Phenoxazinone synthase activity of two iron(III) complexes comprising the same Schiff base ligand: Biomimetic functional model and mechanistic investigation

Article abstract of DOI:10.1016/j.ica.2018.01.024

A new 4,4′-bipyridine (4,4′-byp) mediated 1D- polymeric Fe^III complex (complex 1) of Schiff base ligand H₂L, a 1:2 condensation product of 1,2-diaminopropane and salicylaldehyde, has been synthesized. Complex 1 is structurally characterized by single crystal X-ray diffraction. A phenoxo bridged dinuclear Fe^III complex (complex 2) of analogous ligand has been synthesized also. Dioxygen activation in terms of Phenoxazinone synthase activity using o-aminophenol (OAPH) as a model substrate catalyzed by both the complexes are thoroughly investigated here. ESI-MS spectral study reveals that polynuclear complex 1 dissociates into mononuclear units while dissolve in methanol during catalytic study. The kinetic study illustrates that both the complexes have well competence towards o-aminophenol oxidation where dinuclear Fe^III species demonstrate higher activity than mononuclear intermediate species. Important finding from the mass spectral and electrochemical study provide significant information of the mechanistic pathway of the functioning phenoxazinone synthase like activity of synthesized iron complexes.

Full text of DOI:10.1016/j.ica.2018.01.024

Accepted Manuscript
Phenoxazinone synthase activity of two iron(III) complexes comprising the
same Schiff base ligand: Biomimetic functional model and mechanistic inves-
tigation
Sourav Chatterjee, Dipankar Sukul, Priyabrata Banerjee, Jaydeep Adhikary
PII:
S0020-1693(17)31749-8
https://doi.org/10.1016/j.ica.2018.01.024
ICA 18098
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 November 2017
20 January 2018
23 January 2018
Please cite this article as: S. Chatterjee, D. Sukul, P. Banerjee, J. Adhikary, Phenoxazinone synthase activity of two
iron(III) complexes comprising the same Schiff base ligand: Biomimetic functional model and mechanistic
investigation, Inorganica Chimica Acta (2018), doi: https://doi.org/10.1016/j.ica.2018.01.024
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‘Revised’
Old title: Phenoxazinone synthase activity of di and polynuclear iron(III)
complexes of analogous Schiff base ligand: Biomimetic functional model and
mechanistic investigation
New title: Phenoxazinone synthase
activity of two iron(III) complexes comprising the same Schiff base
ligand: Biomimetic functional model and mechanistic investigation
Sourav Chatterjeea, Dipankar Sukulb, Priyabrata Banerjeec, Jaydeep Adhikary d,e,
*
a Department of Chemistry, Panchakot Mahavidyalaya, Sarbari, Purulia, Pin-723121, India
b Department of Chemistry, National Institute of Technology Durgapur, Durgapur 713 209, India
c Department of Surface Engineering and Tribology, CSIR-CMERI, Durgapur 713 209, India
d Department of Chemistry, University of Calcutta, 92 A. P. C. Road, Kolkata-700 009, India
e Department of Chemical Sciences, Ariel University, Ariel 40700, Israel
Corresponding author:
E-mail address: adhikaryj86@gmail.com (J. Adhikary).
ABSTRACT
A new 4,4′-bipyridine (4,4΄-byp) mediated 1D- polymeric FeIII complex (complex 1) of Schiff
base ligand H2L, a 1:2 condensation product of 1,2-diaminopropane and salicylaldehyde, has
been synthesized. Complex 1 is structurally characterized by single crystal X-ray diffraction. A
phenoxo bridged dinuclear FeIII complex (complex 2) of analogous ligand has been synthesized
also. Dioxygen activation in terms of Phenoxazinone synthase activity using o-aminophenol
(OAPH) as a model substrate catalyzed by both the complexes are thoroughly investigated here.
ESI-MS spectral study reveals that polynuclear complex 1 dissociates into mononuclear units
while dissolve in methanol during catalytic study. The kinetic study illustrates that both the
complexes have well competence towards o-aminophenol oxidation where dinuclear FeIII species
demonstrate higher activity than mononuclear intermediate species. Important finding from the
1

