Design and catalytic studies of structural and functional models of the catechol oxidase enzyme

Article abstract of DOI:10.1007/s00775-020-01791-2

Abstract: The catechol oxidase activity of three copper/bicompartmental salen derivatives has been studied. One mononuclear, [CuL] (1), one homometallic, [Cu₂L(NO₃)₂] (2), and one heterometallic, [CuMnL(NO₃)

Full text of DOI:10.1007/s00775-020-01791-2

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-020-01791-2
ORIGINAL PAPER
Design and catalytic studies of structural and functional models
of the catechol oxidase enzyme
Aarón Terán1 · Aida Jaafar1 · Ana E. Sánchez‑Peláez1 · M. Carmen Torralba1 · Ángel Gutiérrez1
Received: 10 February 2020 / Accepted: 24 April 2020
© Society for Biological Inorganic Chemistry (SBIC) 2020
Abstract
The catechol oxidase activity of three copper/bicompartmental salen derivatives has been studied. One mononuclear, [CuL]
(1), one homometallic, [Cu2L(NO3)2] (2), and one heterometallic, [CuMnL(NO3)2] (3) complexes were obtained using the
ligand H2L=N,N′-bis(3-methoxysalicylidene)-1,3-propanediamine through diferent synthetic methods (electrochemical,
chemical and solid state reaction). The structural data indicate that the metal ion disposition models the active site of type-
3 copper enzymes, such as catechol oxidase. In this way, their ability to act as functional models of the enzyme has been
spectrophotometrically determined by monitorization of the oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-
tert-butyl-o-benzoquinone (3,5-DTBQ). All the complexes show signifcant catalytic activity with ratio constants (kobs
)
lying in the range (223–294)×10–4 min−1. A thorough kinetic study was carried out for complexes 2 and 3, since they show
structural similarities with the catechol oxidase enzyme. The greatest catalytic activity was found for the homonuclear dicop-
per compound (2) with a turnover value (kcat) of (3.89 0.05)×106 h−1, which it is the higher reported to date, comparable
to the enzyme itself (8.25×106 h−1).
Graphic abstract
Keywords Catalytic activity · Biomimetic catalysis · Cu(II) complexes
Dedicated to Professor José Antonio Campo Santillana, in
Introduction
memoriam.
In last decades, one of the main purposes in bioinorganic
chemistry was the focus on the synthesis and study of chemi-
cal models which were able to mimic the structure and func-
tion of the enzyme active sites using coordination chemistry
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00775-020-01791-2) contains
supplementary material, which is available to authorized users.
Extended author information available on the last page of the article
Vol.:(0123456789)
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JBIC Journal of Biological Inorganic Chemistry
compounds [1]. Those models are simpler and, therefore,
easy to study the relationship between structure and enzy-
matic function to establish a mechanism of the catalytic pro-
cesses. Besides, in several cases, some of these reactions can
be implemented in the industry with an adequate catalyst
obtaining compounds under “greener” conditions [2].
Catechol oxidase enzyme is present in plants, animals,
fungi and bacteria. It takes part in the conversion of a large
number of o-catechols into the respective o-benzoquinones,
which subsequently auto-polymerize, resulting in the forma-
tion of melanin, a dark pigment thought to protect a dam-
aged tissue from pathogens. This enzyme is also relevant
in the industry because of its uses as O2 activator [2–4] or
in medical diagnosis of human brain diseases (detection of
catecholamines, noradrenaline and dopa in neurological dis-
orders) [5].
In this work, we report the synthesis and structural char-
acterization of three structural model complexes of catechol
oxidase enzyme obtained using one symmetric bicompart-
mental Schif base ligand (N,N′-bis(3-methoxysalicylidene)-
1,3-propanediamine, H2L). This ligand has an inner site,
N2O2, formed by two imines and two phenol groups and
one outer site, O2O2, from the two bridging phenol groups
and two methoxy groups in 3,3′ positions (Fig. 1a). The
inner site is more suitable to accommodate 3d metal ions
[14–19], while the external one is capable to accept diverse
metal ions such as alkali [20], alkali earth [21], transition
metals [22, 23] and lanthanides [24–26] as well as small
molecules like water [27, 28] or ammonium cation [29]. We
have also studied the catalytic activity of these complexes to
check whether they can act not only as structural model but
as functional models of the enzyme. The studies have been
carried out using UV–Vis spectroscopy to follow the oxi-
dation process of 3,5-di-tert-butylcathechol (3,5-DTBC) to
3,5-di-tert-butyl-o-benzoquinone (3,5-DTBQ) in an aerobic
methanolic solution bufered at pH 8.
The type-3 active site of the enzyme consists of a dinu-
clear copper center where each copper is coordinated by
three histidine nitrogen atoms and one hydroxo bridge, in
the native met state [6]. Since the discovery of the nature of
this active site, a great number of dicopper(II) complexes
have been designed to mimic the structure and function of
the enzyme [7].
