Superoxide dismutase inspired immobilised Ni(II)-protected amino acid catalysts - Synthesis, characterisation, and catalytic activity

Article abstract of DOI:10.1016/j.molcata.2014.07.033

Covalently anchored Ni(II)-C-protected amino acid (l-histidine, l-cysteine, and l-cystine) complexes inspired by the active site of the Ni-superoxide dismutase enzyme were synthesised using chloropropylated silica gel as support. The structural features of the surface complexes were studied by the Kjeldahl method and ICP-MS, mid/far IR, UV-vis diffuse reflectance, and X-ray absorption spectroscopies. The enzyme-like activities of the materials were determined in a biochemical test reaction. Covalent grafting and building the complex onto the surface of the support were successful in all cases. It was found that in many instances the structures obtained and the coordinating groups substantially varied upon changing the conditions of the syntheses. All the covalently immobilised Ni(II)-complexes displayed enzyme-like activity. They also were active in the liquid-phase oxidation of cyclohexene, providing the epoxide with high selectivity.

Full text of DOI:10.1016/j.molcata.2014.07.033

Journal of Molecular Catalysis A: Chemical 395 (2014) 93–99
Contents lists available at ScienceDirect
Journal of Molecular Catalysis A: Chemical
journal homepage: www.elsevier.com/locate/molcata
Superoxide dismutase inspired immobilised Ni(II)–protected amino
acid catalysts—Synthesis, characterisation, and catalytic activity
Zita Csendesa,e, Gábor Vargaa,e, Hajnal Schmehla,e, Zita Timára,e, Stefan Carlsonb,
Sophie E. Cantonb, Éva G. Bajnóczic,e, Dániel Sebo˝ kd, Imre Dékányd, Gábor Eleka,e
,
Pál Siposc,e, István Pálinkóa,e,∗
a Department of Organic Chemistry, University of Szeged, H-6720 Szeged, Hungary
b Max-Lab, Lund University, SE-223 63 Lund, Sweden
c Department of Inorganic and Analytical Chemistry, University of Szeged, H-6720 Szeged, Hungary
d Nanostructured Materials Research Group, University of Szeged, H-6720 Szeged, Hungary
e Material and Solution Structure Research Group, Institute of Chemistry, University of Szeged, H-6720 Szeged, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 July 2014
Received in revised form 28 July 2014
Accepted 29 July 2014
Available online 13 August 2014
Covalently anchored Ni(II)–C-protected amino acid (l-histidine, l-cysteine, and l-cystine) complexes
inspired by the active site of the Ni-superoxide dismutase enzyme were synthesised using chloropropy-
lated silica gel as support. The structural features of the surface complexes were studied by the Kjeldahl
method and ICP-MS, mid/far IR, UV–vis diffuse reflectance, and X-ray absorption spectroscopies. The
enzyme-like activities of the materials were determined in a biochemical test reaction. Covalent grafting
and building the complex onto the surface of the support were successful in all cases. It was found that in
many instances the structures obtained and the coordinating groups substantially varied upon changing
the conditions of the syntheses. All the covalently immobilised Ni(II)-complexes displayed enzyme-like
activity. They also were active in the liquid-phase oxidation of cyclohexene, providing the epoxide with
high selectivity.
Keywords:
Biomimetic catalysis
Silica immobilised Ni(II)-complexes
XAS
Catalytic activities
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
supports; then, not only the work-up procedure becomes easy, but
the catalyst can be recovered and, after some sort of regeneration,
The most active and selective catalysts in nature are the
enzymes. They catalyse a wide-range of organic transformations,
however, they are only active under relatively mild conditions (at
near atmospheric pressure, in a limited temperature range, and in
physiological aqueous solution). If we want to use them or their
analogues for the catalysis of organic transformations in the labora-
tory, moreover in industry, they have to be transformed to materials
that are more durable. The advantages can be retained, while the
disadvantages can be eliminated by, e.g., preparing a structural or
a functional model of the active site [1]. These models are often
homogeneous complexes [2,3], which may be appreciably active
and selective; however, being in the same phase as the reactants
and the products, they are generally lost during the work-up pro-
cedure, or at least, their recovery is very complicated. However, if
these structural or functional mimics are immobilised over solid
reused.
