Catalytic promiscuity of an iron(II)–phenanthroline complex

Article abstract of DOI:10.1002/aoc.3551

A mononuclear iron(II) complex, [Fe(phen)₃]Cl₂ (1) (phen =1,10-phenanthroline), has been synthesized in crystalline phase and characterized using various spectroscopic techniques including single crystal X-ray diffraction. Crystal structure analysis revealed that 1 crystallizes in a monoclinic system with C2/m space group. Complex 1 acts as a functional model for a biomimetic catalyst promoting the aerobic oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) through radical pathways with a significant turnover number (k_cat =3.55?×?10³?h^?1) and exhibits catechol dioxygenase activity towards the same 3,5-DTBC substrate at room temperature in oxygen-saturated ethanol medium. The existence of an isobestic point at 610?nm from spectrophotometric data indicates the presence of Fe³⁺??3,5-DTBC adduct favouring an enzyme–substrate binding phenomenon. Upon stoichiometric addition of 3,5-DTBC pretreated with two equivalents of triethylamine to the iron complex, two catecholate-to-iron(III) ligand-to-metal charge transfer bands (575 and 721?nm) are observed and the in situ generated catecholate intermediate reacts with dioxygen (k_obs =9.89?×?10^?4?min^?1) in ethanol medium to afford exclusively intradiol cleavage products along with a small amount of benzoquinone, and a small amount of extradiol cleavage products, which provide substantial evidence for a substrate activation mechanism. Copyright

Full text of DOI:10.1002/aoc.3551

Full paper
Received: 8 April 2016
Revised: 6 May 2016
Accepted: 8 June 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/aoc.3551
Catalytic promiscuity of an iron(II)–
phenanthroline complex
Abhranil Dea, Mamoni Garaia, Hare Ram Yadavb,
Angshuman Roy Choudhuryb and Bhaskar Biswasa*
A mononuclear iron(II) complex, [Fe(phen)3]Cl2 (1) (phen =1,10-phenanthroline), has been synthesized in crystalline phase and
characterized using various spectroscopic techniques including single crystal X-ray diffraction. Crystal structure analysis revealed
that 1 crystallizes in a monoclinic system with C2/m space group. Complex 1 acts as a functional model for a biomimetic catalyst
promoting the aerobic oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) through radical pathways with a significant turnover
number (kcat =3.55 × 103 hꢀ1) and exhibits catechol dioxygenase activity towards the same 3,5-DTBC substrate at room temper-
ature in oxygen-saturated ethanol medium. The existence of an isobestic point at 610 nm from spectrophotometric data indicates
the presence of Fe3+ ꢀ3,5-DTBC adduct favouring an enzyme–substrate binding phenomenon. Upon stoichiometric addition of
3,5-DTBC pretreated with two equivalents of triethylamine to the iron complex, two catecholate-to-iron(III) ligand-to-metal charge
transfer bands (575 and 721 nm) are observed and the in situ generated catecholate intermediate reacts with dioxygen
(kobs =9.89 × 10ꢀ4 minꢀ1) in ethanol medium to afford exclusively intradiol cleavage products along with a small amount of
benzoquinone, and a small amount of extradiol cleavage products, which provide substantial evidence for a substrate activation
mechanism. Copyright © 2016 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: catecholase activity; catechol dioxygenase activity; iron(II); phenanthroline; X-ray structure
iron(II) ions.[19] In view of the great importance of oxidation reac-
tions in industrial and synthetic processes and of the ongoing
Introduction
The synthesis and reactivity studies of transition metal complexes
as structural and functional models for metalloenzymes with
oxidase and oxygenase activity are of particular interest for the
development of bio-inspired catalysts.[1–3] A number of important
oxidative transformations are carried out by iron enzymes utilizing
dioxygen as an oxygen source. Iron is therefore ideally suited for its
many roles in biology including the transport and storage of
dioxygen (O2) and the catalysis of numerous reactions that utilize
dioxygen.[4–7] Catechol dioxygenases are a class of non-heme iron
enzymes that catalyse the oxidative cleavage of catechols. They
are divided into two subclasses: the intradiol dioxygenases, which
utilize a non-heme iron(III) cofactor in catalysing the cleavage of
the carbon–carbon bond between the two catechol oxygens; and
the extradiol dioxygenases, which utilize a non-heme iron(II) cofac-
tor in catalysing the cleavage of the carbon–carbon bond adjacent
to the catechol oxygens.[8–17] Even though intradiol cleavage is the
less common route in the biodegradation of aromatic molecules as
compared to extradiol cleavage, extensive investigation of intradiol
dioxygenases has been made due to the rich spectral properties of
the iron(III) active site in the enzymes.[14–16]
search for new and efficient oxidation catalysts, we have synthe-
sized and structurally characterized an iron(II)–phenanthroline
complex. Electron paramagnetic resonance indicates the existence
of a low-spin form of the iron complex at room temperature and
points to its inactive behaviour towards 3,5-di-tert-butylcatechol
(3,5-DTBC) under normal conditions. The complex acts as a func-
tional model of catecholase enzyme using 3,5-DTBC as the model
substrate through radical pathways with a significant turnover
number (kcat = 3.55 × 103 hꢀ1) and exhibits catechol dioxygenase
activity towards the same 3,5-DTBC substrate at room temperature
in oxygen-saturated ethanol medium.
