Bioinspired models for an unusual 3-histidine motif of diketone dioxygenase enzyme

Article abstract of DOI:10.1039/c9dt02518a

Bioinspired models for contrasting the electronic nature of neutral tris-histidine with the anionic 2-histidine-1-carboxylate facial motif and their subsequent impact on catalysis are reported. Herewith, iron(ii) complexes [Fe(L)(CH₃CN)₃](SO₃CF₃)₂1-3 of tris(2-pyridyl)-based ligands (L) have been synthesized and characterized as accurate structural models for the neutral 3-histidine triad of the enzyme diketone dioxygenase (DKDO). The molecular structure of one of the complexes exhibits octahedral coordination geometry and Fe-N11_py bond lengths [1.952(4) to 1.959(4) ?] close to the Fe-N_His bond distances (1.98 ?) of the 3-His triad in the resting state of the enzyme, as obtained by EXAFS studies. The diketonate substrate-adduct complexes [Fe(L)(acac^R)](SO₃CF₃) (R = Me, Ph) of 1-3 have been obtained using Na(acac^R) in acetonitrile. The Fe^2+/3+ redox potentials of the complexes (1.05 to 1.2 V vs. Fc/Fc⁺) and their substrate adducts (1.02 to 1.19 V vs. Fc/Fc⁺) appeared at almost the same redox barrier. All diketonate adducts exhibit two Fe(ii) → acac MLCT bands around 338 to 348 and 430 to 490 nm. Exposure of these adducts to O₂ results in the decay of both MLCT bands with a rate of (k_O2) 5.37 to 9.41 × 10^-3 M^-1 s^-1. The k_O2 values were concomitantly accelerated 20 to 50 fold by the addition of H⁺ (acetic acid), which nicely models the rate enhancement in the enzyme kinetics by the glutamate residue (Glu98). The oxygenation of the phenyl-substituted adducts yielded benzoin and benzoic acid (40% to 71%) as cleavage products in the presence of H⁺ ions. Isotope-labeling experiments using ¹⁸O₂ showed 47% incorporation of ¹⁸O in benzoic acid, which reveals that the oxygen originates from dioxygen. Thus, the present model complexes exhibit very similar chemical surroundings to the active site of DKDO and mimic its functions elegantly. On the basis of these results, the C-C bond cleavage reaction mechanism is discussed.

Full text of DOI:10.1039/c9dt02518a

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Ramasubramanian, K. Anandababu, N. C. Moesch-Zanetti, F. Belaj and M. Ramasamy, Dalton Trans., 2019,
DOI: 10.1039/C9DT02518A.
Volume 46
Number 10
14 March 2017
Pages 3073-3412
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DOI: 10.1039/C9DT02518A
Bioinspired Models for an Unusual 3-Histidine Motif of
Diketone Dioxygenase Enzymes
Ramamoorthy Ramasubramanian,a Karunanithi Anandababu,a Nadia C. Mösch-Zanetti,b
Ferdinand Belaj b and Ramasamy Mayilmurugana*
aBioinorganic Chemistry Laboratory/Physical Chemistry, School of Chemistry, Madurai
Kamaraj University, Madurai- 625021, India. mayilmurugan.chem@mkuniversity.org
bInstitute of Chemistry, University of Graz, Schubertstrasse 1, 8010 Graz, Austria.
Abstract: Bioinspired models for contrasting electronic nature of neutral tris-histidine from the
anionic 2-histidine-1-carboxylate facial motif and their subsequent impact on catalysis is reported.
Herewith, iron(II) complexes [Fe(L)(CH3CN)3](SO3CF3)2 1 - 3 of tris(2-pyridyl)-based ligands(L)
have been synthesized and characterized as the accurate structural models for the neutral
3-histidine triad of the diketone dioxygenases enzyme (DKDO). The molecular structure of one of
the complexes exhibits octahedral coordination geometry and Fe-N11py [1.952(4) - 1.959(4) Å]
bond lengths close to Fe-NHis (1.98 Å) bond distances of the 3-His-triad in the rest state of enzyme
obtained by EXAFS studies. The diketonate substrate-adduct complexes [Fe(L)(acacR)](SO3CF3)
(R = Me, Ph) of 1 - 3 have been obtained using Na(acacR) in acetonitrile. The Fe2+/3+ redox potential
of complexes (1.05 - 1. 2 V vs. Fc/Fc+) and their substrate adducts (1.02 - 1.19 V vs. Fc/Fc+)
appeared at almost the same redox barrier. All diketonate adducts exhibit two Fe(II)  acac MLCT
bands around 338 - 348 and 430 - 490 nm. Exposure of these adducts to O2 results in the decay of
both MLCT bands with the rate of (kO2) 5.37 - 9.41 × 10-3 M-1s-1. The kO2 values have been
concomitantly accelerated 20 - 50 fold by the addition of H+ (acetic acid), which nicely models the
rate enhancement in enzyme kinetics by the glutamate residue (Glu98). The oxygenation of the
phenyl substituted adducts yielded benzoin and benzoic acid (40 - 71%) as cleavage products in
the presence of H+ ions. The isotope-labeling experiments using 18O2 showed 47% incorporation
of 18O in benzoic acid, reveals that oxygen is originated from dioxygen. Thus, the present model
complexes exhibit very similar chemical surroundings as in the active site of DKDO and mimic
its function elegantly. On the basis of these results, C-C bond cleavage reaction mechanism is
discussed.
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Introduction
Dioxygenation is one of the important processes in bioremediation for toxic pollutants by
heme and non-heme iron enzymes.1 Especially, a class of non-heme iron enzymes, like catechol
dioxygenases are capable of breaking aromatic carbon-carbon bonds by incorporating both atoms
of dioxygen.2 Another major class of O2-activating enzymes is -keto acid-dependent
dioxygenases which perform oxidative decarboxylation of -keto acids.3 While the enzymology
and biomimetic chemistry of these enzymes were extensively studied,2,3 those of the recently
discovered non-heme dioxygenases like acireductone dioxygenase (ARD),4-7 quercetin 2,3-
dioxygenase (QD)8 and -diketone dioxygenases (DKDO)9,10 are in a rudimentary stage. They are
known to break more challenging aliphatic C-C bonds using O2. Among these enzymes, DKDO
is unique as its active site consists of a redox-active iron(II) center which is coordinated to a
3-histidine facial triad (His-62, His-64, and His-104; Scheme 1) in contrast to the often noted
2-histidine-1-carboxylate facial motif in several other non-heme iron enzymes.9-17 The remaining
coordination sites at iron are speculated as being water molecules.11-17 The occurrence of the
3-His-triad is surprising because the anionic carboxylate of the 2-histidine-1-carboxylate residue
is known to stabilize high valent iron.3,16 DKDO catalyzes C-C bond cleavage in -diketones by
incorporating O2 and converting them into nontoxic compounds.10-18 In fact, the mechanism is not
well understood. Particularly, an open question remains whether the cleavage of the C-C bond
proceeds via Fe(II)-mediated initial substrate activation or via O2 activation.15-17,19 In addition to
biochemical studies of the enzyme, the development of synthetic models is crucial for the
elucidation of mechanistic details.