Many of the enzymes contain one or more metal ions as
cofactors in their active centres. One of them is superoxide dismu-
tase (SOD), which protects cells from the toxic effects of superoxide
radical anions. Through a dismutation reaction they convert these
reactive species to oxygen and hydrogen peroxide [4]. Based on the
metal cofactors in the active site of SODs four classes (Cu,ZnSOD
[5], FeSOD [6], MnSOD [7] and NiSOD [8]) can be distinguished.
Ni-containing SODs were first discovered in Streptomyces species
[8] and cyanobacteria [9]. This enzyme exists as a homohexamer
consisting of four-helix-bundle subunits. The metal centre cycles
between 2+ and 3+ oxidation states during catalysis. In the reduced
state, Ni2+ has square planar geometry coordinated by two thio-
late groups of Cys2 and Cys6, the deprotonated amide nitrogen of
Cys2 and the amino nitrogen of His1. In the oxidised form, Ni3+ is
found in a square-pyramidal coordination environment with the
axial binding of the imidazole nitrogen of His1 [10–13].
As an attempt to obtain highly active and selective biomimetic
catalysts that can be recycled, Ni(II)–C-protected amino acid (l-
histidine, l-cysteine, and l-cystine) complexes were covalently
∗
Corresponding author at: Department of Organic Chemistry, University of
Szeged, H-6720 Szeged, Hungary. Tel.: +36 62 544288.
E-mail address: palinko@chem.u-szeged.hu (I. Pálinkó).
http://dx.doi.org/10.1016/j.molcata.2014.07.033
1381-1169/© 2014 Elsevier B.V. All rights reserved.

94
Z. Csendes et al. / Journal of Molecular Catalysis A: Chemical 395 (2014) 93–99
anchored onto chloropropylated silica gel. The structural features,
the superoxide dismutase and the electron transfer activities of the
surface complexes were determined in a biochemical test reaction
and in the oxidative transformations of cyclohexene, respectively.
Results of this research are described in the followings.
2.4. X-ray absorption measurements
The measurements were carried out at the K-edge of the nickel
at MaxLab at beamline I811. This is a superconducting multi-
pole wiggler beamline equipped with a water-cooled channel
cut Si(1 1 1) double crystal monochromator delivering at 10 keV,
approximately 2 × 1015 photons/s/0.1% bandwidth with horizontal
and vertical FWHM of 7 and 0.3 mrad, respectively [16]. A beam-
size of 0.5 mm × 1.0 mm (width × height) was used. The incident
beam intensity (I0) was measured with an ionisation chamber filled
with a mixture of He/N2. Higher order harmonics were reduced
by detuning the second monochromator to 30% of the maximum
intensity. Data collection was performed in transmission mode. The
samples were contained in Teflon spacers with Kapton tape win-
dows according to the nickel concentration. The data were treated
by the Demeter program package [17].
2. Experimental
2.1. Materials and method of synthesis
For the syntheses, C-protected (in form of methylester)
l-histidine, l-cysteine and l-cystine, NiCl2·6H2O and chloropropy-
lated silica gel (SG – particle size: 230–400 mesh, BET surface area:
500 m2/g, functionalisation: 8%) were used. These materials as well
as the 2-propanol solvent were the products of Aldrich Chemical Co.
All the chemicals were of analytical grade and were used without
further purification.
Even though the preparation method of these types of anchored
complexes have been described previously [14,15], for the sake of
easier tracking the followings, let us briefly repeat the main points,
and give the notations.
First, the support (0.5 g) was covalently grafted via an N-
alkylation reaction by the C-protected amino acids (1.75 mmol).
Then, it was treated with Ni2+-containing solution (1.75 mmol
Ni-salt dissolved in 60 cm3 2-propanol) and the solid mate-
rial was isolated. At his point, a substance was in our hands
that only contained surface-anchored ligands. This is called a
surface-grafted complex synthesised under ligand-poor condi-
tions, and will be referred as SG–C-protected amino acid–Ni(II). If
this material was further treated in a solution containing excess
amount of amino acid (0.875 mmol), then a surface-grafted com-
plex was obtained under ligand-excess conditions, and will be
referred as SG–C-protected amino acid–Ni(II)–C-protected amino
acid.
