Functional models for enzyme-substrate adducts of catechol dioxygenase enzymes: The Lewis basicity of facially coordinating tridentate phenolate ligands tunes the rate of dioxygenation and product selectivity

Article abstract of DOI:10.1016/j.ica.2011.08.025

A few iron(III) 3,5-di-tert-butylcatecholate (DBC^2-) adducts of the type [Fe(L)(DBC)(CH₃OH)], where L is a tridentate substituted monophenolate ligand such as 2-((N-benzylpyrid-2-ylmethylamino)methyl)phenol (H(L1)), 2-((N-benzylpyrid-2-ylmethylamino)-methyl)-4,6-dimethylphenol (H(L2)), 2-((N-benzylpyrid-2-ylmethylamino)methyl)-4,6-di-tert-butylphenol (H(L3)) and 2-((N-benzylpyrid-2-ylmethylamino)methyl)-4-nitrophenol (H(L4)), have been isolated and characterized by elemental and ESI-MS analysis. The spectral and electrochemical properties and dioxygenase activities of the adducts have been studied in methanol solution. Upon varying the substituents on the phenolate ring from electron-releasing to electron-withdrawing, the redox potential of DBSQ/DBC^2- couple is shifted to a more positive value indicating an increase in covalency of iron(III)-catecholate bonds. All the complexes elicit cleavage of DBC^2- using molecular oxygen to afford both intra- (I) and extradiol (E) cleavage products with the product selectivity (E/I) varying in the range 0.3-1.9. Interestingly, the incorporation of electron-withdrawing substituents facilitates the regioselective extradiol cleavage of catechol while that of electron-releasing substituents facilitate the regioselective intradiol cleavage.

Full text of DOI:10.1016/j.ica.2011.08.025

Inorganica Chimica Acta 378 (2011) 87–94
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Functional models for enzyme–substrate adducts of catechol dioxygenase enzymes:
The Lewis basicity of facially coordinating tridentate phenolate ligands tunes
the rate of dioxygenation and product selectivity
⇑
Kusalendiran Visvaganesan, Somasundaram Ramachitra, Mallayan Palaniandavar
Centre for Bioinorganic Chemistry, School of Chemistry, Bharathidasan University, Tiruchirapalli 620 024, India
a r t i c l e i n f o
a b s t r a c t
A few iron(III) 3,5-di-tert-butylcatecholate (DBC2ꢀ) adducts of the type [Fe(L)(DBC)(CH3OH)], where L is a
tridentate substituted monophenolate ligand such as 2-((N-benzylpyrid-2-ylmethylamino)methyl)phe-
nol (H(L1)), 2-((N-benzylpyrid-2-ylmethylamino)-methyl)-4,6-dimethylphenol (H(L2)), 2-((N-benzylpy-
rid-2-ylmethylamino)methyl)-4,6-di-tert-butylphenol (H(L3)) and 2-((N-benzylpyrid-2-ylmethylamino)
methyl)-4-nitrophenol (H(L4)), have been isolated and characterized by elemental and ESI-MS analysis.
The spectral and electrochemical properties and dioxygenase activities of the adducts have been studied
in methanol solution. Upon varying the substituents on the phenolate ring from electron-releasing to
electron-withdrawing, the redox potential of DBSQ/DBC2ꢀ couple is shifted to a more positive value indi-
cating an increase in covalency of iron(III)–catecholate bonds. All the complexes elicit cleavage of DBC2ꢀ
using molecular oxygen to afford both intra- (I) and extradiol (E) cleavage products with the product
selectivity (E/I) varying in the range 0.3–1.9. Interestingly, the incorporation of electron-withdrawing
substituents facilitates the regioselective extradiol cleavage of catechol while that of electron-releasing
substituents facilitate the regioselective intradiol cleavage.
Article history:
Received 25 March 2011
Received in revised form 22 July 2011
Accepted 11 August 2011
Available online 22 August 2011
Keywords:
Catechol dioxygenase
Synthetic model
Iron(III)–catecholate adduct
Dioxygenation
Regioselectivity
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
either O2 or the catecholate substrate to overcome the kinetic barrier
depending upon their spins.
Ring opening of dihydroxy aromatic compounds is the most sig-
nificant single step in the bacterial assimilation of carbon from aro-
matics [1]. Catechol dioxygenase enzymes in aerobic bacteria
catalyze almost exclusively the ring opening using molecular
oxygen and two atoms of molecular oxygen are inserted. They are
classified into two major groups, namely, the intra- and extradiol-
cleaving dioxygenases on the basis of regiospecificity of ring cleav-
age (Scheme 1) [2–4]. The intradiol dioxygenases contain iron(III)
center ligated by two histidine residues, two tyrosine residues and
a solvent derived molecule and catalyze the cleavage of the catechol
ring between two hydroxylated carbon–carbon double bond [5–10].
