A new heteropentanuclear complex containing the [Fe2 IIIZn3 II(μ-OH)3] structural motif as a model for purple acid phosphatases

Article abstract of DOI:10.1016/j.ica.2019.119280

Herein, we describe the synthesis and X-ray structure of a new heteropentanuclear complex (2) containing a [Fe₂ ^IIIZn₃ ^II(μ-OH)₃] structural unit and the unsymmetrical ligand H₂L²

Full text of DOI:10.1016/j.ica.2019.119280

InorganicaChimicaActaxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
III
II
A new heteropentanuclear complex containing the [Fe2 Zn3 (μ-OH)3]
structural motif as a model for purple acid phosphatases
Filipy Gobbo Maranhaa, Graciela Aparecida dos Santos Silvaa, Adailton J. Bortoluzzia,
Ebbe Nordlanderb, Rosely A. Peraltaa,⁎, Ademir Nevesa,⁎
a Universidade Federal de Santa Catarina, Departamento de Química, Laboratório de Bioinorgânica e Cristalografia – LABINC, Campus Universitário, Trindade,
Florianópolis, SC 88040-900, Brazil
b Inorganic Chemistry Research Group, Chemical Physics, Center for Chemistry and Chemical Engineering, Lund University, Box 124, SE-221 00 Lund, Sweden
A R T I C L E I N F O
A B S T R A C T
Keywords:
Pentanuclear complex
Hydrolase
Herein, we describe the synthesis and X-ray structure of a new heteropentanuclear complex (2) containing a
[Fe2IIIZn3II(μ-OH)3] structural unit and the unsymmetrical ligand H2L2-et. The molecular structure of (2) shows
that it is formed by a basic dinuclear [FeIII(μ-OH)ZnII(L2-et)] unit that is connected to a second dinuclear [FeIII(μ-
OH)ZnII(L2-et)] unit through a hydroxo bridge while a third ZnII ion is coordinated by the pendant 1,2-etha-
nediamine groups of H2L2-et, resulting in the pentanuclear complex. Kinetic studies on the hydrolysis of the
substrate 2,4-BDNPP (bis(2,4-dinitrophenyl)phosphate) reveal that (2) shows diesterase activity. While the ki-
netic activity is comparable to the corresponding dinuclear FeIIIZnII complex containing the same ligand, the
association with 2,4-BDNPP is significantly decreased.
Diesterase
1. Introduction
coordination spheres of the ZnII and FeIII ions [16,17]. The two subunits
in the plant PAPs are linked via a disulfide bridge [1]. Furthermore,
There is considerable research interest concerned with systems in-
spired by the active site of the metalloenzymes purple acid phospha-
tases (PAPs) [1–8]. PAPs participate in numerous biological functions,
including the bone turnover [1,3,9], and iron transport [10].
PAPs belong to the family of dinuclear metallohydrolases (FeIII-MII,
where M = Fe, Zn, or Mn) and catalyze the hydrolysis of a variety of
phosphoester bonds under acidic conditions. They are the only di-
nuclear metallohydrolases where the requirement of a mixed-valence
form as the hydrolytically active site for catalysis has been clearly es-
tablished [1].
To date, the crystal structures of PAPs originating from red kidney
bean (rkbPAP) [7,8], rats [5,6], pigs [3,11], humans [7], and sweet
potato [12] have been reported. In plant PAPs, the metal ion compo-
sition is predominantly FeIII-MII (M = Zn or Mn) centers [4,13–15]. In
the structure of rkbPAP, the ZnII ion is coordinated by two nitrogen
atoms from histidine side-chains and one oxygen atom from an aspar-
agine, and the FeIII ion is coordinated by an oxygen atom from a tyr-
osine residue, a nitrogen atom from a histidine, and an aspartate. The
FeIII-MII ions are bridged by two oxygen atoms, one from the carbox-
ylate group of an aspartate and the other from a modeled µ-hydroxo
group. Two oxygen atoms from a µ-1,3 phosphate group complete the
metallohydrolases with a catalytic centre that accommodates more than
two metal ions, while uncommon, were recently identified (i.e. PhoX
and Rv0805). The enzyme PhoX is an extra-cytoplasmic alkaline
phosphatase which contains five metal ions in its active site, comprising
two antiferromagnetically coupled FeIII ions and three CaII ions
[18–20]. The diesterase Rv0805 from Mycobacterium tuberculosis con-
tains as many as four metal ions in the active site, one similar to that of
the other dinuclear metallohydrolases, and a CaII-specific one, which
can bind in a site distinct from but close to the location that accom-
modates the transition metal ions and acts as an activator of the en-
zymatic activity [19,20].
The synthesis of small transition metal coordination complexes
models (biomimetics) of the active site of PAPs has been pursued as a
benchmark for the study of this system. Such biomimetics can poten-
tially serve as structural and/or functional models aimed at gaining a
better understanding of the structure and the action mode of the active
site. In this regard many symmetrical [21–25] and unsymmetrical
[16,24,26–32] ligands containing a bridging phenoxide moiety and
different N/O donor atoms have been reported in the literature. Al-
though much of the reactivity studies of the PAPs system have been
performed with mixed-valence hetero-dinuclear complexes, we decided
⁎ Corresponding authors.
E-mail addresses: rosely.peralta@ufsc.br (R.A. Peralta), ademir.neves@ufsc.br (A. Neves).
https://doi.org/10.1016/j.ica.2019.119280
Received 2 August 2019; Received in revised form 10 October 2019; Accepted 12 November 2019
Availableonline13November2019
0020-1693/©2019ElsevierB.V.Allrightsreserved.
Pleasecitethisarticleas:FilipyGobboMaranha,etal.,InorganicaChimicaActa,https://doi.org/10.1016/j.ica.2019.119280

F.G. Maranha, et al.
InorganicaChimicaActaxxx(xxxx)xxxx
at their idealized positions with distances and Uiso taken from the de-
fault of the refinement program. The hydrogen atoms of the amine and
hydroxo groups were found from a Fourier difference map and treated
with a riding model. Hydrogen atoms of the disordered water solvate
could not be located. Several atoms apart from the main metal complex
entity (perchlorate counterions, acetonitrile and water solvate) have
abnormal adp values; however, these atoms were refined aniso-
tropically with positive definite tensors, but this generates several hard
alerts in the checkcif report.
2.4. Mass spectrometry
Chart 1. Structure of the ligand H2L2-et [27].