HN
HN
N
N
His62
HN
O
O
His62
HN
O
O
2+
OH2
Fe OH2
O
O
2+
Fe
O2
O
N
O
N
OH2
Methyl glyoxal
Acetic acid
N
His64
His104
His64
N
H2O
NH
N
His104
H
Scheme 1. Active site structure of DKDO and adduct and dioxygenation reaction.
In order to simulate the 3-His-triad hydridotris(3,5-R2-pyrazol-1-yl)borato ligand (TpR2)
has been successfully applied.19-22 Kitajima and co-workers have first reported the iron complex
[Fe(TpiPr2)(acac)] (acac = acetyl-acetonato) without better bioinorganic outlook.19 Later,
2

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pioneering efforts of Limberg and co-workers20 and Fiedler and co-workers,21,22 iron(II)
complexes of TpMe2/Ph2 ligand and its derivatives have been projected as successful models for
DKDO. However, they are utilizing boron and phosphorus in the ligand backbone which are
proposed for stabilizing high valent iron-oxo species.2b,19-22 Recently, we have developed the
biomimetic models for ARD and Ni(II)-substituted DKDO enzymes using 2,6-bis(1-methyl-
benzimidazolyl)pyridine based ligands.23 In this article, we are reporting novel tris(2-pyridyl)
based ligand systems (Scheme 2) that exhibit very similar chemical surroundings as in the active
site geometry of DKDO. Our model systems duplicate the coordination environment of the neutral
3-His-triad in DKDO. Furthermore, we demonstrate reactivity towards aliphatic C-C bond
cleavage in -diketones using molecular oxygen. The three pyridine arms of the ligand are
expected to mimic the role of the neutral histidine residues in the 3-His motif. In fact, the ligand
architecture was reported by us to duplicate structural and functional aspects of cysteine
dioxygenase (CDO), whose active site also containing 3-His motif.23
N
L1,
L2,
L3,
R
R = Me
R = -OH
R= -OMe
N
N
Scheme 2. The structure of ligands used.
Results and Discussion
Synthesis and Characterization: The tris(2-pyridine) based ligands tris(2-pyridyl)ethane
(L1), tris(2-pyridyl)methanol (L2) and tris(2-pyridyl)-methoxymethane (L3) were synthesized
according to the previous reports with suitable modifications at -78˚C in THF.24
The model iron(II)
complexes were synthesized by the reaction of Fe(SO3CF3)2 with the respective ligands in
acetonitrile under an argon atmosphere. The complexes [Fe(L1)(CH3CN)3](SO3CF3)2 1,
[Fe(L2)(CH3CN)3](SO3CF3)2 2 and [Fe(L3)(CH3CN)3](SO3CF3)2 3 were obtained as red-orange
solids in very good yields (74 - 82 %). Recrystallization of complexes 1 - 3 in acetonitrile yielded
pure complexes for further studies. The elemental and mass spectrometry analyses confirm their
formation. The HR-ESI showed prominent molecular ion peaks for 1: [C18H15F3FeN3O3S]+ m/z,
466.01309; 2: [C17H13F3FeN3O4S]+ m/z, 467.99263 and 3: [C18H15F3FeN3O4S]+ m/z, 482.00788
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(Figure S2-S4). The loss of coordinated CH3CN may be due to dissociation in the spectrometer.
The magnetic susceptibility measurements for 1 - 3 by Evan’s method showed µeff = 5.1 - 5.4 µB,
which is close to the spin-only value of a high-spin iron(II) center (S = 2). In fact, they exhibited
paramagnetically shifted broad 1HNMR peaks in CD3CN. However, the use of non-coordinating
solvents CH2Cl2 and in CHCl3 for the synthesis of complexes resulted in the formation of bis-
complex of type [Fe(L)2](CF3SO3)2. The substrate adducts of 1 - 3 were obtained by reaction of
the ligands L1 - L3 with Fe(SO3CF3)2 followed by addition of Na(acacR1) (R1 = Me, Ph) in
acetonitrile under an argon atmosphere (Scheme 3) and the solution was filtered to remove
NaSO3CF3 and then evacuated to yield red-orange solids. The solids were washed with diethyl
ether (3 × 3mL) and dried under vacuum. The complexes were redissolved in CH3CN and
recrystallized by layering with diethyl ether yielded pure adducts with moderate yields (49 - 61%).
All adducts were isolated as brown-red solids and characterized by ESI-MS or HR-ESI mass as
[Fe(L1)(acacMe)](SO3CF3) 1a: m/z, 416.11 for [Fe(L1)(acacMe)]+; [Fe(L1)(acacPh)](SO3CF3) 1b:
m/z, 541.9780 for [Fe(L1)(acacPh)]+; [Fe(L2)(acacMe)](SO3CF3) 2a: m/z, 417.70 for
[Fe(L2)(acacMe)]+; [Fe(L2)(acacPh)](SO3CF3) 2b: m/z, 541.6120 for [FeII(L2)(acacPh)]+ and
[Fe(L3)(acacMe)](SO3CF3) 3a: m/z, 432.0536 for [Fe(L3)(acacMe)]+ (Figure S5-S9). The mass
analysis clearly reveals the formation of adducts with five-coordinate geometry and sixth
coordination may be vacant or occupied by a solvent molecule. It is further confirmed by elemental
analysis. However, isolation of the phenyl substituted adduct of 3 was unsuccessful. All the
adducts showed the characteristic carbonyls stretching frequencies around 1600 – 1630 cm-1
(Figure S10). These complexes 1a – 3a further shown to be EPR silent even at 10 K.
SO3CF3
SO3CF3
N
N
N
N
R1
R1
R
R
ONa
- CH3CN
- NaOSO2CF3
O
N
N
FeII
FeII
N
N
O
O
N
R1
R1
1a, R, R1 = Me, Me
1b, R, R1 = Me, Ph
2a, R, R1 = -OH, Me
2b, R, R1 = -OH, Ph
3a, R, R1 = -OMe, Me
3b, R, R1 = -OMe, Ph
1, R = Me
2, R = -OH
3, R= -OMe
Scheme 3. Synthesis of Fe(II)-complexes and their adducts.
