A synthetic model of the putative Fe(II)-iminobenzosemiquinonate intermediate in the catalytic cycle of o-aminophenol dioxygenases

Article abstract of DOI:10.1021/ja212163t

The oxidative ring cleavage of aromatic substrates by nonheme Fe dioxygenases is thought to involve formation of a ferrous-(substrate radical) intermediate. Here we describe the synthesis of the trigonal-bipyramdial complex Fe(^Ph2Tp)(ISQ^tB

Full text of DOI:10.1021/ja212163t

Communication
pubs.acs.org/JACS
A Synthetic Model of the Putative Fe(II)-Iminobenzosemiquinonate
Intermediate in the Catalytic Cycle of o-Aminophenol Dioxygenases
Michael M. Bittner, Sergey V. Lindeman, and Adam T. Fiedler*
Department of Chemistry, Marquette University, P.O. Box 1881, 535 North 14th Street, Milwaukee, Wisconsin 53201, United States
S
* Supporting Information
proposed mechanism is the superoxo-Fe2+-(iminobenzo)-
ABSTRACT: The oxidative ring cleavage of aromatic
semiquinonate intermediate (B) that is thought to form after
substrates by nonheme Fe dioxygenases is thought to
O2 binding to the enzyme−substrate complex (A). The
involve formation of a ferrous−(substrate radical) inter-
development of radical character on the substrate ligand
mediate. Here we describe the synthesis of the trigonal-
presumably facilitates reaction with the bound superoxide,
bipyramdial complex Fe(Ph2Tp)(ISQtBu) (2), the first
synthetic example of an iron(II) center bound to an
iminobenzosemiquinonate (ISQ) radical. The unique
electronic structure of this S = 3/2 complex and its one-
electron oxidized derivative ([3]+) have been established
on the basis of crystallographic, spectroscopic, and com-
putational analyses. These findings further demonstrate
the viability of Fe2+−ISQ intermediates in the catalytic
cycles of o-aminophenol dioxygenases.
yielding the key Fe2+-alkylperoxo intermediate (C).3 Although
the electronic structure of B remains somewhat controversial,4
evidence in favor of substrate radical character has been
provided by radical-trap experiments5 and DFT calculations,3 as
well as a remarkable X-ray structure of the Fe/O2 adduct of an
extradiol dioxygenase in which the radical character of the
bound substrate was inferred from its nonplanar geometry.6
Despite these biological precedents, synthetic analogues of
intermediate B in which a ferrous center is coordinated to an
(iminobenzo)semiquinone radical, (I)SQ, have been lacking in
the literature, even though numerous ferric complexes with
such ligands exist.7−11 Herein, we report the synthesis and
detailed characterization of an Fe2+−ISQ complex, 2, that
represents the first synthetic model of this important type of
enzyme intermediate. We also examine the geometric and
electronic structures of the species [3]+ generated via one-
electron oxidation of 2.
In our efforts to generate synthetic models of HAD, we have
used the tris(pyrazolyl)borate ligand, Ph2Tp,12 to mimic the
facial His2Glu coordination environment of the enzyme active
site. The reaction of [(Ph2Tp)Fe(OBz)]13 with 2-amino-4,6-di-
tert-butylphenol (tBuAPH2) in the presence of base provided the
light yellow complex [(Ph2Tp)Fe2+(tBuAPH)] (1) in 71% yield.
The X-ray crystal structure of 1 reveals a five-coordinate (5C)
Fe2+ center in which the tBuAPH− ligand binds in a bidentate
fashion (Figure 1; crystallographic details are summarized in
Table S1 in the Supporting Information). The average Fe1−
NTp bond length of 2.15 Å is typical of high-spin Fe2+
complexes with Tp ligands,13,14 while the short Fe1−O1
distance of 1.931(1) Å is consistent with coordination by an
aminophenolate anion (Table 1). The complex adopts a
distorted trigonal-bipyramidal geometry (τ = 0.6115) with the
amino group of tBuAPH− in an axial position trans to N5. To
the best of our knowledge, 1 represents the first synthetic
model of an aminophenol dioxygenase.
