchi, Anal. Chim. Acta 316 (1995) 195–204.
[20] C. Frassineti, L. Alderighi, P. Gans, A. Sabatini, A. Vacca, S. Ghelli, Anal. Bioanal.
Chem. 376 (2003) 1041–1052.
[8] R.C. Fox, P.D. Taylor, Bioorg. Med. Chem. Lett. 8 (1998) 443–446.
[9] V.M. Nurchi, J.I. Lachowicz, G. Crisponi, S. Murgia, M. Arca, A. Pintus, P. Gans, J.
Niclos-Gutierrez, A. Domínguez-Martín, A. Castineiras, M. Remelli, Z. Szewczuk,
T. Lis, Dalton Trans. 40 (2011) 5984–5998.
[21] R.W. Evans, X. Kong, R.C. Hider, Biochim. Biophys. Acta 1820 (2012) 282–290.