The mass spectrum for (2) was obtained on a micrOTOF Q-II (Bruker
Daltonics) equipped with an automatic syringe (KD Scientific) for
sample injection in the CEBIME (Centro de Biologia Molecular
Estrutural) at the UFSC. The high-resolution positive ion ESI-MS ana-
lysis was carried out with acetonitrile/water (1:1 v/v) solution in a
concentration of around 500 ppb. The mass spectrometer into which the
samples were injected was operating with a constant flow rate of
3 µL min−1 and ultrapure solvent. The scan range was from m/z
150–1250. The simulated spectra were calculated using the Mmass
software [44].
to explore the similar catalytic potential using a pentanuclear complex,
with the objective of gaining insights on the metallohydrolases with
catalytic centres that accommodate more than two metal ions [18–20].
Previous studies reported the synthesis of tetranuclear FeIII [33,34] and
ZnII [23,33] complexes models of the PAPs.
Herein, we report a continuation of our research on the preparation
of mixed-valence hetero-dinuclear FeIII-MII complexes as models for the
active site of PAPs [16,24,25,27–29,35–41] and we describe the
synthesis and X-ray structure of a new heteropentanuclear complex (2)
containing the [Fe2IIIZn3II(μ-OH)3] structural unit and the un-
symmetrical ligand H2L2-et (Chart 1).
2.5. Ultraviolet–visible spectroscopy and spectrophotometric titration
2. General procedures
The electronic absorption spectra and spectrophotometric titration
were performed using a Perkin-Elmer Lambda 750 spectrophotometer
in the range of 200–800 nm at 25 °C and quartz cuvettes with a capacity
of 1 mL and optical path of 10 mm. The electronic absorption spectra
were obtained with solutions of CH3CN and CH3CN/H2O (1/1; v/v).
The values of ε are given in L mol−1 cm−1. The pKa values were de-
termined by spectrophotometric titration, with experiment performed
in 25 mL of a CH3CN/H2O (1/1; v/v) solution with ionic strength of
0.10 mol L−1 KCl in the pH range of 3.50–10.00 through the addition of
aliquots of the 0.10 mol L−1 KOH solution. The pH was measured with
a pH meter (model Oakton pH2700) and the pKw of the CH3CN/H2O
(1/1; v/v) solution was 15.40 [45].
All of the starting materials were purchased from Sigma-Aldrich,
Acros, Merck, Riedel and Vetec.
2.1. Elemental analyses
Elemental microanalyses were performed with a C, H, N and S
PerkinElmer analyzer (model 2400) using a PerkinElmer balance
(model AD-4 Autobalance) and a tin capsule. Acetanilide (C8H9NO) was
used as reference for calibrating the elemental analyzer, calcd: C, 71.09;
H, 6.71; N, 10.36; O, 11.84. Found C, 71.16; H, 6.77; N, 10.39.
2.2. Infrared spectroscopy
2.6. Square-wave voltammetry
Infrared spectra (Fig. S1) were obtained on a PerkinElmer Fourier
transform infrared-attenuated total reflection (FTIR-ATR) spectro-
photometer (model Spectrum 100) using a crystal of ZnSe (45°), and a
TGS (triglycine sulfate) detector. The samples were analyzed directly
with the crystal, averaging 18 scans in the range of 4000–400 cm−1 at a
resolution of 4 cm−1. The sample measurements were corrected with
background measurements of the crystal alone without the samples.
The redox behavior of (2) was investigated by square-wave vol-
tammetry using a Basi potentiostat/galvanostat (model Epsilon). The
concentration of the complex was 1.0 × 10−4 mol L−1 in degassed
CH3CN solution (0.1 mol L−1n-Bu4NPF6 as supporting electrolyte) at
25 °C, under an argon atmosphere. The electrolytic cell contained three
electrodes: a glassy carbon working electrode; a platinum wire as a
counter electrode and a Ag/AgCl reference electrode. Ferrocene was
used as an internal standard. Scanning parameters: Step (4.0 mV),
amplitude (25 mV) and frequency (15 Hz).
Room temperature was 25.0
1.0 °C.
2.3. X-ray structure analyses
2.7. Reactivity measurements
Crystallographic analysis of complex (2) was carried out with a
single crystal Bruker KAPPA APEX II DUO diffractometer using gra-
phite-monochromated MoKα radiation (λ = 0.71073 Å). Temperature
of the sample was set at 150 ( 2) K with an Oxford Instruments
Cryostream 700 series device. Crystals of complex (2) showed poor
scattering power, so that the frames were collected with 30 s exposure
by frame and with a maximum resolution of 0.83 Å. A total of 1420
frames were recorded with φ and ω scans method using the APEX2
software package [42]. All data were corrected for Lorentz and polar-
ization effects and for absorption using the SADABS multi-scan method
[42]. The structure was solved by direct methods and refined applying
the full-matrix least-squares method using the SHELXS and
SHELXL2014 programs [43], respectively. All non-hydrogen atoms
were refined with anisotropic displacement parameters, except for a
toluene solvate. Hydrogen atoms attached to carbon atoms were placed
The catalytic activity of (2) towards the activated phosphodiester
bis(2,4-dinitrophenyl)phosphate (2,4-BDNPP) was evaluated spectro-
photometrically on a Varian Cary50 Bio spectrophotometer, at 400 nm
based on the appearance of the 2,4-dinitrophenylphenolate chromo-
phore at 25 °C. The activated substrate 2,4-BDNPP was prepared as the
pyridinium salt [46]. The effect of pH on the hydrolytic cleavage of 2,4-
BDNPP was monitored in the pH range from 4.5 to 10.0. Reactions were
performed using the following conditions: 750 µL freshly prepared
aqueous buffer solution ([buffer]final 5.00 × 10−2 mol L−1), buffer: 2-
(N-morpholino)ethanesulfonic acid (MES; pH 4.00–6.50), 2-[4-(2-hy-
droxyethyl)-piperazin-1-yl]ethanesulfonic acid (HEPES; pH 7.00–8.50),
N-cyclohexyl-2-aminoethanesulfonic acid (CHES; pH 9.00–10.00) with
controlled ionic strength (LiClO4) 0.10 mol L−1; 100 µL of an acetoni-
trile complex solution ([2]final = 2.80 × 10−5 mol L−1) and 550 µL of
2

F.G. Maranha, et al.