On the other hand, the extradiol-cleaving dioxygenases contain iro-
n(II) ligated by 2-His-1-carboxylate facial-triad motif and two water
molecules and catalyze the cleavage adjacent to the hydroxylated
carbon-carbon double bond [11–16]. The reason for different metal
ion (+2 or +3 oxidation state) requirement of the two families of the
enzymes, however, remains a mystery in the enzymatic pathway
[17,18]. Each family of the enzyme is quite specific for yielding the
respective reaction products with a range of substrates and activates
A plenty of iron(III)/iron(II) complexes of tri- and tetradentate li-
gands containing pyridine/imidazole/pyrazole/carboxylate and/or
phenolate moieties have been reported as structural and functional
models for catechol dioxygenase enzymes [19–44]. Most of the
model complexes end up with intradiol cleavage products upon
interacting them with catechol and O2 [19–29]. Though iron(II) cen-
ter is present in the active site of extradiol-cleaving enzymes, small
molecule synthetic analogs containing iron(III) also cleave catechols
into their extradiol cleavage products [17,31–44]. So attention has
been shifted to isolate and study iron(III) complexes as functional
models for extradiol-cleaving catechol dioxygenase enzymes
[17,31–44]. The iron(III) complexes of 1,4,7-triazacyclononane
ir
(TACN) and hydridotris(3,5-diisopropyl-1-pyrazolyl)borate (Tp2
)
[33–35,45] have been shown to convert catechol into 30–97% extra-
diol cleavage products. In contrast, the iron(III) complex of the
meridionally coordinated tridentate ligand 2,20,6,200-terpyridine
(tpy) yields mainly the oxidized quinone and intradiol cleavage
(20%) products [35,39]. Recently, Gebbink and co-workers have iso-
lated [40] iron(II)– and iron(III)–catecholate complexes of a new
family of substituted 3,3-bis(1-alkylimidazol-2-yl)propionate li-
gands to mimic the structure as well as function of extradiol-cleav-
ing catechol dioxygenases but obtained both extra- (E) and intradiol
(I) cleavage products with 1:1 product selectivity (E/I).
⇑
Corresponding author. Tel.: +91 431 2407125; fax: +91 431 2407043.
E-mail addresses: palaniandavarm@gmail.com, palanim51@yahoo.com
(M. Palaniandavar).
0020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2011.08.025

88
K. Visvaganesan et al. / Inorganica Chimica Acta 378 (2011) 87–94
Interestingly, we have found that the electron-releasing substitu-
a. Intradiol-cleaving
Catechol dioxygenase
COOH
COOH
ents such as methyl and tert-butyl groups on the phenolate ring
enhance the yield of intradiol cleavage products while the elec-
tron-withdrawing NO2 group enhances the yield of extradiol cleav-
age products.
a
OH
b
OH
OH
COOH
b. Extradiol-cleaving
Catechol dioxygenase
2. Experimental
CHO
2.1. Materials
Scheme 1. Mode of cleavage of catechol dioxygenases.
Thechemicalsbenzylamine, pyridine-2-carboxaldehyde, sodium
borohydride, 3,4-di-tert-butylcatechol, 2,4-dimethylphenol, 2,4-di-
tert-butylphenol, ferric perchlorate hexahydrate, picolylchloride
hydrochloride and 3,5-di-tert-butylcatechol (Aldrich), 36% formal-
dehyde, piperidine, salicylaldehyde, potassium carbonate, sodium
sulfate, diethylether and dichloromethane (Merck, India) were used
as received. The commercial solvents were distilled and then used
for the preparation of the ligands and complexes. The supporting
electrolyte tetra-N-butylammonium perchlorate was recrystallized
twice from aqueous ethanol.
In our laboratory, we have isolated and studied 1:1 iron(III)
complexes of several tri- [21,27,39,42–44,46] and tetradentate
[30,38] ligands with an aim to mimic the extradiol-cleaving
enzymes. We have observed an increase in yield of the cleavage
product with increase in Lewis acidity of iron(III) center [46]. Very
recently, we have observed the formation of both intra- and extra-
diol cleavage products upon interacting a series of iron(III) com-
plexes of the type [Fe(L)Cl3], where L = N-alkyl substituted
bis(pyrid-2-ylmethyl)amine and (pyrid-2-ylmethyl)ethylenedia-
mine, with H2DBC under O2 atmosphere [42,43]. We found that
the regioselectivity in catechol cleavage is remarkably tuned by
the central as well as terminal N-alkyl groups of the facially coor-
dinating tridentate ligands [42,43]. Very interestingly, we have
achieved the selective formation of extradiol cleavage products
(84–94%) in organized assemblies for the iron(III) complex of the
facially coordinating bis(pyrid-2-ylmethyl)amine ligand [47]. All
these studies clearly show that the presence of a vacant or solvent
coordinated site on the iron(III) center and facial coordination of
tridentate ligands are essential to achieve extradiol cleavage prod-
ucts [34,42–44,48]. Previously, we have isolated and studied
iron(III) complexes of the type [Fe(L)Cl2], where L is tridentate
monophenolate ligands with an aim to mimic structure and func-
tion of protocatechuate-3,4-dioxygenase (3,4-PCD) and catechol-
1,2-dioxygenase (1,2-CTD) enzymes [21,46]. Ogo et al. have shown
that the adduct [Fe(L)(DBC)Cl], where H(L) = 2-(N-benzyl-N-pyrid-
2-ylmethyl)aminophenol afforded, in spite of the facial coordina-
tion of the ligand to iron(III), intradiol cleavage (52%) and oxidized
(45%) products upon reacting it with O2 in DMF solvent [29] and
only intradiol cleavage products (97%), even after removal of the
coordinated chloride ion in the adduct. So, in continuation of our
studies to devise extradiol-cleaving mimics, we have now isolated
DBC2ꢀ adducts of iron(III) complexes of a few 2NO ligands (H(L1)–
H(L4), Scheme 2) containing differently substituted phenolate moi-
eties as models for PCA-bound (PCA = protocatechuic acid) form of
3,4-PCD enzymes. We intend to systematically study the effect of
Lewis acidity of the iron(III) center, as modified by the different
substituents on the phenolate moiety in the primary ligands, on
the regioselectivity of catechol ring cleavage in methanol solvent.