mass spectral and electrochemical study provide significant information of the mechanistic
pathway of the functioning phenoxazinone synthase like activity of synthesized iron complexes.
1. Introduction
Schiff bases are significant ligands and can be facilely synthesized and coordinated with
a series of metal ions. Transition-metal complexes consisting Schiff-base ligands has become
one of the most extensively studied topics in coordination chemistry now a days[1] due to ease of
synthesis, wonderful structural diversities, and potential applications in diverse fields such as
catalysis, medicinal chemistry and material sciences etc. [2]. In recent years, Schiff base type
ligands are extensively used to prepare synthetic analogues of metallobiosites such as galactose
oxidase, cytochrome P-450, phenoxazinone synthase, catechol oxidase, phosphatase, supper
oxide dismutase, urease and many others [3]. Our group has been continuously affianced in
studying Schiff-base complexes of transition and post transition metal ions mainly as small
synthetic analogs of various metalloenzyme like phosphatase, catechol oxidase and cytochrome
P-450 [4]. However, being exceptionally important enzyme, phenoxazinone synthase like
activity has not been investigated yet in our group. Multicopper oxidase phenoxazinone synthase
is naturally found enzyme in Streptomyces antibioticus. This enzyme catalyzes the oxidative
dimerization of two molecules of a wide varieties of substituted 2-aminophenol to the
phenoxazinone chromophore during the biosynthesis of actinomycin D. This naturally occurring
antibiotic is used clinically for the treatment of specific tumors [5]. On the other hand, it is well
known that o-aminophenol is rapidly metabolized to 2-aminophenoxazine-3-one in human
erythrocytes, where oxy and methemoglobin are involved [6]. Thus, it seems that iron containing
enzymes can also promote the oxidative dimerization of 2-aminophenol. Therefore, in the
present study FeIII Schiff base complexes have been selected for metalloenzyme mimicking of
phenoxazinone synthase. In particular, iron complexes of tetradentate Schiff base ligands have
been taken under consideration since they exhibit significant catalytic activity, magnetic
properties, and unique coordination chemistry [7]. Only a few examples have been found for
which iron complexes were used as synthetic analogue of phenoxazinone synthase [8] and no
poly nuclear FeIII Schiff base complex has been explored in phenoxazinone synthase like activity
till date. Consequently, we have synthesized 4,4′-bipyridine mediated poly nuclear FeIII complex
2

(complex 1) and phenoxo bridged dinuclear (complex 2) of a tetradentate Schiff base ligand
(H2L) (Scheme 1). Nevertheless, ESI mass spectrum reflects that the polymeric complex breaks
down into mononuclear units during the dissolution of complex 1 in methanol before catalytic
study. Both the complexes are capable to oxidize o-aminophenol. Detailed mechanistic study has
been investigated using cyclic voltammetry and electrospray ionization mass spectral study.
Finally, a comparison of the activity of our synthesized iron complexes to the previously
reported catalysts strongly suggests that our complexes have excellent catalytic efficiency
towards phenoxazinone synthase like activity.
Scheme 1 Synthetic route of complexes 1 and 2.
2. Experimental Section
2.1. Materials &Methods
All chemicals were obtained from commercial sources and used as received. Solvents
were dried according to standard procedure and distilled prior to use.
Elemental analyses were achieved using a Perkin-Elmer 240C elemental analyzer.
Fourier-transform infrared spectroscopy (FT-IR) (4000–500 cm-1) data were taken at 27°C using
a Perkin-Elmer RXI FT-IR spectrophotometer with KBr pellets. Electronic spectra (900–200 nm)
were recorded using a Shimadzu UV-3101PC at 27°C with methanol as solvent and reference.
The electrospray ionization mass spectra (ESI-MS) were recorded on a MICROMASS Q-TOF
3

