Effect of coordination dissymmetry on the catalytic activity of manganese catalase mimics

Article abstract of DOI:10.1016/j.jinorgbio.2020.111264

Two mixed-valence Mn(II)Mn(III) complexes, [Mn₂L¹(OAc)₂(H₂O)]BPh₄·2.5H₂O and [Mn₂L²(OAc)₂]·4H₂O, obtained with unsymmetrical N₄O₂

Full text of DOI:10.1016/j.jinorgbio.2020.111264

Journal of Inorganic Biochemistry 213 (2020) 111264
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Effect of coordination dissymmetry on the catalytic activity of manganese
catalase mimics
,
Ripul Mehrotraa 1, Micaela Richezzia, Claudia Palopolia, Christelle Hureaub,
,
*
Sandra R. Signorellaa
a IQUIR (Instituto de Quimica Rosario), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Facultad de Ciencias Bioquimicas y Farmaceuticas,
Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina
b
´
CNRS, LCC (Laboratoire de Chimie de Coordination) and UPS, INPT, LCC, Universite de Toulouse, 205 route de Narbonne, F-31077 Toulouse, France
A R T I C L E I N F O
A B S T R A C T
Two mixed-valence Mn(II)Mn(III) complexes, [Mn2L1(OAc)2(H2O)]BPh4⋅2.5H2O and [Mn2L2(OAc)2]⋅4H2O, ob-
tained with unsymmetrical N4O2-hexadentate L1(2ꢀ ) (H2L1 = 2-(N,N-bis(2-(pyridylmethyl)aminomethyl)-6-(N-
(2-hydroxybenzyl)benzylaminomethyl)-4-methylphenol) and N4O3-heptadentate L2(3ꢀ ) (NaH2L2 = 2-(N,N-bis(2-
(pyridylmethyl)aminomethyl)-6-(N′-(2-hydroxybenzyl)(carboxymethyl)aminomethyl)-4-methylphenol sodium
Keywords:
Biomimics
diMn complexes
Unsymmetrical ligands
CAT activity
salt) ligands, have been prepared and characterized. Both complexes share a μ-phenolate-bis(μ-acetate)Mn(II)Mn
Mechanism
(III) core and N3O3-coordination sphere around the Mn(II) ion, but differ in the donor groups surrounding Mn(III)
(NO4(solvent) and NO5). In non-protic solvents, these two complexes are able to disproportionate at least 3600
equiv. of H2O2 without significant decomposition, with first-order dependence on catalyst and saturation kinetics
on [H2O2]. Spectroscopic monitoring of the reaction mixtures revealed the two complexes disproportionate H2O2
Spectroscopy
employing
a different redox cycle, with retention of dinuclearity. The higher catalytic efficiency of
[Mn2L2(OAc)2] was rationalized in terms of the larger labilizing effect of the heptadentate ligand that favors the
acetate-shift and the replacement of the non-coordinating benzyl arm of L1 by a carboxylate arm in L2 which
facilitates the formation of the catalyst-H2O2 adduct, placing [Mn2L2(OAc)2] as the most efficient among the
phenolate-bridged diMn catalysts based on the kcat/KM criterion.
1. Introduction
membranes [3,4]. Therefore, low molecular weight enzyme mimics
constitute an alternative to overcome these limitations [5–7], some of
H2O2 is a by-product of aerobic metabolism in respiratory and
photosynthetic electron-transport chains and a product of enzymatic
activity [1]. Excess of H2O2 and hydroxyl radical, its decomposition
product formed via a Fenton-type reaction, are harmful for almost all
cell components. Thus, the rapid and efficient removal of H2O2 is
essential to all aerobically living prokaryotic and eukaryotic cells.
Catalase (CAT) enzymes are present in most aerobic forms of life and are
responsible for the decomposition of H2O2 into molecular oxygen and
water [2]. In many pathological conditions and diseases, including
cancer, neurological disorders, atherosclerosis, hypertension, ischemia/
perfusion, diabetes, etc., the endogenous antioxidant systems can be
overwhelmed. Administration of exogenous enzymes for the treatment
of oxidative stress is limited by their short half-life, antigenicity, high
cost, and large sizes that disable the enzymes to cross the cell
which have proven to protect cells from oxidative damage in animal
models [8].
Manganese catalases (MnCAT) contain two Mn ions at their active
site and catalyze the disproportionation of H2O2 through a Mn(II)2 and
Mn(III)2 redox cycle [9–11]. The two metal ions of the diMn active site
are triply bridged through a μ1,3-carboxylate of a glutamate residue and
two solvent-derived bridges (either aqua, hydroxo, or oxo ligands) and
terminally bound to His and Glu [12,13]. Both Mn subsites are six-
coordinate but only one of them is bound to a labile water molecule
where initial substrate binding is presumed to occur. This coordination
dissymmetry around the two metal ions could be essential for catalysis.
However, most diMn complexes tested as MnCAT mimics have been
prepared with symmetrical dinucleating ligands with a central alco-
holate or phenolate group that afford synthetic models with identical
* Corresponding author.
E-mail address: signorella@iquir-conicet.gov.ar (S.R. Signorella).
1
Present address: Department of chemistry, Govt. College Chhapara, District-Seoni, Madhya Pradesh, India.
https://doi.org/10.1016/j.jinorgbio.2020.111264
Received 2 August 2020; Received in revised form 9 September 2020; Accepted 21 September 2020
Available online 28 September 2020
0162-0134/© 2020 Elsevier Inc. All rights reserved.

R. Mehrotra et al.
Journal of Inorganic Biochemistry 213 (2020) 111264
environment around the two metal centers [14–18], while only a few
ones were made from unsymmetrical ligands [19–21].
spectrum (δ, MeOD, ppm): 8.49 (d, 1H, Ar-HPy), 8.38 (d, 1H, Ar-HPy),
–
–
–
7.69 (dt, 1H, Ar H), 7.61 (dt, 1H, Ar H), 7.39 (m, 2H, Ar H), 7.23
–
–
With the aim of reproducing the singularities of the biosite, we report
here the synthesis, properties and CAT activity of two diMn complexes
obtained with unsymmetrical ligands bearing a central phenolate group:
2-(N,N-bis(2-(pyridylmethyl)aminomethyl)-6-(N-(2-hydroxybenzyl)
benzylaminomethyl)-4-methylphenol (H2L1) and 2-(N,N-bis(2-(pyr-
idylmethyl)aminomethyl)-6-(N′-(2-hydroxybenzyl)(carboxymethyl)
aminomethyl)-4-methylphenol sodium salt (NaH2L2), and compare the
kinetic results to other diMn mimics. These ligands offer two different
coordination compartments to build bimetallic sites with different
environment around each metal center and will be used for assessing the
influence of the ligand dissymmetry on the CAT activity of the diMn
complexes.