InorganicaChimicaActaxxx(xxxx)xxxx
acetonitrile were added to a 1 cm path-length cell. The reaction was
initiated by the addition of 100 µL of an acetonitrile substrate solution
([2,4-BDNPP]final = 1.40 × 10−3 mol L−1) and monitored between 2%
and 5% of reaction at 25 °C. The relevant molar absorption coefficients
of the reaction product 2,4-dinitrophenolate (2,4-DNP) were de-
termined at each pH under the same experimental conditions as those of
the rate measurements [40]. The kinetic experiments under conditions
of excess substrate were performed as follows: 750 µL of freshly pre-
pared aqueous HEPES buffer solution (at pH 7.00), [buf-
fer]final = 5.00 × 10−2 mol L−1, 100 µL of an acetonitrile complex so-
lution ([2]final = 1.25 × 10−5 mol L−1) and 600–0 µL of acetonitrile
were added to a 1 cm path-length cell. The reaction was initiated with
the addition of 2,4-BDNPP solution ([2,4-BDNPP]final = 6.00 × 10−4 to
7.80 × 10−3 mol L−1). Correction for the spontaneous hydrolysis of
2,4-BDNPP was carried out by direct difference using a reference cell
under identical conditions without adding the catalyst. The initial rate
was obtained from the slope of the absorbance versus time plot over the
first 10 min of the reaction. The conversion of the reaction rate units
was carried out using ε = 12100 L mol−1 cm−1 for 2,4-DNP (at pH
7.00) and the initial concentration of the complex [40]. A kinetic
treatment using the Michaelis–Menten equation approach was applied
[47]. Isotopic effects of deuterium on the hydrolysis of 2,4-BDNPP
promoted by complex (2) was evaluated following two reactions with
identical conditions (vide experiments on the effect of pH) using the
buffer solutions HEPES pH 7.00 (H2O) and MES pD 6.60 (D2O) to de-
termine the relation kH/kD [9]. The reactions were monitored at 400 nm
and 25 °C. The number of molecules of the substrate that are hydrolyzed
per molecule of complex was monitored at 445 nm (ε = 3600 L mol−1
cm−1) under a 50-fold substrate excess ([2,4-BDNPP]final = 2 × 10−3
mol L−1), relative to the complex ([2]final = 4 × 10−5 mol L−1), at pH
7.00 and 25 °C. The monoesterase-like activity of (2) was monitored
with the monoester substrate, 2,4-dinitrophenyl phosphate (2,4-DNPP),
directly with 1, 2, 4, 6, 8, 10 and 12 equivalents of 2,4-DNPP, and over
a period of 5 h at 24 °C. After this time, 4 equivalents of the diester 2,4-
BDNPP was added to the reaction mixture. Reactions were performed
using freshly prepared aqueous buffer HEPES solution (at pH 7.00,
Fig. 1. ORTEP plot of the cation of complex (2). Hydrogen atoms were omitted
for clarity.
to the stable heterodinuclear complex [FeIII(μ-OH)ZnII(L2-et)](ClO4)2
(1). This complex was characterized by spectroscopic methods (infrared
and ultraviolet–visible), ESI-MS, elemental analysis and square-wave
voltammetry [27]. Interestingly, when we increase the amounts of Zn
(ClO4)2·6H2O, the unprecedented pentanuclear [Fe2IIIZn3II(μ-OH)3(L2-
et)2](ClO4)5 complex (2) with the unusual coordination of a third ZnII
ion by the pendant 1,2-ethanediamine groups of H2L2-et was obtained.
After recrystallization with stoichiometric amounts of the triphenyl-
phosphine oxide in CH3CN/CH3OH/toluene (0.1/0.9/1; v/v) solution,
single crystals suitable for X-ray analysis were obtained. The triphe-
nylphosphine oxide was used because it is a popular reagent to induce
the crystallization of chemical compounds. Its rigidity and the basicity
of the oxygen center make this species an alternative crystallization
agent when it is difficult to crystallize molecules. This artifice is ap-
plicable to molecules that have acidic hydrogen atoms, e.g. phenols
[50].
[buffer]final = 5.00 × 10−2 mol L−1
, I
final = 5.0 × 10−2 mol L−1
(LiClO4), [2]final = 1.20 × 10−4 mol L−1). The monoester 2,4-DNPP
was obtained as the lutidinium salt [49].
Single purple crystals suitable for X-ray analysis were obtained after
slow evaporation of complex (2) in an CH3CN/H2O (0.9/0.1; v/v)
solvent mixture. The molecular structure of the pentanuclear cation in
complex (2) is shown in Fig. 1.
2.8. Synthesis
To a 30 mL methanolic solution of H2L2-et [27] (64.6 mg, 0.1 mmol,
645.84 g mol−1
)
was added, under stirring, 74.5 mg (0.2 mmol,
372.38 g mol−1) of Zn(ClO4)2‧6H2O. To this solution was added, drop-
wise, methanolic solution (30 mL) containing 0.1 mmol of Fe
(ClO4)3‧9H2O (51.6 mg, 0.1 mmol, 516.33 g mol−1
and 119.9 mg
It can be observed from the molecular structure of (2) that it is
formed by two dinuclear [FeIII(μ-OH)ZnII(L2-et)] units [27]. These units
are connected through a hydroxo bridge while a third ZnII ion is co-
ordinated by the pendant 1,2-ethanediamine groups of H2L2-et, re-
sulting in the pentanuclear complex. To the best of our knowledge, (2)
a
)
(3 mmol, 39.99 g mol−1) of NaOH in 10 mL of water. The stirring was
maintained for approximately 15 min and then NaClO4 (122.4 mg,
1 mmol, 122.44 g mol−1) was added. The solution was filtered off and
left to stand. The resulting purple product was collected and washed
with water, CH2Cl2 and diethyl ether (Yield: 45.3 mg, 20%). Single
crystals suitable for X-ray analysis were obtained after recrystallization
with 5 µmol of triphenylphosphine oxide and 5 µmol of (2) in 2 mL of
the CH3CN/CH3OH/toluene (0.1/0.9/1; v/v) solution. Anal. Calc. for
represents
a new example of a
heteropentanuclear [Fe2IIIZn3II(μ-
OH)3(L2-et)2]5+ cationic complex with this asymmetric structure.
Crystallographic parameters for (2) are summarized in Table 1 and
selected bond distances and angles are given in Table S1. In the struc-
ture of (2), the iron centers Fe(1) and Fe(1′) show an N2O4 coordination
set and Zn(1) and Zn(1′) show an N3O2 coordination set. Both Zn ions
possess distorted geometries intermediate between square pyramidal
and trigonal bipyramidal with Addison parameters (τ) equal to 0.4945
and 0.58, for Zn(1) and Zn(1′), respectively [51]. The third zinc ion, Zn
(2), shows an N4 distorted tetrahedral coordination environment. In
each half of the [Fe2IIIZn3II(μ-OH)3(L2-et)2]5+ cation, the Fe(1) and Fe
(1′) ions are facially coordinated by the hard tridentate pendant arm of
(L2-et)−2 containing the amine (N1, N1′) and pyridine nitrogen (N32,
N32′) and the phenolate oxygen (O20, O20′) atoms, while Zn(1) and Zn
(1′) are coordinated by the soft side of (L2-et)2− through the amine (N4,
N4′) and pyridine (N42, N52, N42′, N52′) nitrogen atoms. The bridging
phenolate (O10, O10′) and the hydroxo (O2, O2′) oxygen atoms
C
78H93N14O7Fe2Zn3(CH3OH)2(H2O)5(ClO4)5: C, 41.81; H, 4.87; N,
8.53%. Found C, 41.64; H, 4.61; N, 8.17%. MM = 2297.91 g mol.−1
FTIR-ATR, cm−1 (Fig. S1c): ν(OH) 3550; ν(C-HAr and C-Haliph
)
2959–2867; ν(C]N and C]C), 1610–1444; ν(CeO) 1276; ν(Cl-O)
1073; δ(C-HAr) 767; δ(C]C) 623.