2.5. FT-IR spectroscopy
Structural information on each step of the synthesis proce-
dure was obtained by far- and mid-range infrared spectroscopy.
Mid-range spectra were recorded with a Bio-Rad Digilab Division
FTS-65 A/896 FT-IR spectrophotometer with 4 cm−1 resolution,
measuring diffuse reflectance. The 3800–600 cm−1 wavenumber
range was investigated. 256 scans were collected for each spectrum.
300 mg KBr and 10 mg sample were combined and finely ground.
Spectra were evaluated by the Win-IR package. They were baseline-
corrected, smoothed (if it was necessary) and the spectra of the
supports were subtracted. The 3800–600 cm−1 wavenumber range
was investigated. The comparison of the difference mid-IR spectra
of the anchored amino acid derivatives with and without metal ion,
and the spectra of the pristine amino acid derivatives were used to
obtain indirect information on the coordinating groups.
Far-range spectra were recorded with a Bio-Rad Digilab Division
FTS-40 vacuum FT-IR spectrophotometer with 4 cm−1 resolution.
256 scans were collected for each spectrum. The Nujol mull
technique was used between two polyethylene windows (the sus-
pension of 10 mg sample and a drop of Nujol mull). Spectra were
evaluated by the Win-IR package. They were baseline-corrected
and smoothed (if it was necessary), and the spectra of the supports
were subtracted. The spectra in the far IR region were applied to
acquire direct information on metal ion–functional group coordi-
nation.
2.2. Analytical measurements
The amount of metal ions on the surface modified silica gel
was measured by an Agilent 7700× ICP-MS. Before measurements,
the anchored complexes (3 mg) were digested by adding cc. H2SO4
(1 cm3), then they were diluted with distilled water to 50 cm3 and
filtered.
The nitrogen content of the samples was determined by the
Kjeldahl method. 5 cm3 cc. H2SO4 and 1 cm3 30% solution of H2O2
were added to 100 mg of the grafted complexes. CuSO4·5H2O was
used as catalyst to increase the boiling point of the medium. The
reaction mixture was boiled for some hours, to obtain colourless
mixture from the initially dark-coloured suspension. Then it was
diluted with 40 cm3 distilled water and was distilled with a 20%
solution of sodium hydroxide in the presence of phenolphthalein
indicator. The released ammonia was absorbed in 0.1 mol/dm3
solution of HCl. Then, the remainder acid was titrated with
0.1 mol/dm3 solution of NaOH in the presence of methyl orange
indicator.
2.6. Testing the enzyme-like activity
The enzyme-like activity was tested by the Beauchamp–
Fridovich reaction [18]. For this biochemical test reaction riboflavin,
l-methionine, and nitro blue tetrazolium (NBT) were used as was
described previously [14]. During their reaction, superoxide radi-
cal anions are formed, which gives blue adducts with NBT. When
the enzyme mimic is present, it dismutates the superoxide radical
anion, the photoreduction of NBT is inhibited (its measure is IC50
in mM), i.e., the enzyme mimic works the better when the colour
change (measured by the absorbance), i.e., the value of IC50, is the
smaller.
2.3. UV–vis diffuse reflectance spectroscopy
2.7. Catalytic oxidation of cyclohexene
For the optical characterisation of the samples, UV-diffuse
reflectance apparatus was used. A Micropack HPX-2000 High Power
Xenon Lamp was the light source and an Ocean Optics ADC1000-
USB diode array photometer was applied as detector. Spectra were
recorded in the range of 350–850 nm using a Micropack ISP508RGT
integration sphere. Approximately 100 mg of samples were used
for each measurement and their spectra were recorded without
any additional sample preparation.