(m, 2H, Ar H), 7.02 (m, 2H, Ar H), 6.85 (m, 2H, Ar-H), 6.78 (s, 2H,
Ar-H), 3.84 (s, 4H, Arpy-CH2-N), 3.74 (s, 2H, -CH2-), 3.72 (s, 2H, CH2),
3.68 (s, 2H, CH2), 3.56 (s, 2H, -CH2-CO2), and 2.23 (s, 3H, CH3). 13C
NMR spectrum (δ, MeOD, ppm): 168.93, 157.96, 157.92, 156.45,
154.03, 148.22, 148.01, 137.44, 137.22, 132.73, 131.75, 131.17,
128.29, 128.06, 127.57, 123.68, 123.51, 123.45, 123.18, 122.44,
122.40, 119.62, 115.12, 59.34, 58.64, 58.28, 58.12, 56.40, 55.90,
54.85, 54.32, and 18.94. Significant IR bands (ATR,
ν
cmꢀ 1): 3420,
2075, 3010, 2917, 2847, 1636, 1591, 1435, 1381, 862, and 760.
2.2. Synthesis of complexes
2.2.1. Complex [Mn2L1(OAc)2(H2O)]BPh4⋅2.5H2O (6⋅2.5H2O)
A solution of H2L1 (113.2 mg, 0.208 mmoL) in CH3OH (2 mL) was
added to a methanolic solution of manganese(II) acetate tetrahydrate
(102 mg, 0.416 mmoL) and left with stirring at room temperature for 2
h. The addition of a methanolic solution (2 mL) of NaBPh4 (71.1 mg)
caused the formation of a brown precipitate, which was collected by
filtration, washed with methanol and hexane and dried under vacuum.
Yield: 0.1920 g (0.167 mmol), 80.0%. Thin layer chromatography (TLC)
of 6 using different mobile phases (in different ratios) and three different
stationary phases (silica, alumina and cellulose) showed only one spot.
Anal. calcd. for BC63H62Mn2N4O7⋅2.5H2O: C 65.6, H 5.9, Mn 9.5, and N
4.9, %; Found: C 65.5, H 5.6, Mn 9.2, and N 5.3%. Molar conductivity
2. Experimental part
2.1. Synthesis of the ligands
The secondary amines, bis(2-pyridylmethyl)amine, N-(2-hydrox-
ybenzyl)benzylamine and N-(2-hydroxybenzyl)glycine chlorohydrate
were prepared as reported previously [22–24]. Intermediates (6-methyl-
2-phenyl-2,4-dihydro-1,3-benzodioxin-8-yl)methanol (1), 8-(bromo-
methyl)-6-methyl-2-phenyl-2,4-dihydro-1,3-benzodioxine (2), 8-(bis(2-
pyridylmethyl)aminomethyl)-6-methyl-2-phenyl-2,4-dihydro-1,3-ben-
zodioxine (3), 2-(N,N-bis(2-(pyridylmethyl)aminomethyl)-6-(hydrox-
ymethyl)-4-methylphenol (4) and 2-(N,N-bis(2-(pyridylmethyl)
aminomethyl)-6-(chloromethyl)-4-methylphenol (5), were synthesized
following procedures described in references [23, 25–27]. Synthetic
details for the obtention of these compounds are described in the sup-
plementary information.
(CH3OH) = 129 Ωꢀ 1 cm2 molꢀ 1. UV–vis, λmax nm (
ε
Mꢀ 1 cmꢀ 1) in N,N-
dimethylformamide (DMF): 275 (20800), 365 (4168), 450 (sh), and 608
(264). Significant IR bands (KBr,
ν
cmꢀ 1): 3445 (broad), 3055, 3030,
2920, 2853, 1625, 1595, 1560, 1431, 864, 734, 705, 644, and 611. The
content of 2.5 molecules of non-coordinated water per complex mole-
cule was confirmed by thermogravimetric analysis of the complex which
showed 3.9% mass loss below 120 ◦C.
2.1.1. 2-(N,N-Bis(2-(pyridylmethyl)aminomethyl)-6-(N-(2-
hydroxybenzyl)benzylaminomethyl)-4-methylphenol (H2L1)
To a solution of 5 (533.6 mg, 1.45 mmoL) and N-(2-hydroxybenzyl)
2.2.2. Complex [Mn2L2(OAc)2]⋅4H2O (7⋅4H2O)
A solution of NaH2L2 (100 mg, 0.187 mmol) in 8 mL CH3OH was
mixed with a solution of Mn(OAc)2⋅4H2O (92 mg, 0.378 mmol) in 2 mL
CH3OH, and the red brown mixture was left with stirring for 3 h. Then
the volume was reduced to 2 mL under vacuum, 10 mL ethyl ether were
added, and the mixture cooled to 4 ◦C. After 3 h, the formed brown
precipitate was filtered off, washed with cool CH3OH and ether and
dried under vacuum. Yield: 108 mg, 78.3%. TLC of 7 using different
mobile phases (in different ratios) and three different stationary phases
(silica, alumina and cellulose) showed only one spot. Anal. calcd. for
benzylamine (309 mg, 1.45 mmoL) dissolved in THF (15 mL), 200 μL of
Et3N were added and left with stirring at room temperature for 6 days.