2.2. Physical measurements
Elemental analyses were performed on a Perkin Elmer Series II
CHNS/O analyzer 2400. 1H NMR spectra were recorded on a Bru-
ker 400 MHz NMR spectrometer. ESI-MS analysis was performed
on a Q-TOF micromass (YA-105) spectrometer. Electronic spectral
measurements in methanol solutions of the iron–DBC2ꢀ com-
plexes were made with an Agilent 8453 diode array spectropho-
tometer. Cyclic voltammetry (CV) and differential pulse
voltammetry (DPV) were performed at 25.0 0.2 °C using
a
three-electrode cell configuration. A platinum sphere, a platinum
plate and Ag(s)/AgNO3 were used as working, auxiliary and refer-
ence electrodes respectively. The Ag(s)/Ag+ reference electrode
consists of a Ag wire immersed in a solution of AgNO3 (0.01 M)
and tetra-N-butylammonium perchlorate (0.1 M) in an acetoni-
trile placed in a tube fitted with a vycor plug. The instruments
utilized included an EG & G PAR 273 Potentiostat/Galvanostat
and P-IV pentium computer along with EG & G M270 software
to carry out the experiments and to acquire the data. The temper-
ature of the electrochemical cell was maintained by a cryocircu-
lator (HAAKE D8-G). The E1/2 observed under identical
conditions for Fc/Fc+ couple in an acetonitrile was 0.10 V with
respect to Ag(s)/Ag+ reference electrode. The experimental solu-
tions were deoxygenated by bubbling research grade nitrogen
and an atmosphere of nitrogen was maintained over the solution
during measurement. The redox potential (E1/2) was calculated
from the anodic (Epa) and cathodic peak (Epc) potentials of CV
traces as (Epa + Epc)/2. The redox potentials were also estimated
from the DPV peak potentials Ep using the relation,
Vacant
Site
H
HN
(a)
(b)
His460
OCH3
COOH
N
O
O
N
O
O
Fe
R'
Fe
N
N
O
O
R, R'
N
N
HO
R
NH
His462
[Fe(L)(DBC)(CH3OH)]
H(L1) : R, R' = -H
Tyr408
H(L2) : R, R' = -CH3
H(L3) : R, R' = -C(CH3)3
H(L4) : R = -H, R' = -NO2
Scheme 2. (a) Schematic drawings of tridentate phenolate ligands and their iron(III)–catecholate adducts. (b) Active site structure of PCA-bound form of 3,4-PCD enzyme.

K. Visvaganesan et al. / Inorganica Chimica Acta 378 (2011) 87–94
89
E1=2 ¼ Ep þ
DE=2
36% aqueous formaldehyde (532
l
L, 7.5 mmol) in methanol
(10 mL) was stirred at room temperature for 3 days. The mixture
was cooled in the freezer overnight, filtered and washed thoroughly
with ice cold methanol to give the ligand L1 as colorless powder
which could be further purified by recrystallization from methanol.
The crude product was used for complex preparation without fur-
ther purification. Yield 1.55 g (93%). 1H NMR (200 MHz, CDCl3): d
7.6–8.1 (m, 2H), 6.5–7.3 (m, 9H), 3.82 (s, 2H), 3.71 (s, 2H), 3.65 (s,
2H), 2.2 (s, 3H), 1.9 (s, 3H).
where E1/2 is the equivalent of the average of Epc and Epa in CV
experiments and E is the pulse amplitude.
The products were analyzed by using Hewlett Packard (HP)
6890 Gas Chromatograph (GC) series equipped with a FID detector
D
and a HP-5 capillary column (30 m ꢁ 0.32 mm ꢁ 2.5
lm) with the
following temperature program: initial temperature, 50 °C; heat-
ing rate 5 °C minꢀ1; final temperature 250 °C; injector temperature
150 °C; and FID temperature 280 °C. GC–MS analysis was per-
formed on a Perkin-Elmer Clarus 500 GC–MS instrument using a
PE-5 (HP-5 equivalent) capillary column under conditions that
are identical to that used for GC analysis.
2.3.3. 2-((N-Benzylpyrid-2-ylmethylamino)methyl)-4,6-di-tert-
butylphenol H(L3)
This ligand was synthesised as reported earlier [52–54]. Yield
1.97 g (95%). 1H NMR (200 MHz, CDCl3): d 7.3–7.7 (m, 2H), 6.6–
7.2 (m, 9H), 3.84 (s, 2H), 3.74 (s, 2H), 3.69 (s, 2H), 1.3 (s, 9H), 1.1
(s, 9H).
2.3. Syntheses of ligands
2.3.1. 2-((N-Benzylpyrid-2-ylmethylamino)methyl)phenol H(L1)
This ligand was synthesized in two steps [49]. The first step in
the synthesis was the preparation of N-benzylaminomethylphenol
which was then reacted with 2-chloromethylpyridine in the sec-
ond step to obtain the ligand H(L1).
2.3.4. 2-((N-Benzylpyrid-2-ylmethylamino)methyl)-4-nitrophenol
H(L4)
To a cold tetrahydrofuran solution (15 mL) containing N-ben-
zylaminomethylpyridine (0.79 g, 4.0 mmol) and triethylamine
(557 lL) was added dropwise 2-chloromethyl-4-nitrophenol
(0.75 g, 4.0 mmol) in tetrahydrofuran (10 mL) with rapid stirring.