n biochemical pathways, the oxidative ring cleavage of
Isubstituted aromatic compounds, such as catechols and o-
aminophenols, is generally performed by mononuclear non-
heme iron dioxygenases.1 While these enzymes are usually
found in bacteria, some play important roles in human metab-
olism: for instance, a key step in tryptophan degradation in-
volves the O2-mediated ring cleavage of 3-hydroxyanthranilate
(HAA) by HAA-3,4-dioxygenase (HAD; Scheme 1).2 With the
Scheme 1. Reaction Catalyzed by HAA Dioxygenase (HAD)
exception of the intradiol catechol dioxygenases, the ring-
cleaving dioxygenases share a common O2-activation mecha-
nism, illustrated in Scheme 2.1 A notable feature of this
Scheme 2. Catalytic Cycle of Ring-Cleaving Dioxygenases
Reaction of 1 with 1 equiv of 2,4,6-tri-tert-butylphenoxy
radical (TTBP•) at RT in CH2Cl2 gives rise to a distinct
chromophore, 2, with a broad absorption manifold centered at
715 nm (εmax = 0.76 mM−1 cm−1; see Figure 2). Addition of
MeCN, followed by cooling to −30 °C, provides pale green
Received: December 29, 2011
Published: March 14, 2012
© 2012 American Chemical Society
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Communication
(2.150 Å), suggesting minimal change in Fe charge. Metric
parameters for the O,N-coordinated ligand, however, are
dramatically different in the two structures. In the structure
of 1, the six C−C bonds of the tBuAPH− ring are approximately
equidistant (1.40 0.02 Å), reflecting its closed-shell, aromatic
nature. In contrast, the corresponding C−C bond distances in 2
exhibit the “four long/two short” distortion commonly
observed for quinoid moieties (Table 1).7−11 The short O1−
C1 and N7−C2 distances of 1.285(3) and 1.328(4) Å,
respectively, are also characteristic of ISQ− ligands, as amply
demonstrated by Wieghardt8−10 and others.7 Thus, the X-ray
crystallographic data strongly support the formulation of 2 as
[(Ph2Tp)Fe2+(tBuISQ)]. This assignment rationalizes the
absorption spectrum of 2, which closely resembles those
reported for Co3+ and Ni2+ complexes with a lone ISQ−
ligand.9a
Figure 1. Synthesis and thermal ellipsoid diagram of complex 1. For
the sake of simplicity, the 5-Ph substituents of the Ph2Tp ligand have
been omitted and only the amino hydrogens are shown. Selected bond
lengths are provided in Table 1.
Table 1. Selected Bond Distances (Å) for Complexes 1−3
The X-band EPR spectrum of 2 displays an intense peak at
g = 6.5, along with a broad derivative-shaped feature centered
near g = 1.8 (Figure 3). Such spectra are typical of S = 3/2
a
1
2
[3]SbF6
Fe1−N1
Fe1−N3
Fe1−N5
Fe1−O1
Fe1−N7
O1−C1
N7−C2
C1−C2
C2−C3
C3−C4
C4−C5
C5−C6
C1−C6
2.101(1)
2.127(1)
2.223(1)
1.931(1)
2.214(1)
1.345(2)
1.451(2)
1.398(2)
1.388(2)
1.388(2)
1.403(2)
1.394(2)
1.420(2)
2.108(2)
2.087(2)
2.216(2)
2.095(2)
1.982(2)
1.285(3)
1.328(4)
1.469(5)
1.413(4)
1.363(4)
1.427(4)
1.375(4)
1.440(4)
2.071(7)
2.038(7)
2.134(6)
2.082(6)
2.017(8)
1.26(1)
1.33(1)
1.47(1)
1.42(1)
1.35(2)
1.43(2)
1.37(2)
1.44(1)
Figure 3. X-band EPR spectrum of 2 at 20 K. The derivative-shaped
feature at g = 4.3 ( ) arises from a minor ferric impurity, while the
feature at g = 2.0 (*) is due to a residual TTBP radical. Parameters
used to generate the simulated spectrum are provided in the text.
▼
a
The bond distances listed here represent the average distance in the
two independent units of [3]+, while the uncertainty is taken to be the
larger of the two σ-values.
systems with large and rhombic zero-field splitting parame-
ters.9,16 The simulated spectrum in Figure 3 assumed a negative
D-value (with |D| ≫ hν), an E/D-ratio of 0.24, and g-values of
2.36, 2.30, and 2.17. Significant E/D strain was incorporated to
adequately account for the broadness of the higher-field
features. The combined experimental results therefore indicate
that 2 contains a high-spin Fe2+ center (S = 2) antiferro-
magnetically coupled to a tBuISQ radical anion.
Further evidence in favor of a ligand-based radical was
obtained from density functional theory (DFT) calculations.
Two geometry-optimized models of 2 with S = 3/2 were
computed that differ with respect to their electronic
configurations. Analysis of the geometric and electronic
structure of the first model (2A) indicates that it contains an
intermediate-spin Fe3+ center coordinated to a closed-shell
imidophenolate ligand, tBuAP2−. The optimized structure of 2A
features a square-pyramidal geometry (τ = 0.18) with very short
Fe−O1 and Fe−N7 distances of ∼1.87 Å, in poor agreement
with the experimental structure (Table S2). Furthermore, the
computed bond distances for the tBuAP2− ligand deviate sharply
from the distances found experimentally for 2, with nearly all
such differences being significantly greater than the estimated
error (3σ) in the crystallographic data. The second model (2B)
was generated via a broken-symmetry calculation in order to
obtain the [(Ph2Tp)Fe2+(tBuISQ)] electronic configuration
described above. The resulting structure accurately reproduces
Figure 2. Electronic absorption spectra of 1 ( - -), 2 (), and
[3]SbF6 (- - -) measured in CH2Cl2 at RT.
crystals of 2 suitable for crystallographic analysis. As with 1, the
X-ray structure of 2 features a neutral 5C Fe complex with a
distorted trigonal-bipyramidal geometry (τ = 0.58), although
O1 now occupies an axial position instead of N7 (Figure S1).