The solid formed was removed by filtration. After evaporation of THF, a
red yellow oil was obtained. This oil was further dissolved in 5 mL
CH2Cl2 and washed with phosphate buffer (pH 7.0). The organic layers
were collected, dried over Na2SO4 and filtered. After solvent evapora-
tion the solid product was obtained. Yield: 380.2 mg, 48.1%. 1H NMR
spectrum (δ, CDCl3, ppm): 8.56 (d, 2H, Ar-Hpy); 7.60–7.54 (td, 2H,
–
–
–
Ar H); 7.45 (d, 1H, Ar H); 7.36–7.31 (m, 5H, Ar H); 7.28–7.24 (m,
–
–
–
C
34H35Mn2N4O8⋅4H2O: C 50.4, H 5.4, Mn 13.6, and N 6.9, %; Found: C
1H, Ar H); 7.16–7.12 (t, 2H, Ar H); 7.10–7.09 (m,1H, Ar H);
50.1, H 5.1, Mn 13.7, and N 6.7%. UV–vis, λ
nm (ε
Mꢀ 1 cmꢀ 1) in
–
–
–
6.99–6.97 (d, 1H, Ar H); 6.91 (s, 1H, Ar H); 6.80–6.70 (t, 2H, Ar H);
6.74–6.71 (dd, 1H, Ar-H); 3.86 (s, 4H, Arpy-CH2-N); 3.79 (s, 2H, –CH2);
3.74 (s, 2H, –CH2); 3.72 (s, 2H, –CH2); 3.68 (s, 2H, –CH2); and 2.22 (s,
3H, CH3). 13C NMR spectrum (δ, CDCl3, ppm): 158.23, 157.7, 154.15,
148.92, 136.73, 131.42, 130.44, 129.71, 128.90, 128.41, 128.30,
127.46, 127.24, 126.55, 123.27, 122.67, 122.18, 118.82, 115.98, 59.16,
max
DMF: 279 (21350), 345 (4105), 462 (1665), and 694 (293). Significant
IR bands (KBr,
ν
cmꢀ 1): 3435 (broad), 3065, 3032, 3004, 2921, 2859,
1624, 1590, 1574, 1422, 806, 761, 654, and 630. The content of 4
molecules of non-coordinated water per complex molecule was
confirmed by thermogravimetric analysis of the complex which showed
8.9% mass loss below 120 ◦C.
57.93, 57.13, 56.70, and 20.43. Significant IR bands (ATR,
3356, 2914, 1589, 1454, 1433, 1357, 746, and 696.
ν
cmꢀ 1):
2.3. Analytical and physical measurements
2.1.2. 2-(N,N-Bis(2-(pyridylmethyl)aminomethyl)-6-(N′-(2-
hydroxybenzyl)(carboxymethyl) aminomethyl)-4-methylphenol sodium salt
Infrared spectra were recorded on a Perkin-Elmer Spectrum One
FTIR spectrophotometer in the 4000–400 cmꢀ 1 range. UV–visible
spectra were recorded on a Jasco V-550 spectrophotometer, with ther-
mostated cell compartments. Metal content was determined with an
Inductively coupled plasma mass spectrometer (ICP-MS) Perkin Elmer
NexION 350×. Electron Paramagnetic Resonance (EPR) spectra were
obtained at 115 K on an Elexsys E 500 Bruker spectrometer, operating at
a microwave frequency of approximately 9.5 GHz. Thermogravimetric
analysis (TGA) measurements were conducted on a Perkin-Elmer Dia-
mond TG/DTA Instrument. The compound was heated at the rate of
10 ◦C/min between RT and 800 ◦C. Conductivity measurements were
(NaH2L2)
A solution of 5 (1.36 mmol, 500 mg) in 15 mL THF was added to N-
(2-hydroxybenzyl)glycine chlorohydrate (296 mg, 1.36 mmol) in 5 mL
of 0.55 M NaOH aqueous solution. The mixture was left with stirring for
48 h. THF was removed under vacuum and 20 mL CH2Cl2 added to the
aqueous residue. The organic layer was separated and washed with
phosphate buffer of pH 7.0 (3 × 10 mL), dried over Na2SO4 and filtered.
After concentration of the filtrate, hexane was added and a yellow solid
formed when cooled at 4 ◦C. Recrystallization from 2:8 CHCl3:hexane
mixture afforded a bright yellow solid. Yield: 139 mg (19.2%). 1H NMR
2

R. Mehrotra et al.
Journal of Inorganic Biochemistry 213 (2020) 111264
performed using a Horiba F-54 BW conductivity meter, on 1.0 mM so-
lutions of the complexes in methanol. Electrospray ionization (ESI) mass
spectra were obtained with a Thermo Scientific LCQ Fleet. The solutions
for electrospray were prepared from solutions of complex or reaction
mixtures diluted with acetonitrile to a final ~10ꢀ 5 M concentration. 1H
NMR spectra were recorded on a Bruker AC 300 NMR spectrometer at
ambient probe temperature (ca. 25 ◦C). Chemical shifts (in ppm) are
referenced to tetramethylsilane and paramagnetic NMR spectra were
acquired employing superWEFT sequence, with acquisition time of 50
ms. The electrochemical experiments were performed with a computer-
controlled Princeton Applied Research potentiostat, VERSASTAT II
model, with the 270/250 Research Electrochemistry Software. Studies
were carried out under Ar, in N,N-dimethylformamide (DMF) solution
using 0.1 M Bu4NPF6 as a supporting electrolyte and ≈10ꢀ 3 M of the
complexes. The working electrode was a glassy carbon disk, and the
reference electrode was a calomel electrode isolated in a fritted bridged
with a Pt wire as the auxiliary electrode. All potentials are referred to the
saturated calomel electrode (SCE). Under these conditions, E(ferrocene/
ferrocenium) = 474 mV, in DMF.
thermostated H2O2 was injected through the septum to the stirred
complex solution, and the evolved O2 was measured with the burette.
The initial reaction rates were obtained by fitting the [O2] versus time
data to a polynomial expression and calculating the slope of the tangent
at time zero. Each rate constant reported here represents the mean value
of multiple determinations that fall within ±5%. All experiments were
carried out at 25 ◦C. Blanc experiments performed with 100 mM H2O2 in
DMF without the catalyst showed no decomposition after 1 h.
3. Results and discussion
3.1. Synthesis and characterization of complexes 6 and 7
The synthesis of the unsymmetrical ligands H2L1 and NaH2L2 is
shown in Scheme 1.