Experimental
Materials
High purity 1,10-phenanthroline (phen; Aldrich, USA), ferric chloride
(Merck, India), 3,5-DTBC (Sigma Aldrich Corporation, St Louis, MO,
Many [FeN6]2+ systems are based on the cationic low-spin [Fe
(2,2′-bipyridine)3]2+ or [Fe(2,2′:2′,6′-terpyridine)2]2+ ions, where the
critical field strength to reach the crossover condition is fulfilled
by introducing sterically hindering groups adjacent to the donor
atoms or by replacing the pyridine rings by five-membered
heterocycles.[18] Examples are the spin crossover systems tris(6-
methyl-2,2′-bipyridine)iron(II) and bis(2,4-bis(pyridin-2-yl)thiazole)
*
Correspondence to: Bhaskar Biswas, Department of Chemistry, Raghunathpur
College, Purulia 723133, India. E-mail:icbbiswas@gmail.com; mr.
bbiswas@rediffmail.com
a Department of Chemistry, Raghunathpur College, Purulia 723133, India
b Department of Chemical Sciences, Indian Institute of Science Education and Re-
search Mohali, Sector 81, S. A. S. Nagar, Manauli PO, Mohali 140306, India
Appl. Organometal. Chem. (2016)
Copyright © 2016 John Wiley & Sons, Ltd.

A. De et al.
USA) and other materials were obtained from commercial sources
and used as purchased. All other chemicals and solvents were of
analytical grade and were used as received without further
purification.
value for each concentration of the substrate and each experiment
was repeated thrice.
Detection of presence of hydrogen peroxide in catalytic oxida-
tion of 3,5-DTBC
Preparation of [Fe(phen)3]Cl2 (1)
To detect the formation of hydrogen peroxide during the
catecholase reaction of 1, we followed a reported method.[24] Reac-
tion mixtures were prepared as in the kinetic experiments. During
the course of the oxidation reaction, the solution was acidified with
H2SO4 to pH 2 to stop further oxidation after a certain time and an
equal volume of water was added. The formed quinone was
extracted three times with dichloromethane. To the aqueous layer
were added 1 ml of a 10% solution of KI and three drops of a 3%
solution of ammonium molybdate. The formation of I3ꢀ could be
monitored spectrophotometrically because of the development
of the characteristic I3ꢀ band (λmax = 353 nm).
A methanolic solution (10 ml) of phen (0.540 g, 3 mmol) was added
dropwise to a solution of FeCl3 (0.163 g, 1 mmol) in the same
solvent (10 ml) and the solution was refluxed for 30 min. The red
coloured solution was kept in air for slow evaporation to obtain
crystalline product.
Yield: 0.127 g (77.9% based on metal salt). Anal. Calcd for
C36H24N6Cl2Fe (1) (%): C, 64.79; H, 3.62; N, 12.59. Found (%): C,
64.71; H, 3.68; N, 12.64. IR (KBr, cmꢀ1): 1618, 1602 (νC¼N).
UV–visible (λmax, nm): 227, 266, 294, 323,512. EI-MS (m/z): 597.34
[1 + H+]+.
Physical measurements
Catechol dioxygenase activity of complex 1
Elemental analyses (carbon, hydrogen and nitrogen) were per-
formed with a PerkinElmer 2400 CHNS/O elemental analyser. Fou-
To investigate the catechol dioxygenase activity of the complex, a
10ꢀ3 M solution of 1 in ethanol solvent was treated with a
10ꢀ3 M solution of DTBC in oxygen-saturated ethanol medium at
room temperature. Absorbance versus wavelength (wavelength
scans) of the solution was recorded at regular time intervals for
6 h in the wavelength range 200–900 nm. It may be noted here that
a blank experiment without catalyst did not show the formation of
any cleavage products up to 12 h in ethanol. The solvent was equil-
ibrated at the atmospheric pressure of O2 at 25 °C using a known
procedure with modifications.[25,26] Investigation of dioxygen reac-
tivity of the in situ generated Fe(III)–catecholate adduct was carried
out in oxygen-saturated ethanol medium at 25 °C. Kinetic
analyses[27,28] of the catechol cleavage reactions were carried out
by time-dependent measurement of the disappearance of the
lower energy DBC2ꢀ-to-iron(III) ligand-to-metal charge transfer
(LMCT) band at ambient temperature (25 °C) by exposure to molec-
ular oxygen.
rier transform infrared (FT-IR) spectra (KBr discs, 300–4000 cmꢀ1
)
were recorded using a Shimadzu FTIR-8400S spectrophotometer.
Ground-state absorption measurements were made with a JASCO
model V-730 UV–visible spectrophotometer. 1H NMR spectra were
recorded with a Bruker DPX 500 MHz spectrometer. Electron para-
magnetic resonance (EPR) spectra were recorded with a Bruker
EMX-X band spectrometer. Electrospray ionization (EI) mass spectra
were recorded using a Q-tof-micro quadruple mass spectrometer.