Materials and methods
Synthesis of complexes
Synthesis of H2L
The Schif base-type ligands provide great environments
to obtain dinuclear copper(II) complexes. In particular,
bicompartmental salen (N,N′-disalicylideneethylenediamine)
and its derivatives can be easily synthesized by condensation
of an aldehyde or ketone with a primary amine [8]. Several
modifcations can be introduced in this type of ligands to
modify their electronic and structural properties such as fex-
ibility, electronic nature or steric characteristics resulting in
an exceptionally rich coordination chemistry [9–11].
The best catechol oxidase model reported to date with
copper complexes exhibit kcat values around 104 h−1, at least
two orders of magnitude lower than those of the enzymes iso-
lated from diferent sources (kcat =8.25×106 h−1 from Ipo-
moea batatas (sweet potatoes) [12] and kcat =5.7×105 h−1
from Lycopus europaeu [13]). In that sense, due to the great
importance of this enzyme, it is of big interest to develop
new models in order of improving their catecholase activity.
This ligand H2L, (N,N′-bis(3-metoxysalicylidene)-1,3-
propanediamine), was obtained by previously reported
procedures [30]. A mixture of 3-methoxy-2-hydroxy-
benzaldehyde (4.30 g, 28 mmol) and 1,3-diaminopropane
(1.2 mL, 14 mmol) in methanol (20 mL) by refuxing for
6 h. A yellow powder appears on cooling. The solid was fl-
tered of, washed with methanol and air-dried. Yield 3.67 g
(77%). Anal. C19H22N2O4 (342.39 g mol–1) Calcd (%): C,
66.65; H, 6.48; N, 8.18. Found (%): C, 66.27; H, 6.32; N,
8.19. IR (cm−1): 3421, 2940, 2948, 2902, 2880, 2839, 1631,
1491, 1469, 1441, 1414, 1384, 1359, 1337, 1271, 1256,
1171, 1129, 1095, 1081, 1051, 1032, 1006, 975, 961, 881,
Fig. 1 Coordination environ-
ment resemblance between the
bicompartmental ligand H2L (a)
and the active site of catechol
oxidase enzyme (b)
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JBIC Journal of Biological Inorganic Chemistry
1
837, 785, 742, 730, 623, 570, 507. H-NMR (300 MHz,
CDCl3): δ=13.92 ppm (s, 2H, OH), δ=8.37 ppm (s, 2H,
CH=N), δ = 7.25-6.80 ppm (m, 6H, ArH), δ = 3.91 ppm
(s, 6H, OCH3), δ = 3.76–3.71 ppm (td, 4H, N–CH2),
δ = 2.16–2.06 ppm (q, 2H, CH2–CH2–CH2). UV–Vis
(CH3OH): λmax/nm (ε/dm3 mol−1 cm−1)=263 (15,280), 296
(6196), 326 (3324), 420 (3321).
gents and leaving the product as a brown solid, which
was fltered, washed with cold methanol and dried
over P4O10. Yield 0.12 g (52%).
Anal. C19H20Cu2N4O10 (591.47 g mol−1) Calcd (%):
C, 38.12; H, 3.50; N, 9.36. Found (%): C, 37.82; H,
3.34; N, 9.26. IR (cm−1): 2993, 2949, 2925, 1627, 1613,
1559, 1519, 1494, 1477, 1466, 1440, 1422, 1409, 1384,
1353, 1335, 1307, 1297, 1245, 1233, 1199, 1173, 1101,
1079, 1070, 1015, 1011, 990, 983, 955, 934, 894, 855,
839, 805, 784, 762, 745. UV–Vis (CH3OH): λmax/nm (ε/
dm3 mol−1 cm−1)=226 (47,027), 279 (24,284), 360 (6360),
608 (78).
Synthesis of [CuL] (1)
H2L (0.66 g, 2 mmol) was dissolved in methanol (140 mL)
and placed in an electrochemical cell with a sacrifcial cop-
per anode and an inert platinum cathode using Et4NBr as
electrolyte. The intensity value was set up at 40 mA and the
output was 100 V. The reaction time was 2 h and 41 min
at room temperature. The green solid obtained was fltered
of, methanol washed and vacuum dried. The Ef value was
0.5 mol F–1 which corresponds to the oxidation of metallic
copper to Cu(II). Yield 0.48 g (60%). Anal. C19H20CuN2O4
(403.90 g mol–1) Calcd (%): C, 56.50; H, 4.99; N, 6.94.