In the reaction a vial capped with septum was loaded with the
catalyst (25 mg), acetone (10 cm3), cyclohexene (5 mmol) and per-
acetic acid (2.5 mmol). After 3 h of continuous stirring at room
temperature (298 K), the mixture was analysed quantitatively by
gas chromatography (Hewlett-Packard 5890 Series II gas chromato-
graph equipped with a flame ionisation detector) using an Agilent
HP-1 column. The products were identified by GC–MS.

Z. Csendes et al. / Journal of Molecular Catalysis A: Chemical 395 (2014) 93–99
95
Scheme 1.
Table 1
Amino acid and Ni2+ content of the samples obtained from Kjeldahl type N-
determination and ICP-MS measurements.
Sample
Amino acid content
(mmol/g)
Ni2+ content
(mmol/g)
SG–His-OMe–Ni(II)
SG–His-OMe–Ni(II)–H-His-OMe
SG–Cys-OMe–Ni(II)
SG–Cys-OMe–Ni(II)–H-Cys-OMe
SG–(Cys-OMe)2–Ni(II)
SG–(Cys-OMe)2–Ni(II)–H-(Cys-
OMe)2
0.576
2.296
0.554
1.091
0.505
0.664
0.320
0.319
0.345
0.343
0.384
0.383
C
Main edge position
eV
A
B
C
8349.9
8349.6
8348.3
The reaction scheme indicating the observed products is seen in
Scheme 1.
B
A
3. Results and discussion
3.1. Analytical measurements
8320
8340
8360
8380
8400
8420
Energy (eV)
The results of the Kjeldahl method and the ICP-MS measure-
ments are displayed in Table 1. Covalent anchoring of the amino
acids was successful in all cases. The conversion of the immo-
bilisation is ranged from 72 to 82%, since the chloropropylated
silica gel contains 0.705 mmol/g active chlorine. Under ligand-
poor conditions the metal ion to amino acid ratio is 1:1.8 for
SG–His-OMe–Ni(II), 1:1.6 for SG–Cys-OMe–Ni(II) and 1:1.3 for
SG–(Cys-OMe)2–Ni(II). It means that complexes formed on the sur-
face are not uniform, there are complexes having 1:1 as well as 1:2
metal to ligand ratios. Under ligand-excess conditions, this ratio
is 1:7.2 for SG–His-OMe–Ni(II)–H-His-OMe, suggesting that there
are non-coordinating histidine molecules on the surface of the sil-
ica gel even after intense washing with the solvent at room as
well as at reflux temperature. For SG–Cys-OMe–Ni(II)–H-Cys-OMe
the ratio is 1:3.2; while for SG–(Cys-OMe)2–Ni(II)–H-(Cys-OMe)2 is
only 1:1.7 indicating that cystine molecules can saturate the coordi-
nation sphere of nickel ions more effectively, since they have more
coordinating groups.
Fig. 1. The Ni K-edge XANES spectra of A–SG–His-OMe–Ni(II), B–SG–(Cys-
OMe)2–Ni(II), C–SG–Cys-OMe–Ni(II).
3.3. X-ray absorption measurements
Ni K-edge XAS spectra were recorded only for the ligand-poor
complexes. The XANES spectra of the materials are displayed in
Fig. 1. The spectra are roughly superimposable, indicating a local
structure of Ni(II) that is more or less independent of the type of
coordinating ligand. A very small pre-edge peak around 8332 eV
corresponding to 1s → 3d transition, the main absorption edge
around 8350 eV, assigned to the 1s → 4p transition, and significant
oscillation in the 8360–8430 eV range can be observed. In square
pyramidal and tetrahedral complexes the pre-edge feature is more
intense, while for square planar geometry the splitting of the main
edge peak can be observed due to the 1s → 4pz and the 1s → 4px,y
transitions [20]. Accordingly, these results suggest, that the Ni(II) in
the immobilised complexes is in an octahedral coordination envi-
ronment. This result is in accordance with the diffuse reflectance
UV–vis observations.
It is known that the position of the main edge can provide infor-
mation about the nature of the coordinating atoms. Substitution of
O or N donor atoms by S-donor ligands induces a shift (∼2 eV) in
the edge energy to lower values [21]. Since the Ni K-edge position
is shifted towards lower energies for SG–Cys-OMe–Ni(II) relative
to the other two grafted complexes, it indicates the coordination of
the thiolate group.
