Synthesis and spectroscopic characterization of ternary copper(ii) complexes containing nitrogen and oxygen donors as functional mimics of catechol oxidase and phenoxazinone synthase

Article abstract of DOI:10.1039/c9nj06131b

Two ternary copper(ii) complexes 1 [(Cu₂Me₄en)₄(EDTA)] and 2 [(CuMe₄en)₂(MIDA)] containing the mixed ligand system of 1,1′,4,4′-tetramethylethylenediamine (Me₄en) (L) and N-methyliminodiace

Full text of DOI:10.1039/c9nj06131b

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Synthesis and Spectroscopic Characterization of Ternary Copper(II)
Complexes Containing Nitrogen and Oxygen Donors as Functional
Mimics of Catechol Oxidase and Phenoxazinone Synthase
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Abd El-Motaleb. M. Ramadana*, Shaban. Y. Shabana, Mohamed. M. Ibrahima,b, Adel A.
Nassarc, Shehab A. Sallamd, Sami A. El-Harbie, Walid Omara
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aDepartment of Chemistry, Faculty of Science, Kafr El-Sheikh University, Kafr El-
Sheikh, Egypt
bDepartment of Chemistry, Faculty of Science, Taif University, Taif, Saudi Arabia
cDepartment of Chemistry, Faculty of Science, El-Menuofia University, Egypt
dDepartment of Chemistry, Faculty of Science, Suez Canal University, Ismailia, Egypt
eDepartment of Chemistry, University College in Al-Jamoum, Umm Al-Qura University,
Makkah, Saudi Arabia
Abstract
Two ternary copper(II) complexes 1 [Cu2Me4en)4(EDTA)] and 2 [CuMe4en)2(MIDA)]
containing mixed ligand system of 1,1′,4,4′- tetra methyl ethylenediamine (Me4en) based
(L) and N-methyl iminodiacetic (MIDAH2) (Lʹ) or the ethylene diamine tetra acetic acid
(EDTAH4) (Lʹ) were synthesized. Complete structural figuration was achieved by many
spectroscopic, electrochemical and magnetic measurements. In addition, spectral data of
PXRD with Expo 2014's structural solution software were utilized in the structural
illustration of homobinuclear complex 1. Furthermore, the structural formulation of
complex 2 was affirmed by structural analysis of single-crystal X-rays. Square pyramidal
stereochemistry is suggested for both the homobinuclear and mononuclear 1 and 2. The
oxidase catalytic activities of complexes 1 and 2 were tested towards a series of catechols
and o-aminophenol and found to be promising candidates as functional mimics of
catechol oxidase and phenoxazinone synthase. Tendency of the studied phenols to
oxidize and transform to the oxidation products is correlated with their binding affinity to
current complexes and substrates structure. Driving force (λ) or the free energy change, –
ΔGº of the studied oxidation processes were computed from the electrochemical results.
The plausible catalytic reactions pathways were suggested in the light of spectral,
electrochemical and stopped-follow kinetic measurements.
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*Corresponding author: Ramadanss@hotmail.com
33516 Kafr El-Sheikh, Egypt; Tel: 00201273604106; Fax: 0020473215175
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Keywords: Ternary copper(II) complexes, electrochemical, X-ray, catechol oxidase,
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phenoxazinone synthase, biomimetic, catalytic activity, stopped-follow kinetic
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Introduction
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Catalytic aerobic oxidation of phenolic compounds leads to formation of biologically
significant products like o-quinones and o-benzoquinone monoimine (o-BQMI) [1].
Quinones are of great significance in biological systems; especially due to their capability
to undergo redox reactions [2]. Because of its high chemical activity, o-quinones perform
many vital functions in mammals, animal and plant kingdoms, as well as humans such as
antibiotics, defensive secretions, balanid adhesion and pigmentation [2]. Ortho quinones
are highly sensitive to nucleophilic addition reactions and this feature makes them as
worthy cross - linking agents. The greatest benefit of quinones is the formation of
melanin pigment which provides protection for all living organisms from sun damage.
Furthermore, melanin in the brain helps in electron transfer processes and in the eyes
excited by UV-light to produce tyrosyl radicals, which appear to play a role in visual
perception [3].
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Related to o-quinones is the o-benzoquinone monoimine (o-BQMI) the precursor for
formation of the vital biological component o-amino-3H-phenoxazine-3-ones (APX) core
[4]. Ortho-amino-3H-phenoxazine-3-ones is the aerobic oxidative dimerization reaction
constitute which contained in the final step of the bacterial synthesis of the antibiotic
(Streptomyces antibiotic), Actinomycin D [4]. Furthermore, o-amino-3H-phenoxazine-3-
ones chromophore has been proven to be substantial constituent in both xanthommatin
and cinnabarin [5]. Clinical trials have proven that the natural product antibiotic
Actinomycin D is promising medicine in treatment many critical diseases such as
Choriocarcinoma, Wilm’s tumors, rhabdomyosarcoma, and Kaposi’s sarcoma [5]. In this
regard pharmaceutical experiences displayed that these cytotoxic products are potent
antineoplastic agents [5].
In the biological systems the aforementioned vital quinones are obtained catalytically
by the aerobic oxidation of phenols in presence of the oxidoreductase metalloproteins. Of
these native enzymes is the multi-copper containing proteins that include tyrosinase,
catechol oxidase and phenoxazinone synthase. This family of copper enzymes is
characterized by the capability to reversibly bound molecular oxygen (O2) at ambient
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conditions. [6]. Tyrosinase and catechol oxidase can catalyze the four electron oxidation
of catechols to the corresponding light absorbing o-quinones. Tyrosinase catalyzes the
hydroxylation of mono phenolic substrates to catechols followed by the aerobic oxidation
of the resulting catechols to the corresponding o-quinone. On the other hand, catechol
oxidase catalyzes only the aerobic oxidation of catechols to o-quinone without acting on
the monophenols [7]. This is the main difference between these two oxidase copper
proteins. In the same context phenoxazinone synthase the oligomeric multicopper oxidase
protein catalyzes the aerobic transformation of o-aminophenols into the oxidation product
o-amino-3H-phenoxazine-3-ones chromophore through the six-electron oxidative
dimerization process.