Found (%): C, 56.42; H, 4.91; N, 6.94. IR (cm−1): 3431,
3053, 2999, 2941, 2830, 1629, 1545, 1471, 1441, 1403,
1327, 1243, 1221, 1167, 1101, 1082, 1008, 977, 959, 939,
857, 786, 743, 619, 560, 452. UV–Vis (CH3OH): λmax/nm
(ε/dm3 mol−1 cm−1)=282 (24,773), 371 (7172), 550 (197).
Synthesis of [CuMnL(NO3)2] (3)
This compound has been prepared following the previously
reported procedure [22]. The mononuclear complex (1)
(0.10 g, 0.5 mmol) was dissolved in MeOH:MeCN (1:4)
(125 mL), Mn(NO3)2·4H2O (0.06 g, 0.5 mmol) was added
and the resulting solution was stirred for 0.5 h. After 4 days
of slow evaporation at low temperature, a dark green powder
and single crystals suitable for X-ray data collection were
obtained. The product was fltered of, washed with cold
methanol and dried in vacuum. Yield 0.19 g (66%). Anal.
C19H20CuMnN4O10 (582.87 g mol–1) Calcd (%): C, 39.15;
H, 3.46; N, 9.61. Found (%): C, 39.36; H, 3.58; N, 9.27. IR
(cm−1): 1617, 1561, 1472, 1445, 1384, 1302, 1236, 1172,
1101, 1073, 1030, 988, 953, 855, 786, 745, 638, 574, 539,
466. UV–Vis (CH3OH): λmax/nm (ε/dm3 mol−1 cm−1)=226
(44,254), 276 (19,559), 356 (4630), 618 (33).
Synthesis of [Cu2L(NO3)2] (2)
Three diferent procedures have been followed for the syn-
thesis of the title compound.
(i) A methanolic solution (40 mL) of Cu(NO3)2·3H2O
(0.80 g, 3.3 mmol) was added to an stirring solution
of H2L (0.36 g, 1.1 mmol) in methanol (15 mL). The
reaction mixture was stirred at room temperature for
12 h. The solid appeared as a brown powder that was
fltered of and washed with methanol. Single crys-
tals suitable for X-ray data collection were obtained
by slow evaporation of the fltered solution. Yield
0.41 g (65%).
Physical methods and materials
All chemicals of analytical grade were purchased from
Sigma-Aldrich and used without further purifcation.
Elemental analysis (carbon, hydrogen and nitrogen) were
carried out by the Microanalytical Service of the Universi-
dad Complutense de Madrid (UCM) using a LECO CHNS-
932 analyser. FTIR spectra (4000–650 cm−1) of solid powder
samples were recorded using a Perking Elmer spectropho-
tometer with a universal ATR accessory and FTIR KBr-
dispersion spectra (4000–400 cm−1) were recorded using a
THERMO NICOLET 200 spectrophotometer. Mass spectra
were recorded using electrospray ionization (ESI–MS) in
DMSO and MeOH with the HCTultraPTM Discovery Sys-
tem mass spectrometer equipped with a conventional ESI
source. 1H-NMR spectra were collected in the UCM Nuclear
Magnetic Resonance Service using a Burker AVIII300
(300 MHz) spectrophotometer. Electronic spectra in oxygen-
ated methanol (200–1000 nm) were registered in a Cary-5G
spectrophotometer. The measurements of the catalytic oxi-
dation of 3,5-di-tert-butylcatechol (3,5-DTBC) were carried
(ii) H2L (0.17 g, 0.5 mmol) and Cu(NO3)2·3H2O (0.14 g,
0.56 mmol) were dissolved in methanol (105 mL)
in an electrochemical cell with a sacrifcial copper
anode and an inert platinum cathode. The intensity
value was set up at 40 mA and the output was 100 V.
The reaction time was 41 min at room temperature.
The product was fltered of, washed with methanol
and dried. The Ef value was 0.5 mol F–1. Yield 0.13 g
(43%).
(iii) A solid mixture of Cu(NO3)2·3H2O (0.10 g,
0.4 mmol) and H2L (0.14 g, 0.4 mmol) was ground
for 30 min at room temperature. The mixture was
suspended in methanol to extract the excess of rea-
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JBIC Journal of Biological Inorganic Chemistry
out in a JASCO V-630 spectrophotometer at a constant
wavelength of 400 nm under aerobic conditions at 20 °C
and in bufered solution at pH 8 with tris(hydroxymethyl)
aminomethane (Tris). Variable-temperature magnetic sus-
ceptibility measurements in the temperature range 2–300 K
were performed on a Quantum Design MPMSXL SQUID
magnetometer using a constant magnetic feld of 0.5 T. All
susceptibility data were corrected for the diamagnetic con-
tribution of the sample holder, while the molar diamagnetic
corrections from the sample were calculated using the Pas-
cal constants. Cyclic voltammetry (CV) measurements were
performed using an EmStat3 blue potentiostat controlled by
PSTrace software. A three-electrode assembly comprising
a gold working electrode, platinum auxiliary electrode and
Ag/AgCl electrode as reference electrode were used. The
cyclic voltammetry study was carried out at room tempera-
ture in MeCN:H2O (2:1) solution under Argon with 50 mM
Tris bufer (pH 8) at a scan rate of 50 mV s−1 in a potential
range from+0.8 to − 0.8 V.