Measurements also revealed that there was negligible loss of
nickel ions on altering the synthesis conditions from ligand poor to
ligand excess.
3.2. UV–vis diffuse reflectance spectroscopy
The solid-state electronic spectra of all the immobilised Ni(II)
complexes exhibit two broad bands at 14,300 and 22,500 cm−1
3
3
3
3
assigned to the A2g(F) → T1g(F) (␯2) and A2g(F) → T1g(P) (␯3)
transitions, respectively, which are typical for the hexacoordinated
(octahedral) geometry [19].
Table 2
Parameters deduced from the fitted EXAFS spectra (N – coordination number, R – bond length, ꢀ2 – Debye–Waller factor, ꢁE0 – energy shift, R factor – goodness of fit).
2
Sample
(Ni2+ )X
N
ꢀ2 (A )
ꢁE0 (eV)
R factor
˚
R (A)
˚
SG–His-OMe–Ni(II)
SG–(Cys-OMe)2–Ni(II)
O/N
O/N
6
6
2.07
2.07
0.0048
0.0045
0.61
0.37
0.0312
0.0182
O/N
S
4.5
1.5
2.12
2.43
0.0106
0.0035
0.85
0.0157
SG–Cys-OMe–Ni(II)

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Z. Csendes et al. / Journal of Molecular Catalysis A: Chemical 395 (2014) 93–99
C
C
B
A
B
A
0
2
4
6
8
10
12
0
2
4
k (Å-1)
Radial distance (Å)
Fig. 2. k3-Weighted Ni K-edge EXAFS spectra of A–SG–His-OMe–Ni(II), B–SG–(Cys-
OMe)2–Ni(II), C–SG–Cys-OMe–Ni(II), red line – fit, black line – experimental. (For
interpretation of the references to color in this figure caption, the reader is referred
to the web version of this article.)
Fig. 3. Fourier transformed EXAFS data (without phase correction) of A–SG–His-
OMe–Ni(II), B–SG–(Cys-OMe)2–Ni(II), C–SG–Cys-OMe–Ni(II), red line – fit, black line
– experimental. (For interpretation of the references to color in this figure caption,
the reader is referred to the web version of this article.)
The EXAFS data were k3-weighted and Fourier transformed
coordinated by six O atoms [22]. The EXAFS measurement indicated
that six oxygen/nitrogen atoms form the first coordination sphere
of SG–(Cys-OMe)2–Ni(II) as well with a Ni(II) O/N bond length of
in the range of k = 2–12 A−1. The ranges for the backtransform
˚
˚
were 1–3 A for all complexes. The fitted parameters included
˚
the amplitude reduction factor (S02), interatomic distances (R),
Debye–Waller factors (ꢀ2) and energy shift (ꢁE0). The coordina-
tion numbers (N) were kept constant during each optimisation,
but a range of coordination numbers were used to find the best
fit. The k3-weighted EXAFS spectra and their non-phase corrected
Fourier transforms for the ligand-poor complexes are shown in
Figs. 2 and 3, respectively, and the results of the fitting are reported
in Table 2. For SG–His-OMe–Ni(II), the EXAFS analysis showed that
the first coordination shell contained 6 oxygen/nitrogen atoms (by
these measurements the difference between oxygen and nitro-
gen cannot be estimated clearly, they scatter the photoelectrons
nearly the same way, due to their similar atomic masses), where
2.07 A. For SG–Cys-OMe–Ni(II) two different interatomic distances
can be separated in the first shell. The first one is related to the
˚
Ni(II) O/N distance (R = 2.12 A) with a coordination number of 4.5,
˚
while the second peak corresponds to a Ni(II) S distance of 2.43 A,
and a coordination number of 1.5.
3.4. FT-IR spectroscopy
The coordinating groups were identified through comparing the
difference mid-IR spectra (the spectrum of the support was sub-
tracted) of the silica gel grafted amino acids and the difference
spectra of the immobilised complexes prepared under ligand-
poor conditions or the spectra of the pristine amino acids and
the difference spectra of the anchored complexes prepared under
ligand-excess conditions.