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In the last decades scientists have paid considerable attention for dinucleating ligand
systems that have ability to bind two metal cations in the same time [8]. Importance of
this subject is owing to existence of the bimetallic heart in a numerous of metalloproteins.
Dinucleating ligands were utilized for synthesis abundant number of bimetallic chelates
with the aim of mimicking dinuclear native metalloenzymes [9]. More attention is paid to
this class of metal chelates for their characteristic magnetic and catalytic properties [10].
In this regard copper(II) complexes are the lighthouse of the bioinorganic models which
mimic assorted metalloproteins and also serve as magic catalysts in several catalytic
synthetic oxidation reactions [11]. There are a huge number of research articles for
dinuclear and mononuclear copper(II) complexes as functional models of catechol
oxidase [1,8,12]. On the other hand the available metal complexes as functional mimics
of phenoxazinone synthase are few as compared to catechol oxidase [1,13,14].
In seeking a deeper understanding of some chemical and biological oxidation
processes, we have resorted to modeling small synthetic molecules to gain valuable
insights into these oxidation processes. In this context, we present in this work the
synthesis and characterization of two ternary copper(II) complexes as functional models
of the active sites of the native copper oxidases catechol oxidase and phenoxazinone
synthase. As well the kinetic studies for the catalytic aerobic oxidation of some
biologically significant phenols such as catechols and o-aminophenol will be carried out.
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2. Experimental
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2.1. Chemicals and reagents
The chemicals and reagents used in the present work are of analytical grade. Ligands
1,1′,4,4′-tetramethylethylenediamine (Me4en), N-methyl iminodiacetic acid (Me-IDA)
and ethylenediaminetetraacetic acid (EDTA) were purchased from Merck Chemical
Company and used without further refinement. The studied substrates catechol, 4-methyl
catechol, 4-nitro catechol, 3,5-di-tert-butyl catechol and o-amino phenol were also
purchased from Merck Chemical Company and was used as such.
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Caution: Perchlorate salts are likely to be explosive and should be handled in small
quantities only carefully.
2.2. Synthesis of copper(II) complexes 1 and 2
Two and four milimole of aqueous NaOH was added to the N-methyl iminodiacetic acid
and ethylenediaminetetraacetic acid respectively in distilled water and the reaction
mixture was stirred for 10 minutes. To these mixtures copper(II) salt (CuClO4)2·6H2O) (1
mmol) in MeOH was slowly added with constant stirring followed by slow addition of
1,1′,4,4′-tetramethylethylenediamine (1.0 mmol) in methanol. After one hour of the
constant stirring of the reaction mixtures at room temperature the microcrystalline blue
precipitates were formed which were filtered off, washed with MeOH, ether and finally
kept in an evacuated desiccator for one week.
2.3. Physical, measurements, catalytic and kinetic experiments were performed as
previously described [13a,b,15-16].
3. Results and Discussion
3.1. Synthesis and formulation
The present ternary copper(II) chelates
1
and
2
containing 1,1′,4,4′-
tetramethylethylenediamine (Me4en) based mixed ligands were synthesized facilely at
room temperature by mixing the methanolic solutions of copper(II) perchlorate and the
sodium salt of the N-methyl iminodiacetic (MIMDAH2) or the ethylenediaminetetraacetic
acid followed by the addition of 1,1′,4,4′-tetramethylethylenediamine in an equimolar
amounts of the reactants. Copper and chlorine content were determined by EDXRF
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technique while carbon, hydrogen and nitrogen were determined by the usual elemental
analysis methods. The suggested molecular formulae given in Table 1 were assigned
based on the analytical data and molar conductance measurements. The full structural
characterizations were achieved by many spectroscopies, electrochemical and magnetic
measurements. In addition, spectral data of PXRD with Expo 2014's structural solution
software were utilized in the structural illustration of homobinuclear 1. Further, the
structural formulation of complex 2 was confirmed by X-ray structural analysis for a
single crystal.
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For the copper(II) complex [Cu2L2L′] (NaClO4)2 2H2O (1) the coordinated mixed
ligand system (L2L′) in which L′ is the ethylenediaminetetraacetato anion which binds to
the two copper(II) ions in a dibasic tridentate pattern per copper(II) ion through the
donors sites NO2. The remaining coordination sites of the penta coordinate conformation
are provided by the bidentate based ligand 1,1′,4,4′-tetramethylethylenediamine (L) as
depicted in scheme 1. Concerning complex [CuL2L′](NaClO4)3 (2) the coordinated mixed
ligand system (L2L′) in which L is 1,1′,4,4′-tetramethylethylenediamine based ligand
coordinates to the copper(II) center in a bidentate pattern via its two nitrogen atoms. On
the other hand L′ is the N-methyl iminodiacetato anion which binds to the copper(II)
center in a dibasic fashion via the coordination chromophore NO2.
The solid form of the current copper(II) chelates is microcrystalline shows shades of
blue colors (Table 1). The complexes exhibit good solubility in H2O, DMF, MeOH,
EtOH and CH3CN while in less polar organic solvents a low solubility is observed.
Several attempts were made to obtain the single crystal suitable for X-ray structural
analysis and only succeeded in the case of complex 2.
Study the electrolytic conductance of the present copper(II) chelates in the methanolic
solution (1 × 10-3M) at the room temperature (22 °C) confirms the presence of NaClO4 in
the crystal lattice of the solid microcrystalline forms of these metal chelates. Based on the
analytical data these copper(II) chelates are electrically neutral because they are deprived
of the counter anions and consequently a nonelectrolytic behavior is expected in solution.
However the electrical conductance measurements indicate their electrolytic nature. This
finding was inferred from the electrical conductivity values of 150 and 155 and Ω−1 mol−1
cm2 in the case of complexes 1 and 2 respectively which are distinctive to 1:1 electrolytic
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behavior [17]. These detected conductance values are attributed to the electrolytic
dissociation of the crystal lattice NaClO4 content as a result of dissolution the
microcrystalline solid complexes in MeOH.