X-ray diffraction study
Single-crystal X-ray diffraction data of the iron(II) complex were col-
lected using a Rigaku XtaLABmini diffractometer equipped with a
Mercury CCD detector. The data were collected with graphite
monochromated Mo Kα radiation (λ = 0.71073 Å) at 100 K using ω
scans. The data were reduced using CrystalClear suite 2.0[20] and
space group determination was done using Olex2. The structure
was resolved by direct method and refined by full-matrix least-
squares procedures using the SHELXL-2014/7[21] software package
through the Olex2 suite.[22] The crystallographic bond distances
and bond angles were calculated using PARST.[23]
Determination of 3,5-DTBC cleavage products
An amount of 0.2548 g (0.04 mmol) of the iron(II) complex 1 was
reacted with 0.088 g (0.04 mmol) of 3,5-DTBC in oxygen-saturated
ethanol (100 ml) at ambient condition and then allowed to stir for
12 h. The red solution slowly turned green. The residue was then
treated with 15 ml of 3 M HCl and the catechol cleavage products
were extracted with diethyl ether (3 × 10 ml) and dried over sodium
sulfate. The catechol cleavage products were analysed using
electrospray ionization (ESI)-MS and were quantified using 1H
Catecholase activity of complex 1
In order to examine the catecholase activity of the complex, a
10ꢀ4 M solution of 1 in ethanol solvent was treated with 100 equiv.
of 3,5-DTBC under aerobic conditions in the presence of
triethylamine at room temperature. Absorbance versus wavelength
(wavelength scans) of these solutions was recorded at a regular
time interval of 5 min in the wavelength range 300–1000 nm. It
may be noted here that a blank experiment without catalyst did
not show formation of the quinone up to 6 h in ethanol. Kinetic
experiments were performed spectrophotometrically with complex
1 and the substrate 3,5-DTBC in ethanol at 25 °C. An amount of
0.04 ml of the complex solution, with a constant concentration of
1 × 10ꢀ4 M, was added to 2 ml of 3,5-DTBC of a particular concen-
tration (varying from 1 × 10ꢀ3 to 1 × 10ꢀ2 M) to achieve an ultimate
concentration of the complex of 1 × 10ꢀ4 M. The conversion of 3,5-
DTBC to 3,5-DTBQ (quinone band maxima) was monitored with
time at a wavelength of 400 nm (time scan) in ethanol medium.
The initial rate method was applied to determine the rate constant
1
NMR spectroscopy. H NMR data for 3,5-DTBC cleavage products
(500 MHz, CDCl3, δ, ppm): 3,5-di-tert-butyl-5-(carboxymethyl)-2-
furanone: 6.89 (s, 1H); 3,5-di-tert-butyl-2-pyrone: 6.05 (m, 2H);
4,6-di-tert-butyl-2-pyrone: 7.23 (d,1H), 7.25 (d, 1H); 3,5-di-tert-
butylbenzoquinone: 6.18 (d, 1H), 6.76 (d, 1H).
Results and discussion
Synthesis of Fe(II) complex 1 and spectroscopic
characterization
The mononuclear iron(II)–phenanthroline complex was synthesized
and isolated in crystalline phase from FeCl3 with mixing of phen in
an aqueous methanol medium under refluxing condition. The
wileyonlinelibrary.com/journal/aoc
Copyright © 2016 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. (2016)

Catalytic Promiscuity of a Fe(II)-Phenanthroline Complex
geometry of the metal complex (1) was determined using single-
crystal X-ray diffraction along with routine spectroscopic and analyt-
ical techniques.
Table 2. Selected bond distances (Å) and angles (°) for 1
Bond distances
The FT-IR spectrum of 1 (Fig. S1) contains characteristic strong
bands for the 1,10-phen ligands, which shows characteristic bands
N1–Fe1
1.968(4)
1.967(4)
1.988(4)
N4–Fe1
N5–Fe1
N6–Fe1
1.981(3)
1.968(3)
1.971(3)
N2–Fe1
for the α-diimine ligands at ca 1600 cm .
ꢀ1 [29] In the higher-energy
N3–Fe1
region of the optical spectrum, 1 shows characteristic bands at
266, 294 and 323 nm which are assignable to π–π* transition of
the C¼N chromophore in phen while a low-intensity broad band
centred at 512 nm is observed in the visible region (Fig. S2), sug-
gesting octahedral geometry around the iron centre.[30] The struc-
tural integrity in solution state for the iron(II) complex was
determined using ESI-MS analysis. The mass spectrum of 1 in etha-
nol medium exhibits a molecular ion peak at m/z 597.34 as [[Fe
(phen)3]2+ ꢀ H+]+ (Fig. S3). The stability of the primary zone of coor-
dination in the form of tris(phenanthroline)iron complex is also
evident from the ESI-MS spectrum.