For catalytic activity studies, 2×10–5 M solutions of com-
plexes 1–3 were treated with 2 × 10–3 M solutions of the
substrate 3,5-DTBC. The reaction progress was followed by
UV/Vis spectroscopy observing the increase in the charac-
teristic quinone (3,5-DTBQ) absorption band at 400 nm. The
data were taken at intervals of 60 s and the conversion was
measured up to 60 min in each case. All data were fxed to
a frst-order kinetic according to Eq. (1), where A∞ denotes
absorbance at t = ∞, At is the variation absorbance value
with time and kobs is the rate constant.
(
)
A∞
log
= kobs ⋅ t.
(1)
A
∞ − At
Enzymatic kinetic experiments for the oxidation of 3,5-
DTBC to 3,5-DTBQ were performed using complex 2 and 3
as catalysts. The experimental procedure was carried out by
the initial rate method monitoring by UV–Vis spectroscopy.
The experiments were carried out by keeping constant the
fnal catalyst concentration at 2×10–5 M and changing the
substrate concentration between 2×10–3 and 2×10–2 M.
Crystallographic studies
A good quality crystal of (2) was directly collected from the
reaction vessel and mounted on a Bruker Smart-CCD dif-
fractometer using graphite monochromated Mo Kα radiation
(λ = 0.71073 Å) operating at 50 kV and 25 mA at 293 K.
Data were collected over a reciprocal space hemisphere by
combination of three exposure sets. Each frame exposure
time was 20 s covering 0.3° in ω. The cell parameters were
determined and refned by least-squares ft of all refections
collected. The frst 50 frames were recollected at the end of
the data collection to monitor crystal decay, and no appreci-
able decay was observed. The structure was solved by direct
methods and refned by applying full-matrix least-squares on
F2 with anisotropic thermal parameters for the non-hydrogen
atoms. The hydrogen atoms were included with fxed iso-
tropic contributions at their calculated positions determined
by molecular geometry. All the calculations were carried
out using SHELXT program for solution and SHELXL for
refnement [31] working into the OLEX2 software package
program [32]. A summary of the fundamental data can be
found in the supplementary material Table S1.
Results and discussion
Synthesis of complexes
The synthetic procedures followed in this work are sum-
marized in Scheme 1.
The mononuclear complex [CuL] (1) has been obtained
by an electrochemical procedure, an unusual synthetic route
for this type of compound, where a copper wire electrode is
oxidized by an electric current to form copper(II) ions that
coordinate into the inner cavity of the anionic bicompart-
mental ligand. This procedure allows a precise stoichiomet-
ric control of the reacting copper(II) amount, obtaining purer
derivatives with a lower formation of by-products.
The efficiency of the electrochemical process is
0.5 mol F–1, indicative of a two electron transfer according
to the following half-reactions:
Anode ∶ L2− + Cu0 → [CuL] + 2e−
Catalytic activity and kinetic studies
Cathode ∶ H2L + 2e− → H2(g) + L2−
.
Catecholase-like activity and kinetic studies of complexes
1–3 were evaluated in aerobic condition spectrophoto-
metrically by monitoring the oxidation of 3,5-DTBC to
3,5-DTBQ at 400 nm as a function of time. The reaction
was studied in a thermostated cell with 1 cm path length at
20.0 0.1 °C. The solvent used to prepare all solutions was
Tris bufer (100 mM, pH 8) in methanol medium saturated
with atmospheric dioxygen during several minutes.
The homonuclear [Cu2L(NO3)2] (2) has been synthetized
by three diferent routes:
(i) electrochemical synthesis in presence of excess
Cu(NO3)2·3H2O;
(ii) one-pot chemical reaction by mixing methanolic
solutions of H2L and Cu(NO3)2·3H2O in a 1:2 stoi-
chiometric ratio;
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JBIC Journal of Biological Inorganic Chemistry
Scheme 1 Synthetic routes used for the preparation of complexes 1–3
(iii) solid state grinding of H2L and Cu(NO3)2·3H2O in a
1:2 ratio.
associated to ν(N=O), νas(NO2) and νs(NO2), respectively,
which are indicative of the presence of coordinated nitrate
groups [34] (see supplementary material Figure S1).
All procedures lead to similar results in purity and yield
of the dinuclear derivative.