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Table 1: Molecular formulae, physical prosperities, and elemental analysis data of
copper(II) complexes 1 and 2
ΛM
Found (Calcd.)
Ω-1cm2 mol-1
Complex
Color
%C
%H
5.19
%N
%Cl
7.64
%Cu
1.[Cu2L2L′].(NaClO4)2 2H2O
Deep
Blue
150
155
28.48
9.08
13.69
(28.44) (5.17) (9.05) (7.64) (13.69)
2.[CuLL′].(NaClO4)3
Blue
19.06
3.31
6.08
15.36
9.18
(19.07) (3.32) (6.07) (15.38) (9.18)
For the two complexes 1 and 2, L is 1,1′,4,4′- tetra methyl ethylenediamine, for complex
1, L′ is ethylene diamine tetra acetato and for complex 2, L′ is N-methyl iminodiacetic
acetato
3.2. Mode of bonding
In order to figure out the mode of bonding of the studied ligand systems the FTIR
spectral technique was employed. For this purpose IR spectra of the synthesized
copper(II) complexes and the free components of the current ligand systems were
measured as KBr disk. Assignment of the observed peaks in both high and low frequency
regions are listed in Table 2 and the spectral charts are given in the supplementary
materials (S1-S6). Spectrum of 1,1′,4,4′- tetra methyl ethylenediamine based ligand
displays medium to weak band in the region 2762-2939 cm-1 due to the C–N stretching
vibration of the tertiary amine [18]. As a result of coordination to copper(II) ion these
bands were shifted to 3294 and 3220 cm-1 in the spectra of complexes 1 and 2
respectively [19].
Concerning the free N-methyl iminodiacetic acid (NMIDA) and the ethylene diamine
tetra acetic acid their spectra exhibit the characteristic frequencies for both tertiary amine
and the carboxylate moieties at the regions of 3007-3023 and 1614-1683 cm-1
respectively [19]. On complex formation the characteristic peak of C-N is shifted to
lower wavenumber in the spectra of the complexes 1 and 2 indicating participation of the
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tertiary amine nitrogen in coordination to copper(II) center [19]. Concerning the
carboxylate groups their υ(OH) peak was disappeared in the spectra of the synthesized
copper(II) complexes 1 and 2 indicating deprotonation of the COOH group followed by
its binding to copper(II) ion via the carboxylate oxygen [20]. In addition the distinctive
bands at 1614 - 1683 and 1472 - 1474 cm-1 ascribed to υasym(COO–) and υsym(COO–)
respectively appeared at 1589 - 1622 and 1498 indicating involvement of (COO–) in
coordination to copper(II) center [20]. These spectral features are characteristic to the
tridentate dibasic fashion of both the N-methyl iminodiacetato anion (MIMA2−) and the
ethylene diamine tetra acetato anion (EDTA4-) per copper(II) ion. This finding is further
supported from the X-ray structural analysis in the case of complex 2. For both MIMA2−
and EDTA4− the unidentate fashion of (COO–) was deduced from the computed value of
the frequency difference Δυ(cm-1); where Δυ = υasym(COO–) – υsym(COO–) =91 - 124 cm-1.
In the low frequency region the newly found stretching vibrations bands within the
ranges of 520 - 590 and 485 – 500 cm-1 attributed to the ν(M-N) and ν(M-O) respectively
are further evidence for the assigned structures given in scheme 1. Based on the
analytical data, molar conductance measurements and FTIR spectral investigations the
newly synthesized copper(II) complexes 1 and 2 are depicted in scheme 1.
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Table 2: FTIR spectra (cm-1) of the ternary copper(II) complexes 1- 2
Compound* ν(OH) ν(C−N)
νas(COO-) νs(COO-) Δν (cm-1) M-N M-O
(CH3)4en
2762-2939
-
-
-
-
-
-
-
EDTA
3490
3106
3450
3023
2915
3007
2887
1614
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1683
1622
1474
1498
1472
1498
-
1
91
-
520 500
MIDAH2
-
-
2
124
530 485
*MIDAH2 is N-methyl iminodiacetic acid.
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Complex 1
Complex 2
Scheme 1: Structure of complexes 1 and 2
3.3. Electronic absorption spectra
Due to lacking of a proper single crystal for X-ray structural analysis in the case of
complex 1, comparative spectral measurements in the UV-Vis region were performed and
correlated with complex 2 to explain the structural aspects of 1. In this regard, the
ultraviolet and visible spectra of the present copper(II) chelates were measured as a
concentrated solution in MeOH. The recorded spectra as shown in Figure 1 display
similar features of the two penta coordinated copper(II) complexes 1 and 2. On the other
hand X-ray structural analysis of 2 demonstrated square pyramidal stereochemistry and
therefore we expect square-pyramidal geometry for complex 1. Further corroboration for
these geometrical predictions could be getting from the incoming: i) ESR spectra
measurements; ii) the obtained results of the XRD spectral data processing by the
structural solutions program - Expo 2014 in the case of complex 1.
Figure 1 shows that complex 1 and 2 exhibit almost identical band position in the
visible region at about 535 nm. Three d-d electronic transitions are usually observed for
copper(II) ion in the square-pyramidal environment (C4v) which agree with dxz, dyz →
dx2–y2, dxy → dx2–y2 and dz2 → dx2–y2 transitions [21].
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Figure 1(a) and (b): Electronic absorption spectra in methanol for saturated solution of
the copper(II) complexes 1 and 2
The spectra of the current copper(II) complexes display a strong intensity peak at 525
nm may arise from the d-d electronic transitions dxz, dyz → dx2–y2. The other band at ≈
650 nm is a broad and weak may be assigned to the two superimposed transitions dxy →
dx2–y2 and dz2 → dx2–y2. These spectral features are distinguishing for copper(II) center
in the square-pyramidal stereochemistry in a harmony with the previously reported for
other penta coordinated copper(II) complexes in a square-pyramidal geometry [22].