The key step in the synthesis for introduction of dissymmetry is the
formation of the cyclic acetal 1 to protect one of the terminal hydrox-
ymethyl groups of the starting 2,6-bis(hydroxymethyl)-4-methylphenol
[25]. Next, bromination and alkylation of bis-2-picolylamine afforded
the N3-branch. Acid hydrolysis of the acetal, followed by chlorination
and subsequent alkylation of a second amine bearing terminal benzyl/
phenol or carboxylate/phenol groups afforded the N4O2-hexadentate
(H2L1) and N4O3-heptadentate (H2NaL2) ligand, respectively.
Dimanganese complexes 6 and 7 were prepared from 1:2 mixtures of
the corresponding ligand and Mn(OAc)2⋅4H2O in methanol. Acetate
facilitates the deprotonation of the ligand for coordination to the metal
[28] and complexation occurs immediately after mixing, as evidenced
2.4. Evaluation of CAT activity
Reaction rates were determined by volumetric measurement of the
O2 evolved after addition of excess of H2O2 to a DMF solution of the
complex. A round-bottom flask with a rubber septum, containing the
degassed solution of the complex, was thermostated at 25 ◦C and con-
nected to a gas-measuring burette (precision of 0.1 mL). Previously
N
N
N
i
ii
iii
N
N
O
O
OH OH OH
OH
O
O
Br
O
O
N
3
1
2
iv
v
N
N
OH OH
N
N
OH Cl
N
N
N
OH
5
4
HO
NHCl
OH
O
vi
vii
ONa
O
N
OH
N
N
N
OH
HO
N
N
N
HO
N
H2L1
NaH2L2
Scheme 1. Synthesis of unsymmetrical ligands. (i) PhCH(OMe)2, p-MeC6H4SO3H, DMF, 50 ◦C; (ii) CBr4, PPh3, DMF; (iii) NaH, CH2Cl2; (iv) HCl, THF, 0 ◦C; (v) Cl2SO,
35 ◦C; (vi) Et3N, THF; (vii) NaOH, THF
3

R. Mehrotra et al.
Journal of Inorganic Biochemistry 213 (2020) 111264
by the change of color of the reaction mixture from pale yellow to
brown. Complex 6 was precipitated as a brown solid from the reaction
mixture upon addition of the bulky BPhꢀ4 , while complex 7 separated
from the solution as a brown solid after addition of ether at 4 ◦C.
The IR spectra of powdered 6 and 7 (Fig. S1) exhibit strong phenolate
and pyridyl ring absorptions between 1625 and 1575 cmꢀ 1 as well as
absorption bands at 1560–1574 cmꢀ 1 and 1431–1422 cmꢀ 1 attributable
to the antisymmetrical and symmetrical stretching vibrations of
carboxylate groups. For 6, the three bands at 611, 705 and 734 cmꢀ 1
together with a strong and sharp stretching band at ~3055 cmꢀ 1 can be
assigned to non-coordinated BPh4ꢀ counter anion, in agreement with its
molecular structure. In line with the loss of H2O evidenced by the
thermogravimetric analysis, the two compounds display a broad band at
≈3440 cmꢀ 1 assigned to water molecules.
spectrum of complex 6 shows only one peak at m/z = 319 correspond-
ing to BPhꢀ4 anion, in agreement with the molar conductivity of 129 Ωꢀ 1
cm2 molꢀ 1 for 6 in methanol, a value expected for a complex that be-
haves as a 1:1 electrolyte in solution.
Fig. 1(b) illustrates the X-band EPR spectra of the two powdered
complexes at 120 K. These spectra exhibit two features, one around g = 2
which dominates the spectra and a low-field component at g = 5 for 6
and 6.8 for 7, which can be associated with spin transitions in S > ½ spin
states of the weakly coupled phenolate-bridged Mn(II)Mn(III) center, as
already observed for other Mn(II)Mn(III) complexes [29–30].
The electronic spectra of complexes 6 and 7 (Fig. 1(c)) recorded in
DMF exhibit an intense absorption at 275 and 279 nm, respectively,
assigned to
intense bands in the region 330–550 nm attributed to ligand-to-metal
charge transfer (LMCT) transitions from p orbital of phenolate (either
π-π* transitions within the ligands, and two moderately
ESI-mass spectra of complexes 6 and 7 in CH3CN (Fig. 1(a))
confirmed their chemical composition as well as retention of their
nuclearity in solution. For complex 6, the parent peak is observed at m/z
= 811.2 in the positive mode ESI-mass spectra and originates from the
[Mn2L1(OAc)2(CH3CN)]+ monocation. Other peaks generated during
the electrospray correspond to the exchange of AcOꢀ by HCO2ꢀ
([Mn2L1(OAc)(HCO2)]+, m/z = 757.2) and the loss of Mn(OAc)2
([MnL1H]+, m/z = 598.2). Complex 7 affords a mass spectrum with the
major peak at m/z = 755.1 ([Mn2L2(OAc)2(H2O)]+) and two minor
peaks that can be assigned to [Mn2L2(OAc)]+ at m/z = 678.1 and
[MnL2Na]+ at m/z = 587.1. The isotopic patterns of these peaks match
very well their simulated spectra. In addition, negative ion ESI-mass
π
bridging or terminal phenolate) to partially filled dσ* and dπ* orbitals of
Mn(III) [17,19,31,32] with an additional contribution of carboxylate-to-
Mn(III) CT on the lower energy band [33]. The low intensity broad band
observed around 608 nm (
ε
= 265 Mꢀ 1 cmꢀ 1) for 6 and at 698 nm (
ε =
294 Mꢀ 1 cmꢀ 1) for 7, corresponds to d-d transitions of the Mn(III) ion in
agreement with reported values for related Mn(II)Mn(III) complexes
[31,34]. The red shift of the d-d band can be interpreted in terms of the
effect of the axial or equatorial position of the terminal phenolate group
in the two complexes (Fig. 2). The more distant binding of phenolate to
the Mn(III) ion on the elongation Jean-Teller axis weakens the metal-
phOax bond reducing the d-d energy gap and this can account for red
Fig. 1. (a) ESI-mass spectra of 6 and 7, in MeCN, positive mode. (b) EPR spectra of solid complexes,
ν = 9.5 GHz, T = 120 K, microwave power = 0.5 mW. (c)
Electronic spectra of 6 and 7 in DMF. [complex] = 0.125 mM; l = 1 cm. (d) 1H NMR spectra of 6 in DCCl3 and 7 in CD3OD. [complex] = 15 mM.