Bond angles
N1–Fe1–N2
N1–Fe1–N3
N1–Fe1–N4
N1–Fe1–N5
N1–Fe1–N6
N3–Fe1–N4
N3–Fe1–N5
N3–Fe1–N6
82.92(14)
91.71(13)
172.28(13)
94.48(13)
94.25(14)
82.62(13)
171.67(14)
91.38(13)
N2–Fe1–N3
N2–Fe1–N4
N2–Fe1–N5
N2–Fe1–N6
N4–Fe1–N5
N4–Fe1–N6
N5–Fe1–N6
91.73(13)
91.98(13)
94.55(13)
175.85(13)
91.70(13)
91.14(14)
82.62(13)
to form a distorted octahedral environment. The Fe1–N3 bond
length, 1.988(6) Å, is slightly longer than that of the other Fe–N
bonds; the average Fe–N bond length is 1.973 Å, in agreement with
previously reported results for analogous compounds.[31] The mean
value of the N–Fe–N bite angle in the bidentate ligands is 82.77° and
the average trans angle for opposite N atoms in the coordination
polyhedra is 175.85°, which is consistent with those found in similar
complexes.[31] The bond lengths of all C = C & C = N in phen fall
within the range of literature values.[32]
EPR spectroscopy experiments on 1 in ethanol medium at room
temperature remain silent (Fig. S4) and indicate the low-spin con-
formation of complex 1. The 1H NMR spectrum of the iron complex
1
in DMSO-d6 testifies to its diamagnetic nature. The H NMR spec-
trum of the complex (Fig. S5) contains characteristics peaks for aro-
matic protons in the region from ca 7 to 6 ppm for the phen ligands
which further confirms its solution stability.
Description of crystal structure
The molecular structure of iron(II) complex 1 was determined using
the single-crystal X-ray diffraction technique. Crystallographic data
and details of data collection and refinement for 1 are presented in
Table 1. Important bond lengths and angles are summarized in
Table 2. Single-crystal analysis of complex 1 reveals that the com-
pound crystallizes in the monoclinic system with C2/m space group.
The ORTEP diagram of the complex cation of 1 is shown in Fig. 1.
Each Fe(II) ion is coordinated by six N atoms of three phen ligands
Enzyme kinetics study including spectrophotometric
investigation
In order to investigate the efficiency of catecholase activity of com-
plex 1, 3,5-DTBC with two bulky t-butyl substituents on the ring and
low quinone–catechol reduction potential has been chosen as sub-
strate. These properties of the substrate help it to undergo easy ox-
idation (Scheme 1) to the corresponding o-quinone, 3,5-DTBQ,
which is highly stable and shows a maximum absorption in the
wavelength range 380–420 nm.[24,33–35]
Table 1. Crystallographic refinement parameters of [Fe(phen)3]Cl2 (1)
Crystal parameter
1
Empirical formula
Formula weight
Temperature
C36H24N6Cl2Fe
637.41
100.0(2) K
0.71073 Å
Monoclinic
C2/m
Wavelength
Crystal system
Space group
Unit cell dimensions
a = 23.694(11) Å; α = 90.0°
b = 20.786(16) Å; β = 106.533(16)°
c = 15.304(6) Å; γ = 90.0°
Volume
7226(5) Å3
Z
8
Density (calculated)
Absorption coefficient
F(000)
1.162 Mg mꢀ3
0.522 mmꢀ1
2600
Reflections collected
Independent reflections
R(int)
34 847
8496
0.066
Completeness to θ
Goodness-of-fit on F2
R indices (all data)
Largest diff. Peak and hole
99.4%
1.085
R1 = 0.0813, wR2 = 0.2637
1.68 and ꢀ0.56 e Åꢀ3
Figure 1. ORTEP diagram of the crystal structure of 1.
Appl. Organometal. Chem. (2016)
Copyright © 2016 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/aoc

A. De et al.
Scheme 1. Catalytic oxidation of 3,5-DTBC to 3,5-DTBQ in ethanol medium.
The catecholase activity of iron(II) complex 1 was studied using
3,5-DTBC as a convenient model substrate, in air-saturated ethanol
solvent at room temperature. For this purpose, a 1 × 10ꢀ4 M solution
of this complex was treated with 1 × 10ꢀ2 M (100 equiv.) 3,5-DTBC
and the course of the reaction was followed by recording the UV–
visible spectra of the mixture at intervals of 5 min for 2 h. At 25 °C
under aerobic conditions, complex 1 shows inactive behaviour
towards the model substrate, but instantly exhibits catechol oxida-
tion activity in the presence of triethylamine. Spectral bands at
227, 266, 294, 323 and 512 nm appear in the electronic spectrum
of complex 1 in ethanol, whereas 3,5-DTBC shows a single band at
284 nm. After the addition of triethylamine, there is a gradual
decrease in intensity of the band at 284 nm due to the catechol[36]
and a new band maximum is formed at 400 nm (Fig. 2) along with
the generation of an isobestic point at ca 610 nm (Fig. 3). The exper-
iment unequivocally proves catalytic oxidation of 3,5-DTBC to 3,5-
DTBQ by the mononuclear Fe(II)–phen complex. The formed qui-
none derivative 3,5-DTBQ was purified by column chromatography
using hexane–ethyl acetate eluent mixture. The product was iso-
lated by slow evaporation of the eluent and was identified using
H1 NMR spectroscopy. 1H NMR (CDCl3, 400 MHz, δH, ppm): 1.16 (s,
9H), 1.20 (s, 9H), 6.15 (d, J = 2.4 Hz, 1H), 6.86 (d, J = 2.4 Hz, 1H).
The kinetics of the oxidation of 3,5-DTBC were determined by the
method of initial rates and involved monitoring the growth of the
quinone band at 400 nm as a function of time.[37] The complex
shows saturation kinetics where a mechanism based on the
Michaelis–Menten model seemed to be appropriate. The values of
the Michaelis binding constant (Km), maximum velocity (Vmax) and
rate constant for dissociation of substrate (i.e. turnover number,
Figure 3. Increase of Fe(II)–semiquinone band with decrease of Fe(III)–
catecholate band in the expansion of the wavelength range showing the
presence of an isobestic point during the investigation of catecholase
activity.
the Fe(II) complex is 3.55 × 103 hꢀ1 in ethanol. The kinetic parame-
ters of 1 are also presented in Table 3.