The N7 atom in 2 is monoprotonated, confirming that 2 is
generated via abstraction of a H-atom from the −NH2 group of 1.
Interestingly, the average Fe1−NTp bond distance observed
for 2 (2.136 Å) is nearly identical to the value found for 1
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Journal of the American Chemical Society
Communication
the overall trigonal-bipyramidal geometry of 2 and provides
reasonably consistent Fe−ligand distances. Most importantly,
the computed and experimental tBuISQ− bond distances exhibit
remarkable agreement, with an rms deviation of merely 0.007 Å
(Table S2). Model 2B is also 9 kcal/mol more stable than 2A,
indicating an energetic preference for the Fe2+−tBuISQ form.
To the best of our knowledge, the electronic structure of 2
has no precedent among synthetic complexes. While Fe2+−SQ
intermediates are often invoked in the mechanisms of catechol
dioxygenases, all relevant models to date feature unambiguous
[Fe3+−catecholate]+ units.17,18 Similarly, the Fe3+−ISQ com-
plexes generated by Wieghardt and co-workers exclusively
undergo ligand-based reductions to give the corresponding
Fe3+−AP species.8,9 The unique Fe2+−ISQ configuration of 2 is
likely due to the presence of a high-spin, 5C Fe ion, whereas related
complexes prepared by Wieghardt (such as [(L)Fe3+(RISQ)]+,
and Fe2+−tBuIBQ limits. Detailed spectroscopic studies are
currently underway to better understand the electronic
structure of [3]+.
Complexes 1−3 replicate key structural and electronic
aspects of the proposed o-aminophenol dioxygenase mecha-
nism. In particular, the conversion of 1→2 mimics the
transformation of the enzyme−substrate complex (A) into a
ferrous−ISQ species (B) via coupled proton and electron
transfers. Our results therefore provide a synthetic precedent
for the existence of Fe2+−ISQ intermediates in enzymatic
catalysis. Of course, complex 2 is an imperfect model of
intermediate B, since it lacks the coordinated superoxo ligand.
Attempts are currently in progress to characterize species
formed during the reaction of 1 and 2 with O2 (and its
surrogate, NO). These studies will yield further insights into
the role of noninnocent ligands in ring-cleaving dioxygenase
mechanisms.
t
where L = cis-cyclam and R = H or Bu) generally feature low-
spin, 6C Fe centers.8 Thus, changes in spin state and coordina-
tion geometry are capable of shifting the delicate balance
between the Fe2+−ISQ and Fe3+−AP valence tautomers.
Reaction of 2 with 1 equiv of an acetylferrocenium salt in
CH2Cl2 provides a dark green species, [3]+, with intense
absorption features at 770 and 430 nm (Figure 2). Treatment
of [3]+ with 1 equiv of reductant (such as Fe(Cp*)2) fully
regenerates 2 (Figure S2), indicating that the two species are
related by a reversible one-electron process. EPR experiments
with frozen solutions of [3]+ failed to detect a signal in either
perpendicular or parallel mode, indicative of an integer-spin
system. Indeed, the magnetic moment of [3]+ was found to be
5.0(1) μB at RT, close to the spin-only value for an S = 2
paramagnet.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details, computational methods and models,
crystallographic structures and data (CIFs), and absorption
spectra of the interconversion of 2 and [3]+. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
adam.fiedler@marquette.edu
Notes
The authors declare no competing financial interest.
X-ray quality crystals of [3]SbF6 were prepared by vapor
diffusion of pentane into a concentrated dichloroethane
solution. The resulting structure (Figure S3) contains two
symmetrically independent Fe units, each featuring a distorted
square-pyramidal geometry (τ = 0.42 and 0.38). Despite the
difference in charge, complexes [3]+ and 2 have identical
atomic compositions. Yet the average Fe−NTp bond distance
shortens from 2.132 to 2.081 Å upon conversion of 2 to [3]+,
suggesting an increase in Fe-based charge. While the structural
parameters of the bidentate O,N-donor ligand of [3]+ are
consistent with a tBuISQ− radical, it was not possible to rule out
a neutral iminobenzoquinonate ligand (tBuIBQ) due to sizable
uncertainties in the bond distances.
We therefore turned to DFT calculations to further explore
the electronic structure of [3]+. The resulting geometry-
optimized model, [3DFT]+, exhibits good agreement with the
crystallographic data, although the DFT structure is more dis-
torted toward the trigonal-bipyramidal limit (τ = 0.64; Table S3).
The computed Fe−ligand bond distances nicely match the
experimental values (rms deviation = 0.022 Å), indicating that
ACKNOWLEDGMENTS
■
We thank Dr. Brian Bennett for generously allowing us to
perform EPR experiments at the National Biomedical EPR
Center (supported by NIH P41 Grant EB001980), and for
assistance with the simulation. We are also grateful to the NSF
(CAREER CHE-1056845) and Marquette University for
financial support.
REFERENCES
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