4

R. Mehrotra et al.
Journal of Inorganic Biochemistry 213 (2020) 111264
+
O
w
N
O
N
N
O
N
Mn
O
Mn
Mn
O
N
Mn
O
O
N
O
O
N
O O
O
O
N
O
6+
7
Fig. 2. Schematic structure formulas of the diMn complexes under study. w = water.
shift of this band in the spectrum of 7 relative to 6 [35]. The contribution
of other electronic transitions disable direct comparison of the energy of
the phenolate-to-Mn(III) CT bands. Electronic spectra of the two com-
plexes registered at different times after preparing the DMF solutions (up
to 24 h), showed identical molar absorption coefficients, indicating that
both are stable in this solvent.
oxidation to generate a Mn(II)Mn(III)-phenoxyl radical [40]. In the
case of complex 7, three non-reversible anodic processes are detected at
Epa1 ≈ 0.4 V, Epa2 ≈ 0.52 V and Epa3 ≈ 0.79 V vs SCE (Fig. S3(d)), which
might be attributed to the formation of higher oxidation state complexes
with concomitant formation of oxo-bridges. It must be observed that
while reduction and first oxidation processes of 6 take place at potentials
differing in 0.38 V, 7 shows electrochemical stability over ΔE = 0.56 V.
Therefore, the introduction of carboxylate instead of the non-
coordinating benzyl group in the ligand framework plays a significant
role in the stabilization of the mixed valence Mn(II)Mn(III) state in dry
DMF. An still larger ΔE (0.965 V) was reported for another triply
phenolate-bis(acetate)-bridged Mn(II)Mn(III) complex where the benzyl
group is replaced by pyridine, a donor stronger than carboxylate, with
redox processes taking place at Ered = ꢀ 0.057 V and Eox = 0.908 V vs
SCE in MeCN [19].
The 1H NMR spectrum of solutions of 6 and 7 (Fig. 1(d)) show res-
onances outside the diamagnetic region spanning from 70 to ꢀ 30 ppm.
For the two complexes the signals appear in the same spectral region
according to a weak magnetic coupling between the metal ions; how-
ever, resonances are much narrower for 7 as a consequence of the
different spatial arrangement of the ligands in the two compounds. For
6, the spectral pattern comprises a set of broad signals at 60, 48, 29, 20
and ꢀ 13 ppm (linewidth in the range of 2500 to 3500 Hz) ascribed to
phenolate/pyridine ring protons with even broader peaks of methylene
protons spanning the full spectral range [36]. For 7, the spectrum is
better resolved showing intense resonances at 62, 49, 47 and 45 ppm
(w1/2 = 1200 Hz), and relatively sharp resonances at 26, 19, ꢀ 9, ꢀ 12
and ꢀ 14.5 ppm (w1/2 = 600 Hz), and ꢀ 26 ppm (w1/2 = 300 Hz). These
peaks can be attributed to phenol and pyridine ring protons shifted
down- and upfield as a consequence of spin-delocalization and dipolar
effects, the last playing an important role in this kind of mixed-valence
Mn(II)Mn(III) complexes [37].
3.3. Catalase activity studies
3.3.1. Kinetics
In the previous sections it was shown that the replacement of the
non-coordinating benzyl arm by the carboxylate in the ligand influences
both the ligand-field splitting and the redox potentials. Thus, it is ex-
pected that this ligand changes also affect the catalase activity of the
resulting complexes. The ability of complexes 6 and 7 to catalyze H2O2
disproportion was tested in DMF, CH3CN, and CH3OH. For both com-
plexes, an immediate vigorous evolution of dioxygen was observed after
addition of H2O2 to a solution of the catalyst. Turnovers as high as 3600
without significant decomposition were measured for the two complexes
in DMF, and up to 600 and 300 equivalents of H2O2 were decomposed in
CH3CN and CH3OH, respectively. Besides, in the last two solvents, the
rate of O2 evolution diminished after successive additions of H2O2. It is
likely that the bridging ligands of the complexes serve as internal bases
facilitating deprotonation of the peroxide coupled to the redox reaction.
Therefore, in DMF, a non protic solvent (αDMF = 0) [41], these catalysts
disproportionate H2O2 with only slight loss of activity (Fig. S4). How-
ever, in the protic CH3OH solvent (αMeOH = 0.93) or CH3CN, a solvent
with intermediate proton donor ability (αMeCN = 0.19), protonation of
the bridges could inactivate the catalyst resulting in moderate to low
turnover numbers. Therefore, DMF was chosen as solvent for evaluation
of catalase activity.
The 1–10 ppm region of the 1H NMR spectra of the complexes
(Fig. S2) show peaks belonging to the solvent, and BPhꢀ4 in the case of
complex 6, without traces of the ligand, indicating the compounds are
stable towards metal dissociation. Besides, no signal attributable to
terminally coordinated acetate (broad signal at around 1.9 ppm [38]) is
observed, which suggests that the most plausible structure for the two
complexes in solution contains two bridging acetate ligands as shown in
Fig. 2.
3.2. Electrochemical studies
The electrochemical properties of complexes 6 and 7 were investi-
gated by cyclic voltammetry in DMF solutions containing 0.1 M Bu4NPF6
(Fig. S3). The two complexes exhibit one quasi-reversible reduction
wave at E1/2 = 0.058 V (6) and ꢀ 0.164 V (7). Linear voltammetry
confirmed that these waves correspond to reduction processes. W1/2
values of 0.12 V for 6 and 0.13 V for 7 in the square-wave voltammetry
(SWV) experiments (Fig. S3(a–b)) suggest that these reductions corre-
spond to one-electron processes and may be attributed to the Mn(II)Mn
(III)/Mn(II)Mn(II) redox couple.