3.4. Magnetic moment measurements and EPR spectra
The EPR spectra and magnetic susceptibility measurements are beneficial tools for
designate the stereochemistry of the present copper(II) complexes. Accordingly we have
measured the magnetic susceptibility of the two copper(II) complexes at room
temperature and the computed magnetic moments are given in Table 3.
The observed magnetic moments values of 1.97 and 2.12 BM (Table 3) are slightly
greater than the spin only value of 1.73 BM for d9 configuration in a magnetically non
concentrated environment and indicate to magnetic dilute character of the copper(II)
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complexes 1 and 2. This finding ruled out any spin coupling interactions between the
neighboring CuII ions [23] in the powder samples and reflects the monomeric nature of
the current newly synthesized copper(II) complexes.
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Room temperature powder-EPR measurements of copper(II) complex 1 and 2 have
been carried out in the X-band region at scan rate of 1000. The strength of the applied
magnetic field is 5000 gauss along with a frequency of 9100 megacycles per second. The
EPR-spectral parameters, g||, g⊥, gav and G, were determined and listed in Table 3 and the
recorded spectra are presented in S7.
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The spectra of complexes 1 and 2 are similar and characteristic to the rhombic
symmetry (Fig. 3) which yields three different g-values, i. e., gx ≠ gy ≠ gz. The indexed
geometrical quotient, (R), where, R = (gx – gy)/(gy – gz), is beneficial distinguish
geometry for the penta coordinated copper(II) complexes [13b]. In the case of R > 1 the
dz2 is the ground state but when R < 1 the dx2-y2 is the ground state which is the most
common for copper(II) complexes in the square pyramidal stereochemistry [13b]. The
obtained values of R are 0.845 and 0.618 for complexes 1 and 2 respectively which
confirm the square pyramidal geometry. As well the numerical value of the indexed
geometrical quotient (R) may use as a measure of the geometry distortion of the metal
complex. Accordingly the ideal square pyramidal geometry should exhibit R value of
zero thus data in Table 3 indicate that complex 1 (R = 0.845) is more distorted than
complex 2 (R = 0.618). Anyway, the electronic absorption spectra investigations indicate
square pyramidal geometry for the studied copper(II) complexes 1 and 2 and this finding
was also corroborated by the EPR study.
The g-values (Table 3) that are estimated from the relation: gav =1/3(g|| + 2g⊥) refer to
the predominant covalence in the binding of the coordinated ligand system to copper(II)
ion [24]. In the same vein, the EPR parameter (G) that measures the coupling of adjacent
spinning centers in the polycrystalline samples was calculated from the relation [25]: G =
(g|| - 2)/(g
- 2). The computed G-values, 8.974 and 5.774, for complexes 1 and 2
⊥
respectively are greater than 4 excluded any spin – spin coupling between the adjacent
copper(II) centers in the solid complexes [26] in a consistence with the aforementioned
for the magnetic moment values (Table 3).
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Table 3: EPR spectral parameters and magnetic moments of copper(II) complexes 1-2
7
8
Complex* gx (g||)
gy
gz
gav
G
R
μeff
(g⊥)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
2
2.179
2.175
2.086
2.089
1.976
1.950
2.031 2.080
2.019 2.061
5.774 0.845
8.974 0.618
2.08
1.97
*Complexes details are as listed in Table 1.
3.5. Powder X-ray Diffraction
Nowadays, powder X-ray diffraction (PXRD) along with an appropriate
computational study is a scientific technique for full structural determination of low
molecular mass polycrystalline materials such as organic, organometallic and metal
complexes [27]. Many trials were carried out to obtain a suitable single crystal for
structure determination of the copper(II) complexes which succeeded only in the case of
complex 2. Accordingly, the XRD measurements were performed for appropriate
microcrystalline sample of the copper(II) chelates 1 and the resulting XRD patterns are
given in Figure 2.
Figure 2 The powder XRD pattern of the homobinuclear copper(II) complex 1
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The quality of fit between the experimental and the calculated PXRD patterns in the
final Rietveld refinement that displayed in Figure 3 provides strong validation of the
crystal structure of the examined complex 1. The Rietveld methodology is a
computational treatment of the powder X-ray diffraction data that allows precise structure
determinant of the metal complex. In this context the computer program Expo 2014 was
utilized to perform all the proceedings of the crystal structure solution process from the
PXRD data by the Rietveld technique [28].
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49
50
51
52
53
54
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57
58
59
60
Figure 3: The quality of fit between the experimental powder XRD pattern of complex 1 and the
calculated data in the final Rietveld refinement
Table 4 contains the crystallographic data a while selected bond angles and bond
length are listed in Tables 5 and 6 respectively. The data in Table 4 showed that complex
1 crystallizes in the monoclinic space group P 1 21 1 with the unit cell dimensions, a
=14.34426 Å, b = 12.96501 Å, c = 5.50817 Å, α = 90°, β = 95.293° and γ = 90°.
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Table 4: Crystallographic data of the homobinuclear copper(II) complex 1
Complex (1) [Cu2L2L'](NaClO4)2 2H2O
7
8
9
Empirical formula
Formula weight
T (K)
Cu2C22H48N6O18 Na2Cl2
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
928.14
295
λ (Å)
1.529040
Monoclinic
P 1 21 1
Centric
4
Crystal system
Space group
Centro symmetry
Space Group Number
Unite cell dimensions:
a (Å)
14.34426
12.96501
5.50817
90
b (Å)
c (Å)
α (°)
β (°)
95.293
90
γ (°)
Cell volume (Å3)
Volume per atom (Å3)
Calculated density (g/cm3)
θ range for data collection (°)
Total reflection
Rietveld results:
Rp
1020.01
51.00
1.43
10.025 - 30.031
677
12.383
18.953
6.374
Rwp
R-Bragg
R-F
4.329
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Figure 4 illustrates the optimum structure of complex 1 which has the molecular
formula [(Cu2L2L′)]. L′ is the hexadentate ligand (EDTA) and L is the bidentate nitrogen
base, 1,1′,4,4′-tetra methyl ethylenediamine.