The mixture was allowed to warm to room temperature, and
then heated to reflux for 4 h. After rotaevaporation, the ligand
2-((N-benzylpyrid-2-ylmethylamino)methyl)-4-nitrophenol as an
yellow oil. The product was used as such for the complex prep-
aration. Yield 1.38 g (98%). 1H NMR (200 MHz, CDCl3): d 8.52 (dd,
1H), 7.90–8.00 (m, 2H), 7.5–7.6 (dt, 1H), 6.6–7.6 (m, 8H), 3.80 (s,
2H), 2.20 (s, 2H), 1.85 (s, 2H).
2.3.1.1. Step 1. Synthesis of N-benzylaminomethylphenol. A solution
of benzylamine (0.54 g, 5.0 mmol) in 4 mL ethanol and 99% salicyl-
aldehyde (0.62 g, 5.0 mmol) in 4 mL ethanol were mixed. Heat was
evolved and the color darkened. The solution was stirred for 12 h
and boiled gently 30 min. Then 7.5 mmol of sodium borohydride
was added at 0 °C. The mixture was stirred overnight and rota-
evaporated. Then it was extracted by CH2Cl2 and dried by Na2SO4.
Yield 0.90 g (85%).
2.4. Synthesis of mononuclear iron(III)–DBC2ꢀ complexes
2.3.1.2. Step 2. Synthesis of ligand H(L1). To
N-benzylaminomethylphenol (0.90 g, 4.2 mmol) and Et3N (585
a
solution of
L,
l
2.4.1. [Fe(L1)(DBC)(CH3OH)] 1
4.2 mmol) in 10 mL of ethylacetate was added 10 mL of an ethanolic
solution of 2-chloromethylpyridine obtained by neutralization of
the monohydrochloride (0.69 g, 4.2 mmol) with a 10% excess of a
2 mL saturated aqueous K2CO3 solution with vigorous stirring. The
mixture was allowed to stir at room temperature for 5 days. After fil-
tration, the solvent was removed under reducedpressure to give yel-
lowish oil. To this was added 20 mL of ethylacetate, the mixture was
filtered and the volume of filtrate was reduced to approximately
5 mL by rotary evaporation. The remaining solution was extracted
with dichloromethane and dried over Na2SO4. Yield 1.26 g (99%).
1H NMR (200 MHz, CDCl3): d 8.52 (dd, 1H), 7.90–8.00 (m, 2H), 6.6–
7.2 (m, 10H), 3.95 (s, 2H), 3.80 (s, 2H), 3.72 (s, 2H).
To a methanolic solution (4 mL) of Fe(ClO4)3ꢂ6H2O (17.0 mg,
0.5 mmol), was added a methanolic solution (4 mL) of ligand L1
(0.5 mmol) pretreated with an equivalent amount of piperidine
under nitrogen atmosphere then 0.5 mmol of H2DBC pretreated
with an equivalent amount of piperidine in methanol (3 mL) was
added and stirred for 30 min. The resulting solution was layered
with hexane and kept under 4 °C, after 2 days dark purple colored
complex was obtained. Yield 0.22 g (73%). Anal. Calc. for
C
35H43FeN2O4: C, 68.74; H, 7.09; N, 4.58. Found: C, 68.64; H,
+
7.19; N, 4.51%. ESI-MS m/z = 580.22 FeC34H39N2O3
.
2.4.2. [Fe(L2)(DBC)(CH3OH)] 2
This complex was isolated as a dark purple colored product in a
manner analogous to that described for isolating [Fe(L1)(DBC)]
using L2 as ligand. Yield 0.21 g (68%). Anal. Calc. for C37H47FeN2O4:
C, 69.48; H, 7.41; N, 4.38. Found: C, 69.42; H, 7.35; N, 4.32%. ESI-MS
m/z = 607.25 [FeC36H43N2O3]+.
2.3.2. 2-((N-Benzylpyrid-2-ylmethylamino)methyl)-4,6-
dimethylphenol H(L2)
This ligand was synthesized in two steps by a modified proce-
dure reported already [50,51]. The first step in the synthesis was
the preparation of N-benzylaminomethylpyridine which was then
reacted with 2,4-dimethylbutylphenol in the second step to obtain
the ligand (L2).
2.4.3. [Fe(L3)(DBC)(CH3OH)] 3
Complex 3 was prepared by using the procedure employed for
[Fe(L1)(DBC)]. Yield 0.26 g (75%). Anal. Calc. for C43H59FeN2O4: C,
71.36; H, 8.22; N, 3.87. Found: C, 71.26; H, 8.30; N, 3.82%. ESI-
MS, m/z = 691.32 [C42H55FeN2O3]+.
2.3.2.1. Step 1. N-Benzylaminomethylpyridine. A solution of benzyl-
amine (0.54 g, 5.0 mmol) in 4 mL ethanol and pyridine-2-carboxy-
aldehyde (0.54 g, 5.0 mmol) in 4 mL ethanol were mixed. Heat was
evolved and the color darkened. The solution was stirred for 12 h
and boiled gently for 30 min. Then 7.5 mmol of sodium borohy-
dride was slowly added with stirring at 0 °C. The mixture was stir-
red overnight and rotaevaporated. Then it was extracted by CH2Cl2
and dried by Na2SO4. Yield 0.95 g (96%).
2.4.4. [Fe(L4)(DBC)(CH3OH)] 4
This complex was prepared by using the procedure adopted for
[Fe(L1)(DBC)]. Yield 0.24 g (76%). Anal. Calc. for C35H42FeN3O6: C,
64.03; H, 6.45; N, 6.40. Found: C, 64.18; H, 6.23; N, 6.45%. ESI-MS
+
m/z = 624.20 FeC34H38N3O5
.