˚
the Ni(II) O/N bond distance was fitted to be 2.07 A. This value
is close to that was obtained from XAFS measurements for the
octahedral [Ni(H2O)6]2+ complex (it was 2.05 A), where Ni(II) was
˚
1759
D
1757
1737
C
1610
1403
B
1582
1412
A
1800
1600
1400
1200
1000
800
600
-1
Wavenumber (cm )
Fig. 4. Difference IR spectra of (A) SG–His-OMe, (B) SG–His-OMe–Ni(II), (C) SG–His-OMe–Ni(II)–H-His-OMe, and (D) H-His-OMe. (The spectrum of SG is subtracted.)

Z. Csendes et al. / Journal of Molecular Catalysis A: Chemical 395 (2014) 93–99
97
1746
1734
B
D
1744
1731
260
260
355
C
B
1613
1596
A
1406
355
1411
1400
500
450
400
350
300
250
200
A
Wavenumber (cm-1)
1800
1600
1200
1000
800
600
Wavenumber (cm-1)
Fig. 5. Difference far IR spectra of (A) SG–His-OMe–Ni(II) and (B) SG–His-
OMe–Ni(II)–H-His-OMe. (The spectrum of SG is subtracted.)
Fig. 7. Difference IR spectra of (A) SG–(Cys-OMe)2, (B) SG–(Cys-OMe)2–Ni(II), (C)
SG–(Cys-OMe)2–Ni(II)–H-(Cys-OMe)2, and (D) H-(Cys-OMe)2. (The spectrum of SG
is subtracted.)
1741
D
[24], in which the far IR spectra of complexes with one possible
coordination site – among others, Ni-imidazole complex – were
registered and analysed, and on Ref. [25].
1605
1408
The difference spectrum of SG–His-OMe–Ni(II)–H-His-OMe
(Fig. 4, trace C) is very similar to the spectrum of C-protected
histidine (trace D), the carbonyl band is split, indicating that the
carbonyl oxygen appears in the coordination sphere of the metal
ion, but there are non-coordinating amino acids as well. This obser-
vation is supported by the analytical measurements (N and Ni(II)
1738
C
1606
1598
1409
B
A
contents). The Ni(II)–Nimidazole vibration is observed at 260 cm−1
,
the bands above 400 cm−1 can be assigned as coordinated water
(originating from crystal water) vibrations (Fig. 5, trace B).
1408
1400
As far as SG–Cys-OMe–Ni(II) is concerned (Fig. 6, trace B), ꢃ
is 1606–1409 cm−1 = 197 cm−1 indicating monodentate ligation of
the carboxylate group. In the far IR range (not shown), the band
at 355 cm−1 confirms the complexation of the carboxylate oxygen.
Under ligand-excess conditions (trace C), the carbonyl band is not
coordinated to the nickel ion, since the position of the band does
not shift relative to the pristine amino acid (trace D). The lack of
the S H vibration proves that the thiolate group takes part in the
complexation, as it was also learnt from the XAS measurement.
For SG–(Cys-OMe)2–Ni(II) (Fig. 7, trace B) the carboxylate group
was involved in the coordination as monodentate ligand, since ꢃ
increased from 185 cm−1 (trace A) to 207 cm−1. In the far IR range
the peak at 354 cm−1 corresponds to the Ni Ocarboxylate vibration
and a new band appeared at 373 cm−1 due to the coordination of
amino nitrogen [26]. Adding C-protected cystine in excess (trace
C) resulted in the rearrangement of the surface-grafted ligand-
poor complex. The carbonyl oxygens are not coordinated, since the
1800
1600
1200
1000
800
600
Wavenumber (cm-1)
Fig. 6. Difference IR spectra of (A) SG–Cys-OMe, (B) SG–Cys-OMe–Ni(II), (C) SG–Cys-
OMe–Ni(II)–H-Cys-OMe, and (D) H-Cys-OMe. (The spectrum of SG is subtracted.)