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Molecular Structure of 1 and Relevance to DKDO: The suitable single crystal of 1 for X-
ray studies were obtained from recrystallization and exhibits an octahedral coordination geometry
around the FeII-center. There are two crystallographically independent complex molecules with
the same chemical formulae present in the asymmetric unit cell, both exhibiting the same
coordination geometry but slightly different bond lengths and bond angles are observed. The three
pyridine nitrogen atoms of L1 and three acetonitrile molecules occupy six coordination sites via
facial coordination mode. The structure exhibited pseudo-centrosymmetric: If the structure is
shifted in the unit cell that the cations are arranged around an inversion center then the cations and
two anions (including S1, S2) showed a centrosymmetric sub-structure. The bond angles at N(11)-
Fe(1)-N(1), N(21)-Fe(1)-N(2) and N(31)-Fe(1)-N(3) are 179.01(18)˚, 177.98(19)˚ and 178.70(18)˚
respectively and are slightly deviated from 180˚, which reveals a slight distortion from perfect
octahedral geometry. The equatorial bonds Fe-N11py, 1.959(4) Å and Fe-N31py, 1.958(4) Å are
slightly longer than the axial bonds Fe-N21py, 1.952(4) Å (Table 1). Most interestingly, the Fe-Npy
bond lengths are almost identical to the Fe-NHis bond distances of the DKDO active site (Fe-NHis
1.98 Å) determined by EXAFS analysis for the enzyme isolated from Burkholderia xenovorans
bacteria.25 In the absence of available X-ray structural parameters for the DKDO enzyme, therefore
here presented information is extremely valuable. Furthermore, the
Fe-N bond lengths
of 1 are shorter than those of the Fe(II)Tp complexes and their derivatives (2.096 - 2.260 Å).20-22
Thus, 1 represents the first example of a structurally well-defined iron(II) complex with similar
coordination geometry and chemical surroundings as in DKDO. The similarity of the FeII model
with that of the enzyme is visualized by the superposition of 1 on the active site structure of DKDO
(Figure 1). The main difference between the two geometries is the torsion around the Fe-N axes,
enforced by the rigidity of the tripodal structure of the ligand.
Electronic Spectral and Redox Properties: The complexes 1 - 3 and their substrate adducts
(1a - 3a) were subjected to UV-Vis spectroscopic analysis (Table 2, Figure S11-S15). 1 - 3 showed
Fe(II)-to-ligand charge transfer (MLCT) transitions at 418 - 438 and 345 - 386 nm14-17 along with
the ligand-based transitions around 205 - 341 nm. However, the complex-substrate adducts showed
MLCT transitions around 430 - 490 nm and 348 - 317 nm. They possibly originate from d-orbitals
of iron(II)-to p-orbitals of the -keto-enolate and trispyridine. Similar to the enzyme-acac adduct,
the positive charge on iron(II)-center decreases the energy of the d-orbital manifold and results in
the shift to the higher wavelength of its Fe(II)-to-acac (acac-monoanionic) MLCT transitions.14,15
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Further, the energies of MLCT transitions are significantly affected by substituents on the
substrate. The electron-withdrawing phenyl groups in 1b and 2b are expected to decrease the
energy gap of d-orbitals of iron(II) and p-orbitals of acacPh. This results in a redshift of MLCT
band compared to their respective methyl-substituted adducts. The electronic spectra of these
adducts are consistent with native DKDO enzyme with similar iron(II)-to-enolate MLCT transition
transitions at 355 and 420 nm for the acacMe substrate.14 The Fe2+/Fe3+ redox potential of complexes
was measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in
acetonitrile using Pt-disc and Ag/Ag+ as working and reference electrodes respectively. The Fe2+/3+
(vs. Fc/Fc+) redox potential of 1 (1.05 V) is slightly lower than those of 2 (1.32 V) and 3 (1.20 V)
(Table 2, Figure S16-S18). In CV, the Fe2+/3+ redox couples appear far away from reversibility.
The Fe2+/3+ redox potential of the complexes and their respective adducts 1a-3a (E1/2, 1.03 - 1.32
V vs. Fc/Fc+) was found to be very similar and have irreversible redox couple (Table 2, Figure
S19-S22). These redox potentials are much higher than a reported redox barrier for the O2-
activation by non-heme iron centers (less than -0.10 V).26 Furthermore, these Fe2+/3+ redox
potential of adducts are more positive than those of reported for Fe(II)Tp-adducts (-0.34 to -0.58
V).21,22
Dioxygenation Reactions and Kinetic Studies: The reactivity of adducts toward dioxygen
was investigated by monitoring the decay of the Fe(II)  acac MLCT band (max 430, 490 nm) in
acetonitrile/acetate buffer. The freshly prepared substrate adducts in acetonitrile were used
(formation was confirmed by ESI-MS before O2 treatment). On treating the methyl-substituted
adduct (1a - 3a) with excess O2 in acetonitrile at 25˚C, the intensity of FeII acacMe MLCT band
decreased with pseudo-first-order reaction rate kobs = 3.83 - 7.34 × 10-5 s-1 (Table S2, Figure S23-
S25). This decay of the MLCT transition indicates the progress of the dioxygenation reaction. The
kO2 (= kobs/[O2]) values were calculated by including dissolved oxygen concentration in
acetonitrile, 8.1 × 10-3 M.28 The adduct 1a showed a higher kO2 value (9.41 × 10-3 M-1s-1) than 2a
(4.92 × 10-3 M-1s-1) and 3a (5.37 × 10-3 M-1s-1) (Table S2, Figure S26-S28) around pH, 7.4. No
such decrease in intensity of the MLCT band was noticed for phenyl substituted adducts (1b - 3b).
Interestingly, the dioxygenation reaction rates of 1a - 3a have been increased to 20 – 50 fold (kO2,
110.24 - 460 × 10-3 M-1s-1) by the addition of 0.5 equivalent H+ (acetic acid), whereupon the pH
of reaction mixtures decreased to around 5.3 - 5.4 (Table S2, Figure 2, S29-S32) in
acetonitrile/acetate buffer. Furthermore, the less reactive bulky phenyl substituted adducts 1b -3b
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turn to be reactive at this acidic pH, however, the reaction rates (kO2, 0.08 - 3.32 × 10-4 M-1 s-1) are
slower than those of 1a -3a (Table S2, Figure S33-S36). This H+ assisted enhancement of reaction
rates is very interesting because a glutamate (Glu98) residue is believed to enhance the
dioxygenation reaction rate in DKDO enzyme kinetics. In DKDO, Glu98 is located in the
secondary coordination sphere of the iron center and promoting both substrate binding and O2
reduction.13-15 The Glu98 is connected to the nitrogen of His104 via H-bond at a distance of 2.86
Å.15b
The dioxygenation reaction rates were observed for 1a – 3a many-fold higher than those of
Fe(II)Tp-adducts in toluene.21-22 Nevertheless, these rates are lower than those of the native
enzyme (kcat, 8.5 s-1).10,11 The kO2 value for 1a was measured over a wide range of pH (5.4 - 7.4),
which is increased by lowering the pH of the acetonitrile/acetate buffer solution (Table S3, Figure
2B, S37 - S41). The reaction rate saturated at pH 5.4 where it showed the highest kO2 value of 466
× 10-3 M-1 s-1. On the other hand, dioxygenation was completely inhibited at pH values higher than
7.0. It is interesting that the plot of pH vs kO2 is similar to that obtained for the DKDO enzyme
kinetics (Figure 2B).15a This result consistent with the impact of acid-base catalysis by secondary
coordination residues (hydrophobic and hydrophilic) as in native enzyme which alters the rates of
reactions.15 Furthermore, adducts of Fe3+, Mn2+, Cu2+, and Zn2+ using ligand L1 exhibited no
dioxygenation reaction kinetics and no cleavage products formation were observed even after