7
8
9
10
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14
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22
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
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58
59
60
Figure 4: The optimized structure of the homobinuclear copper(II) complex 1
The coordination chromophore of the present mixed ligand system per copper(II) ion
is N3O which occupies the equatorial plane of the square pyramidal structure while the
oxygen atom of the acetato portion of the non-protonated EDTA compartment exists in
the apical position. To identify the stereochemistry of the inspected five coordinated
copper(II) chelate 1 the geometrical index τ was determined from the relation τ =
(β−α)/60; where α and β being the two largest angles around the copper(II) center. It has
been reported that for the penta-coordinated copper(II) complexes if τ equals zero the
hybridization of the copper(II) ion is dsp3 (d is dx2-y2 ) of the perfect square pyramidal
geometry while if τ is one the corresponding geometry is perfect trigonal-bipyramidal
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[29]. The determined values of τ are 0.0920 and 0.0917 for (Cu1) and (Cu2) respectively
which demonstrate the distorted square pyramidal stereochemistry. This structural
formulation based on the PXRD crystallographic analysis is roughly consistent with the
current spectral guides to determine the final structure of 1.
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As shown in Table 5 the bond distances (Cu-N) or (Cu-O) of both EDTA
compartment and the nitrogen base, 1,1′,4,4′- tetra methyl ethylenediamine are in the
usual values [13c, 22b, 30]. Table 5 shows that the angles of the bond around CuII centers
in the equatorial and apical regions are almost in agreement with those reported for
similar copper(II) complexes [13c, 22b, 30].
Table 5: Selected bond length (Å) of the homobinuclear copper(II) complex 1
N1 − Cu1
Cu1 − O1
Cu1 − O3
N2 − Cu1
N3 − Cu1
2.09467
1.82011
1.84752
1.95729
1.99603
N4 − Cu2
Cu2 − O5
Cu2 − O7
N5 − Cu2
N6 − Cu2
1.98737
1.88153
1.87240
2.06104
1.90816
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Table 6: Selected bond angles (°) of the homobinuclear copper(II) complex 1
5
6
Type
Angle
Type
Angle
7
8
9
C2-N1-Cu1
C4-N1-Cu1
Cu1-N1-C11
N1-Cu1-O1
N1-Cu1-O3
N1-Cu1-N2
N1-Cu1-N3
C1-O1-Cu1
O1-Cu1-O3
O1-Cu1-N2
O1-Cu1-N3
C3-O3-Cu1
O3-Cu1-N2
O3-Cu1-N3
C5-N2-Cu1
C6-N3-Cu1
C9-N2-Cu1
C10-N2-Cu1
N2-Cu1-N3
C7-N3-Cu1
C8-N3-Cu1
85.96
C12-N4-Cu2
C14-N4-Cu2
C16-N4-Cu2
N4-Cu2-O5
N4-Cu2-O7
N4-Cu2-N5
N4-Cu2-N6
C13-O5-Cu
O5-Cu2-O7
O5-Cu2-N5
O7-Cu2-N6
C15-O7-Cu2
O7-Cu2-N5
O7-Cu2-N6
C17-N5-Cu2
C18-N6-Cu2
C21-N5-Cu2
C22-N5-Cu2
N5-Cu2-N6
C19-N6-Cu2
C20-N6-Cu2
108.76
109.78
104.59
86.89
107.72
143.10
85.61
10
11
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13
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17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
85.77
83.28
169.01
93.95
101.10
164.63
111.73
170.13
84.18
100.27
163.49
86.51
90.91
88.04
115.43
99.91
109.75
96.85
103.72
101.20
101.76
113.18
114.78
93.81
101.69
101.69
103.74
113.57
124.46
92.80
110.35
122.75
111.18
110.80
3.6. Description of the crystal structure of complex 2
The final figuration of the ternary copper(II) complex 2 was realized by conducting a
structural X-ray analysis of the appropriate blue crystal. The suitable crystal was obtained
by the slow evaporation of the methanolic solution of complex 2 at room temperature.
The crystalline data are shown in Table 7 where the orthorhombic crystalline system of
the space group Pbca is reported and the ORTEP scenery of the asymmetric unit is
displayed in Figure 5.
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5: Presentation of the single crystal X-ray structure of copper(II) complex 2, showing the
labeling scheme of non-hydrogen atoms
Tables 8 and 9 report representative bond angles, and bond distances around
copper(II) ion. The stereochemistry of the inspected copper(II) complex 2 is proved
based on the determined value of the geometrical index τ. The determined τ-value of 0.05
N1
indicates that it is better to describe the stereochemistry of copper(II) complex, 2, as a
slightly distorted square pyramid.
O1
The coordination polyhedron around the copper center shows that the equatorial plane
N3
Cu
is occupied by both the nitrogen donors of the two tertiary nitrogen atoms of the 1,1′,4,4′-
tetra methyl ethylenediamine compartment in addition to the N-methyl tertiary amine
N2
O2
nitrogen donor and one of the oxygen atoms of the carboxylate moiety of the coordinated
N-methyl iminodiacetic acid. On the other hand the apical position is occupied by the
iminodiacetic oxygen (O1) in the square pyramidal conformation. Data in Tables 8 and 9
demonstrate that the bond distances between the copper center and the oxygen and
nitrogen donors are in the normal values [13c, 30, 31]. As well as the values of the bond
angels around the central copper(II) ion are characteristic to the proven current
stereochemistry.