Electronic spectra
The heteronuclear derivative, 3, can only be obtained in
good yields by reaction of methanolic solutions of 1 with
Mn(NO3)2·4H2O. The use of other synthetic procedures
lead to a competition between the Cu(II) and Mn(II) ions
to occupy the coordinative cavities of the salen ligand, the
main product being 2.
The electronic spectrum for the ligand, H2L, shows three
bands in the 260–330 nm region that are ascribed to intrali-
gand π→π* and n→π* transitions. Additionally, the ligand
shows an intense absorption band at 420 nm, assigned to a
charge transfer between the ligand and the methanol solvent
(MSCT), since this band was not observed when the spectra
were collected in acetonitrile solution. The formation of the
complexes causes a bathochromic shift of the intraligand
transitions along with the appearance of a low-intensity
absorption band centred at 600 nm that can be assigned to a
d–d transition for the copper(II) ion due to the 2T2g ←2E2g
transition. In the dinuclear complexes, a charge transfer
between the metal ion and the ligand (MLCT) at about
360 nm for 2 and 356 nm for 3 is also observed. A board
low-intensity absorption band at 608 nm for the Cu–Cu com-
plex and 618 nm for the Cu–Mn one is also attributed to the
d–d transition of the copper(II) ion [35] (see supplementary
material Figure S2).
Infrared spectra
The IR spectra indicate that the coordination of a metal center
to the salen ligand slightly shifts the characteristic vibration
bands, ν(C=N)=1630 cm−1, ν(C–O)phenoxo =1255 cm−1 and
νs(C–OCH3)=1081 cm−1. Thus, these bands appear respec-
tively at 1629, 1239 and 1070 cm−1 for 1, 1613, 1245 and
1070 cm−1 for 2 and 1617, 1246 and 1070 cm−1 for 3. The
shift to lower frequencies is indicative of the lower electron
density on the donor atoms because of the coordination of
the metal atoms [33]. The two latter compounds also show
characteristic bands around 1470, 1310 and 1030 cm−1
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JBIC Journal of Biological Inorganic Chemistry
results in small diferences in the metal–oxygen distances
(2.41–2.42 Å for the Cu–Cu derivative and 2.37–2.38 Å
for the Cu–Mn one), leading to metal–metal separations
of Cu···Cu=3.232(1) Å and Cu···Mn=3.322(1) Å, respec-
tively. The former distance is very similar to the Cu···Cu dis-
tance observed in the met state of catechol oxidase enzyme
(3.032 Å). A comparison between the coordinative environ-
ment of the dinuclear complexes and that of the active site
of met state of this enzyme is observed in Fig. 3.
X‑ray crystal structure of (2)
Single crystals suitable for X-ray diffraction have been
obtained from methanolic solutions of the dinuclear
complexes.
The crystal structure of 2 indicates that it is isomor-
phous with the previously characterized complex 3 (Fig. 2)
[22]. Both complexes show a copper(II) ion coordinated
to the inner cavity, N2O2, in a square-planar geometry.
The presence of a second coordination site, O2O2, allows
the coordination of a second metal ion, copper(II) in 2 or
manganese(II) in 3. In both cases, this ion is heptacoordi-
nated by the four oxygen atoms from the outer site of the
ligand and by three oxygen atoms from two nitrate anions,
in a bidentate and monodentate fashion. The bond distances
between the copper ion and the donor atoms in the inner
cavity are very similar, about 1.9 Å, in both complexes. The
diference in size of the metal ion located in the outer cavity
Magnetic properties
The magnetic susceptibility data for 2 and 3 have been meas-
ured in the temperature range 2–200 and 2–300 K, respec-
tively. The temperature dependence of the χT values has
been plotted in Fig. 4 for the two complexes.
The high temperature value of χT for 2 (Fig. 4a) is 0.554
cm3 Kmol–1, below the expected value for two isolated
S=½ spins (0.75 cm3 Kmol–1). On cooling, the χT values
decrease to a minimum value of 0.343 cm3 Kmol–1 at 2 K.
This behaviour can be interpreted in terms of an antifer-
romagnetic coupling between the two copper(II) ions in the
same molecular unit. In this sense, the magnetic data for the
dicopper complex have been ftted to Heisenberg’s dimers
model according to Hamiltonian H=−J·S1·S2, where J is the
coupling constant between both spins, with the addition of a
temperature independent paramagnetism term. The best ft
(solid line) was obtained when g=2.305(2), J=− 1.45(3)
cm–1 and Nα = 2.7(1) × 10–4 cm3 mol–1, this results agree
with the antiferromagnetic behaviour between the two metal
ions.