2.5. Reactivity studies
2.3.2.2. Step 2. 2-((N-Benzylpyrid-2-ylmethylamino)methyl)-4,6-
dimethylphenol H(L2). A solution of 2,4-dimethylphenol (0.61 g,
5.0 mmol), N-benzylaminomethylpyridine (0.99 g, 5.0 mmol), and
The following typical procedure was employed to study the
dioxygenases activity of model compounds. solution of
A

90
K. Visvaganesan et al. / Inorganica Chimica Acta 378 (2011) 87–94
iron(III)–DBC2ꢀ adduct (0.05 mmol) was left in contact with oxygen
at room temperature for 3 days. The solvent was then removed
under vacuum at 35 °C and the resultant residue was acidified with
HCl to pH 3. The organic compounds were extracted from the aque-
ous solution with diethylether, dried over Na2SO4, and then rota-
evaporated. The product mixture was then subjected to GC–MS
and GC analysis.
supported by ESI-MS analysis (Fig. 1), which reveals the presence
of [Fe(L)(DBC)]ꢂ+ species for all the adducts, the coordinated solvent
molecule being detached from the coordination sphere in the gas
phase [55]. Also, it is interesting to note that the 2NO ligands are
facially bound to iron(III). The electronic environment around
iron(III) center of the present adducts closely mimics the intra-
diol-cleaving 3,4-PCD enzyme adducts (Scheme 2) and is expected
to confer interesting spectral and chemical properties. The vacant
site on iron(III) center of the adducts is possibly the site for molec-
ular oxygen binding during oxygenation. The differently substi-
tuted phenolic moieties on the ligands are expected to impose
varying steric and electronic effects on the iron(III) center and
hence the reactivity of the adducts with molecular oxygen.
3. Results and discussion
3.1. Synthesis of ligands and iron(III)–DBC2ꢀ adducts
The linear tridentate monophenolate ligands H(L1)–H(L4) were
synthesized according to known procedures [49–54], which involve
reductive amination and Mannich base condensation. The N–H
hydrogen atom of N-benzylaminomethylpyridine can be consid-
ered to have been substituted with differently substituted phenolic
moieties to give H(L1)–H(L4). The ligand H(L1) was synthesized by
reductive amination of salicylaldehyde and benzylamine using so-
dium borohydride as the reducing agent, followed by reaction with
picolylchloride hydrochloride in presence of a base. The ligands
H(L2) and H(L3) were prepared by reductive amination of pyri-
dine-2-carboxaldehyde and benzylamine using sodium borohy-
dride as reducing agent followed by Mannich base condensation
of the product N-benzylaminomethylpyridine with the correspond-
ing phenol and formaldehyde. The ligand H(L4) was synthesized by
reacting 2-chloromethyl-4-nitrophenol with N-benzylaminometh-
ylpyridine in presence of one equiv. of triethylamine. Attempts to
isolate the mononuclear iron(III) complexes of the phenolate li-
gands failed to yield well-defined products. So, in this study we
have isolated the DBC2ꢀ adducts (1–4) of the iron(III) complexes
in good yields by reacting the iron(III) complexes generated by mix-
ing equimolar amounts of Fe(ClO4)3ꢂ6H2O, the ligand and one
equivalent of piperidine and then treating with H2DBC pretreated
with two equivalents of piperidine in methanol under nitrogen
atmosphere (Scheme 2). Based on the elemental analysis all of the
present adducts are formulated as [Fe(L)(DBC)(CH3OH)], which is
3.2. Electronic absorption spectra of iron(III)–DBC2ꢀ adducts
The absorption spectral data of the present iron(III)–catecholate
adducts are collected in Table 1 and a typical electronic absorption
spectrum is displayed in Fig. 2. The electronic spectra of 1–4 in
methanol are dominated by two moderately intense absorption
bands in the regions 400–480 and 600–790 nm, which are charac-
teristic of catecholate-to-iron(III) charge-transfer bands [19–28].
The two LMCT bands are attributed to catecholate-to-iron(III) LMCT
transitions involving two different catecholate frontier orbitals and
d
p⁄ orbital of iron(III) center. The energies of the low-energy LMCT
band follow the trend 3 > 2 > 1 > 4 indicating that the Lewis acidity
of iron(III) center and hence the covalency of iron(III)–catecholate
bond increases along this series [42–44]. Thus, upon incorporating
the electron-releasing tert-butyl groups on the phenolate ring in 1
to obtain 3, the electron density on iron(III) center increases, the ir-
on(III) dp⁄ orbital is destabilized [19,42,56] and hence the energy
gap between the dp⁄ orbital and the catecholate orbitals increases
leading to a large blue-shift (160 nm) of the LMCT band. Similarly,
as expected, on incorporating the methyl groups to obtain 2, a much
lower blue-shift (20 nm) is observed. On the other hand, on incor-
porating the electron-withdrawing group -NO2 on the phenolate
ring in 1 to obtain 4, the Lewis acidity of the iron(III) center and
Fig. 1. Electrospray ionization-mass spectrum of complex 2 in methanol.

K. Visvaganesan et al. / Inorganica Chimica Acta 378 (2011) 87–94
91
Table 1
200
Electronic spectral data (kmax in nm; emax in Mꢀ1 cmꢀ1 in
paranthesis) for iron(III)–DBC2ꢀ adductsa in methanol solution.