3. Results and discussion
The reaction between the ligand H2L2-et [27] and stoichiometric
amounts of Zn(ClO4)2·6H2O and Fe(ClO4)3·6H2O in the presence of six
equivalents of NaOH and two equivalents of NaClO4 in methanol leads
3

F.G. Maranha, et al.
InorganicaChimicaActaxxx(xxxx)xxxx
Table 1
Crystal data and structure refinement for (2).
Empirical formula
C87H107Cl5Fe2N15O28.50Zn3
Formula weight
Temperature
2303.93
150(2) K
Wavelength
0.71073 Å
Crystal system
Space group
Monoclinic
P 21/n
Unit cell dimensions
a = 14.4012(15) Å
b = 44.515(5) Å
c = 16.5153(17) Å
β = 111.2860(10)°
9865.2(18) Å3
Volume
Z
4
Density (calculated)
Absorption coefficient
F(0 0 0)
1.551 Mg m−3
1.226 mm−1
4756
Crystal size
Theta range for data collection
Index ranges
0.260 × 0.240 × 0.040 mm3
1.585 to 25.407°.
−13 ≤ h ≤ 17, −53 ≤ k ≤ 52,
−19 ≤ l ≤ 19
Fig. 3. Electronic absorption spectrum of complex (2) in CH3CN and CH3CN/
H2O (1/1; v/v). [2] = 1.5 × 10−3 mol L−1. λmax nm (ε mol−1 cm−1) in the
solvent: CH3CN = 500 nm (ε = 4570 L mol−1 cm−1) and 295 nm (ε = 18837 L
mol−1 cm−1); CH3CN/H2O (1/1; v/v) = 496 nm (ε = 4462 L mol−1 cm−1) and
292 nm (ε = 19968 L mol−1 cm−1).
Reflections collected
75,370
18,147 (Rint = 0.0279)
100.0%
Semi-empirical from equivalents
0.7452 and 0.6634
Full-matrix least-squares on F2
18,147/184/1282
1.121
Independent reflections
Completeness to theta = 25.242°
Absorption correction
Max. and min. transmission
Refinement method
hydroxo ions bound to the metal centers, two water molecules of hy-
dration, two methoxide ions and one perchlorate counterion. In addi-
tion, peaks corresponding to fragmentation of the pentanuclear com-
plex are also apparent, including peaks at m/z = 470.1, 879.2 and
1041.2 assigned to the dinuclear [FeIII(μ-OH)ZnII(L2-et)(H2O)3(Li)
(ClO4)]2+, [FeIII(μ-OH)ZnII(L2-et)(ClO4)]+ and trinuclear [FeIII(μ-OH)
ZnII(L2-et)ZnII(CH3O)2(H2O)2(ClO4)]+ species, respectively. Peaks at-
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
Extinction coefficient
R1 = 0.0617, wR2 = 0.1391
R1 = 0.0740, wR2 = 0.1450
n/a
Largest diff. peak and hole
1.376 and −0.895 e Å−3
tributed to the fragments of the ligand ([C15H16N2O2Na]+
, m/
z = 279.1; [C32H40N6O3Na]+, m/z = 579.2) are also present.
complete the distorted bipyramidal N3O2 coordination of Zn(1) and Zn
(1′) while the distorted octahedral N2O4 coordination of Fe(1) and Fe
(1′) is complemented by the bridging phenolate oxygen (O10, O10′)
and the bridging hydroxo groups (O2, O2′ and O1). The Zn(2) ion ex-
hibits a distorted tetrahedral geometry with an N4 donor set in which N-
donors are provided by two secondary and two primary amine atoms of
the 1,2-ethanediamine groups. The Fe-µ-OH (Fe(1)-O(2) = 1.935(3) Å;
Fe(1)-O(1) = 1.971(3) Å; Fe(1′)-O(2′) = 1.937(3) Å; Fe(1′)-O
(1) = 1.962(3) Å) and Zn-µ-OH (Zn(1)-O(2) = 1.984(3) Å; Zn(1′)-O
(2′) = 2.008(3) Å; Zn(1′)-O(2′) = 1.937(3) Å; Zn(1′)-O(1) = 1.962(3)
Å) distances in (2) are somewhat shorter than the corresponding metal-
μ-OH distances in rkbPAP, which have been modeled to be between 2.0
and 2.2 Å. Furthermore, the Fe+3‧‧‧Zn+2 distances (Fe(1)-Zn
(1) = 3.0967(9) Å and Fe(1′)-Zn(1′) = 3.0951(8) Å) are shorter but
comparable to the distance of 3.20 Å found in rkbPAP [17].
The UV − vis absorption spectrum of complex (2) in CH3CN/H2O
(1/1; v/v) solution shows an absorption band at 496 nm (ε = 4462 L
mol−1 cm−1, ca. 2200 L mol−1 cm−1 by dinuclear unit of the com-
position [FeIII(μ-OH)ZnII(L2-et)]), similar to the dinuclear complex (1)
[27]), which is assigned to a ligand-to-metal charge transfer (LMCT)
process, from the pπ(phenolate) → dπ*(FeIII). A second band was also
observed at 292 nm (ε = 19968 L mol−1 cm−1) which is attributed to
the intra-ligand transition of the pyridine and phenolic rings (Fig. 3).
The UV–Vis spectrum in acetonitrile is very similar, with an absorption
band at 500 nm (ε = 4570 L mol−1 cm−1) and a second one at 295 nm
(ε = 18837 L mol−1 cm−1), showing that in both media the species are
the same.
The spectrophotometric titration (Fig. 4) in CH3CN/H2O (1/1; v/v)
revealed two pKa values for (2) (4.52 and 6.96). The first constant pKa1
can be attributed to the deprotonation of the FeIII-bound H2O water
molecule (Scheme 1b), while pKa2 can be attributed to the deprotona-
tion of the terminally coordinated water molecule at the ZnII site
(Scheme 1c) [52].