seven days (Figure S42-S45). Similar non-reactive behavior of acac substrates was found with a
solution of L1 in the absence of metal. Thus, only the redox-active Fe2+ center appears to facilitate
C-C bond breaking, which is similar to the enzyme kinetics reported by Straganz and Nidetzky.13-15
Additional mechanistic information has been obtained by using stronger oxidizing sources
such as tert-butyl hydroperoxide (t-BuOOH) and PhI(OAc)2 as spectroscopic probes. The
treatment of adducts 1a - 3a with 10 equivalents of t-BuOOH in acetonitrile at 25˚C showed the
decrease in intensity of the FeII  acac MLCT bands with pseudo-first-order reaction rates kobs,
63.25 - 159.29 × 10-5 s-1 (Table S2, Figure 3, S46-S49). Upon addition of 0.5 equivalent H+ (acetic
acid) the rate increased approximately 3-fold kobs 156.6 – 412.3 × 10-5 s-1. Less reactivity has been
observed for the adducts 1b - 3b with t-BuOOH, but again, they turned to be reactive by adding
0.5 equivalent of H+ (acetic acid) and showed kobs values of 1.48 - 12.8 × 10-5 s-1 (Table S2, Figure
S50-S55). As expected, the reaction rates for bulkier 1b - 3b are drastically lower as compared to
1a - 3a. However, all kobs values obtained using t-BuOOH are relatively higher than those of O2
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kinetics. Whereas, treatment of 1 - 3 with t-BuOOH led to an immediate color change to green
which can be assigned to a Fe3+-alkylperoxido species with a t-BuOO-  Fe3+ LMCT transition
around 687-690 nm (Figure 3, S56-S59). Its intensity started to decrease with kobs values of 53.72
- 66.67 × 10-5 s-1 at 25˚ C. The ESI-MS analysis of 2 with t-BuOOH revealed a mass cluster at m/z
= 727.09 corresponds to [Fe(L2)(t-BuOO-)(SO3CF3)2]Na+ (Figure S60). The EPR spectra of 3
with t-BuOOH measured in methanol showed signals for high-spin FeIII (S = 5/2) (g, 7.42,
4.29), a rhombic low-spin FeIII (S = ½) (g, 2.47, 2.15, 1.92) (Figure S61). The high-spin
FeIII is possibly formed via rapid decay of low-spin species. Interestingly, while treating
complex-substrate adducts (1a-3a) with t-BuOOH showed no such LMCT transition and EPR
-
signals, which is indicating that the absence of the Fe3+-O2 type species formation. Furthermore,
very similar kinetic information has been obtained on treating complex-substrate adducts with
PhI(OAc)2 as an oxidizing source (Table S2, Figure S62-S69). The kobs values were obtained in the
range of 1.27 - 140.3 × 10-5 s-1 but they are lower than those of the O2 kinetics.
Product Analysis and Dioxygenation Mechanism: In separate experiments, 1b treated with
excess O2 in the presence of acetic acid over 12 hours. The EPR analysis of the dioxygenation
reaction of 1b at 77 K found silent during the course of the reaction.30b After that the reaction
mixture was analyzed by ESI-MS and showed products benzoin (A) and benzoic acid (B) bound
iron(II) complexes, bis-iron(II) complex (Figure S70, S71) along with unidentified products. The
time-dependent differential pulse voltammetry studies for 3a with dioxygen showed no
concomitant changes in redox potential was observed over 1 hour (Figure S72). Therefore, these
results invariably suggest that the oxidation state of Fe(II) center does not varied during the course
of the dioxygenation reaction. A very similar observation was reported by Limberg and co-workers
for the reaction of [Fe(TpMe/Ph)(acac)] with dioxygen.20 On the contrary, in the absence of O2 even
with an excess of H2O, no cleavage products were isolated under the identical condition, hence
hydrolytic type mechanistic pathway could be eliminated.
Further, the cleavage products were separated as A (42%) and B (29%) (Scheme 3) and
1
characterized by H/13C NMR and GC-MS spectra (Figure S73-S77). The adduct 1b yielded a
higher amount of cleavage products (71%) than 2b (50%) and 3b (40%). The product yield has
been increased (B, 42%; benzil C, 46%) for 1b on using t-BuOOH over 3 hours. Further addition
of acetic acid to reaction results in the formation of A (47%) along with B (41%) for 1b, which is
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higher than 2b A(31%) along with B (34%)and 3b A(26%) along with B (28%). The addition of
the radical scavenger TEMPO (2,2,6,6-tetramethylpiperidin-1-yl-oxyl) to the reaction of 1b with
O2 showed no perceptible change in the product yields under identical reaction condition, which
eliminates the possibility of radical based reaction pathways.27 In two identical dioxygenation
reactions were performed for 1b and their headspaces were purged separately into a saturated
solution of Ca(OH)2 and PdCl2 leads to formation CaCO3 and Pd precipitate respectively, which
confirms CO2 and CO formation during the reaction.7,23a Evolution of CO2(g) from the reaction
mixture attributes to the formation of benzoic acid from the oxidative decomposition of phenyl
glyoxal due to excess dioxygen used (Scheme 4; Path 1). Time-dependent product analysis for 1b
with dioxygen was performed over various time intervals (Table S5) and showed yields A, 17%,
B, 11%; A, 29%, B, 19%; A, 36%, B, 25% over 3, 6 and 9 hours respectively. The yields of benzoic
acid and benzoin are seemingly independent over these reaction time under similar conditions.
This result suggests that a remote possibility for the conversion of benzoin into benzoic acid.
Further, the reaction of equimolar benzoin and complex 1 in the presence of dioxygen showed
almost no formation of benzoic acid, which rules out the possibility of benzoic acid formation via
benzoin. The source of oxygen is verified by using 18O2 instead of 16O2 and exhibited a shift of two
mass units for benzoic acid (GC-MS, m/z, 124) with 47% isotopic incorporation (16O/18O = 53:47)
18
(Figure 4).28 Thus, only one of the atoms of the O2 molecule has been incorporated into the
product benzoic acid and another one presumably eliminated as labeled CO/CO2 (Scheme 3). The
18
18
low incorporation of O may be due to hydrolysis of triketone intermediate or exchange of O
18
with formed carboxylic acid.29 Similarly when the reaction performed using an excess of H2 O
and in presence of dioxygen has shown only 2.7% of labeled benzoic acid (Figure S78). This little
incorporation of 18O into formed (16O) benzoic acid must be resultant from partial
hydrolysis/exchange of 18O over the longer reaction time.29a Benzoin formation (A) from
dioxygenation reaction invariably attributes that triketone being one among transient intermediate,
which on subsequent decay by Lewis acid promoting benzoyl migration with the evolution of CO
(Scheme 4).7,30a Thus, these results are suggesting that C-C bond breaking presumably proceeds
via the initial formation of FeII-organoperoxidate intermediate.14 Earlier studies on the enzyme
were also suggesting a very similar mechanism for C-C bond cleavage.13-15 In contrast to this,
Solomon and co-workers have reported a Fe-mediated O2 activation mechanism by the
involvement of higher oxidation (FeIV=O) intermediate formed via the Fe3+-O2-. species.16 On the
9

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other hand, the involvement of multiple spin states of the metal center and its crucial role in
controlling the reactivity via magnetic exchange coupling with bound oxygen have been
established computationally for biomimetic models of other dioxygenase enzymes.31 However, the
accurate prediction of key intermediate and nature of the spin state of iron center needs further
experimental and computational investigations.