125
50
ꢀ1
Complex
kmax nm (
e
M
cmꢀ1
)
max
[Fe(L1)(DBC)(CH3OH)]
462 (2600)
758 (2910)
472 (2210)
740 (2375)
409 (1430)
596 (1290)
476 (1345)
787 (1970)
-25
[Fe(L2)(DBC)(CH3OH)]
[Fe(L3)(DBC)(CH3OH)]
[Fe(L4)(DBC)(CH3OH)]
-100
-175
-250
a
Concentration iron(III)–DBC adducts, 4 ꢁ 10ꢀ4 M.
1000
700
400
100
E (V)
-200
-500
-800
-1100
Fig. 3. Cyclic voltammogram of
2 in methanol solution at 25 °C. Supporting
electrolyte: 0.1 M TBAP. Scan rate: 50 mV sꢀ1
.
1.2
1
1
2
40
30
20
10
0
0.8
0.6
0.4
0.2
4
3
300
400
500
600
700
800
900
Wave length (nm)
Fig. 2. Electronic absorption spectra of iron–DBC adducts 1–4 (2 ꢁ 10ꢀ4 M) in
1000
700
400
100
E (V)
-200
-500
-800
-1100
methanol solution.
Fig. 4. Differential pulse voltammogram of
2 in methanol solution at 25 °C.
Supporting electrolyte: 0.1 M TBAP. Scan rate: 5 mV sꢀ1
.
hence the covalency of iron(III)–catecholate bond increases leading
to the red-shift (47 nm) of the LMCT band. Hence, the position of
the low-energy LMCT band exhibits a remarkable dependence on
the Lewis basicity of the phenolate ligands, as modified by the
phenolate ring substituents and reflects the covalency of the iro-
n(III)–catecholate bonds as modified by the tridentate ligands.
Upon adding N-methylimidazole (N-MeIm) or pyridine in excess,
the low-energy LMCT band of iron(III)–DBC2ꢀ adducts are shifted
to higher energies (4–12 nm) with decrease in absorptivity for
1–4, which is mainly due to the increase in electron density on
the iron(III) center by coordination of N-MeIm [46]. This suggests
that a labile coordination site is available on iron(III) center of the
present iron (III)–DBC2ꢀ adducts, which may be utilized for O2 bind-
ing during oxygenation.
Table 2
Electrochemical dataa for [Fe(L)(DBC)(CH3OH)] in methanol at 25 0.2 °C using a scan
rate of 50 mV/s (CV) and in 5 mV/s (DPV).
Complex
E1/2 (V)
Process
CV
DPV
[Fe(L1)(DBC)(CH3OH)]
[Fe(L2)(DBC)(CH3OH)]
[Fe(L3)(DBC)(CH3OH)]
[Fe(L4)(DBC)(CH3OH)]
–
0.916
DBQ ? DBSQ
DBSQ ? DBC
FeIII ? FeII
DBQ ? DBSQ
DBSQ ? DBC
FeIII ? FeII
DBQ ? DBSQ
DBSQ ? DBC
FeIII ? FeII
DBQ ? DBSQ
DBSQ ? DBC
FeIII ? FeII
ꢀ0.023
ꢀ0.027
ꢀ0.587
0.826
ꢀ0.034
ꢀ0.552
0.815
ꢀ0.044
ꢀ0.532
0.924
–
–
ꢀ0.038
–
–
ꢀ0.040
–
–
3.3. Electrochemical behavior of Iron(III)–DBC2ꢀ adducts
0.043
–
0.040
ꢀ0.605
The electrochemical features of the iron(III)–DBC2ꢀ adducts 1–4
were investigated in methanol solution by employing cyclic (CV)
and differential pulse voltammetry (DPV) on a stationary Pt
electrode. All the four [Fe(L)(DBC)(CH3OH)] adducts exhibit three
electrochemical responses (Figs. 3 and 4), which are assigned to iro-
n(III)/iron(II), DBSQ/DBC2ꢀ and DBQ/DBSQ redox couples. The redox
potentials of Fe(III)/Fe(II) couple fall in the more negative region
ꢀ0.532 to ꢀ0.605 V (Table 2) due to strong coordination of the phe-
nolate ligands and DBC2ꢀ dianion and follow the trend 3 > 2 > 1 > 4,
reflecting an increase in interaction between catecholate anion and
the iron(III) center along this series. Thus, upon incorporating the
electron-releasing tert-butyl groups on the phenolate ring in 1 to ob-
tain 3, the Fe(III)/Fe(II) redox potential becomes more positive illus-
trating the weaker interaction of catecholate anion with iron(III)
center. On the other hand, the incorporation of the electron-with-
drawing –NO2 group in 1 to obtain 4, the Fe(III)/Fe(II) redox potential
a
Potential measured vs. Ag(s)/AgNO3 (0.01 M, 0.10 M TBAP); add 0.544 V to
convert to NHE.
becomes more negative illustrating the stronger interaction of
DBC2ꢀ with iron(III) center. This means that the electron-transfer
[57] from catecholate adducts to dioxygen is thermodynamically
more unfavorable in 4 than in 1–3, due to the higher covalency of ir-
on(III)–DBC2ꢀ bonds. The redox potentials of the DBSQ/DBC2ꢀ cou-
ple (ꢀ0.040 to +0.043 V) for the [Fe(L)(DBC)(CH3OH)] is higher
than that for the free DBSQ/DBC2ꢀ couple (Epc, ꢀ1.274 V) [30],
revealing the significant stabilization of coordinated DBC2ꢀ anion
toward oxidation. They follow the trend 3 < 2 < 1 < 4, revealing that
the stabilization of coordinated DBC2ꢀ toward oxidation increases
along theseries. On incorporatingtert-butylgroups onthe phenolate

92
K. Visvaganesan et al. / Inorganica Chimica Acta 378 (2011) 87–94
Table 3
ring in 1 to obtain 3, the electron density on iron(III) increases lead-
ing to a weaker iron (III)–DBC2ꢀ interaction and so the DBSQ/DBC2ꢀ
redox couple is located in the more negative region. Similarly, on
incorporating two methyl groups on the phenolate ring to obtain
2, the DBSQ/DBC2ꢀ redox couple is slightly shifted to the negative re-
gion. The incorporation of electron-withdrawing –NO2 group shifts
the DBSQ/DBC2ꢀ redox coupleto a more positive value revealingthat
electron-transfer from catecholate adducts to dioxygen is made dif-
ficult and hence less reactive than that in 1. The trend in the redox
potentials of DBQ/DBSQ couple (0.815-0.924 V) is the same as that
observed for DBSQ/DBC2ꢀ couple. It may be noted that all the ob-
served trends in E1/2 values of DBSQ/DBC2ꢀ couple are consistent
with the above spectral results.