Mechanistic pathway for catecholase activity
Complex 1 in ethanol medium is a d6 low-spin system and exhibits
chemical inertness in ethanolic solution. But, upon addition of 3,5-
DTBC to 1 in the presence of an organic base, triethylamine, the
effective in situ generation of the Fe(III)–catecholate adduct is
observed which is also evident from EPR spectral analysis of the
reaction mixture at room temperature. Though the EPR spectrum
of the iron(II) complex in ethanol medium remains silent (Fig. S4)
under normal conditions, upon addition of 3,5-DTBC to 1 in the
presence of triethylamine, it displays high-spin (S = 5/2) rhombic
ferric signals[38,39] at g = 7.9, 4.3, 2.1 (Fig. S4). Triethylamine, being
an organic base, helps to abstract the protons from phenolic-OH
groups and enhances the nucleophilicity on oxygen atoms. As a
result, the substrate now acts as a better chelating ligand and facil-
itates the formation of iron(III)–catecholate adduct. To establish the
pathway for the catalytic reaction, we initially detected the pres-
ence of hydrogen peroxide following a reported procedure[24]
and observed the band at 353 nm which is due to I3ꢀ (see Experi-
mental section; Fig. S7). To obtain a mechanistic inference of the
catecholase activity of 1 and to confirm possible complex–
substrate intermediates, we recorded the ESI-MS spectrum for a
1:100 mixture of the complex and 3,5-DTBC within 10 min of mixing
in an ethanol solvent. ESI-MS analysis (Fig. S8) of the reaction
kcat) were calculated for the complex 1 from graphs of 1/V versus
1/[S] (Fig. S6), known as Lineweaver–Burk graphs, using the equa-
tion 1/V = {Km/Vmax}{1/[S]} + 1/Vmax. The turnover number (kcat) for
Table 3. Kinetic parameters for the oxidation of 3,5-DTBC catalysed by
1 in ethanol
Molecular formula
V
max (M sꢀ1
)
Km (M)
kcat (hꢀ1
)
Ref.
C
C
36H24N6Cl2Fea
42H44N6O10Fe2
9.87 × 10ꢀ5 1.06 × 10ꢀ2 3.55 × 103 This work
2.08 × 10ꢀ5 4.38 × 10ꢀ4 7.51 × 102
[40]
8.91 × 10ꢀ5 3.15 × 10ꢀ3 3.21 × 103
[41]
C75H42O71Fe11
C35H49Cl3N4O11Fe2
—
—
7.30 × 10ꢀ3 3.43 × 104 [42
7.10 × 10ꢀ2 4.16 × 104
[43]
C35H36N4O3Fe2
aStandard error for Vmax (M sꢀ1) = 1.24 × 10ꢀ5; standard error for Km
(M) = 3.21 × 10ꢀ3
Figure 2. Increase of quinone band at 400 nm after addition of 100 equiv.
of 3,5-DTBC to a solution containing 1 (10ꢀ4 M) in ethanol at 25 °C. The
spectra were recorded every 5 min.
.
wileyonlinelibrary.com/journal/aoc
Copyright © 2016 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. (2016)

Catalytic Promiscuity of a Fe(II)-Phenanthroline Complex
mixture reveals that enzyme–substrate binding occurs in solution
and the presence of an isobestic point at ca 610 nm is further evi-
dent. The mass spectrum of the reaction mixture supports the exis-
tence of mononuclear Fe(III)–catecholate adduct, {[Fe(phen)2
(DTBC)]H+} at m/z 637.34, {[Fe(O2)(phen)2(DTBC)]H+} at m/z 670.25
and [Na(DTBQ)]+ at m/z 243.10 in ethanol solution. During the
course of catechol oxidation, an intense catecholate-to-iron(III)
LMCT band at ca 718 nm[39] with gradual decrease in intensity
(Fig. 3) and a Fe(II)–semiquinone band[38] with gradual increase in
intensity at ca 548 nm (Fig. 3) are also observed for 1. This spectro-
photometric investigation indicates that the Fe(III)–catecholate
species and Fe(II)–semiquinone species form a valence tautomer-
ism, and the reactive intermediate species exist in an equilibrium
which is also confirmed by the presence of an isobestic point at
ca 610 nm. Benzoquinone as major product is a result of this course
of catalysis. Moreover, in the catalytic reaction, when performed in
an inert atmosphere, no 3,5-DTBQ formation is observed. However,
upon exposure of the reaction mixture to dioxygen atmosphere,
immediately 3,5-DTBQ formation is noticed. This observation in-
deed indicates that dioxygen is one of the active participants in
the catalytic cycle and it converts the semibenzoquinone radical
to the quinone with hydrogen peroxide. The catalytic cycle
favouring the catecholase activity is shown in Scheme 2.