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Table 7: Crystallographic data and structure refinement for complex 2
5
6
7
8
Empirical formula
Formula weight
Temperature [K]
Wavelength [Å]
Crystal system
Crystal color
C11 H23 Cl3 Cu N3 Na3 O16
692.07
9
150(2)
10
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18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.71073
Orthorhombic
Blue
Crystal size [mm3]
Space group
0.160 x 0.060 x 0.040
Pbca
Volume [Å3],
5057.0(18), 2
Z
8
Unit cell dimensions [Å; °]
a = 8.3550(17); α = 90
b =19.633(4); β= 90
c = 30.829(6) ; γ = 90
1.866
Density(calc.) [g/cm3]
Absorption coefficient [mm-1]
F(000)
1.315
2888
Theta range for data collection [°]
Index ranges of h, k, l
Refinement method
1.321 to 25.755°
10<=h<=9, -23<=k<=23, -37<=l<=35
Full-matrix least-squares on F2
33098
Reflections collected
Independent reflections
Completeness to theta
Data /restraints / parameters
Goodness-of-fit on F2
Final R indices [I > 2 sigma(I)]
R indices (all data)
4777 [R(int) = 0.0831]
25.000°
4777 / 3 / 356
0.993
100.0 %
R1 = 0.0384, wR2 = 0.0904
R1 = 0.0678, wR2 = 0.1073
n/a
Extinction coefficient
Largest diff. peak [e.Å-3] and hole
0.447 and -0.580
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Table 8: Selected bond lengths [Ẳ] of complex 2
Bond lengths [Ẳ]
7
8
9
Cu(1)-O(1)
Cu(1)-O(3)
Cu(1)-N(3)
Cu(1)-N(2)
Cu(1)-N(1)
1.981(3)
1.983(3)
1.994(3)
2.018(3)
2.314(3)
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14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
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60
Table 9: Selected bond angles [º] of complex 2
Bond angles [º]
O(1)-Cu(1)-O(3)
O(1)-Cu(1)-N(3)
O(3)-Cu(1)-N(3)
O(1)-Cu(1)-N(2)
O(3)-Cu(1)-N(2)
N(3)-Cu(1)-N(2)
O(1)-Cu(1)-N(1)
O(3)-Cu(1)-N(1)
N(3)-Cu(1)-N(1)
N(2)-Cu(1)-N(1)
Cu(1)-O(1)-Na(3)1
Cu(1)-O(3)-Na(2)2
C(7)-N(1)-Cu(1)
C(8)-N(1)-Cu(1)
C(6)-N(1)-Cu(1)
C(11)-N(2)-Cu(1)
C(10)-N(2)-Cu(1)
C(2)-N(3)-Cu(1)
C(3)-N(3)-Cu(1)
C(5)-N(3)-Cu(1)
164.75(10)
82.83(11)
82.59(11)
97.19(11)
95.47(11)
163.23(13)
96.26(11)
93.19(11)
111.28(12)
85.43(12)
157.72(13)
162.75(13)
115.5(2)
98.4(2)
114.1(2)
112.9(2)
110.4(2)
107.2(2)
105.6(2)
107.9(2)
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3.7. Electrochemical studies
5
6
7
8
All the reported studies for the oxidation of catechols or o-aminophenol catalyzed by
copper(II) oxidase models or native copper proteins demonstrated the reduction of
copper(II) to copper(I) by the concerned phenol. This is followed by oxidation of the
generated monovalent copper by the atmospheric oxygen during the catalytic aerobic
oxidation cycle. Accordingly the redox potentials for both complexes 1, 2 and the studied
substrates is a key master for the current catalytic oxidation processes.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The electrochemical behavior of complexes 1 and 2 at a carbon paste electrode was
studied by using the cyclic voltammetry technique in phosphate buffer of pH value of 7.0
in a potential range from -1.2 to +1.2 V (vs. Ag/AgCl) using a potential scan rate of 50
mVs-1. The methanolic solutions of 1 × 10-3 M of the studied complexes 1 and 2 were
used for performing the present cyclic voltammetric measurements. The cyclic
voltammograms of both complexes 1 and 2 showed a quasi-reversible systems and well -
oxidation peaks (Epa) were appeared at + 0.0 V while the cathodic peaks (Epc) were
appeared between -0.33 and -0.35 V (Figure 6 and S8).
Fig 6: (a) Cyclic voltammogram for complex 1 at CPE at phosphate buffer pH 7 using scan rate
of 50 mV/s; (b) Effect of scan rate on the peak current height of 1 × 10-3 M complex 1 using
cyclic voltammetry at CPE at phosphate buffer pH 7
The separation of the anodic and cathodic peak potentials (∆E) was found to be lower
than 0.35 and 0.33 V for complexes 1 and 2 respectively (Table 10). The formal potential
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E1/2 was taken as the average of Epc and Epa which is found to be + 0.175 and + 0.165 V
in the case of 1 and 2 respectively.
7
8
9
The detected redox peaks of cyclic voltammograms are due to the reversible
reduction and oxidation behavior of Cu(II)/Cu(I) couple. Subsequently the redox
behavior of the current oxidase models can be summarized in scheme 2. In addition to the
quasi-reversible system, one cathodic broad peak was appeared at more negative value (-
0.73V). The effect of scan rate on the peak currents was examined at different scan rate
values which extended from 10 to 70 mV s-1. For both complexes 1 and 2 as the scan rate
increases both the anodic and cathodic peak current also increases (Figure 6 and S8).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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27
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31
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40
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43
44
45
46
47
48
49
50
51
52
53
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58
59
60
Scheme 2: The square scheme for the observed cyclic voltammograms of copper(II) complexes 1
and 2
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The electrochemical behavior of 3,5 DTBCH2 as a representative of the studied
catechols, was also examined at a carbon paste electrode in a phosphate buffer of pH
value of 7. The applied potential range is from -1.0 to +1.5 V (vs. Ag/AgCl) at the
potential scan rate of 50 mVs-1. The cyclic voltammogram for 1 × 10-3 M 3,5 DTBCH2
showed well-defined anodic peak current (Epa) at + 0.34V and one cathodic peak (Epc) at
-0.1 (S9). The oxidation reaction of 3,5 DTBCH2 is illustrated in reaction 1, where 3,5-
DTBCH2 is oxidized to the corresponding light absorbing 3,5-DTBQ [32]. The
electrochemical oxidation product 3,5-DTBQ was extracted by CH2Cl2 and its spectrum
was measured which exhibited the distinctive peak of quinone at 400 nm.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In the same regard the electrochemical behavior of o-aminophenol at a carbon paste
electrode was examined using phosphate buffer of pH value of 9.5 in a potential range
from -1.0 to +1.5 V (vs. Ag/AgCl) using a potential scan rate of 50 mVs-1. The cyclic
voltammogram for 1 × 10-3 M o-APH3 displays well-defined anodic peak current (Epa) at
+ 0.36V and one cathodic peak (Epc) at -0.62 (S10). Oxidation of o-APH3 leads to
formation of o-benzoquinone monoimine (o-BQMI) (reaction 4) the precursor for
production of o-amino-3H-phenoxazine-3-ones (APX) [33]. After the electrochemical
oxidation of o-APH3 the oxidation product was extracted by CH2Cl2. The extracted
product was designated as o-aminophenoxazin-3-one (APX) based on TLC analysis and
comparison with an authentic sample of APX prepared by chemical oxidation of o-APH3.