The high-resolution positive ion ESI-MS for complex (2) (Fig. 2) was
recorded in acetonitrile/water (1:1; v/v). A group of peaks at m/z 919.2
with a 2+ charge was observed. This signal can be assigned to the
system [Fe2IIIZn3II(μ-OH)3(L2-et)2(CH3O)2(H2O)2(ClO4)]2+, which cor-
responds to the molecular ion of complex (2) having three terminal
Fig. 2. The high-resolution positive ion ESI-MS
spectrum of (2) in acetonitrile/water (1:1; v/v) so-
lution and its isotopic distribution pattern. m/
z = 919.1967 (experimental – black line) and m/
z = 919.1975 (simulated – red line). Molecular for-
mula: [C78H93N14O7Fe2Zn3(H2O)2(CH3O)2 (ClO4)].2+
(For interpretation of the references to colour in this
figure legend, the reader is referred to the web ver-
sion of this article.)
4

F.G. Maranha, et al.
InorganicaChimicaActaxxx(xxxx)xxxx
Fig. 5. Square-wave voltammogram of (2) ([2] = 1.0 × 10−4 mol L−1) in
CH3CN solution and 0.1 mol
Electrodes: a glassy carbon working electrode; a platinum wire as a counter
electrode and Ag/AgCl reference electrode. Scanning parameters: Step
L
−1n-Bu4NPF6 as supporting electrolyte.
a
(4.0 mV), amplitude (25 mV) and frequency (15 Hz).
(BPBPMPF)2− [35].
Thus, we suggest that in pure CH3CN solution the structural unit
[FeIII(µ-OH)FeIII] is maintained as observed in the X-ray structure of (2).
Nevertheless, it should be emphasized that the electrochemical beha-
vior of (2) becomes much more difficult to interpret when square-wave
voltammograms are obtained in CH3CN/H2O solution (not shown),
most probably due to the equilibria proposed in Scheme 1.
We investigated the catalytic activity of complex (2) in the hydro-
lysis of the substrate 2,4-BDNPP (bis(2,4-dinitrophenyl)phosphate) by
following spectrophotometrically the absorbance increase of the re-
leased 2,4-dinitrophenolate anion (λmax = 400 nm), under conditions
of excess substrate. The pH effect on the catalytic activity of complex
(2) was evaluated from pH 4.5 to 10. The dependence of the initial rate
on the pH shows a bell-shaped profile, with maximum activity around
pH 7 (Fig. 6). This is similar to the results previously reported for other
dinuclear FeIII-ZnII complexes with similar coordination environments
[27,29] and to that found for rkbPAP [17].
The data were fitted using Eq. (S1) [34], with γ = 0.3, and a sig-
moidal fit of the curve revealed pKa1 4.88 and pKa2 7.57, values which
are in reasonable agreement with the results of pKa1 4.52 and pKa2 6.96
obtained from the spectrophotometric titration experiment. The pKa
values determined spectrophotometrically are lower than the kinetic
pKa values due to the absence of the substrate.
Fig. 4. Spectrophotometric titration in different pH ranges of ca. 3.5–5.5 (a)
and 5.5–8.5 (b). Addition of 0.1 mol L−1 NaOH solution in a CH3CN/H2O (1/1;
v/v) solution of complex (2) with I = 0.1 mol L−1 (KCl); [2] = 5.4 × 10−4 mol
L−1
.
These pKa values are similar to those found for dinuclear FeIII(µ-OH)
ZnII model complexes containing terminally bound FeIII-OH2 and ZnII-
OH2 units [27,29].
The square-wave voltammogram (Fig. 5) of (2) in acetonitrile so-
lution reveals two quasi-reversible redox processes: E1/21 = −0.468
and E1/22 = −0.964 V (relative to the ferrocenium/ferrocene couple)
which are attributed to the FeIIIFeIIIZn3II/FeIIFeIIIZn3II and FeIIFeIIIZn3
/
II
FeIIFeIIZn3 couples, respectively. The E1/2 is in close agreement with
the second redox (-0.89 V versus Fc+/Fc) process observed for the di-
nuclear FeIIFeIII complex containing the unsymmetrical ligand
II
2
The shapes of the curves in Fig. 6 indicate that two active species are
present in the pH range of 4.5–10: the first deprotonation step at pH ≅ 5
generates an active species; deprotonation of this species at pH ≅ 7.5
Scheme 1. Proposed equilibria for (2) in CH3CN/H2O (1/1; v/v) solution.
5

F.G. Maranha, et al.
InorganicaChimicaActaxxx(xxxx)xxxx
decreasing the association constant of the substrate. In the dinuclear
species [27], the KM value is of 1.61 × 10−3 mol L−1 while with the
formation of the pentanuclear species and the presence of the third ZnII
ion the KM value is of 9.85 × 10−3 mol L−1. Although it was speculated
that the presence of the third ZnII may be a reflection of the catalytic
activity in the case of complex (2), this metal may have a structural
influence and catalytic relevance in the hydrolysis of the substrate 2,4-
BDNPP (higher KM and lower kcat/KM values). In this sense, the Rv0805
metallohydrolase is fully active in the “conventional” metal site, but the
CaII-specific dinuclear site acts as a modulator of the activity of Rv0805.
The CaII can bind in a site distinct from but close to the dinuclear
transition-metal-ion binding site, and can alter the level of its reactivity
depending on the presence or absence of CaII [19]. Finally, as shown in
Table 2, complex (2) shows higher kcat and kcat/KM values when com-
II
pared with a tetranuclear Zn4 complex for which the same substrate
(2,4-BDNPP) and pH optimum (8.5) were employed [23].
Fig. 6. Dependence of the initial reaction rate (V0) on pH for the hydrolysis of
2,4-BDNPP enhanced by complex (2). Conditions: [2]final = 2.80 × 10−5 mol
In order to elucidate the mode of interaction between 2,4-BDNPP
and the pentanuclear complex (2), we followed the spectral change of
the reaction mixture at pH 7.0 over a period of 24 h at 25 °C in the
presence of excess substrate (Fig. S2). The intensity of the phenolate to
FeIII LMCT band (λmax = 498 nm) is only slightly affected, thus strongly
suggesting a monodentate coordination mode of the substrate to the
ZnII metal center. On the other hand, the absorption at ca. 400 nm
continues to increase due to the formation of the product 2,4-dini-
trophenolate (2,4-DNP).
L
−1; [2,4-BDNPP]final = 1.40 × 10−3 mol L−1; [buffer] = 5.0 × 10−2 mol L−1
(HEPES, pH = 7.00); I = 5.0 × 10−2 mol L−1 (LiClO4) in CH3CN/H2O (1/1; v/
v) at 25 °C. The distributions of the corresponding species (b), (c) and (d) (see
Scheme 1) are shown as colored lines.
lowers its activity. The decrease in the activity upon deprotonation of
ZnII-H2O indicates that the substrate replaces the bound H2O. A hy-
droxide would be less labile than an aqua ligand, resulting in a lower
leaving tendency of the ZnII-OH group [31].