Figure 4. Absorption spectral changes during the reaction of the in situ
generated adduct [Fe(phen)2(DBC)]+ with O2 (the spectra were recorded
every 12 min).
by monitoring the decay of the low-energy DTBC2ꢀ-to-Fe(III) LMCT
band (Fig. 4). The presence of an isobestic point during the investi-
gation of catecholase activity further supports the effective forma-
tion of iron–catecholate intermediate which also remains one of
the fundamental aspects for the conversion of substrate to product
and catalytic efficacy of the metal complex. The red solution of 1 re-
acts with dioxygen in bio-friendly ethanol medium at ambient con-
ditions over a period of 6 h and two new visible bands with
maximum absorption at 575 and 721 nm (Fig. 4) are observed for
the catecholate adduct of complex 1. The lower energy visible
bands decreasing in absorbance are attributed to 3,5-DTBC2ꢀ-to-
iron(III) LMCT transitions involving two different catecholate ligand
orbitals.[5–8,28,38,39,44] As reported in many cases, the energy of the
LMCT transitions strongly depends on the nature of the ligands,
and it reflects the Lewis acidity of the iron centre in the
complex.[5,6,45] The disappearance of the lower energy
catecholate-to-iron(III) LMCT band (Fig. 4) on oxygenation exhibits
pseudo first-order kinetics, as judged from the linearity of a plot
of [1 + log(absorbance)] versus time[39,44] (Fig. 5), and the value of
To investigate the efficiency of the catalytic activity we draw
a comparison between our complex 1 and some recently re-
ported iron complexes which show efficient catecholase activity
(Table 3).[40–43] This comparison also indicates that complex 1 acts
as a better and effective catalyst towards catecholase activity than
the reported ones.[40–43]
Catechol dioxygenase activity of complex 1
The oxygenation reactions with the iron(II) complex were carried
out using 3,5-DTBC as the model substrate in bio-friendly ethanol.
The advantages of using the latter as substrate are the relatively
high stability of the main cleavage product and the fast reaction
of the catecholate complex with dioxygen at room temperature.
The DTBC2ꢀ adducts of the iron(II) complex were generated in situ
in ethanol solution, and their reactivity towards O2 was investigated
k
obs is obtained from the slope of the plot. The pseudo first-order
rate constant is determined as kobs = 9.89 × 10ꢀ4 minꢀ1
.
Further, the oxidized products (Scheme 3) from catechol-bound
Fe(III) species were identified and quantified using 1H NMR
Scheme 2. Mechanistic aspects in favour of catecholase activity by iron
complex.
Figure 5. Plot of [1 + log(absorbance)] versus time for the reaction of [Fe
(phen)2(DTBC)]+ with O2 at 25 °C in ethanol solution.
Appl. Organometal. Chem. (2016)
Copyright © 2016 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/aoc

A. De et al.
point at 610 nm from spectrophotometric data supports the
presence of Fe3+–3,5-DTBC adduct favouring enzyme–substrate
binding and the occurrence of a valence tautomerism between
Fe(II)–semiquinone and Fe(III)–catecholate species. Upon stoichio-
metric addition of 3,5-DTBC pretreated with two equivalents of
triethylamine to the iron complex, two catecholate-to-iron(III)
LMCT bands (575 and 721 nm) are observed and the in situ
generated catecholate intermediate reacts with dioxygen
(kobs = 9.89 × 10ꢀ4 minꢀ1) in ethanol medium to afford exclusively
intradiol cleavage products along with a small amount of benzoqui-
none and a small amount of extradiol cleavage products which pro-
vide substantial evidence for the substrate activation mechanism.
Complex 1 is a new addition to the class of oxidase and
dioxygenase enzymes with good catalytic activity and functional
biomimicking efficacy.
Scheme 3. Oxygenation products of DTBC for iron complex in ethanol.
spectroscopy (Fig. S9) and ESI-MS (Fig. S10). The distribution of
catechol-derived products is found to be mainly 3,5-di-tert-butyl-
5-(carboxymethyl)-2-furanone as intradiol cleavage product in
major amount and 3,5-di-tert-butylbenzoquinone as minor product,
while 4,6-di-tert-butyl-2-pyrone and 3,5-di-tert-butyl-2-pyrone, as
extradiol cleavage products, are found in trace quantities. In the
reaction of (1 + DTBC) with O2 in bio-friendly ethanol, 73% of
intradiol cleavage product is obtained along with the formation of
small amounts of extradiol cleavage products (5.3% + 2.4%) as side
products. The amount of minor oxidation product, 3,5-di-tert-
butylbenzoquinone, is also found to be small (11.4%). The
amount of organic product from catechol cleavage accounts for
ca 93% of 3,5-DTBC. The remaining 7% is accounted for
unreacted substrate.
Acknowledgements
The work was supported financially by the Science & Engineering
Research Board (SERB), New Delhi, India under Fast Track Scheme
for Young Scientist (no. SB/FT/CS-088/2013 dtd.21/05/2014). BB is
1
grateful to IACS, Kolkata for providing EPR analysis, H NMR and
The formation of intradiol cleavage products for the catecholate
adduct of the iron(II) complex is expected of the six-coordinate
geometry, which favours a substrate-activation[46,47] rather than
dioxygen-activation[48–50] pathway as the latter requires a vacant
coordination site on the catecholate adduct for oxygen coordina-
tion followed by activation. The observation of smaller amounts
of extradiol products for this iron(II) complex may be explained by
invoking the partial displacement of the phen ligand from the
coordination sphere containing more Lewis basic ligand donors
(ethanol), which facilitates dioxygen attack on iron(III) for extradiol
cleavage to occur.