Table 10: Electrochemical data (mV) of copper(II) complexes 1, 2, 3,5-DTBCH2 and
o-APH3
Complex/substrate
Epc
EPa
ΔEp
E1/2
1.[CuL2 H2O].Cl2
2.[ CuLL' Cl].H2O
3,5-DTBCH2
o-APH3
− 350
− 330
− 100
− 620
0.00
0.00
340
360
350
330
444
980
175
165
120
− 130
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DOI: 10.1039/C9NJ06131B
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3.8. Catalytic oxidation of biologically significant phenols
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The current copper(II) complexes were synthesized with the aim of the functional
mimicking of the multicopper proteins catechol oxidase and phenoxazinone synthase in
the aerobic catalytic oxidation of various phenols to the corresponding o-quinone.
Catechol oxidase catalyzes the aerobic transformation of catechol to o-quinone via four
electrons oxidation of catechol or its derivatives with concomitant reduction of O2 to two
H2O as a by product as depicted in reaction 1.
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OH
OH
O
O
Catalyst
+ O2
(1)
2
2
+ 2H2O
3,5-DTBQ
3,5-DTBC
The reaction catalyzed by the other copper(II) enzyme phenoxazinone synthase is a
six-electron oxidation of o-aminophenol to o-amino-3H-phenoxazine-3-one which
commonly known as phenoxazinone chromophore (APX) with formation of water as
shown in reaction 2.
OH
N
NH2
O
Catalyst
3/2 O2
2
3H2O
+
(2)
+
NH2
O
OAP
APX
Within the framework of the current study, five phenols (Scheme 3) were studied to
investigate the effect of phenol substituents on the catalytic efficiency of the copper(II)
complexes under study.
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OH
OH
OH
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OH
3,5-di-tert-butylcatechol
OH
H3C
OH
O2N
OH
4-methyl catechol
4-nitro catechol
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OH
OH
NH2
Pyrocatechol
o-amino phenol
Scheme 3: Structure of the current studied phenols
Study the catalytic oxidation reactions were performed in methanol by mixing the
catalyst solution (1 × 10-4M) with the particular phenol solution (1 × 10-2M). Oxidation
products formation was monitored spectrophotometrically at the wavelengths of 400 and
433 nm distinctive to 3,5-DTBQ and APX respectively. After 24 hours from the
beginning of the oxidation reaction, spectral monitoring showed formation of a single
oxidation product excluding further oxidative cleavage of 3,5-DTBQ or APX.
Fig. 7: UV/Vis spectral changes recorded for the catalytic oxidation reaction of both (a) 3,5-
DTBCH2 and (b) o-APH3 at concentration of (10−2 M) in MeOH at 296 K in presence complex 1
(10−4 M). Inset shows the course of the absorbance at 400 for (a) and 433 nm for (b) verses time
in second.
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New Journal of Chemistry
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DOI: 10.1039/C9NJ06131B
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Figure 7 shows peak growing of 3,5-DTBQ and APX in presence of complex 1 as a
representative spectral profile of the studied catalytic reactions. The substrates being
studied are pyrocatechol (CatH2), 3,5-di-tert-butylcatechol (3,5-DTBCH2), 4-methyl-
catechol (4-Me-CatH2), 4-nitro-catechol (4-NO2-CatH2) in addition to o-aminophenol (o-
APH3).
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To confirm the catalytic activity of the studied copper(II) complexes, a typical
catalytic experiment was performed in the absence of the catalyst. The resulting amount
of oxidation product can be neglected when compared to that produced in the presence of
the catalyst.
Kinetic investigations
The kinetic measurements were performed using the stopped–flow instrument and the
kinetic details showed that in presence of catalyst aerobic oxidation of the current
phenols takes place via the formation of binary [substrate-catalyst] and ternary [substrate-
catalyst-O2] intermediates. Accordingly the whole oxidation process can be represented
in the reactions 3 and 4 for both 3,5-DTBCH2 and o-APH3 as representative substrates.
k1
O2
[CuIILL')2-3,5-DTBC]
2 [CuIILL'X] 3,5-DTBC
+
3,5-DTBQ
O
(3)
(4)
H2O2
+
k-1
k2
O2
k1
[(CuIILL')-o-APH2]
[CuIILL'X]
H2O2
o-APH3
+
+
k2
k-1
NH
(o-BQMI)
It is worth mention that earlier studies [33,5] displayed that o-benzoquinone
monoimine (o-BQMI) is the precursor to APX formation which converts to the final
product o-amino-3H-phenoxazine-3-ones (APX) via further uncatalyzed reactions.
Figure 8 shows that the kinetics trace of 3,5-DTBCH2 oxidation precedes in two
steps: i) the first step is the fast step in which the catalyst binds to the substrate molecule
in a reversible reaction to form [catalyst-substrate] intermediate. Binding the catalyst to
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the substrate renders the catalyst sensitive to react with O2 and leads to formation of the
ternary adduct [catalyst-substrate-O2].
ii) The second step is the irreversible slow step where the ternary intermediate
spontaneously converts to the oxidation product with releasing the catalyst in its original
form. This step occurs slowly in successive stages to form the oxidation products, and
therefore is the rate determining step. However, under anaerobic conditions this slow step
was not achieved indicating the essential involvement of O2 in the catalytic cycle.