mass spectrometer. The cyclic voltammetric measurements were performed in dry methanol
solutions with 0.2M TBAP as supporting electrolyte (scan rate = 10 mVs−1) employing a PAR
potentiostat/ galvanostat model Versa Stat-II. A three-electrode system was used here in which
the counter and working electrodes were platinum foils and the reference electrode was a
saturated calomel electrode.
2.2. Syntheses
2.2.1. Synthesis of [{FeL(4,4΄-byp)ClO4}]n(1)
An ethanolic solution (5 mL) of 1,2-diaminopropane (0.109 g, 1 mmol) was added
dropwise to ethanolic solution (10 mL) of salicylaldehyde (0.244 g, 2 mmol) and the resulting
mixture was stirred for half an hour. Then, an ethanolic solution (10 mL) of iron(III) perchlorate
(0.354 g, 1 mmol) was added under continuous stirring condition and followed by the addition of
5mL ethanolic solution of 4 4'-bipyridine (0.156 g, 1 mmol). Resulting solution was then allowed
to stir for one hour and filtered. Deep brown colored X-ray suitable square shaped crystals were
obtained from the filtrate. Yield: 85%. Anal. Calcd. C, 54.74; H, 4.05; N, 9.46. Found: C, 54.73;
-
H, 4.03; N, 9.44; FTIR: ν(C=N) = 1614 cm-1, ν(skeletal vibration) = 1545cm-1, ν(ClO4 ) =
1087cm1; UV–Vis (methanol): 235, 322 and 509 nm.
2.2.2. Synthesis of [FeLCl]2 (2)
Complex 2 was synthesized following the similar procedure reported by Hai-Liang et
al.[9]. Yield: 85%. Anal. Calcd. C, 54.89; H, 4.34; N, 7.53. Found: C, 54.83; H, 4.29; N, 7.52;
FTIR: ν(C=N) = 1622 cm-1, ν(skeletal vibration) = 1544cm-1; UV–Vis (methanol): 259, 304 and
473 nm.
Caution! Transition metal perchlorate complexes are potentially explosive and should be handled
in lesser amounts and with necessary precautions.
2.3. X-ray data collection and structure determination
Diffraction data for complex 1 was collected at room temperature (293 K) on a Bruker
Smart CCD diffractometer equipped with graphite monochromated MoKα radiation
(λ=0.71073Å). Cell refinement, indexing and scaling of the data set were done using Bruker
SMART APEX and Bruker SAINT package [10]. The structure of the complex 1 was solved by
4

direct methods and subsequent Fourier analyses [11] and refined by the full-matrix least-squares
method based on F2 with all observed reflections using SIR-92 and SHELX-97 [12], software.
For the complex, all non-hydrogen atoms were refined with anisotropic thermal parameters and
the hydrogen atoms were fixed at their respective positions riding on their carrier atoms and
refined anisotropically. All the calculations were made using the WinGX System, Ver 1.80.05
[13], PLATON99 [14], ORTEP3 [15] programs. Selected crystallographic data and refinement
details are displayed in Table 1.
Table 1 Crystallographic data and details of structure refinement for the complex 1.
Empirical formula
Formula mass
Crystal system
Space group
a (Å)
C27H24FeN4O6Cl
591.80
Triclinic
P-1(No. 2)
9.796(2)
11.466(2)
15.055(3)
100.231(2)
104.842(2)
106.828(2)
1505.5(5)
2
b (Å)
c (Å)
α (°)
β (°)
γ (°)
V (Å3)
Z
T (K)
293
µ(MoKα) (/mm)
Dcalc (Mg/m3)
F(000)
0.633
1.306
610
θ max (deg)
24.9
Tot., Uniq. Data, Rint 10105, 5096, 0.024
Observed (I > 2σ(I))
4210
Nref, Npar
5096, 391
5

R, wR2, S
0.0646, 0.1927, 1.08
residual extrema (e/Å3) -0.44, 0.63
2.4. Catalytic oxidation of o-aminophenol
Phenoxazinone synthase like activity of our synthesized complexes (1 and 2) was
investigated by the reaction of 1.0 × 10–4 M methanolic solutions of the complexes with 0.01 M
solution of o-aminophenol (OAPH) in dioxygen saturated methanol at room temperature. The
reaction was monitored spectrophotometrically by monitoring the enhancement of the
absorbance as a function of time at ca. 433 nm which is typical band of the phenoxazinone
chromophore [16]. Determination of various kinetic parameters for the catalytic activity was
carried out by using the same procedure reported earlier [16]. In addition, to confirm the rate
dependency on catalyst concentrations similar set of experiments were performed at a fixed
concentration of substrate with varying amount of catalyst. Rate of a reaction was estimated from
the initial rate method. All measurements were performed in triplicate, and the average values
were adopted.
3. Results and discussions
3.1. Syntheses and characterization
Addition of Fe(ClO4)3 to the ethanolic solution of H2L followed by the addition of 4,4′-
bypiridine afforded complex 1. The crystal structure was determined by X-ray analysis (vide
infra). Crystal structure reveals that complex 1 has 1D polymeric structure. Addition of FeCl3 to
the ethanolic solution of H2L provided phenoxo bridged dinuclear FeIII complex (complex 2).
Unfortunately, the crystal structure of 2 was reported by another group [9]. The complexes 1 and
2 were characterized by routine physiochemical analyses. The complexes exhibit characteristic
FTIR bands at the range of 1614-1622 cm-1 and 1544−1545 cm−1assigned to C=N and skeletal
vibrations (Fig. S1-2, Supplementary information), respectively. Complex 1 also shows broad
FTIR band centered in the range of 1068-1087 cm-1 due to presence of the perchlorate ion [16b].
The electronic spectra for 1 and 2 in methanol show a band at 473-509 nm can be assigned as
charge-transfer transitions from the in-plane pπ orbital of the phenolate to the half-filled dπ*
orbital of iron(III) (Fig. S3, Supplementary information). Other two electronic spectral bands
6