Kinetic data for oxidative cleavage of DBC2ꢀ in iron(III)–DBC2ꢀ adducts in methanol
and the cleavage products.
kO Mꢀ1 sꢀ1
Complex
Cleavage products
2
(ꢁ10ꢀ2
)
Extradiol Intradiol Total E/I
ratio
[Fe(L1)(DBC)(CH3OH)] 5.03
[Fe(L2)(DBC)(CH3OH)] 6.26
[Fe(L3)(DBC)(CH3OH)] 7.18
[Fe(L4)(DBC)(CH3OH)] 5.50
35.3 (5,
6)
28.6 (5,
6)
20.3 (5,
6)
55.9 (5,
6)
45.7 (7,
8)
62.6 (7,
8)
70.1 (7,
8)
28.3 (7,
8)
81.0
91.2
90.4
84.2
0.8
0.5
0.3
1.9
3.4. Dioxygenase activity of Iron(III)–DBC2ꢀ adducts
The catecholate adducts 1–4 were reacted with dioxygen over
48 h (t1/2, 1.3–1.8 h) to afford 80–90% of cleavage products and
the remaining 10–20% is detected as H2DBC. The formation of both
intradiol (I, 38–70%) and extradiol (E, 20–46%) cleavage products
(Scheme 3, Table 3) is observed with the product selectivity (E/I)
falling in the range 0.3–1.2. This is interesting because only intra-
diol cleavage products (97%) have been observed previously for
[Fe(L1)(DBC)(DMF)] in DMF solvent [29]. Also, the yield of extradiol
cleavage products follow the trend 3 (20.3) < 2 (28.6) < 1 (35.3) < 4
(55.9%) whereas that of intradiol cleavage products follow the re-
verse trend 3 (70.1) > 2 (62.6) > 1 (45.7) > 4 (38.3%). All these obser-
vations can be illustrated by invoking both substrate [19,20,58,59]
and oxygen activation [60,61] mechanisms proposed for the en-
zymes. It is well-known that substrate activation pathway, which
involves the attack of dioxygen on the activated substrate, that is,
the semiquinonate radical of iron(II)–DBSQ species, leads to the for-
mation of intradiol cleavage products. On the other hand, dioxygen
activation mechanism, which involves the attack of dioxygen on the
vacant or solvent coordinated site on iron(II) bound to catecholate,
leads to the formation extradiol cleavage products. Also, in both the
cases the same alkylperoxoiron(III) intermediate (Scheme 4) in
which the iron(III) center is bridged with the semiquinonate carbon
through a peroxy group. An acyl migration in this alkylperoxo-
iron(III) intermediate gives intradiol cleavage products while alke-
nyl migration yields extradiol cleavage products. Very recently, the
key peroxide intermediate and the O2 adduct precursor and the
product complex successor to the substrate-alkylperoxo-Fe2+ inter-
mediate are found [57] to be simultaneously present in the X-ray
crystal structures of different subunits of the homoprotocatechuate
2,3-dioxygenase enzyme reacted with 4-nitrocatechol as the sub-
strate. Upon incorporating the electron-releasing tert-butyl group
on the phenolate ring in 1 to obtain 3, the electron density on iro-
n(III) center increases illustrating that O2 attack on the activated
substrate rather than on the iron center is facilitated leading to
the formation of intradiol cleavage products more for 3 (70.1%) than
for 1 (45.7%). Similarly, on incorporating the electron-releasing
methyl group in 1 to obtain 2, only a lower increase in intradiol
cleavage (62.6%) products is observed, as expected. On the other
hand, on incorporating the electron-withdrawing -NO2 group on
the phenolate ring in 1 to obtain 4, the electron density on iron(III)
center decreases leading to facilitate O2 attack on the iron center
and hence the yield of extradiol cleavage product is higher for 4
(55.9%) than for 1–3. Thus, for the present catecholate adducts,
the Lewis basicity of the primary ligand, as modified by the substit-
uents on the phenolate ring, dictates the choice of substrate or oxy-
gen activation mechanism, and hence the regioselectivity of
catechol cleavage.
The iron(III)–catecholate adducts 1–4 were reacted with molec-
ular oxygen in methanol solution. The disappearance of the low-
energy catecholate-to-iron(III) LMCT band (Fig. 5) on oxygenation
exhibits pseudo-first order kinetics as judged from the linearity
of the [1 + log(absorbance)] versus time plot and the value of kobs
was calculated from the slope of the plot (Fig. 6). The second order
rate constant was then derived (Table 3) by using the equation,
kO ¼ kobs=½O2ꢃ
2
The products of cleavage of H2DBC in methanol solvent (Table
3) are identified (5–8, Scheme 3) by GC–MS and 1H NMR tech-
niques and quantified by GC analysis.