The initial rate of H2O2 disproportionation by complexes 6 and 7 in
DMF was measured as a function of the complex (Fig. S5) and substrate
concentrations at 25 ◦C. As shown in Fig. 3, for both complexes, at
constant [H2O2]0 = 148 mM, the initial rate (ri) varies linearly with the
[catalyst], and the first-order rate constants k61 = 24.5(3) minꢀ 1 and k71
= 51.2(2) minꢀ 1 were obtained from the slope of the straight line.
At constant [catalyst] = 0.25 mM, complexes 6 and 7 exhibit satu-
ration kinetics with [H2O2]0 (Fig. 3) and the ri/[catalyst] vs [H2O2]0
data could be fitted to the Michaelis-Menten equation (Eq. (1)). In this
equation, kcat is the catalytic rate constant (turnover number) and KM is a
measure of the complex affinity for H2O2 (the lower the KM value, the
On the oxidative side, 6 shows two anodic waves at Epa1 = 0.44 V
(from deconvolution of the SWV wave) and E1/2(2) = 0.54 V vs SCE (ΔEp
= 0.14 V) (Fig. S3(c)). These two one-electron oxidation processes might
correspond to the formation of Mn(III)2 and Mn(II)Mn(III)-phenoxyl
radical. The formation of Mn(II)Mn(III)-phenoxyl radical and Mn(III)2
occurs at close potentials and are difficult to distinguish [39]. However,
the growth of the relative intensity of the second oxidation wave with
increasing scan rate suggests it corresponds to the ligand-centered
5

R. Mehrotra et al.
Journal of Inorganic Biochemistry 213 (2020) 111264
Fig. 3. Effect of [catalyst]0 (green and orange lines; [H2O2] = 148 mM) and [H2O2]0 (brown and blue lines; [catalyst] = 0.25 mM) on the initial rate of H2O2
disproportionation catalyzed by (a) 6 and (b) 7 at 298 K, in DMF.
higher affinity for H2O2).
at 322 nm. The intensity of bands at longer wavelengths changed
slightly during the reaction. The same spectral changes were observed
upon successive additions of 100 equiv. of H2O2 to the DMF solution of
the catalyst, and the O2 production rate measured after each new
addition was essentially constant, suggesting that the catalyst is stable in
these reaction conditions.
The values of catalytic turnover numbers k6cat = 2.3(1) sꢀ 1 and kc7at
=
1.22(2) sꢀ 1, and Michaelis constants KM6 = 6.8(4) × 10ꢀ 1 M and K7M = 7.3
(5) × 10ꢀ 2 M, were determined from the non-linear fit of experimental
data to Eq. 1 (solid wine and blue lines in Fig. 3).
ri
kcat [H2O2]
After addition of 100 equiv. of H2O2 to a solution of 6 in DMF, the
low-temperature X-band EPR spectra exhibit a broad signal (Fig. 4(a),
inset) consistent with a weakly coupled Mn(II)Mn(III) center [17]
overlapped to a six-line signal centered at g = 2 with hyperfine splitting
of ≈ 93 G. This 6-line signal departs from that of the solvated Mn2+ ion
and is characteristic of a Mn(II) center bound to the ligand that could
arise from a Mn(III)Mn(II) species where the phenolate-bridge disrupted
through protonation [36,37]. This cleavage of the phenolate bridge
makes the diMn unit appear as the individual ions: an EPR silent Mn(III)
and a mononuclear Mn(II) with a six-line signature. This was confirmed
by addition of p-toluensulphonic acid to the solution of 6 in DMF giving
the same EPR spectrum. This spectral pattern was kept at the end of the
reaction. Retention of the complex nuclearity during the reaction was
confirmed by mass spectrometry. ESI-mass spectra obtained after addi-
tion of 100 equiv. H2O2 to a DMF solution of 6 showed the parent peak at
m/z 811.2 and other peaks already appearing in the spectrum of the
starting solution and a new peak at m/z 773.2 corresponding to
[Mn2L1(OAc)(HCO2)(OH)]+ (Fig. S6(a)), thus providing a clear indica-
tion that the diMn complex persisted during the catalytic cycle.
During the spectrophotometric monitoring of the reaction of 7 with
excess H2O2 in DMF, the intensity of the CT band at 345 nm decreased
=
(1)
[catalyst] KM + [H2O2]
Kinetics results show that electron transfer between 6 and H2O2 is
more effective (larger kcat) than for 7, but 6 is 10-times less efficient at
binding the substrate (KM6 > KM7 ), so that can reach a maximal rate (Vmax
= kcat [catalyst]) at [H2O2] much higher than 7. Based on the kcat/KM
6
k7cat
criterion, k = 3.38 Mꢀ 1 sꢀ 1 and
= 16.7 Mꢀ 1 sꢀ 1, the catalytic effi-
cat
KM6
KM7
ciency of 7 is ≈ 5-times higher than for complex 6. The higher efficiency
of 7 to disproportionate H2O2 is related to its larger affinity for binding
peroxide, probably because the presence of the terminal carboxylate
facilitates H2O2 binding through hydrogen bonding favoring the for-
mation of the catalyst-H2O2 adduct [18,42].
3.3.2. Spectroscopic monitoring of the catalase reaction
In order to get insight into the mechanism of the H2O2 dispropor-
tionation catalyzed by complexes 6 and 7 in DMF, the reaction was
monitored by using a combination of electronic and EPR spectroscopies
and ESI-MS. UV–visible absorption spectra taken in DMF during the
progress of the reaction of complex 6 with excess H2O2 exhibited an
increase of the absorption band at 275 nm concurrently with the
decrease of the absorbance at 365 nm (Fig. 4(a)) with an isosbestic point
Fig. 4. UV–vis spectra of (a) 6 (0.125 mM) and (b) 7 (0.1 mM) after addition of 100 equiv. of H2O2, in DMF. T = 25 ◦C. Inset: X-band EPR spectra of 1 mM catalyst
+100 equiv. H2O2 a few minutes after mixing.
ν
= 9.5 GHz, T = 110 K, microwave power = 0.5 mW.