The dependence of the hydrolysis rate on the substrate concentra-
tion (2,4-BDNPP) was determined at pH 7.0, in which the most cata-
lytically active species of complex (2) is present. The data presented in
Fig. 7 shows saturation behavior and were analyzed using the Mi-
chaelis-Menten scheme. The Michaelis-Menten equation v0 = Vmax([S]/
([S] + KM) was used to calculate the kinetic parameters kcat, KM and
kcat/KM which are listed and compared with other PAP model com-
plexes in Table 2.
We tested the activity of (2) in the hydrolysis of the monoester
substrate 2,4-dinitrophenyl phosphate (2,4-DNPP) (Fig. S3); directly
with 1, 2, 4, 6, 8, 10 and 12 equivalents of the 2,4-DNPP and over a
period of 5 h at 24 °C only the background reaction was observed. After
this time, 2,4-BDNPP was added to the reaction mixture and im-
mediately the absorbance at 400 nm started to increase (Fig. S3), in-
dicating that only the diester can be hydrolyzed. The number of cata-
lytic cycles, estimated from a reaction that was monitored over 24 h at
25 °C and 445 nm (ε = 3600 L mol−1cm−1) with a 50:1 2,4-BDNPP/
complex ratio, was found to be 9 turnovers. Furthermore, the measured
kinetic isotope effect (kH/kD ≅ 0.8) for (2) suggests that no proton
transfer is involved in the rate-limiting step of the reaction [48].
Based on the X-ray structure, solution and kinetic studies, we pro-
pose a mechanism for the hydrolysis of 2,4-BDNPP by complex (2) as
shown in Scheme 2. In the proposed mechanism at pH 7, the penta-
nuclear active species is composed of two dinuclear units of the com-
position [(HO)FeIII(μ-OH)ZnII(H2O)(L2-et)], connected to a third ZnII
ion by the 1,2-ethanediamine groups. The mechanism can be described
analogously to the mechanism proposed in the literature
[1,16,17,40,53] for dinuclear complexes, where the [(HO)FeIII(μ-OH)
ZnII(H2O)(L2-et)] units of (2) are responsible for the hydrolysis of two
2,4-BDNPP substrate molecules. In brief, monodentate binding of the
substrate to ZnII is followed by a nucleophilic attack by the terminal,
FeIII-bound hydroxide and the concomitant release of 2,4-dini-
trophenolate. The µ-1,3-coordinated DNPP intermediate undergoes
substitution by water/OH− molecules from the environment and re-
generates the active site for the next catalytic cycle.
The kcat value for (2) is of similar magnitude when compared to
heterodinuclear FeIIIZnII complexes and lower than that of a tetra-
nuclear Fe4III complex. In fact, the higher KM value (lower association of
the substrate) of (2) significantly decreases its catalytic efficiency (kcat
/
KM) under similar pH conditions [16,27]. At present we do not have a
clear explanation for this behavior of (2) but steric constraints within
the structure of the pentanuclear complex could contribute to
In summary, reaction of the dinucleating ligand (H2L2-et) with Zn
(ClO4)2·6H2O and Fe(ClO4)3·6H2O results in the formation of the het-
erodinuclear [FeIII(μ-OH)ZnII(L2-et)](ClO4)2 complex (1), as expected
[27]. However, in the presence of an excess of Zn(ClO4)2·6H2O, the
recrystallization of this complex yields the unexpected pentanuclear
[Fe2IIIZn3II(L2-et)2(μ-OH)3](ClO4)5 complex (2), which has been struc-
turally characterized. Complex (2) is
a new heteropentanuclear
Fe2IIIZn3II complex that is a functional model for the active site of PAPs,
capable of cleaving diester bonds in the model substrate 2,4-BDNPP.
Fig. 7. Dependence of the initial reaction rate (V0) on the 2,4-BDNPP con-
centration in the hydrolysis reaction promoted by complex (2). Conditions:
Declaration of Competing Interest
[2]final = 1.25 × 10−5
mol
7.80 × 10−3 mol L−1; [buffer]final = 5.0 × 10−3 mol L−1 (HEPES, pH = 7.00);
final = 5.0 × 10−2 mol L−1 (LiClO4) in CH3CN/H2O (1/1; v/v) at 25 °C.
L−1
;
[2,4-BDNPP]final = 6.00 × 10−4
to
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
I
6

F.G. Maranha, et al.
InorganicaChimicaActaxxx(xxxx)xxxx
Table 2
Kinetic parameters of reported di- and tetranuclear iron and zinc model complexes for 2,4-BDNPP hydrolysis.
Complex
pH
kcat (s−1
)
KM (mol L−1
)
kcat/KM (mol−1 L s−1
)
Ref
(2)
(1)
7.0
7.0
6.5
6.5
8.5
8.6 × 10−4
(9.85
(1.61
3.49) × 10−3
1.91) × 10−3
0.087
0.696
0.217
0.216
0.029
This study
[27]
[16]
[34]
[23]
(11.2
5.56) × 10−4
[L(OH2)FeIII-(µ-OH)ZnII]2+
[Fe4III(HPBA)2(μ-OAc)2(μ-O)-(μ-OH)(OH2)2]2+
[Zn4II(TPPNOL)2(OAc)3]3+
9.13 × 10−4
4.20 × 10−3
(16
0.2) × 10−4
(7.4
(4.4
0.6) × 10−3
1) × 10−3
1.28 × 10−4
(1) = H2L2-et = 2-(((2-aminoethyl)amino)methyl)-6-(((2-hydroxy-5-methyl-3-(((2-(pyridin-2-yl)ethyl)(pyridin-2-ylmethyl)amino)methyl)benzyl)(pyridin-2-yl-
methyl)amino)methyl)-4-methylphenol.
(H2L) = 2-bis[{(2-pyridylmethyl)-aminomethyl}-6-{(2-hydroxybenzyl)-(2-pyridyl methyl)}aminomethyl] −4-methylphenol.
H3HPBA = 2-((2-hydroxy-5-methyl-3-((pyridin-2-ylmethylamino)methyl)benzyl)(2-hydroxybenzyl)amino) acetic acid.
HTPPNOL = N,N,N’-tris-(2-pyridylmethyl)-1,3-diaminopropan-2-ol.
doi.org/10.1016/j.jmb.2005.04.014.
[8] D. Feder, L.R. Gahan, R.P. McGeary, L.W. Guddat, G. Schenk, ChemBioChem 20
(2019) 1536–1540, https://doi.org/10.1002/cbic.201900077.