Regarding the mid-IR difference spectra of the silica gel
anchored amino acids (Figs. 4, 6 and 7trace A) it is clear that in
all cases carboxylate vibrations appeared. It means that the C-
protecting group hydrolysed upon covalent grafting due to the basic
conditions and is present as carboxylate group in the samples.
The ꢂasym(COO
and
SG–(His-OMe) are observed at 1582 cm−1 and 1412 cm−1, respec-
tively (Fig. 4, trace A). To assign the vibrations,
Raman spectra (not shown) were registered as well, since
␯
stretching frequencies of
)
sym(COO )
␯
sym(COO
)
these vibrations are more intense there. The difference
ꢃ
[ꢃ = ␯
) − ꢂsym(COO )] was 170 cm−1 here, while it was
asym(COO
Table 3
found to be 207 cm−1 [ꢃ = 1610–1403 cm−1] for the ligand poor
complex (Fig. 4, trace B). This difference indicates that the car-
boxylate group was involved in the coordination as monodentate
ligand [23]. In the far IR spectrum of the complex (Fig. 5, trace A)
the vibration at 260 cm−1 confirms the coordination of the imid-
azole nitrogen, the band at 355 cm−1 belongs to the Ni Ocarboxylate
vibration, while the bands above 400 cm−1 can be assigned as the
Ni–coordinated water (originating from the crystal water) vibra-
tions. The assignations are based on our recently published work
The SOD activities of the surface grafted complexes.
IC50 (␮M)
Cu,ZnSOD enzyme
SG–His-OMe–Ni(II)
0.4
10
SG–His-OMe–Ni(II)–H-His-OMe
SG–Cys-OMe–Ni(II)
59
61
SG–Cys-OMe–Ni(II)–H-Cys-OMe
SG–(Cys-OMe)2–Ni(II)
170
79
SG–(Cys-OMe)2–Ni(II)–(H-Cys-OMe)2
91

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Z. Csendes et al. / Journal of Molecular Catalysis A: Chemical 395 (2014) 93–99
Table 4
The conversion and selectivity results of the oxidation of cyclohexene after 3 h (catalyst: 25 mg, acetone: 10 cm3, cyclohexene: 5 mmol, peracetic acid: 2.5 mmol, temperature:
298 K).
Catalyst
TOF (1/min) [conversion (%)]
Epoxide (%)
Alcohol (%)
Ketone (%)
Diol (%)
–
nr [21]
64
87
88
88
88
87
87
4
3
2
4
5
1
3
2
0
2
4
3
2
0
30
10
8
4
4
SG–His-OMe–Ni(II)
SG–His-OMe–Ni(II)–H-His-OMe
SG–Cys-OMe–Ni(II)
SG–Cys-OMe–Ni(II)–H-Cys-OMe
SG–(Cys-OMe)2–Ni(II)
SG–(Cys-OMe)2–Ni(II)–(H-Cys-OMe)2
1.01 [29]
0.97 [28]
0.97 [30]
0.97 [30]
0.87 [30]
0.75 [26]
10
10
stretching vibrations of the carbonyl bands (1746 and 1734 cm−1
did not shift to lower wavenumbers. This is the reason why the
added C-protected cystine molecules were coordinated with their
amino nitrogens.
Putting together all the information, structural proposals for the
surface-bound complexes are offered as follows:
SG–His-OMe–Ni(II)
)
selectivity results of the immobilised catalysts on the oxidation of
cyclohexene with peracetic acid in acetone are reported in Table 4.
The expected reactions are the epoxidation, the isomerisation,
and the oxidative ring opening of the epoxide ring producing diols.
In order to be able to study the oxidative transformations of
cyclohexene, we needed to avoid the decomposition of peracetic
acid. Acetone was found the solvent of choice – peracetic acid did
not decompose in this solvent, even when the immobilised com-
plexes were (but cyclohexene was not) present.
Peracetic acid alone (without any catalyst) is a known epoxida-
tion agent. It reacts stoichiometrically, and interestingly, we found
that its epoxide selectivity was far from being excellent (Table 4,
first entry); appreciable amount of diol, as the major ring-opening
product, could be detected. It is to be noted that after 1 h reaction
time the epoxide selectivity was even lower (54%).