Compound 3 shows a similar behaviour (Fig. 4b), with
a room temperature χT value at of 3.99 cm3 Kmol–1, lower
than the expected for one S=½ and one S=5/2 independent
spins (4.38 cm3 Kmol–1). The χT values decrease on cooling
until ca. 40 K, when they reach a plateau at 2.89 cm3 Kmol–1,
very close to the expected value of 3.00 cm3 Kmol–1 for the
two spins coupling antiferromagnetically. Below 15 K, the
χT values decrease once more to a minimum value of 2.47
Fig. 2 ORTEP view (50% probability ellipsoids) and labelling
scheme of the asymmetric unit for 2
Fig. 3 Metal–metal distances and angles of dinuclear complexes, 2 (a) and 3 (b), and the coordination sphere of the dinuclear copper(II) centre
of catechol oxidase from sweet potato in the met state (PDB ID: 1BT3) (c)
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JBIC Journal of Biological Inorganic Chemistry
Fig. 4 Temperature dependence
of χT for complexes a 2 and b 3.
The solid lines represent the ft
of the data using the parameters
described in the text
cm3 Kmol–1 at 2 K; this fact can be attributed to the zero
feld splitting of the manganese ion, observable at very low
temperatures, indicative of a small anisotropy distortion
in the metal environment. To simplify the ftting, we have
treated the data in two steps:
D values obtained from the previous ftting where kept con-
stant and the ft (solid line in Fig. 3) afords the fnal values
gCu = 2.350(2), J = − 70.6(5) cm–1 and Nα = 4.6(2) × 10–4
cm3 mol–1, as expected for the antiferromagnetic coupling
predicted between the two metal spins.
The evaluation of the zero feld splitting contribution for
the manganese has been evaluated by ftting the data below
30 K, with the use of the Hamiltonian H = D ⋅ Sz2, where D
is the zero feld splitting parameter for the manganese ion.
The best ft corresponds to gMn =1.962(2) and D=1.52(7)
cm–1.
Catechol oxidase activity
The synthesized complexes in this research have been
designed as structural and functional models of the catechol
oxidase enzyme, so it is necessary to evaluate their ability to
oxidize a catechol (3,5-DTBC) to the corresponding quinone
(3,5-DTBQ). The reaction progress was followed by UV/Vis
spectroscopy (Fig. 5) observing the increase in the charac-
teristic quinone (3,5-DTBQ) absorption band at 400 nm as
function of time in all three cases (see supplementary mate-
rial Figure S3) [3, 36].
In a second step, the whole set of data were ftted, using a
Heisenberg’s dimers model and the zero feld splitting cor-
rection for the Mn(II) cation anisotropy, along with a tem-
perature independent paramagnetism term, usually present
when the Cu(II) ion is involved. The resulting Hamiltonian
was H = −J ⋅ SCu ⋅ SMn + D ⋅ Sz2, where J is the coupling
constant between copper and manganese ions. The gMn and
Fig. 5 Variation of 3,5-DTBQ
absorbance at 400 nm after
addition of complex 2. The
spectra were recorded at inter-
vals of 60 s during 60 min
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JBIC Journal of Biological Inorganic Chemistry
As a previous step, to take into account the possible
autoxidation of the catechol, diferent blank experiments
were performed. The autoxidation of the 3,5-DTBC sub-
strate was measured in the absence of Tris bufer and with-
out oxygenation of the solvent and checked that the autoxi-
dation process does not occur if the medium has not been
previously oxygenated and that the highest autoxidation rate
was obtained when the pH was bufered at 8.
process. The dinuclear compounds have a better catalytic
activity than the mononuclear complex and the homonuclear
dicopper derivative shows the best behaviour, since it is the
most structural and compositionally analogous to the active
site of catechol oxidase enzyme, which it is a type-3 copper
enzyme.
According to these results, the enzymatic kinetic study
was carried out with the dinuclear compounds, 2 and 3,
because of their quick conversion processes. These com-
pounds have been chosen to elucidate a possible mechanism
involved in the catalytic process as well, using two analogue
compounds that show diferences in the composition of the
active site.
In all three complexes, the kobs values for a frst-order
kinetic indicate a remarkable catalytic oxidation process to
form the quinone (Table 1).
The obtained ratio constant values are of the same mag-
nitude order, making difcult the identifcation of the com-
plex having the best catalytic activity and the observation
of what structural and compositional diferences improve
or fall away the catalytic process. For this reason, an evalu-
ation of the conversion percentage of catechol to quinone
was made to check which compound shows the best catalytic
behaviour. To do this, a calibration curve was made using
methanol-oxygenated 3,5-DTBC solutions bufered at pH
8 at three diferent concentrations, 0.005 M, 0.010 M and
0.015 M. The calibration plot allows the interpolation of the
amount of quinone formed in the presence of the catalyst
during the frst 30 min of reaction. The UV–Vis absorbance
at 400 nm for each solution was recorded after 24 h to ensure
that all 3,5-DTBC had been converted to 3,5-DTBQ. The
results are shown in Fig. 6.