From ESI-MS analysis of the reaction mixture it is revealed that, at
the primary stage, 3,5-DTBC behaves as a bidentate chelator
towards mononuclear Fe(II) complex and turns into an octahedral
Fe(III)–catecholate adduct, which exhibits no preference for molec-
ular oxygen activation. But the existence of iron(II)–semiquinone
species in solution facilitates a substrate activation mechanism
and provides one electron from Fe(II) to the anti-bonding orbital
of dioxygen generating oxo-species in solution. Further, the acyl
group migration produces the intradiol cleavage products and
hydrogen peroxide. Probably, the intradiol catechol cleavage reac-
tion proceeds by an iron(III) peroxo intermediate that undergoes
1,2-Criegee rearrangement to yield the intradiol catechol cleaved
products analogous to the native enzyme.
mass spectrometry. HRY thanks UGC for a fellowship and ARC
thanks IISER Mohali for the use of the departmental X-ray facility.
References
[1] R. H. Prince, in Comprehensive Coordination Chemistry, Vol. 5 (Eds: G.
Wilkinson, R. Gillard, J. A. McCleverty), Pergamon, Oxford, 1987, p. 925.
[2] B. Biswas, M. Mitra, J. Adhikary, G. R. Krishna, P. P. Bag, C. M. Reddy,
N. Aliaga-Alcalde, T. Chattopadhyay, D. Das, R. Ghosh, Polyhedron
2013, 53, 264.
[3] B. Biswas, A. Al-Hunaiti, M. T. Räisänen, S. Ansalone, M. Leskelä, T. Repo,
Y.-T. Chen, H.-L. Tsai, A. D. Naik, A. P. Railliet, Y. Garcia, R. Ghosh, N. Kole,
Eur. J. Inorg. Chem 2012, 4479.
[4] A. L. Feig, S. J. Lippard, Chem. Rev. 1994, 94, 759.
[5] E. L. Hegg, L. Que, Jr., Eur. J. Biochem. 1997, 250, 625.
[6] J. D. Lipscomb, A. M. Orville, in Metal Ions in Biological Systems, Vol. 28
(Eds: H. Sigel, A. Sigel), Marcel Dekker, New York, 1992, p. 243.
[7] L. Que, Jr., R. Y. N. Ho, Chem. Rev. 1996, 96, 2607.
[8] L. Que, Jr., J. D. Lipscomb, E. Münck, J. M. Wood, Biochim. Biophys. Acta
1977, 485, 60.
[9] L. Que, Jr., in Iron Carriers and Iron Proteins (Ed: T. M. Loehr), VCH, New
York, 1989, p. 467.
[10] D. H. Ohlendorf, J. D. Lipscomb, P. C. Weber, Nature 1988, 336, 403.
[11] D. H. Ohlendorf, A. M. Orville, J. D. Lipscomb, J. Mol. Biol. 1994, 244, 586.
[12] M. P. Valley, C. K. Brown, D. L. Burk, M. W. Vetting, D. H. Ohlendorf,
J. D. Lipscomb, Biochemistry 2005, 44, 11024.
[13] R. W. Frazee, A. M. Orville, K. B. Dolbeare, H. Yu, D. H. Ohlendorf,
J. D. Lipscomb, Biochemistry 1998, 37, 2131.
[14] M. W. Vetting, D. A. D’Argenio, L. N. Ornston, D. H. Ohlendorf,
Biochemistry 2000, 39, 7943.
[15] M. W. Vetting, D. H. Ohlendorf, Structure 2000, 8, 429.
[16] J. W. Whittaker, J. D. Lipscomb, J. Biol. Chem. 1984, 259, 4487.
[17] A. M. Orville, J. D. Lipscomb, D. H. Ohlendorf, Biochemistry 1997, 36,
10052.
[18] P. Gütlich, Y. Garcia, H. A. Goodwin, Chem. Soc. Rev. 2000, 29, 419.
[19] B. J. Childs, D. C. Craig, M. L. Scudder, H. A. Goodwin, Inorg. Chim. Acta
1998, 274, 32.
[20] CrystalClear 2.0, Rigaku Corporation, Tokyo, Japan.
[21] G. M. Sheldrick, Acta Crystallogr. A 2008, 64, 112.
[22] O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, H.
Puschmann. J. Appl. Crystallogr 2009, 42, 339.
Conclusions
A mononuclear iron(II)–phrenanthroline complex was synthesized
and characterized using various spectroscopic methods. Single-
crystal X-ray diffraction analysis revealed that 1 crystallizes in a
monoclinic system with C2/m space group. The iron(II) complex is
stable in ethanolic medium, but upon addition of 3,5-DTBC
pretreated with two equivalents of triethylamine, the diamagnetic
iron(II) compound converts into a mononuclear paramagnetic Fe
(III)–catecholate species. The complex acts as a functional model
for a biomimetic catalyst promoting the aerobic oxidation of 3,5-
DTBC through radical pathways with a significant turnover number
(kcat = 3.55 × 103 hꢀ1) and exhibits catechol dioxygenase activity to-
wards the same substrate, i.e. 3,5-DTBC, at room temperature in
oxygen-saturated ethanol medium. The existence of an isobestic
[23] M. Nardelli, Comput. Chem. 1983, 7, 95.