+
SO3CF3
N
R
N
R1
R1
N
N
N
N
N
FeII
N
N
N
N
N
N
N
H+
O-
-CH3CN
H+
N
FeII
FeII
O
FeII
Fe
N
O
O
O
O
O
O2
O
O
O
N
N
-H2O
R1
R1
R1
R1
R1
R1
R1
HO
R1
O
O
O
O
R1 = Ph/Me
Fe(L)
Triketone formation
N
N
N
Path 1
Fe
Benzoyl
2
Path
migration
N
O
O
O
O-
B
Ph
Ph
N
N
OH
H+
-C
FeII
-CO2
R1
O
O
O
R1
O2
O
R1
R1
A
O
Scheme 4. The proposed reaction pathway.
Summary
In summary, the structurally well-defined models for the iron(II)-dependent DKDO
enzyme have been synthesized and characterized. The molecular structure of the complexes and
their electronic spectral signatures of substrate adducts showed a very similar coordination
environment as similar to the active site and substrate adduct of native Fe(II)-DKDO enzyme. The
Fe2+/3+ redox potentials of complexes and their substrate adducts showed higher redox potentials
than a reported redox barrier for dioxygen activation. The dioxygenation reaction has been
accelerated 20 – 50 fold by the addition of H+ (acetic acid). This is unambiguously exhibiting a
relevant correlation with the rate of dioxygenation by native DKDO enzyme, where the glutamate
residue (Glu98) facilitates the binding of substrate and its dioxygenation reactions from secondary
coordination sphere as a pH regulating channel. Interestingly, the oxidation state of Fe(II) center
is unaltered during the dioxygenation reaction as proposed similar to the native enzyme. Thus,
kinetic studies and product analyses are revealing that the mechanism possibly proceeds via initial
formation of FeII-organoperoxidate intermediate pathway as suggested for the DKDO enzyme.
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The dioxygenation reaction of the phenyl substituted adducts yielded benzoin and benzoic acid as
a major cleavage product with the evolution of CO and CO2.
Experimental Section
Materials: The chemicals 2-ethylpyridine, 2-fluoropyridine, di-2-pyridylketone, n-butyl-
lithium (2 M in hexane), iron(II) trifluromethane-sulfonate, iron(III) perchlorate hydrate, zinc(II)
perchlorate hexahydrate, copper(II) trifluoromethane sulfonate, manganese(II) trifluromethane-
sulfonate, acetic acid, acetylacetone, 1,3-diphenylpropane-1,3-dione, (diacetoxy-iodo)benzene
18
PhI(OAc)2, H2 O and tert-butyl hydroperoxide (t-BuOOH) were purchased from Sigma-Aldrich.
18O2 was purchased from Cambridge Isotope Laboratories. The supporting electrolyte
tetrabutylammonium perchlorate (TBAP) was prepared in water and recrystallized twice from
aqueous ethanol. Oxidant iodosobenzene (PhIO) was synthesized from PhI(OAc)2. Anhydrous
acetonitrile, 2-bromo pyridine, iodomethane, (Alfa Aesar), Tetrahydrofuran (THF) (Merck, India)
were used as received. Dry THF obtained by literature method,32 THF was refluxed for 1 hour over
sodium metal and benzophenone and then distilled under N2 atmosphere. The distilled solvent was
degassed and stored under argon over molecular sieves.
Experimental conditions and physical measurements: All workups were carried out under dry
argon using a standard Schlenk line or glove box techniques for the reactions involving air-
sensitive synthesis and experiments. All NMR spectra were recorded on a Bruker 300 MHz
spectrometer. Chemical shift values are given in parts per million (ppm). High-resolution electron
impact mass spectra (HR-ESI MS) have been measured on a Bruker 12 Tesla APEX-Qe FTICR-
MS (or) HRMS-ESI-Q-TOF LC/MS system. Elemental analyses were carried out using a Heraeus
Vario Elemental automatic analyzer. UV-Vis spectra were recorded on an Agilent 8453
spectrometer with a cooling unit by Unisoku (Osaka, Japan). The Electrochemical data were
recorded in Biologic SP-150 Electrochemical workstation. The potential was externally calibrated
against the ferrocene/ferrocenium couple prior to analysis. The ESI-MS measurement was carried
out on Thermo Fischer LC-MS. The ESI-MS & GC-MS measurement was carried out on Thermo
Fischer LC-MS & Agilent 5977E GCMSD respectively. All pH measurements are made with the
addition of 10% of aqueous acetonitrile solution. FT-IR was recorded on a Thermo Nicolet 6700
FT-IR spectrometer. The EPR experiments were made with a Biospin ELEXSYS E500
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spectrometer (Bruker, Karlsruhe, Germany). The EPR spectrometer was equipped with a
continuous-flow liquid He cryostat and an ITC503 temperature controller made by Oxford
Instruments.
Evans Method Measurements: The effective magnetic moment was determined by using
Evans’method.33 In a typical experiment, an oxygen-free solution of a complex in d6-DMSO,
containing 5% tert-butanol by volume was placed in an NMR tube, while a reference solution of
5% tert-butanol (v/v) in d6-DMSO, was placed into NMR tube insert. Then 1HNMR experiments
were performed on the Bruker 300 MHz spectrometer and magnetic moment values were obtained
by fitting the chemical shift values in the standard equation of Evan’s method.
Crystal Structure Determination of 1: All the measurements were performed using
+2
monochromatized Mo K radiation at 100K: (C23H24FeN6 )2(CF3O3S–)4 · C2H3N, Mr 1517.99,
triclinic, space group P 1, a = 11.8586(6)Å, b = 12.4140(6)Å, c = 12.6961(6)Å,  = 60.817(2)°,
 = 83.284(2)°,  = 73.222(2)°, V = 1561.69(14)Å3, Z = 1, dcalc = 1.614g cm-3  = 0.704
mm-1. A total of 13837 reflections were collected (max = 28.0°), from which 10107 were
unique (Rint = 0.0227), with 9675 having I > 2(I). The structure was solved by direct methods
(SHELXS-97) and refined by full-matrix least-squares techniques against F2 (SHELXL-
2014/6).34,35 Since racemic twinning was detected the structure was refined as a 2-component
inversion twin resulting in a scale factor of 0.301(20) between the two unequal components. The
size of the voids occupied by the four anions and the solvent molecule was computed to approx.
114, 110, 111, 136, and 104 Å3, respectively. The CF3 group of the anion in the largest void
(including S4) was disordered over two orientations and was refined with site-occupation factors
of 0.597(6) and 0.403(6), respectively. The same anisotropic displacement parameters were refined
for equivalent atoms in the disordered part, and the equivalent bonds were restrained to have the
same lengths. The other non-hydrogen atoms were refined with anisotropic displacement
parameters without any constraints. The H atoms of the pyridine rings were put at the external
bisectors of the X–C–C angles at C–H distances of 0.95Å and common isotropic displacement
parameters were refined for the H atoms of the same ring. The H atoms of the methyl groups were
12

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refined with common isotropic displacement parameters for the H atoms of the same group and
idealized geometries with tetrahedral angles, enabling rotation around the C–C bond, and C–H
distances of 0.98Å. For 870 parameters final R indices of R1 = 0.0400 and wR2 = 0.1129 (GOF =
1.045) were obtained. The largest peak in a difference Fourier map was 0.846eÅ-3. CCDC-
1522488 contains the supplementary crystallographic data for this paper.