[9] G.W. Oddie, G. Schenk, N.Z. Angel, N. Walsh, L.W. Guddat, J. de Jersey,
A.I. Cassady, S.E. Hamilton, D.A. Hume, Bone 27 (2000) 575–584, https://doi.org/
10.1016/S8756-3282(00)00368-9.
[10] P.R. Nuttleman, R.M. Roberts, J. Biol. Chem. 265 (1990) 12192–12199.
[11] L.W. Guddat, A.S. McAlpine, D. Hume, S. Hamilton, J. de Jersey, J.L. Martin,
Structure 7 (1999) 757–767, https://doi.org/10.1016/S0969-2126(99)80100-2.
[12] G. Schenk, L.R. Gahan, L.E. Carrington, N. Mitić, M. Valizadeh, S.E. Hamilton, J. de
Jersey, L.W. Guddat, Proc. Natl. Acad. Sci. 102 (2005) 273–278, https://doi.org/10.
1073/pnas.0407239102.
[13] G. Schenk, Y. Ge, L.E. Carrington, C.J. Wynne, I.R. Searle, B.J. Carroll, S. Hamilton,
J. de Jersey, Arch. Biochem. Biophys. 370 (1999) 183–189, https://doi.org/10.
1006/abbi.1999.1407.
[14] A. Durmus, C. Eicken, B.H. Sift, A. Kratel, R. Kappl, J. Hüttermann, B. Krebs, Eur. J.
Biochem. 260 (1999) 709–716, https://doi.org/10.1046/j.1432-1327.1999.
00230.x.
[15] J.L. Beck, L.A. McConachie, A.C. Summors, W.N. Arnold, J. De Jersey, B. Zerner,
Biochim. Biophys. Acta BBA – Protein Struct. Mol. Enzymol. 869 (1986) 61–68,
https://doi.org/10.1016/0167-4838(86)90310-9.
[16] A. Neves, M. Lanznaster, A.J. Bortoluzzi, R.A. Peralta, A. Casellato, E.E. Castellano,
P. Herrald, M.J. Riley, G. Schenk, J. Am. Chem. Soc. 129 (2007) 7486–7487,
https://doi.org/10.1021/ja071184l.
[17] T. Klabunde, N. Sträter, R. Fröhlich, H. Witzel, B. Krebs, J. Mol. Biol. 259 (1996)
737–748, https://doi.org/10.1006/jmbi.1996.0354.
[18] S.C. Yong, P. Roversi, J. Lillington, F. Rodriguez, M. Krehenbrink, O.B. Zeldin,
E.F. Garman, S.M. Lea, B.C. Berks, Science 345 (2014) 1170–1173, https://doi.org/
10.1126/science.1254237.
[19] M.M. Pedroso, J.A. Larrabee, F. Ely, S.E. Gwee, N. Mitić, D.L. Ollis, L.R. Gahan,
G. Schenk, Chem. Eur. J. 22 (2016) 999–1009, https://doi.org/10.1002/chem.
201504001.
[20] M. Monteiro Pedroso, C. Selleck, J. Bilyj, J.R. Harmer, L.R. Gahan, N. Mitić,
A.J. Standish, D.L. Tierney, J.A. Larrabee, G. Schenk, Dalton Trans. 46 (2017)
13194–13201, https://doi.org/10.1039/C7DT01350G.
[21] C. Pathak, D. Kumar, M.K. Gangwar, D. Mhatre, T. Roisnel, P. Ghosh, J. Inorg.
Scheme 2. Proposed mechanism for the hydrolysis of 2,4-BDNPP promoted by
Biochem. 185 (2018) 30–42, https://doi.org/10.1016/j.jinorgbio.2018.04.018.
complex (2).
[22] C. Pathak, M.K. Gangwar, P. Ghosh, Polyhedron 145 (2018) 88–100, https://doi.
org/10.1016/j.poly.2018.01.029.
[23] L.L. Mendes, D. Englert, C. Fernandes, L.R. Gahan, G. Schenk, A. Horn, Dalton
Trans. 45 (2016) 18510–18521, https://doi.org/10.1039/C6DT03200A.
[24] T.P. Camargo, F.F. Maia, C. Chaves, B. de Souza, A.J. Bortoluzzi, N. Castilho,
T. Bortolotto, H. Terenzi, E.E. Castellano, W. Haase, Z. Tomkowicz, R.A. Peralta,
A. Neves, J. Inorg. Biochem. 146 (2015) 77–88, https://doi.org/10.1016/j.
jinorgbio.2015.02.017.
influence the work reported in this paper.
Appendix A. Supplementary data
[25] A. Neves, M.A. de Brito, I. Vencato, V. Drago, K. Griesar, W. Haase, Inorg. Chem. 35
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ica.2019.119280.
(1996) 2360–2368, https://doi.org/10.1021/ic950456v.
[26] C. Pathak, S.K. Gupta, M.K. Gangwar, A.P. Prakasham, P. Ghosh, ACS Omega 2
(2017) 4737–4750, https://doi.org/10.1021/acsomega.7b00671.
[27] G.A. dos S. Silva, A.L. Amorim, B. de Souza, P. Gabriel, H. Terenzi, E. Nordlander,
A. Neves, R.A. Peralta, Dalton Trans. 46 (2017) 11380–11394, https://doi.org/10.
1039/C7DT02035J.
[28] C. Pereira, G. Farias, F.G. Maranha, N. Castilho, G. Schenk, B. de Souza, H. Terenzi,
A. Neves, R.A. Peralta, J. Biol, Inorg. Chem. 5 (2019) 675–691, https://doi.org/10.
1007/s00775-019-01680-3.
[29] T.P. Camargo, A. Neves, R.A. Peralta, C. Chaves, E.C.P. Maia, E.H. Lizarazo-Jaimes,
D.A. Gomes, T. Bortolotto, D.R. Norberto, H. Terenzi, D.L. Tierney, G. Schenk,
Inorg. Chem. 57 (2018) 187–203, https://doi.org/10.1021/acs.inorgchem.
7b02384.
References
[1] N. Mitić, S.J. Smith, A. Neves, L.W. Guddat, L.R. Gahan, G. Schenk, Chem. Rev. 106
(2006) 3338–3363, https://doi.org/10.1021/cr050318f.
[2] G. Schenk, N. Mitić, L.R. Gahan, D.L. Ollis, R.P. McGeary, L.W. Guddat, Acc. Chem.
Res. 45 (2012) 1593–1603, https://doi.org/10.1021/ar300067g.
[3] C. Selleck, D. Clayton, L.R. Gahan, N. Mitić, R.P. McGeary, M.M. Pedroso,
L.W. Guddat, G. Schenk, Chem. Eur. J. 23 (2017) 4778–4781, https://doi.org/10.
1002/chem.201700866.