at ∼235-259 and ∼304-322 nm can be attributed to π-π* and ligand to metal charge transfer
(LMCT) transitions, respectively, as reported earlier [3e].
3.2. Description of structure of complex 1
The perspective view of the monomeric unit of complex 1 is illustrated in Fig. 1. The
structure consists of distorted octahedral iron(III) center coordinated equatorially by the
ictetradentate Schiff base ligand which provides a N2O2 donor set. The [Fe(III)(salen)]+ moieties
are then bridged by the two connecting 4,4′-bipyridyl ligand, to form infinite 1-D chain (Fig. 2).
Each [Fe(III)(salen)]+ species is neutralized by one perchlorate counter anion which is not
involved in polymerization. Selected bond lengths and angles of complex 1 are depicted in Table
2. The Fe(1)-O(1) and Fe(1)-O(2) bond distances are found to be 1.886(3) and 1.889(3) Å, and
are very similar with those of related iron(III)(salen) compounds which are in the range from
1.887(6) to 1.894(7)Å with pertaining to the phenoxy axial ligand [17-19]. The Fe(1)-N(1) and
Fe(1)-N(2) bond distances are found to be 2.092(5) and 2.094(4)Å, which are similar to those
observed in related Fe(salen) type compound [20]. Axial positions of metal center are occupied
by N atoms of two 4,4′-bipyridyl groups having Fe(1)-N(3) and Fe(1)-N(4) bond distances are
2.205(4) and 2.230(4), respectively. Successive Fe…Fe distance is 11.466 Å.
Fig. 1 Crystal structure of complex 1. Hydrogen and perchlorate anion are omitted for clarity.
7

Fig. 2 Representation of 1D polymeric chain of complex 1. Hydrogen atoms and distorted
perchlorate anions are omitted for clarity.
Table 2 Selected bond distances (Å) and angles (°) for the complex 1.
Fe -O1
Fe -O2
Fe -N1
Fe -N2
Fe -N3
Fe -N4
1.886(3)
1.889(3)
2.092(5)
2.094(4)
2.205(4)
2.230(4)
O1-Fe-O2
O1-Fe -N1
O1-Fe -N2
O1-Fe -N3
O1-Fe -N4
O2-Fe-N1
O2-Fe-N2
O2-Fe-N3
O2-Fe-N4
N1-Fe -N2
N1-Fe -N3
N1-Fe -N4
N2-Fe -N3
N2-Fe-N4
N3-Fe-N4
106.25(13)
89.11(16)
165.54(17)
89.08(15)
89.72(14)
164.31(16)
88.12(16)
91.12(15)
87.07(14)
76.65(19)
92.37(19)
89.84(18)
89.04(17)
92.67(15)
177.46(14)
3.3. Phenoxazinone synthase like activity
8