[24] D. Dey, S. Das, H. R. Yadav, A. Ranjani, L. Gyathri, S. Roy, P. S. Guin,
D. Dhanasekaran, A. R. Choudhury, M. A. Akbarsha, B. Biswas,
Polyhedron 2016, 106, 106.
[25] D. T. Sawyer, Oxygen Chemistry, Oxford University Press, New York,
1991.
wileyonlinelibrary.com/journal/aoc
Copyright © 2016 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. (2016)

Catalytic Promiscuity of a Fe(II)-Phenanthroline Complex
[26] P. Mialane, L. Tehertanov, F. Banse, J. Sainton, J. Girerd, Inorg. Chem.
2000, 39, 2440.
[40] M. Mitra, A. K. Maji, B. K. Ghosh, P. Raghavaiah, J. Ribas, R. Ghosh,
Polyhedron 2014, 67, 19.
[27] P. Halder, S. Paria, T. K. Paine, Chem. Eur. J 2012, 18, 11778.
[28] R. Mayilmurugan, K. Visvaganesan, E. Suresh, M. Palaniandavar, Inorg.
Chem. 2009, 48, 8771.
[41] S. K. Mal, M. Mitra, B. Biswas, G. Kaur, P. P. Bag, C. M. Reddy,
A. R. Choudhury, N. A. Alcalde, R. Ghosh, Inorg. Chim. Acta 2015,
425, 61.
[29] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination
Compounds: Part B, Applications in Coordination, Organometallic and
Bioinorganic Chemistry, John Wiley, New York, 1997, p. 116.
[30] J. G. Solé, L. E. Bausá, D. Jaque, An Introduction to the Optical
Spectroscopy of Inorganic Solids, John Wiley, Chichester, 2005.
[31] Q. X. Wang, K. Jiao, W. Sun, F. F. Jian, X. Hu, Eur. J. Inorg. Chem 1838,
2006.
[32] Y. P. Tian, C. Y. Duan, X. X. Xu, X. Z. You, Acta Crystallogr. C 1995, 51,
2309.
[33] D. Dey, A. De, H. R. Yadav, P. S. Guin, A. R. Choudhury, N. Kole, B. Biswas,
ChemistrySelect 2016, 01, 1910.
[42] T. P. Camargo, F. F. Maia, C. Chaves, B. de Souza, A. J. Bortoluzzi,
N. Castilho, T. Bortolotto, H. Terenzi, E. E. Castellano, W. Haase,
Z. Tomkowicz, R. A. Peralta, A. Neves, J. Inorg. Biochem. 2015,
146, 77.
[43] R. J. Souza, Síntese, Caracterização e Avaliação da Promiscuidade
Catalítica de Complexos Binucleares Bioinspirados, Chemistry
Department, Universidade Federal de Santa Catarina, Florianópolis,
2010.
[44] M. Merkel, F. K. Müller, B. Krebs, Inorg. Chim. Acta 2002, 337, 308.
[45] J.-Y. Xu, J. Astner, O. Walter, F. W. Heinemann, S. Schindler, M. Merkel,
B. Krebs, Eur. J. Inorg. Chem 1601, 2006.
[34] S. Pal, B. Chowdhury, M. Patra, M. Maji, B. Biswas, Spectrochim. Acta A
2015, 144, 148.
[46] M. Pascaly, M. Duda, F. Schweppe, F. Zurlinden, K. Muller, B. Krebs,
J. Chem. Soc. Dalton Trans. 2001, 828.
[35] D. Dey, A. De, S. Pal, P. Mitra, A. Ranjani, L. Gayathri, S. Chandraleka,
D. Dhanasekaran, M. A. Akbarsha, N. Kole, B. Biswas, Indian J. Chem. A
2015, 54, 170.
[36] D. Dey, G. Kaur, A. Ranjani, L. Gyathri, P. Chakraborty, J. Adhikary,
J. Pasan, D. Dhanasekaran, A. R. Choudhury, M. A. Akbarsha, N. Kole,
B. Biswas, Eur. J. Inorg. Chem 2014, 3350.
[47] W. O. Koch, H.-J. Kruger, Angew. Chem. Int. Ed. Engl. 1995, 34, 2671.
[48] M. Ito, L. Que, Jr., Angew. Chem. Int. Ed. Engl. 1997, 36, 1342.
[49] T. Funabiki, A. Mizoguchi, T. Sugimoto, S. Tada, M. Tsuji, H. Sakamoto,
S. Yoshida, J. Am. Chem. Soc. 1986, 108, 2921.
[50] A. Die, D. Gatteschi, L. Pardi, Inorg. Chem. 1993, 32, 1389.
[37] D. D. Cox, S. J. Benkovic, L. M. Bloom, F. C. Bradley, M. J. Nelson,
L. Que, Jr., D. E. Wallick, J. Am. Chem. Soc. 1988, 110, 2026.
[38] T. Kurahashi, K. Oda, M. Sugimoto, T. Ogura, H. Fujii, Inorg. Chem. 2006,
45, 7709.
[39] R. Mayilmurugan, M. Sankaralingam, E. Suresh, Palaniandavar, Dalton
Trans 2010, 39, 9610.
Supporting information
Additional supporting information may be found in the online ver-
sion of this article at the publisher’s web-site.
Appl. Organometal. Chem. (2016)
Copyright © 2016 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/aoc