Enzymatic kinetic experiments for the oxidation of
3,5-DTBC to 3,5-DTBQ were performed using 2 and 3 as
catalysts, setting the fnal concentration at 2×10–5 M. The
initial rate (Vo) is calculated for each substrate concentra-
tion (between 2×10–3 and 2×10–2 M) from the slope of the
frst-order ftting data at the beginning of the reaction (only
the frst three measurements), when the reverse reaction is
insignifcant.
The obtained data were analysed using the Michae-
lis–Menten approach of enzymatic kinetic. The kinetic
parameters, Vmax, KM and kcat, were calculated from the
Lineweaver–Burk double reciprocal plot. The observed ini-
tial rate (Vo) versus the concentration of 3,5-DTBC plot and
the Lineweaver–Burk plot for 2 and 3 are shown in Fig. 7.
The typical asymptotic representation of the Michae-
lis–Menten kinetic plot was not observed in Fig. 7a because
the maximum rate, Vmax, is not reached. This parameter rep-
resents the amount of substrate molecules converted into
the product by an enzyme molecule per unit time when the
enzyme is fully saturated with substrate, so in this case,
the saturation of the catalysts is not achieved at the meas-
ured substrate concentrations indicating that the catalytic
process must occur very quickly. The Michaelis constant
(KM), which it is inversely related with x-intercept of the
graphs (Fig. 7b), represents the substrate concentration at
The presence of any of the three metallic compounds
improves the catalytic process relative to the autoxidation
Table 1 Values of kobs obtained using the complexes described in this
work as catalysts
Catalyst
[C] (mol L−1
)
kobs (min−1
)
1
2
3
2×10–5
2×10–5
2×10–5
(258 4)×10–4
(294 4)×10–4
(282 2)×10–4
Fig. 6 a Calibration curve
of 3,5-DTBC. b Percentage
conversion of 3,5-DTBC to 3,5-
DTBQ in the presence of the
catalysts, 1 (red), 2 (green), 3
(black) and autoxidation (blue)
during the frst 30 min reaction
time
1 3

JBIC Journal of Biological Inorganic Chemistry
Fig. 7 a Plot of initial rate vs.
substrate concentration for
the oxidation of 3,5-DTBC by
complexes 2 (red) and 3 (blue).
b Lineweaver–Burk plot of
complexes 2 (red) and 3 (blue)
Table 2 Kinetic parameters
for the dinuclear complexes as
catalysts
[Catalyst] (M) Vmax (M·min−1
)
KM (mM)
kcat (h−1
(3.89 0.05)×106
892
)
kcat/KM (mM−1 h−1
)
2
3
0.039
0.003
36.6 0.6
106,059
36
25
3
7
half-maximum reaction rate and it is dependent on both the
enzyme and the substrate, as well as on conditions such as
temperature and pH. KM is correlated with the dissociation
constant of the enzyme–substrate complex, so a higher value
means a low afnity in the enzyme–substrate complex.
The kinetic parameters for both complexes are summa-
rized in Table 2. The higher KM and kcat values found for the
dicopper derivative suggest the best catalytic behaviour for
this compound.
A study of the pH influence on the catalytic pro-
cess has been carried out for compound 2. The enzy-
matic kinetic experiments were performed at pH 7 in the
absence of bufer. In this case, the kcat value obtained was
(2.93 0.03)×104 h−1, one order of magnitude lower than in
the bufered experiment. These kinetic parameters are con-
sistent with an improvement of the catalytic activity associ-
ated with a greater substrate–catalyst afnity favoured by the
coordination of the deprotonated catechol to the copper(II)
metal ions. As observed in Fig. 8, there is a substantial
increase of nearly 30% in the substrate oxidation after
30 min of reaction with the increase of pH.
Fig. 8 Ratio of 3,5-DTBC conversion in Tris bufer medium (black)
and without bufer (red) for 2. Catalyst to 3,5-DTBC molar ratio
1:400
Through comparison with published catalytic constant
(KM and kcat) values for other metal compounds with similar
catechol oxidase activity (summarized in Table 3), it is clear
that 2 is one of the best catalyst models for this oxidation
process and that the kcat value is one of the highest reported,
being comparable to that measured for the catechol oxidase
enzyme.
coordination of a bridging catechol molecule during the
catalytic process.
In the case of the Cu–Mn compound, where the only
diference with the dicopper complex is the presence of a
manganese(II) atom of the outer cavity, the percentage con-
version values are very low. So, it can be deduced that the
presence, in the case of 3, of a second metal ion does not
have a prodigious infuence on the catalytic process because
of the low tendency of Mn(II) to be reduced, preventing a
more efcient catalytic process.