0.9
0.7
0.5
0.3
350
450
550
650
750
850
wavelength (nm)
Fig. 5. Progress of the reaction of adduct [Fe(L2)(DBC)(CH3OH)] with O2 in
methanol solution. The disappearance of the DBC2ꢀ-to-iron(III) charge-transfer
band is monitored.
1
0.9
0.8
0.7
0.6
0
10
20
30
40
50
60
70
80
Time (minutes)
It may be noted that the redox potential of DBC2ꢀ/DBSQ couple
is more positive for 4 than for others illustrating that the removal
of electron from or the oxidation of the bound catecholate is diffi-
cult; in other words, substrate activation pathway is not facilitated
Fig. 6. Plot of [1 + log(absorbance)] versus time for the reaction of adduct
[Fe(L2)(DBC)] with O2 at 25 °C in methanol solution. The Concentration of adduct,
4 ꢁ 10ꢀ4 M.

K. Visvaganesan et al. / Inorganica Chimica Acta 378 (2011) 87–94
93
O
Extradiol
Intradiol
O
+
O
O
5
6
O2
[Fe(L)(DBC)]
O
O
O
O
O
+
N
COOCH3
8
7
Scheme 3. Products of catechol cleavage of H2DBC of [Fe(L)(DBC)] adducts using molecular oxygen: 4,6-di-tert-butyl-2H-pyran-2-one (5), 3,5-di-tert-butyl-2H-pyran-2-one
(6), methyl-2-(2,4-di-tert-butyl-5-oxo-tetrahydrofuran-2-yl)acetate (7), 3,5-di-tert-butyl-5-(2-oxo-2-(piperidin-1-yl)ethyl)-dihydrofuran-2(3H)-one (8).
N
O
O
N
O
O
III
Fe
II
Fe
O
O
N
N
O
O2
DBC2-
Substrate
activation
O
OH
Fe
O
N
O
O
N
O
III
S
Sol
Fe
O
Fe
Acyl
Sol
Sol
N
N
O
O
migration
Intradiol
product
N
O
N
Alkenyl
migration
OH
Fe
O
N
Extradiol
product
O
O
O
N
Scheme 4. Proposed mechanisms for both extra- and intradiol cleavage products.
and oxygen activation is encouraged leading to extradiol cleavage
pathway. Also, the higher negative redox potential of DBC2ꢀ/DBSQ
couple for 4 encourages oxygen attack on the substrate leading to
facilitate intradiol rather than extradiol cleavage product. Also, it is
to be noted that the adduct [Fe(L1)(DBC)(DMF)] [29] and the ad-
ducts of the type [Fe(L)(DBC)Cl], where L is a tridentate 3N ligand,
afford only intradiol cleavage products in DMF solvent [27,42,43].
This type of selective formation of intradiol cleavage products is
because the substitution of strongly coordinated DMF solvent by
molecular oxygen is made difficult and hence the substrate activa-
tion mechanism is preferred in DMF solvent.
is observed, which is mainly because the intradiol cleavage pathway
is facilitated more. Similarly, on incorporating the electron-releas-
ing methyl groups in 1 to obtain 2, only a slight increase in reaction
rate is observed. On the other hand, on incorporating the electron-
withdrawing group –NO2 in 1 to obtain 4, though most of the adduct
species follow the extradiol cleavage (dioxygen activation) path
way, the rate of the reaction is increased mainly due to the higher Le-
wis acidity of iron(III) center conferred upon by nitro group and
hence the enhanced O2 attack [20,23,45,62]. Thus the nature of sub-
stituents on the phenolate ring of the ligands controls the reaction
rate as well as product selectivity.
The second order rate constants observed for the [Fe(L)(DBC)
(CH3OH)] adducts 1–4 fall in the range 5.0–7.2 ꢁ 10ꢀ2 Mꢀ1 sꢀ1
(Table 3), which corresponds to the involvement of both intradiol
and extradiol cleavage mechanisms to different extents. The reac-
tion rate constants follow the trend 3 > 2 > 1 < 4. Upon incorporating
the electron-releasing tert-butyl groups on the phenolate ring in 1 to
obtain 3, the electron density on iron(III) center increases and hence
the rate of dioxygenation to give extradiol product would be
expected to decrease, but, in contrast, an increase in reaction rate
4. Conclusions
A few catecholate adducts of iron(III) complexes of facially
coordinating tridentate phenolate ligands with different ring sub-
stituents have been isolated and studied as models for enzyme-
substrate adducts of catechol dioxygenase enzymes. The adducts
show two DBC2ꢀ ? iron(III) LMCT bands in the visible region,
which are similar to those observed for the enzyme–substrate

94
K. Visvaganesan et al. / Inorganica Chimica Acta 378 (2011) 87–94
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ration of an electron-releasing substituent on the phenolate ring
shifts the energy of the low-energy LMCT band to higher energy
and facilitates regioselective intradiol cleavage of catechol. On
the other hand, the incorporation of an electron-withdrawing
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Acknowledgements
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We sincerely thank the Council of Scientific and Industrial Re-
search, New Delhi for Senior Research Fellowship to K.V. This work
is supported also by the Indo-French Centre (IFCPAR) (Scheme No.
IFC/A/4109-1/2009/993). Professor M. Palaniandavar is a recipient
of DST Ramanna Fellowship (Scheme No. SR/S1/RFIC-01/2007 and
SR/S1/RFIC-01/2010). We thank Prof. Krishna P. Kaliappan, Indian
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