Catalytic oxidation of o-aminophenol (OAPH) was examined spectrophotometrically in
dioxygen saturated methanolic solution at room temperature. In order to confirm the ability of
our synthesized complexes to oxidize OAPH, 1.0 × 10-4 M methanolic solutions of the
complexes were treated with a 1 × 10-2 M (100 equiv) of OAPH under aerobic conditions. The
catalytic studies were performed in the absence of any added base to minimize the possibility of
auto oxidation of OAPH. The course of the reaction was followed by UV–Vis spectroscopy, and
the time dependent spectral scans of the two complexes are depicted in Fig. 3 and 4. The spectral
scan reveals the development of peak intensity at ca. 433 nm, characteristic of phenoxazinone
chromophore, suggesting the catalytic oxidation of OAPH to 2-aminophenoxazine-3-one in
aerobic condition [16]. A blank experiment without catalyst in an identical condition does not
show any increment in the band intensity at 433 nm. These spectral behaviors clearly reveal that
both the complexes are active catalysts for the aerial oxidation of OAPH to the corresponding
phenoxazinone chromophore.
Reaction kinetics was performed to understand the extent of the catalytic efficiency of the
complexes. The concentration of OAPH was always kept 10 times higher than that of the
complex and the rise of respective 2-aminophenoxazine-3-one concentration were determined at
433 nm wavelength for each complex. Solutions of OAPH of concentration ranging from 0.001
to 0.05 mol dm–3 were prepared from a concentrated stock solution in methanol. 2 mL
methanolic solution of the substrate were taken in a 1 cm spectrophotometer quartz cell
thermostated. 0.04 mL of 0.005 mol dm–3 complex solution was then quickly added to it so that
the ultimate concentration of the complex becomes 1 × 10–4 mol dm–3. The dependence of the
initial rate on the concentration of substrate was spectrophotometrically monitored at 433 nm.
The initial rates method shows a first-order dependence on complex concentration and exhibited
saturation kinetics at higher substrate concentrations. This type of saturation rate dependency on
the concentration of the substrate can be treated with the Michaelis–Menten model, which on
linearization gives a double reciprocal Lineweaver–Burk plot to analyze the values of maximum
velocity (Vmax), binding constant (KM), and rate constant (kcat) (Fig. S4-S7, Supplementary
information). All these kinetic parameters of both the complexes for OAPH oxidation are
demonstrated in Table 3. Table 4 represents the kcat values of o-aminophenol oxidation of some
transition metal complexes reported in recent years [8,16b,21]. On comparing Tables 3 and 4 it
9

may be stated that complexes 1 and 2 belongs to the highly efficient catalysts group where the
order of their activity is 2>1.
1.0
2-aminophenoxazine-3-one
0.8
0.6
0.4
0.2
0.0
Complex 1
OAPH
300
400
500
600
700
800
900
Wavelength(nm)
Fig. 3 UV–Vis spectral scans showing the increase in phenoxazinone chromophore band at 422
nm after the addition of o-aminophenol (0.001 M) to a solution of complex 1 (1×10–4 M) in
methanol at 25oC. The spectra were recorded for the period of 2h.
2.5
2.0
1.5
1.0
0.5
0.0
2-aminophenoxazine-3-one
Complex 2
OAPH
300
400
500
600
700
800
Wavelength(nm)
10

Fig. 4 UV–Vis spectral scans showing the increase in phenoxazinone chromophore band at 422
nm after the addition of o-aminophenol (0.001 M) to a solution of complex 2 (1×10–4 M) in
methanol at 25oC. The spectra were recorded for the period of 2 h.
Table 3 Kinetic parameters for complex 1 and 2.
Catalyst
Vmax (Ms-1)
8.989×10-7
5.449×10-6
KM(M)
kcat (h-1)
32.36
2.22×10-3
8.19×10-3
1
2
196.18
Table 4 Kinetic data for phenoxazinone synthase like activity of different metal complexes.
Catalysta
Solvent
DMF
kcat(h-1)
Ref.year
8a 2014
21a2014
21b2013
21c2013
21d2013
8b2012
1.
[FeCl2(L5)]
[Co(L2)(N3)3]
137
2.
Methanol
Methanol
Methanol
Methanol
Methanol
33.26
13.75
13.68
26.32
56
3.
[Co2(amp)2(µ-imp)2Cl2]Cl2·2H2O
[Co(L1)Cl(H2O)]Cl.H2O
[Mn(L4)Cl2]·H2O
[Fe(L1)Cl3]
4.
5.
6.
7.
[Mn(6΄Me2indH)(H2O)2(CH3CN)](ClO4)2 DMF
2.93
21e2008
21f2016
16b2016
21g2016
21h2017
8.
[Mn(Br4Cat)(Br3pyCat)(py)2]
[Mn(HL)2](ClO4)2
[LCo(L')2]ClO4
DMF
22.39
138.62
11.48
54.0
9.
Methanol
Methanol
Methanol
10.
11.
[Co(L)(N3)2]
a(1) [HL5
=1,3-bis(5′-methyl-2′-thiazolylimino)isoindoline]; (2)[L2=(2-pyridylmethyl)(2-
pyridylethyl)amine]; (3) [amp = 2-aminomethylpyridine; imp = 2-iminomethylpyridine anion];
(4) [L1=N,N′-bis(pyridin-2-ylmethylene)-2,2-dimethylpropane-1,3-diamine]; (5)[ L4=hexahydro-
1-(2-(tetrahydro-2-(pyridin-2-yl)pyrimidin-(2H)-yl)ethyl)-2-(pyridin-2-yl)pyrimidine];
(6)
[L1=N,N′-Bis(2-Methylbenzimidazolyl) pyridinediamide]; (7) [ 6′Me2indH= 1,3-bis(6′-methyl-
2′-pyridylimino)isoindoline]; (8) py = pyridine, Br4CatH2 = tetrabromocatechol and Br3pyCatH2
=
3,5,6-tribromo-4-pyridiniumcatechol; (9) LH
=
4-tert-butyl-2,6-bis-[(2-pyridin-2-yl-
ethylimino)-methyl]-phenol;
(10)
L'=4-aminopyridine,
H2L=
N,N'-bis(3-
11