Synthesis of Ligands
Synthesis of tris(2-pyridyl)ethane (L1): The ligand tris(2-pyridyl)ethane (L1) was
synthesized by the early report with slight modifications.24a The solution of n-BuLi (14 mL, 28.0
mmol of 2 M in hexane) was added dropwise via syringe over 15 minutes to 2-ethyl pyridine (1.6
mL, 12.0 mmol) which was pre-cooled to -78˚C in THF (40 mL) under argon atmosphere. Then it
was stirred vigorously and the temperature maintained below -70˚C. After 1 hour, the solution
turns into a deep red color and then 2-fluoro pyridine (0.5 mL, 6 mmol) was added dropwise at -
20˚C. The temperature was slowly elevated into room temperature. Over 30 minutes and refluxed
for an additional 30 minutes. Further, another equivalent of 2-fluoro pyridine was added at 0˚C
and subsequently refluxed for 2 hours. Finally, the reaction was quenched with water at 0˚C and
organics were extracted with ethylacetate. Volatiles were removed by vacuum. Pure off-white
crystalline solids were obtained by column chromatography using silica (7:3 hexane: ethyl acetate)
1
with a yield of 72% (1.13 g). H NMR (CDCl3, 300 MHz): (ppm) 2.33 (s, 3H, CH3), 7.10 (m,
6H), 7.59 (m, 3H) 8.58 (d, 3H); 13C NMR (CDCl3, 75 MHz) (ppm) 27.26 (CH3), 59.99 (C-CH3),
121.21, 123.43, 135.99, 149.00, 165.72. ESI-Mass, m/z, 261.05.
Synthesis of tris(2-pyridyl)methanol (L2): The ligand (L2)tri(2-pyridyl)methanol was also
synthesized by the early report with slight modifications.24b To a pre-cooled 2-bromopyridine (4.60
g, 28 mmol) in THF (175 mL) n-BuLi (20 mL, 28 mmol of 2.0 M in hexane) was added dropwise
with vigorous stirring at a temperature below -70C under argon atmosphere. This reaction mixture
was stirred for 30 minutes and then di-2-pyridyl ketone (2.54 g, 13.8 mmol) in THF (25 mL) was
added. The further stirring of solution for an additional hour shows a change of color deep red into
violet. At this stage, the reaction was quenched by adding methanol-water mixture at 0˚C and
organics were separated with ethyl acetate. The removal of ethyl acetate under vacuum yielded
1
pure off-white crystalline solid, yield: 92% (3.30 g). H NMR (CDCl3, 300 MHz):  (ppm) 7.19
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(td, 3H), 7.71 (m, 6H) 8.58 (dd, 3H); 13C NMR (CDCl3, 75 MHz)  (ppm) 81.21 (C-OH), 122.27,
122.90, 136.38, 147.77, 162.80. ESI-MS, m/z, 263.05.
Synthesis of tris(2-pyridylmethoxy)methane (L3): The ligand (L3) tris(pyridyl-2-
methoxy)methane has been synthesized by adding NaH (0.83 g, 0.03 mol) to tris-pyridylcarbinol
in DMF and followed by the dropwise addition of iodomethane (0.40 mL, 0.9 g, 0.0065 mol) at
room temperature. The reaction mixture was stirred for 1.5 hours and quenched with a 1:1 mixture
of acetone and 2-propanol. After addition of water, the organic solvents are removed under
vacuum. The product was extracted with CH2Cl2 and recrystallized from acetone to give a
1
crystalline white solid (yield, 48%, 1.5 g). H NMR (CDCl3, 300 MHz):  (ppm) 7.19 (td, 3H),
13
7.71 (m, 6H) 8.58 (dd, 3H); C NMR (CDCl3, 75 MHz) (ppm) 81.21 (C-OH), 122.27, 122.90,
136.38, 147.77, 162.80.
Synthesis of iron(II) complexes: All the iron(II) complexes were synthesized under dry
argon using standard Schlenk line and glove box. The ligand (0.5 mmol) was added to iron(II)
trifluoromethanesulfonate (0.176 g, 0.5 mmol) in acetonitrile (10 mL). The reaction mixture was
stirred for 2 hours and removal of solvent under vacuum yielded red-orange color solid. Then, it
was washed with diethyl ether to remove the free ligand.
[Fe(L1)(CH3CN)3](SO3CF3)2, 1: Yield, 0.30 g, 82%, HR-ESI Mass, m/z, 466.01309 corresponds
to [Fe(L1)(SO3CF3)]+ ion. [Fe(L1)(CH3CN)3](SO3CF3)2, 1: Analytically calculated for
C25H24F6FeN6O6S2: C, 40.66; H, 3.28; N, 11.38%. Found: C, 40.28; H, 3.62; N, 11.47%. 1H NMR
(CD3CN, 300 MHz):  (ppm) 18.66, 15.97, 14.1, 8.98, 4.3.
[Fe(L2)(CH3CN)3](SO3CF3)2, 2: Yield, 0.27 g, 74%, HR-ESI Mass, m/z, 467.99263 corresponds
to [Fe(L2)(SO3CF3)]+ ion. [Fe(L2)(CH3CN)3](SO3CF3)2 2: Analytically calculated for
C24H22F6FeN6O7S2: C, 38.93; H, 3.0; N, 7.54%. Found: C, 38.68; H, 3.20; N, 7.14%.1HNMR
(CD3CN, 300 MHz):  (ppm) 17.88, 15.57, 14.1, 8.88.
[Fe(L3)(CH3CN)3](SO3CF3)2, 3: Yield, 0.28 g, 82%, HR-ESI Mass, m/z, 482.00789 corresponds
to [Fe(L3)(SO3CF3)]+ ion. [Fe(L3)(CH3CN)3](SO3CF3)2 3: Analytically calculated for
C25H24F6FeN6O7S2: C, 39.80; H, 3.21; N, 11.14%. Found: C, 39.58; H, 3.17; N, 11.26%. 1H NMR
(CD3CN, 300 MHz):  (ppm) 19.06, 16.07, 14.3, 9.1, 3.67.
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Synthesis of complex-acac adducts: The complex-acac adducts were synthesized by two
steps as a previously reported method with suitable modifications.20,21 The Na(acacX) (X = Me,
Ph) salts were prepared by the reaction of respective β-diketone (0.52 mmol) and NaH (0.52 mmol)
in dry THF. Then, stirred for 30 minutes and the solvent was removed under vacuum to yield the
Na(acacX) salts as a white solid. In second step, Fe(SO3CF3)2 (0.5 mmol) was added to ligand (0.5
mmol) in acetonitrile (10 mL) and then followed by addition of Na(acacX) in acetonitrile. The
reaction mixture was stirred overnight at room temperature. The solution was filtered to remove
NaSO3CF3 and then evacuated to yield red-orange solids. The solids were washed with diethyl
ether (3 × 3 mL) and dried under vacuum to yield pure adducts.