6

R. Mehrotra et al.
Journal of Inorganic Biochemistry 213 (2020) 111264
and absorbance at 279 nm grew up, with an isosbestic point at 323 nm
(Fig. 4(b)). Interestingly, the intensity of the band at 462 nm (PhO-to-
kinetics with substrate indicates that a catalyst-peroxide adduct is
formed at this step, probably by substitution of the labile solvent
–
molecule bound to Mn(III), which upon O O bond breaking yields the
d
π
Mn(III) CT) also raised, although in a lesser proportion. These spec-
tral changes suggest a different arrangement of phenolate groups around
Mn(III) during catalysis. For this complex, the initial intensity of the
LMCT bands was not restored at the end of the H2O2 disproportionation
reaction. The EPR spectra taken during the 7 + H2O2 reaction course
(Fig. 4(b), inset) showed broad lines expanded over the whole spectral
range characteristic of diMn(II) species with weak anti-ferromagnetic
exchange interactions [37,43,44] which originate from the contribu-
tion of the populated paramagnetic higher excited spin states [45].
Several sets of 11 lines are observed on the top of some of the more
intense signals (i.e. signal centered at 2870 G), with an average hyper-
fine splitting of ≈ 43 G typical of the interaction between the electronic
spin and two Mn(II) nuclear spins (IMn = 5/2). This Mn(II)2 spectrum is
superimposed to a 6-line signal at g ≈ 2 that arises from a small fraction
of monomeric aqueous Mn2+ (up to 5% at the end of the reaction). The
final EPR spectrum of the reaction mixture was identical to those
registered during the reaction meaning the formed Mn(II)2 species
persists in solution, in agreement with absorption spectra. The dispro-
portionation of H2O2 catalyzed by 7 was also followed by ESI-MS. Be-
sides the parent peak at m/z 755 and other peaks already present in the
starting complex solution, another intense peak is observed at m/z =
619.1 that can be attributed to [Mn2L2]+, thus confirming the retention
of dinuclearity of the complex during catalysis (Fig. S6(b)).
μ-oxo-Mn(III)Mn(IV) and water. This oxidized form of the complex re-
acts with another H2O2 molecule in a fast-oxidative half-reaction to
afford O2 and restores the starting complex closing the cycle. This
oxidative half-reaction must be much faster than the reductive one, so
that the μ-oxo-Mn(III)Mn(IV) is not observed by EPR.
For H2O2 disproportionation catalyzed by complex 7, the observa-
tion of a coupled Mn(II)2 species in the EPR spectra taken during and at
the end of the reaction suggests a redox cycle involving Mn(II)2 and Mn
(III)2 oxidation states, as proposed in Scheme 3.
According to this scheme, initial oxidation of the starting Mn(II)Mn
(III) complex by H2O2 affords Mn(III)2(OAc)1(2)(μ-O(H)) species (EPR
silent) in a fast step. In this way, the starting complex should be a pre-
cursor of the active complex. A similar one-electron oxidation of a
precursor phenolate-bridged diMn(II) complex has already been re-
ported [36]. The H2O2 binding to the starting complex can take place
through acetate-shift to yield the
μ-hydroxo-Mn(III)2 species, where
terminally bound acetate can be further protonated and partially dis-
sociates. The higher labilizing effect of L2(3ꢀ ) compared to L1(2ꢀ ) may
favor acetate dissociation as evidenced by the high proportion of the
Mn2L+ species in the mass spectra. The
μ-oxo-Mn(III)2 species is pro-
posed to be the oxidized active form of the catalyst that enters the cat-
alytic cycle of H2O2 dismutation. The absence of a lag-time at the onset
of the reaction is consistent with the oxidation of the mixed valence
complex being faster than the H2O2 disproportionation reaction.
Hydrogen bond to the terminally bound carboxylate group of the ligand
within the catalyst-H2O2 adduct (Fig. S7) may account for the higher
efficiency of this complex for binding peroxide (low KM value) which
renders 7 more efficient to catalyze H2O2 disproportionation than 6.
At pH 7, the electrochemical potentials for the two-electron O2/H2O2
and H2O2/H2O redox couples are +0.04 V and +1.11 V vs SCE,
respectively. If disproportionation of H2O2 occurs through an outer-
sphere mechanism, only diMn complexes with redox couples between
the H2O2 reduction and oxidation potentials should be active. However,
a number of complexes that exhibit redox potentials outside the H2O2
dismutation range react efficiently [7,14], suggesting diMn complexes
dismutate H2O2 through an inner-sphere mechanism with electron
transfer taking place within the catalyst-peroxide adduct, in agreement
with the saturation kinetics observed for a number of diMn mimics. In
Table 1, the catalytic efficiency (kcat/KM values) of complexes 6 and 7 is
3.3.3. Mechanism of the CAT-like reaction of 6 and 7
The two complexes catalyze H2O2 disproportionation with first order
kinetics on [catalyst], rates are essentially constant upon successive
additions of H2O2, and O2 evolution occurs without a time-lag at the
onset of the reaction, all suggesting these complexes are responsible for
the disproportionation of H2O2. Based on spectroscopic results, both
compounds retain their dinuclearity during catalysis but employ a
different redox cycle for H2O2 disproportionation. From the EPR spectra,
the mixed valence Mn(II)Mn(III) complex 6 is present in solution during
the catalytic cycle either in the coupled [Mn(II)(
μ
-PhO)Mn(III)]+ or
uncoupled [Mn(II)(PhOH)Mn(III)]2+ forms. A possible mechanism is
presented in Scheme 2, including the equilibrium of the uncoupled/
coupled diMn species of 6 (observed in the EPR spectra) and species
present in the ESI-mass spectra. It is proposed that 6 is the active reduced
form of the catalyst that reacts with H2O2 in the slow reductive half-
reaction (turnover-limiting step). The observation of saturation
Scheme 2. Proposed catalytic cycle for complex 6.
7

R. Mehrotra et al.
Journal of Inorganic Biochemistry 213 (2020) 111264
Scheme 3. Proposed catalytic cycle for complex 7.
Table 1
Catalytic efficiency of phenolate-bridged diMn complexes for disproportionating H2O2.