This behaviour can be related to the fact that 2 is formed
by a fexible ligand capable of accommodating two metal
centers with a suitable Cu···Cu distance. One of these cent-
ers has coordinative vacancies while the other is coordi-
nated to two nitrate ligands, easily displaced, favouring the
A plausible mechanism for the catalytic process
using complex 2 is given in Scheme 2. In a frst step, the
1 3

JBIC Journal of Biological Inorganic Chemistry
Table 3 Values of KM and
kcat reported for diferent
compounds with Schif base
ligands showing catechol
oxidase activity
Metal
Solvent
KM(mM)
kcat (h−1
)
References
Dinuclear Ni(II)
Dinuclear Cu(II)
[a]
[a]
[b]
[b]
[b]
[a]
[c]
[a]
0.3
1.44×104
[37]
[38, 39]
1.7–2.3
1.09–9.7
0.9–3.7
0.7–8.3
0.1–4.2
9.52
(1.08–3.24)×104
(0.0167—2.16)×104
(2.41–3.60)×103
(0.17–1.80)×104
(2.47–7.20)×103
3.10×106
Dinuclear Mn(III)
Mononuclear Mn(III)
[5]
Mononuclear Mn(II)
Dinuclear Cu(II) (2)
[40]
This work
36.6
3.89×106
[a] MeOH, [b] CH3CN, [c] DMF
Scheme 2. Proposed mecha-
nism for the oxidation process
of 3,5-DTBC to 3,5-DTBQ
3,5-DTBC displaces one nitrate ligand from the coordina-
tion sphere of the Cu(II) ion located in the outer cavity and
coordinates to the two metal ions [6, 41, 42]. Electrospray
ionization mass spectrometry (ESI–MS) measurements of
compound 2 detected a peaks at m/z=232.9 [M−2NO3]2+,
271.9 [M − 2NO3 + DMSO]2+ and 527.9 [M − NO3]+ (see
supplementary material Figure S5). In addition, the UV–Vis
measurements indicate clear diferences between the solu-
tion spectra of 1 and 2. Both facts clearly demonstrate the
stability of the dinuclear derivative, both in solid state and
solution, and it can be assumed that the second metal ion
remains stable on the ligand outer cavity, its presence being
crucial in the catalytic process.
hydrogen peroxide was not formed and the reaction would
directly proceed to the formation of water or, more probably
in our opinion, that we were unable to detect the hydro-
gen peroxide since it would disproportionate very fast in
the reaction media. According to the literature on the cata-
lytic mechanism for cobalt and manganese compounds, the
ROS-involved mechanism has not been probed overall and
it is supported the formation of water [43–46]. This may be
because Mn and Co compounds are remarkable for carrying
out the fast decomposition of H2O2 which leads to the forma-
tion of H2O and O2 [47, 48]. Chaudhuri et al. showed that
lowering temperature (− 25 °C) can help to slow down the
decomposition of H2O2 allowing its detection [46].
The oxidation process of 3,5-DTBC to 3,5-DTBQ by
transfer of two electron from the two Cu(II)/Cu(II) active
site causes their reduction to Cu(I)/Cu(I). Once the quinone
is uncoordinated, the interaction with molecular O2 from the
reaction medium produces the reoxidation of the dimetallic
core giving rise to water molecules and regenerating the
catalyst. The catalytic mechanism described for the most
compounds that have catechol oxidase activity shows the
formation of hydrogen peroxide (H2O2) in the catalysis
process [7]. In this case, an iodometry was carried out to
detect H2O2 during the 3,5-DTBC oxidation but no hydrogen
peroxide was observed. This fact can either indicate that
Due to the ligand fexibility, a distortion of the coordina-
tion environment of the copper (inner or outer) from a square
planar geometry to pseudo-tetrahedral is possible, through
a rotation of the propylene spacer which it would facilitate
the stabilization of Cu(I) ion. Cyclic voltammetric measure-
ments have proven that the redox behaviour of 2 (Fig. 9) is
reversible in the reaction conditions. The voltammogram
plot shows two reduction peaks at − 0.01 V and − 0.63 V
and two oxidation peaks at − 0.32 V and 0.08 V, respec-
tively. These pseudo-reversible processes can by assigned to
the successive reductions from Cu(II) to Cu(I) of the metal
ions. The peak centred at 0.04 V would correspond to the
1 3

JBIC Journal of Biological Inorganic Chemistry
Compliance with ethical standards
Conflict of interest The manuscript was written through contributions
of all authors. All authors have given approval to the fnal version of
the manuscript. The authors declare no competing fnancial interest.
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Afliations
Aarón Terán1 · Aida Jaafar1 · Ana E. Sánchez‑Peláez1 · M. Carmen Torralba1 · Ángel Gutiérrez1
1
* Aarón Terán
aaronter@ucm.es
Departamento de Química Inorgánica, Facultad de
Ciencias Químicas, Universidad Complutense de Madrid,
28040 Madrid, Spain
1 3