NimidazoleHissurfOcarboxylateHissurfNimidazoleHissurfOcarboxylateHissurfH2O,
H2O,
SG–His-OMe–Ni(II)–H-His-OMe
NimidazoleHissurfOcarboxylateHissurfNimidazoleHisOcarbonylHisH2O, H2O,
SG–Cys-OMe–Ni(II)
In the presence of our immobilised nickel complexes, the
conversion increased somewhat, however, more importantly, the
epoxide selectivity increased appreciably, by more than 20% for
each catalyst. The catalysts were reused twice, with simply rinsing
them with acetone between the runs, without loss in activities and
selectivities.
SthiolateCyssurfOcarboxylateCysssurfSthiolateCyssurfOcarboxylateCysssurfH2O,
H2O,
SG–Cys-OMe–Ni(II)–H-Cys-OMe
It is to be noted that neither the conversion nor the selectivity
levels were influenced significantly by either the identity of the
amino acid ligands or the type of the anchored complexes (i.e.,
whether they were prepared under ligand-poor or ligand-excess
conditions). Probably, the role of the ligands is to exert steric influ-
ence on the accessibility of the central ion by the reactants. The
presence of the ligands were necessary though, since silica gel
impregnated with the Ni(II) ions only decomposed the epoxida-
tion agent. In the presence of the immobilised complex; however,
the peracetic acid may have been directly coordinated to the cen-
tral ion, and oxygen transfer to the double bond of cyclohexene
may occur at this stage, to the possibly uncoordinated cyclohexene.
This should be the reason of the outstanding epoxide selectiv-
ity. If both reactants were coordinated, there would be plenty of
time for further reactions. If cyclohexene was coordinated alone,
the situation would not be much different from the stoichiometric
reaction.
SthiolateCyssurfOcarboxylateCysssurfSthiolateCysSthiolateCysH2O, H2O,
SG–(Cys-OMe)2–Ni(II)
Namino(Cys)2surfOcarboxylate(Cys)2surf Namino(Cys)2surfOcarboxylate(Cys)2surf
H2O, H2O,
SG–(Cys-OMe)2–Ni(II)–(H-Cys-OMe)2
Namino(Cys)2surfOcarboxylate(Cys)2surf
Namino(Cys)2H2O, H2O,
Ocarboxylate(Cys)2surf
where ‘surf’ stands for surface.
3.5. Enzyme-like activity
All materials performed catalytic activity, i.e., could catalyse the
dismutation of the superoxide radical anion. Catalytic activities dif-
fered widely though (Table 3).
4. Conclusions
The most active anchored complex was SG–His-OMe–Ni(II), its
catalytic activity was coming close to the activity of the Cu,ZnSOD
enzyme (this was used as reference, since no such measurements
are available for the NiSOD enzyme). The immobilised complexes
prepared under ligand-poor conditions were more active than
those made in the presence of ligand excess were. It seems that
a more strained environment arising under ligand-poor conditions
makes the complex more reactive.
Immobilised Ni(II)–amino acid catalysts inspired by the active
site of the NiSOD enzyme could be prepared successfully both under
ligand-poor and ligand-excess conditions. The coordinating sites
could be identified with UV–vis diffuse reflectance, X-ray absorp-
tion, mid/far IR spectroscopies, and chemical intuition. It was found
that the major coordinating sites are the carboxylate oxygen(s) of
the ligands, the imidazole nitrogen of histidine and the thiolate
sulphur of cysteine. The other coordination sites depend on the
structures of the molecules.
3.6. Oxidative transformations of cyclohexene
All the materials displayed superoxide dismutase activity and
were found to be active in the oxidative transformations of pro-
ducing cyclohexene oxide with outstanding selectivity.
The turnover frequency – molecules reacted over one metal
ion of the anchored complexes per minute – (conversion) and

Z. Csendes et al. / Journal of Molecular Catalysis A: Chemical 395 (2014) 93–99
99
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appreciated.
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