[Fe(L1)(acacMe)]SO3CF3, 1a: Yield, 0.13 g, 61%, ESI-MS, m/z, 416.11 corresponds to
[Fe(L1)(acacMe)]+ ion. 1a: Analytically calculated for C23H22F3FeN3O5S: C, 48.86; H, 3.92; N,
7.43%. Found: C, 48.83; H, 3.90; N, 7.42%.; IR: ῡ(CO) - 1605 cm-1.
[Fe(L1)(acacPh)]SO3CF3, 1b: Yield, 0.16 g, 58%, HR-ESI mass, m/z, 541.9780 for corresponds to
[Fe(L1)(acacPh)]+ ion. 1b: Analytically calculated for C33H26F3FeN3O5S: C, 57.49%; H, 3.80%; N,
6.09%. Found: C, 57.51; H, 3.81; N, 6.07%; IR: ῡ(CO) - 1617 cm-1.
[Fe(L2)(acacMe)]SO3CF3, 2a: Yield, 0.11 g, 53%, ESI-MS, m/z, 417.70 corresponds to
[Fe(L2)(acacMe)]+ ion. 2a: Analytically calculated for C22H20F3FeN3O6S: C, 46.58; H, 3.55%; N,
7.41%. Found: C, 46.56; H, 3.54; N, 7.40%.; IR: ῡ(CO) – 1602 cm-1.
[Fe(L2)(acacPh)]SO3CF3, 2b: Yield, 0.13 g, 49%, HR-ESI mass, m/z, 541.6120 corresponds to
[FeII(L2)(acacPh)]+ ion. 2b: Analytically calculated for C32H24F3FeN3O6S: C, 55.58%; H, 3.50%;
N, 6.08%. Found: C, 55.51; H, 3.48; N, 6.04%; IR: ῡ(CO) - 1619 cm-1.
[Fe(L3)(acacMe)]SO3CF3, 3a: Yield, 0.1 g, 49%, HR-ESI mass, m/z, 432.0536 corresponds to
[Fe(L3)(acacMe)]+. 3a: Analytically calculated for C23H22F3FeN3O6S: C, 47.52; H, 3.81; N, 7.23%.
Found: C, 47.49; H, 3.79; N, 7.12%.; IR: ῡ(CO) - 1593 cm-1
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Kinetics studies and products analysis: The solutions of iron(II) complexes and substrate
were prepared under an argon atmosphere in the Schlenk line. The kinetics of the dioxygenase
reactions monitored by time-dependent measurements of the disappearance of the FeII  (acacX)
MLCT bands at 25˚ C. The freshly prepared adducts were exposed to O2 in acetonitrile solution.
The solubility of O2 in acetonitrile is 8.1 × 10-3 M.28 The dioxygenase activities and product
analysis of the present complexes was carried out in the separate experiments. The phenyl
substituted adducts 1b - 3b (0.1 mmol) were exposed to O2 in the presence of acetic acid and stirred
for 12 hours. The oxygenation reaction was quenched by adding 6 M HCl (5 mL). Then the
products were extracted using ethyl acetate (3
15 mL). The organic layer was dried over
×
anhydrous Na2SO4 at room temperature. Further purification was performed using column
chromatography using silica gel (8:2 hexane: ethyl acetate). The major products were separated
and characterized by NMR and GC-MS analysis. Whereas, the reactions using t-BuOOH and
PhI(OAc)2 as oxidant were performed by an identical procedure over 3 hours only.
GC-MS: Benzoic acid: m/z, 150.13; 1H NMR (CDCl3, 300 MHz):  (ppm) 8.14 (m, 2H), 7.63 (m,
1H) 7.48 (m, 2H); 13C NMR (CDCl3, 75 MHz) (ppm) 128.80, 130.61, 134.20, (C=O) 173.02. 1H
& 13C NMR.
Benzil m/z, 210.05; 1H NMR (CDCl3, 300 MHz):  (ppm) 8.1 (dd, 4H), 7.65 (m, 2H) 7.51
(m, 4H). Benzoin m/z, 212.05; 1H NMR (CDCl3, 300 MHz):  (ppm) 8.14 (m, 4H), 7.63 (m, 6H)
5.6 (s, 1H). These data were good agreement with previously reported data in literature.
18O2 Labeling studies: In the separate experiments, 1b (0.1 mmol) was exposed to 18O2 for
12 hours and stirred. The reaction was quenched with 0.1 mL of HCl and solvent was removed in
vacuo under a nitrogen atmosphere. The products were extracted with ethyl acetate, passed through
a short silica column and analyzed by GC-MS. GC-MS analysis performed on Agilent 5977E
GCMSD using HP- 5 MS ultra-inert (30 m × 250 µm × 0.25 µm) capillary column.
Acknowledgment
We acknowledge Science and Engineering Research Broad (SERB), New Delhi and Board
Research in Nuclear Sciences (BRNS), Mumbai for funding.
Conflict of interest
16

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The authors have no conflict of interest
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29 (a) M. L. Bender, R. R. Stone, and R. S. Dewey, J. Am. Chem.Soc., 1956, 78, 319; (b) S. Paria,
P. Halder and T. K. Paine, Angew. Chem., Int. Ed., 2012, 51, 6195.
30 (a) J. D. Roberts, D. R. Smith, C. C. Lee, J. Am. Chem. Soc. 1951, 73, 618. (b) R. D. Jana, D.
Sheet, S. Chatterjee, T. K. Paine Inorg. Chem. 2018, 57, 8769.
31 (a) S. Rana, J. P. Biswas, A. Sen, M. Clemancey, G. Blondin, J. M. Latour, G. Rajaraman, D.
Maiti, Chem. Sci., 2018, 9, 7843; (b) P. Comba, S. Wunderlich, Chem. Eur. J. 2010, 16, 7293; (c)
A. Ansari, P. Jayapal, G. Rajaraman Angew. Chem. Int. Ed., 2015, 54, 564.
32 B. S. Furniss, A. J. Hannaford, P.W. Smith, A. R. Tatchell, Vogel‘s Textbook of practical
organic chemistry, 5th Edition. PEARSON Education Limited, New Delhi, 2004, p 40.
33 D. F. Evans J. Chem. Soc. 1959, 2003. b) Z. Xue, J. C. Daran, Y. Champouret, R. Poli, Inorg.
Chem. 2011, 50, 11543.
34 C. K. Johnson, ORTEP. Report ORNL-3794. Oak Ridge National Laboratory, Tennessee, USA
(1965)
35 (a) Sheldrick, G. M. A short history of SHELX. Acta Cryst. A64, 2008, 112; (b) Sheldrick, G.
M. Crystal structure refinement with SHELXL. Acta Cryst. C71, 2015, 3.
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Figures and Tables
N21
N31
N11
N3
Fe1
N1
N2
His64
His62
His104
Figure 1. The molecular structure of 1 (50% probability factor of thermal ellipsoids). H atoms and
SO3CF3¯ ions are omitted for clarity (top). Overlay plot of the structures of 1 and DKDO active
site with Zn(II) cofactor (bottom).