[4] G. Schenk, N. Mitić, G.R. Hanson, P. Comba, Coord. Chem. Rev. 257 (2013)
[30] A.E. Roberts, G. Schenk, L.R. Gahan, Eur. J. Inorg. Chem. 2015 (2015) 3076–3086,
473–482, https://doi.org/10.1016/j.ccr.2012.03.020.
https://doi.org/10.1002/ejic.201500351.
[5] J. Uppenberg, F. Lindqvist, C. Svensson, B. Ek-Rylander, G. Andersson, J. Mol. Biol.
290 (1999) 201–211, https://doi.org/10.1006/jmbi.1999.2896.
[6] Y. Lindqvist, E. Johansson, H. Kaija, P. Vihko, G. Schneider, J. Mol. Biol. 291 (1999)
135–147, https://doi.org/10.1006/jmbi.1999.2962.
[7] N. Sträter, B. Jasper, M. Scholte, B. Krebs, A.P. Duff, D.B. Langley, R. Han,
B.A. Averill, H.C. Freeman, J.M. Guss, J. Mol. Biol. 351 (2005) 233–246, https://
[31] S.J. Smith, R.A. Peralta, R. Jovito, A. Horn, A.J. Bortoluzzi, C.J. Noble, G.R. Hanson,
R. Stranger, V. Jayaratne, G. Cavigliasso, L.R. Gahan, G. Schenk, O.R. Nascimento,
A. Cavalett, T. Bortolotto, G. Razzera, H. Terenzi, A. Neves, M.J. Riley, Inorg. Chem.
51 (2012) 2065–2078, https://doi.org/10.1021/ic201711p.
[32] M. Jarenmark, M. Haukka, S. Demeshko, F. Tuczek, L. Zuppiroli, F. Meyer,
E. Nordlander, Inorg. Chem. 50 (2011) 3866–3887, https://doi.org/10.1021/
7

F.G. Maranha, et al.
InorganicaChimicaActaxxx(xxxx)xxxx
C.T. Pich, G.G. Azzolini, R.A. Peralta, L.M. Rossi, A. Neves, Inorg. Chem. 49 (2010)
2580–2582, https://doi.org/10.1021/ic902489j.
ic1020324.
[33] N. Dutta, S. Haldar, G. Vijaykumar, S. Paul, A.P. Chattopadhyay, L. Carrella,
M. Bera, Inorg. Chem. 57 (2018) 10802–10820, https://doi.org/10.1021/acs.
inorgchem.8b01441.
[42] Bruker APEX2, SAINT and SADABS, version 2011.8-0; Bruker AXS Inc., Madison,
Wisconsin, USA.
[34] A. Kantacha, R. Buchholz, S.J. Smith, G. Schenk, L.R. Gahan, J. Biol. Inorg. Chem.
16 (2011) 25–32, https://doi.org/10.1007/s00775-010-0696-0.
[43] G.M. Sheldrick, Acta Crystallogr. Sect. C Struct. Chem. 71 (2015) 3–8, https://doi.
org/10.1107/S2053229614024218.
[35] A. Neves, M. Aires de Brito, V. Drago, K. Griesar, W. Haase, Inorganica Chim. Acta.
237 (1995) 131–135, https://doi.org/10.1016/0020-1693(95)04678-3.
[36] A. Neves, L.M. Rossi, I. Vencato, W. Haase, R. Werner, J. Chem. Soc. Dalton Trans. 5
(2000) 707–712, https://doi.org/10.1039/A908062G.
[44] M. Strohalm, D. Kavan, P. Novák, M. Volný, V. Havlíček, Anal. Chem. 82 (2010)
4648–4651, https://doi.org/10.1021/ac100818g.
[45] M.Á. Herrador, A.G. González, Talanta 56 (2002) 769–775, https://doi.org/10.
1016/S0039-9140(01)00607-5.
[37] R. Jovito, A. Neves, A.J. Bortoluzzi, M. Lanznaster, V. Drago, W. Haase, Inorg.
Chem. Commun. 8 (2005) 323–327, https://doi.org/10.1016/j.inoche.2005.01.
007.
[46] C.A. Bunton, S.J. Farber, J. Org. Chem. 34 (1969) 767–772, https://doi.org/10.
1021/jo01256a001.
[47] D.L. Nelson, M.M. Cox, Principles of Biochemistry, Macmillan, New York, 2013.
[48] K.A. Deal, A.C. Hengge, J.N. Burstyn, J. Am. Chem. Soc. 118 (1996) 1713–1718,
https://doi.org/10.1021/ja952306p.
[38] B. de Souza, R. Heying, A.J. Bortoluzzi, J.B. Domingos, A. Neves, J. Mol. Catal.
Chem. 397 (2015) 76–84, https://doi.org/10.1016/j.molcata.2014.11.006.
[39] A. Horn, I. Vencato, A.J. Bortoluzzi, R. Hörner, R.A.N. Silva, B. Spoganicz, V. Drago,
H. Terenzi, M.C.B. de Oliveira, R. Werner, W. Haase, A. Neves, Inorganica Chim.
Acta. 358 (2005) 339–351, https://doi.org/10.1016/j.ica.2004.09.021.
[40] R.A. Peralta, A.J. Bortoluzzi, B. de Souza, R. Jovito, F.R. Xavier, R.A.A. Couto,
A. Casellato, F. Nome, A. Dick, R. Lawrence, G. Gahan, G.R. Schenk, F.C.S. de
Hanson, E.C. Paula, S. de Pereira-Maia, P. Machado, P.C. Severino, C. Pich,
T. Bortolotto, H. Terenzi, E.E. Castellano, A. Neves, M.J. Riley, Inorg. Chem. 49
(2010) 11421–11438, https://doi.org/10.1021/ic101433t.
[49] G. Rawji, R.M. Milburn, J. Org. Chem. 46 (1981) 1205–1206, https://doi.org/10.
1021/jo00319a032.
[50] M.C. Etter, P.W. Baures, J. Am. Chem. Soc. 110 (1988) 639–640, https://doi.org/
10.1021/ja00210a076.
[51] A.W. Addison, T.N. Rao, J. Reedijk, J. van Rijn, G.C. Verschoor, J. Chem. Soc.,
Dalton Trans. (1984) 1349–1356, https://doi.org/10.1039/DT9840001349.
[52] M. Lanznaster, A. Neves, A.J. Bortoluzzi, B. Szpoganicz, E. Schwingel, Inorg. Chem.
41 (2002) 5641–5643, https://doi.org/10.1021/ic025892d.
[53] A. Erxleben, Front. Chem. 7 (2019), https://doi.org/10.3389/fchem.2019.00082.
[41] C. Piovezan, R. Jovito, A.J. Bortoluzzi, H. Terenzi, F.L. Fischer, P.C. Severino,
8