methoxysalicylidehydene)cyclohexane-1,2-diamine; (11) HL= 2-{[3-(3-Dimethylamino-
propylamino)-propylimino]-methyl}-6-methoxy-phenol.
3.4. Electrochemical study
It is very essential to check the electrochemical behavior of biomimetic models since the
redox potential of the active site of a metalloenzyme plays a vital role in catalyzing the oxidation
of various organic matters. The electrochemical features of the two complexes were investigated
in methanol solution by employing cyclic voltammetry (CV) stationary platinum-sphere
electrode. Typical cyclic voltammogram of two complexes are depicted in Fig. 5.
Electrochemical data for complexes are tabulated in Table 5. The cyclic voltammogram of the
complexes 1–2 exhibit redox waves at negative potentials that can be assigned to a FeIII/FeII
redox couple [22,23]. Two complexes exhibit a cathodic (-0.185 to -0.403 V) as well as an
anodic wave (-0.083 to -0.265V). The values of the peak current ratio (ipa/ipc), which is far from
unity, and the peak potential separation (∆Ep, 103–138 mV) clearly reveal that the redox process
is far from reversible. The E1/2 values of FeIII/FeII redox potentials follow the trend 2 > 1,
reflecting a decrease in Lewis acidity of the iron(III) center of 1 from 2.
It is already demonstrated that the rate of OAPH oxidation of complex 2 is much higher
than complex 1. This fact is now also supported by the electrochemical data that the reduction of
complex 1 is quite difficult as compared to 2 as E1/2 value of FeIII→FeII process for complex 1 is
more negative than that of 2. Consequently, the effectivity towards o-aminophenol oxidation is
higher for complex 2.
15.0µ
Complex 1
10.0µ
Complex 2
5.0µ
0.0
-5.0µ
-10.0µ
-15.0µ
-1.0
-0.5
0.0
0.5
1.0
Potential (V)
12