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A
B
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Figure 2. The progress of the reaction of 1a (6 × 10-4 M) with O2 and 0.5 equiv. of acetic acid) in
ammonium acetate in water/acetonitrile (3:7) buffer at 25˚C. Inset: Plot of time vs absorbance
(435 nm) (A). The plot of ko2 vs pH (B) in ammonium acetate in water/acetonitrile (3:7) buffer.
A
B
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Figure 3. The progress of the reaction of 1a (6 × 10-4 M) with acetic acid (0.5 equiv) (A) in
ammonium acetate in water/acetonitrile (3:7) buffer and 1 (6×10-4 M) (B) in the presence of t-
BuOOH in acetonitrile at 25˚C. Insets: plots of time vs absorbance change.
122
100
105
110
115
m/z
120
125
130
122
124
100
105
110
115
120
125
130
m/z
Figure 4. Oxygen isotopic studies for the reaction of 1b using 16O2 (top) and with 18O2 (bottom).
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Table 1. Selected bond lengths [Å] and angles [°] for 1
1
1’
Fe(1)-N(3)
Fe(1)-N(21)
Fe(1)-N(2)
Fe(1)-N(31)
Fe(1)-N(11)
Fe(1)-N(1)
1.948(4)
1.952(4)
1.954(3)
1.958(4)
1.959(3)
1.961(4)
Fe(2)-N(51)
Fe(2)-N(43)
Fe(2)-N(41)
Fe(2)-N(71)
Fe(2)-N(61)
Fe(2)-N(42)
1.952(3)
1.952(4)
1.954(4)
1.956(4)
1.958(3)
1.965(4)
N(11)-Fe(1)-N(1)
N(21)-Fe(1)-N(2)
N(31)-Fe(1)-N(3)
N(3)-Fe(1)-N(21)
N(3)-Fe(1)-N(2)
N(21)-Fe(1)-N(31)
N(2)-Fe(1)-N(31)
N(3)-Fe(1)-N(11)
N(21)-Fe(1)-N(11)
N(2)-Fe(1)-N(11)
N(31)-Fe(1)-N(11)
N(3)-Fe(1)-N(1)
N(21)-Fe(1)-N(1)
179.01(18)
177.98(19)
178.70(18)
90.48(14)
88.45(15)
88.25(15)
92.83(15)
92.09(15)
88.66(14)
89.69(14)
88.15(14)
88.49(15)
92.12(14)
N(51)-Fe(2)-N(41)
N(61)-Fe(2)-N(42)
N(71)-Fe(2)-N(43)
N(51)-Fe(2)-N(43)
N(43)-Fe(2)-N(41)
N(51)-Fe(2)-N(71)
N(41)-Fe(2)-N(71)
N(51)-Fe(2)-N(61)
N(43)-Fe(2)-N(61)
N(41)-Fe(2)-N(61)
N(71)-Fe(2)-N(61)
N(51)-Fe(2)-N(42)
N(43)-Fe(2)-N(42)
179.08(16)
178.81(16)
179.00(16)
92.32(15)
88.50(15)
88.68(15)
90.51(16)
88.38(14)
91.46(15)
91.16(15)
88.70(15)
90.43(14)
88.53(15)
Standard deviation in parenthesis.
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Table 2. Electronic spectraa and redox datab for iron(II) complexes and their adducts in acetonitrile
solution.
c
Complex
λmax, nm
ʋst
Epa
(V)
Epc
(V)
ΔE
(mV)
E1/2 (V)
DPV
(ε,M-1 cm-1)
CV
(cm-1)
1
470 (724)
1.13
0.98
150
1.05
1.06
378 (1870)
341 (183 ×102)
259 (109 ×105)
-
-
2
3
478 (219)
1.3
1.1
200
190
1.2
1.32
1.2
345 (1250)
341 (189×102)
259 (1112×105)
418 (402)
1.24
1.05
1.14
386 (1744)
340 (183×102)
263 (109×105)
-
1a
1b
2a
430 (305)
1605
1.12
1.08
1.3
0.97
0.96
1.1
-
-
-
1.04
1.02
1.2
1.05
1.03
1.25
348 (507)
280 (146×105)
490 (628)
1617
1602
417 (1837)
341 (155×102)
249 (97×105)
440 (305)
338 (507)
278 (146×105)
221 (119×105)
2b
3a
485 (618)
1619
1593
1.27
1.25
1.12
1.08
-
-
1.12
1.17
-
413 (1878)
339 (135×102)
251 (102×105)
430 (305)
1.2
348 (507)
280 (146×105)
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aConcentration of iron(II) complexes, 1 × 10-3 M; Ligand-based transitions were measured in 1 × 10-7 M.bElectrochemistry:
concentration of complexes:1 ×10-3 M; TBAP (0. 1 M) as supporting electrolyte in acetonitrile. Ag(s)/Ag+ (0.01 M, 0.10 M TBAP)
as reference electrode; add 0.681 V to convert to NHE. Platinum disc and platinum wire were used as working and counter
c
electrodes respectively. Scan rate: 100 mV s-1 calibrated with ferrocene. carbonyl stretching frequency
Table 3. Kinetic data and isolated yields for oxidative cleavage of complex-substrate adducts in
CH3CN
+
SO3CF3
N
O
B
O
O
R
O2
OH
CH3CN
N
N
OH
O
Fe
A
C
O
O
1b,
R= -Me
Adduct
pH
kobs
kO2
t1/2 (h)a
Cleavage
2b
, R = -OH
+ O2
(× 10-5 s-1)
7.34 ±0.02
(× 10-3 M-1 s-1)
9.41 ± 0.09
products (%)b
ND
3b
, R= -OMe
1a
-
7.4
5.4
7.3
5.3
7.4
5.4
5.1
5.0
5.1
2.62
0.05
5.02
0.14
4.58
0.22
74.03
310.4
337.7
H+
-
360.18 ±0.47 460 ± 0.66
3.83 ± 0.014 4.92 ± 0.07
133.33 ±0.36 170.28±0.50
ND
2a
ND
H+
-
ND
3a
3a
1b
2b
3b
4.20 ± 0.01
86.32 ± 0.29 110.24± 0.21
0.26 ± 0.01 0.332 ± 0.08
5.37 ± 0.09
ND
H+
H+
H+
H+
ND
A(42), B (29)
A(26), B (24)
A(18),B (22)
0.062 ± 0.03 0.079 ± 0.23
0.057 ± 0.02 0.0772± 0.15
akO2 = kobs/[O2]. [O2] in CH3CN, 8.1×10-3 M; t1/2= 0.693/ kobs; bIsolated yields. ND – not detected. H+: the half
equivalent of acetic acid
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Bioinspired Models for an Unusual 3-Histidine Motif of Diketone
Dioxygenase Enzymes
Ramamoorthy Ramasubramanian,a Karunanithi Anandababu,a Nadia C. Mösch-Zanetti,b
Ferdinand Belajb and Ramasamy Mayilmurugana*
Iron(II) complexes of tris-pyridine ligands have been showed as accurate models for an
unusual 3-His motif of DKDO. Their diketonate adducts were showed regioselective C-C cleavage
with O2.
His62
His64
His104
O
O
O
O
Ph
Ph
OH
OH
(40 - 71%)
O2
Regioselective Csp3-Csp3 Cleavage