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Fig. 8: Representative kinetic trace obtained by the stopped-flow spectrophotometry
monitored at 400 nm for the reaction of complexes 1 with 3,5-DTBCH2 in MeOH at 296 K
The initial rate method was used to treat the kinetics of current oxidation reactions by
monitoring the characteristic band growth of the oxidation product. The reaction rate
dependence on the concentration of the substrate was determined by the treatment of the
catalyst solution (1 × 10-4 M) by substrate solutions with concentrations of 1 × 10-3 to 8 ×
10-3 M. The obtained results are represented graphically by plotting the catalysis rate, kobs
versus the substrate concentration, [S] for the first and second steps as shown in Figures 9
and 10. On the other hand similar plots related to complex 2 and other substrates such as
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DOI: 10.1039/C9NJ06131B
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pyrocatechol, 4-methyl catechol and 4-nitro catechol are presented in the supplementary
materials S11-S16.
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Fig. 9: Dependence of the initial rate on the concentration of the substrate for the oxidation
reaction of 3,5-DTBCH2 catalyzed by complex 1 in methanol; (a) is the first step and (b) is the
second step.
Figures 9 and 10 show that at low substrate concentrations, the reaction rate (kobs)
changes linearly with [S], and indicates first-order behavior whereas at high
concentrations of the substrate, kobs becomes independent on [S] in reference to the
kinetic saturation pattern. The simplest model explaining the running catalytic behavior is
presented in equations 3 and 4, which corresponds to the Michael Menten method, where
catalytic kinetic parameters such as Vmax, kcat and KM are defined. Based on the plots of
Michael Menten the computed data for the studied substrates are in the ranges of 50 - 490
s-1 for kcat, and for KM is 15 – 37 × 10-4 M, in the case of studied catechols and kcat = 50 -
150 s-1, and KM = 20 – 29 × 10-4 M, for o-APH3 (Table 11).
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Figure 10: Dependence of the initial rate for on the concentration of the substrate for the
oxidation reaction of o-APH3 catalyzed by complex 1 in methanol; (a) is the first step and (b) is
the second step. The concentration of complex was 1.0 ×10−4 M and the reaction was followed at
433 nm
For the reversible reactions 3 and 4 the equilibrium constant K1 (K1 = k1 / k-1), of the
first step is a practical determination of the bonding affinity (coordination) of the studied
substrates towards the current copper(II) complexes. Concerning 3,5-DTBCH2 the
corresponding K1 values are 286 and 75 for complexes 1 and 2 respectively (Table 11). In
the same context o-APH3 exhibits binding affinity values of 243 for complex 1 and 89 in
the case of complexes 2. The difference in the coordination affinity of the studied
substrates could be ascribed to structural causes. However, the values of KM listed in
Table 11 are in almost consistent with values of K1 for both complexes 1 and 2.
The ratio kcat/KM with a unit of M-1 s-1 is a useful index for description the catalytic
efficiency and its values (Table 11) for complexes 1 is greater than 2 and the same trend
holds for the binding affinity of these complexes towards the examined substrates.
Previous studies for catechol oxidation by copper(II) catechol oxidase models reported
that transfer of electrons from the catecholate ring to the copper(II) centers occurs only
after the deprotonated catechol binds copper(II) centers [12]. In this context binding of
the current functional models to the dibasic catecholate anion (3,5-DTBC2‒) or o-APH2¯
is the spark of the beginning catalytic oxidation cycle [1,34]. This is evident from the
data in Table 11 which indicate that the catalytic efficiency of 1 and 2 depends on their
K1 values. The data in Table 11 show that both 3,5-DTBCH2 and o-APH3 exhibit binding
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affinity towards complex 1 greater than complex 2. In the same vein, for the other
substrates under study K1 values follow the order: 4-NO2-CatH2 > 3,5-DTBCH2 > o-
APH3 > CatH2 > 4-Me-CaH2 in the case of complex 1 (Table 11). The same order holds
for the oxidase catalytic efficiency (kcat/KM) of complex 1 (Table 11) except in the case of
4-nitrocatechol.
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The oxidation pattern of both pyrocatechol and 4-methy catechol continued as in the
case of 3,5-DTBCH2 and o-APH3 and marked oxidation products were formed.
Concerning 4-nitro-catechol, its kinetic trace (S14) displayed only the fast step while
the kinetically significant slow step is absent and no oxidation product formed.
Table 11: Catalytic kinetic parameters for oxidation of various substrates
Complex/substrate
CatH2
k1
k-1
k1 /k-1 Vmax M s−1 kcat h-1 KM ×10-4 M kcat/KM ×104
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0.27
89
268
52
0.015
0.049
0.008
150
490
80
37
29
15
4.054
16.896
5.333
3,5 DTBC
4-CH3- CatH2
1
56.2 0.209
19.2 0.369
4-NO2- CatH2 98.2 0.104
944
243
75
-
-
-
-
OAP
51.0 0.210
30.9 0.409
34.1 0.384
0.015
0.0058
0.005
150
58
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7.5
3,5 DTBC
OAP
2.416
1.724
2
89
50
Several dinuclear and mononuclear copper(II) complexes has been synthesized and
used as a catalyst for the oxidation of the widely used substrate 3,5-DTBCH2
[12,13,14,35,36]. A list of reported copper systems (catechol oxidase mimics) with their
kinetic parameters of 3,5-DTBCH2 oxidation are listed in Table 12. The data in Tables 11
and 12 show that the catecholase mimetic catalytic activity of the current copper(II)
complexes are comparable with the other copper based catechol oxidase functional
models. However, the efficiency of the native catechol oxidase enzyme is still the highest
compared to all the functional models studied [37].
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