Fig. 5 Cyclic voltammograms (CV) of the complexes in methanol solution at 25°C. Supporting
electrolyte: 0.1 M TBAP. Scan rate: 50 mV s-1.
Table 5 Electrochemical data of the complexes in methanol at 25°C.
Complex
Ep,a(V)
-0.265
-0.083
Ep,c(V)
-0.403
-0.185
∆Ep(V)
0.138
E1/2(V)
-0.334
-0.134
Redox Process
FeIII→FeII
FeIII→FeII
1
2
0.102
3.5. Electrospray ionization mass spectral study
The electrospray ionization mass spectra (ESI-MS positive) of compounds 1 and 2 were
recorded in methanol solution and are depicted in Fig. S8 and S9(Supplementary information),
respectively. Compound 1 exhibits two assignable peaks at m/z = 336.0352 (base peak) and
689.1639 with line-to-line separation of 1.0 corresponds to [FeL]+ and [ArFeL(4,4΄-byp)2]+,
respectively. The spectrum clearly implies that polymeric moiety dissociates into mononuclear
units in methanol. Complex 2 has also two peaks at m/z = 336.1289 (base peak) and 707.0813
can be assigned as [FeL]+ and [{FeL}2-Cl]+, respectively [line-to-line separation of 1.0]. The
result clearly tells that the base peaks in both complexes are same. However, the rate of o-
aminophenol oxidation of both complexes are different, indicating that the active species for two
complexes are not the identical. In order to find a perception into the nature of plausible
complex–substrate intermediates, the ESI-MS positive spectra of a 1: 50 mixture of the
complexes and o-aminophenol in methanol were recorded after 10 minutes of their mixing. The
spectrum for the compound 2 (most active) is depicted in Fig. 6 and S10 (Supplementary
information). The peaks at 780.1573 and 467.0909 with line-to-line separation of 1.0 can be
assigned as [{FeIIIL}2-(OAP)]+ and [FeIIIL-(OAP)Na]+, respectively. The peak at m/z = 213.1001
can be described to the protonated species of 2-aminophenoxazine-3-one. Base peak at m/z =
336.0561 is already assigned as [FeL]+. The remaining peaks at m/z = 672.1124 and 358.8539
are most interesting and can be assigned as [FeIII FeIIL2]+ and [FeIIL-Na]+, respectively. These
interesting peaks are absent in case of the ESI-MS spectra of sole complex and thus definitely
these species can only generate due to reduction of iron(III) center during the OAPH oxidation.
The ESI-MS positive spectra of a 1: 50 mixture of the complex 1 and o-aminophenol in methanol
13

recorded after 10 minutes of mixing is depicted in Fig. S11 (Supplementary information). The
spectrum exhibits 4 peaks at m/z = 335.9214(base peak), 213.1018, 358.9010 and 466.9208 with
line-to-line separation of 1.0. All those peaks are already assigned before. Peak at m/z =
358.9010 is generated during the oxidation process as happened in case of complex 2. The
experimental and the simulated spectral patterns are in nice agreement with each other,
demonstrating accurate assignment of the species. During the oxidation process for both
complexes one common intermediate, namely [FeIIIL-(OAP)Na]+, was formed but [{FeIIIL}2-
(OAP)]+ was only generated in case of complex 2. And probably this unique intermediate
promotes complex 2 to show higher activity of complex 2 over 1 towards OAPH oxidation.
With the help of some relevant references [8b,21a] and the ESI-MS spectral data, we
have proposed a plausible mechanistic pathway of phenoxazinone synthase like activity of the
most active complex 2 (Scheme 2).
Fig. 6 Electrospray ionization mass spectrum (ESI-MS positive) of a 1: 50 mixture of complex 2
and o-aminophenol in methanol recorded after 10 min of mixing, showing experimental and
simulated isotopic distribution patterns.
14

Fig. 7 Plausible mechanistic pathway showing the formation of 2-aminophenoxazine-3-one in
which complex 2 chosen as model complex.
4. Conclusions
In summary, our study expresses the syntheses and characterization of one polymeric and
one dinulcear FeIII complexes of a ONNO donor Schiff base ligand. The complexes are efficient
15

catalysts for the 2-aminophenol oxidation, which means that the examined systems serve as
functional models for phenoxazinone synthase enzyme. The ESI-MS positive spectra together
with UV-vis spectroscopy clearly suggest the formation of a catalyst–substrate adduct by a
substitution reaction in the catalytic cycle. Higher efficiency of dinuclear iron complex over the
mononuclear species towards the o-aminophenol oxidation process has been elucidated by the
electrochemical and ESI-MS study. Low negative value of reduction potential and generation of
higher number of active species during catalytic process are the reason for higher activity of
complex 2. On the other hand, high negative reduction potential and rigid structure content of the
complex 1 are jointly responsible for the lower activity of 4,4'-bipyridine coordinated complex.
Acknowledgements
The author (J.A) gratefully acknowledge Prof. Debasis Das of University of Calcutta, India for
his valuable suggestions.
Appendix A. Supplementary data
CCDC 1025941 contains the supplementary crystallographic data for the complex 1 reported in
this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif. Supplementary data associated with this article
can be found in the online version, at doi:XXXX.
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Highlights
1)
Dinuclear and polynuclear iron(III) complexes of ONNO donor Schiff base ligand have
been synthesised and characterised.
2)
3)
Phenoxazinone synthase activity of both the complexes was scrutinized thoroughly.
Probable mechanistic pathway involved in the catalytic reaction has been proposed with
the help of cyclic voltammeter and electrospray ionization mass spectrometer.
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21