Complex
kcat/KM or k (Mꢀ 1
Bridging ligands
Ligand terminal
Redox cycle
Mn⋯Mn (Å)
Refs
sꢀ 1
)
groups
1
2
7
16.7
(
μ
(
-phO)(
-phO)(
μ
-OAc)2
-OAc)2
py2/phOꢀ ,COꢀ2
py,COꢀ2 /py,COꢀ2
Mn(II)2/Mn(III)2
Mn(II)Mn(III)/Mn(III)Mn
(IV)
–
This work
[17]
[Mn2(bcpmp)(
μ
-OAc)2]
11.44
μ
μ
3.47
3
4
5
[Mn2L(OH)2(H2O)2]
5.08a
(μ
(μ
(μ
-phO)
-phO)(
-phO)(
(COꢀ2 )2/(COꢀ2 )2
py2/py,phOꢀ
py2/py2
Mn(II)2/Mn(III)2
NR
3.67d
3.497
3.45
[18]
[Mn2(bphpmp)(
μ
-OAc)2(H2O)]+
4.7b
μ
-OAc)2
-OAc)2
[19]
[Mn2(bpbp)(
μ
-OAc)2]2+
3.76
μ
Mn(II)Mn(III)/Mn(III)Mn
(IV)
[17,46]
6
6
3.38
(μ
-phO)(
μ-OAc)2
py2/phOꢀ
Mn(II)Mn(III)/Mn(III)Mn
(IV)
–
This work
7
8
[Mn2(bphba)2(Cl)2]
[Mn2(HBPClNOL)(BPClNOL)
Cl]+
1.1
(
μ
(
-phO)2
-phO)(
py2/py2
Mn(II)2/Mn(III)2
Mn(II)Mn(III)/Mn(III)Mn
(IV)
3.41
3.16
[47,48]
[30]
80.2c
μ
μ-OR)
py,ROꢀ /py,phOꢀ
9
[Mn2(
μ
-OMe)(OAc)(hppentO)]+
106.3
(μ
(μ
(μ
-OR)2(
μ
μ
-OAc)
py,phOꢀ /py,phOꢀ
Mn(II)2/Mn(III)2
Mn(II)2/Mn(III)2
2.95
[49]
10
MnCAT (T. thermophilus)
3.1 × 106
-OH)(
-OH2)
His,Glu/His,Glu,H2O
3.18; 3.03
[50,51,13]
-OAc)
NR = not reported. Bcpmp = 2,6-bis({(carboxymethyl)[(1-pyridyl)methyl]amino}methyl)-4-methylphenol]; bpbp = 2,6-bis{[bis(2-pyridylmethyl)amino]methyl}-4-
tert-butylphenol; bphba
= 2-{(N,N-bis(2-pyridylmethyl)amino)methyl}phenol; bphpmp = 2-[bis(2-pyridylmethyl)aminomethyl]-6-{[(2-hydroxybenzyl)(2-pyr-
idylmethyl)amino]methyl}-4-methylphenol; LH4 = 5-methyl-2-hydroxo-1,3-xylene-
α
,α
-diamine-N,N,N′,N′-tetraacetic acid; H2BPClNOL = N-(2-hydroxybenzyl)-N-(2-
pyridylmethyl)[(3-chloro)(2-hydroxy)]propylamine; H2hppentOH = 1,5-Bis[(2-hydroxybenzyl)(2-pyridylmethyl)amino]pentan-3-ol.
a
Second-order kinetics.
b
c
Calculated from reported data.
In the presence of piperazine, pH 9.7.
d
Calculated from an optimized geometry.
compared to other phenolate-bridged diMn complexes of ligands with
different terminal N/O donors that employ either Mn(II)2/Mn(III)2 or
Mn(II)Mn(III)/Mn(III)Mn(IV) redox cycles during catalysis. For those
complexes with low affinity for the substrate (large KM value) that do not
achieve saturation with [H2O2], second-order rate constants (k2cat) are
given for comparison, considering that when KM ≫ [H2O2], the de-
efficiency lower than 5 Mꢀ 1 sꢀ 1 (entries 4–6), highlighting the role of
carboxylate to facilitate the formation of the adduct with H2O2 through
hydrogen bond. Phenolate-bridged [Mn2L(OH)2(H2O)2] with no acetate
bridges but four carboxylate arms, is placed in between these two
groups, with the carboxylate groups compensating the long intermetallic
separation that disfavors the cooperativity between the two Mn ions for
nominator of Eq. 1 is dominated by KM, and ri = k [catalyst][H2O2] =
cat
the electron transfer process (entry 3). Bis(
μ
-phO)diMn complexes are
KM
even less efficient than any of the (
μ
-phenolate)bis(μ1,3-carboxylate)
k
2cat [catalyst][H2O2].
diMn complexes, one example is shown in entry 7. Enhanced activity is
observed when the two Mn ions are linked by one or two alcoholate
bridges (entries 8–9) where the intermetallic distance is shorter than in
the phenolate-bridged complexes. As can be observed in Table 1, the
The catalytic efficiency of triply bridged (μ-phenolate)bis(μ1,3-
carboxylate)diMn complexes falls into two groups. Complexes bearing
terminal carboxylate that react with efficiency higher than 11 Mꢀ 1 sꢀ 1
(entries 1–2), and those with pyridyl/phenolate arms that react with
8

R. Mehrotra et al.
Journal of Inorganic Biochemistry 213 (2020) 111264
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Mn⋯Mn separation in phenolate-bridged complexes is longer than 3.4
Å, while in the (μ-alcoholate)ndiMn complexes the Mn⋯Mn distance is
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oxidized and reduced forms, respectively, entry 10), strengthening the
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4. Conclusions
Unsymmetrical N4O2-hexadentate H2L1 and N4O3-heptadentate
NaH2L2 ligands afford mixed valence Mn(II)Mn(III) complexes 6 and 7
where each Mn ion is in a different coordination environment. Both
complexes share the coordination sphere of the Mn(II) ion, but differ in
the donor groups surrounding Mn(III). In 6, the sixth coordination po-
sition of Mn(III) is occupied by a water molecule, while in 7 Mn(III) is
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MnCAT of T. thermophilus [50], and, based on the kcat/KM criterion,
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Acknowledgments
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