Radical Rebound Hydroxylation Versus H-Atom Transfer in Non-Heme Iron(III)-Hydroxo Complexes: Reactivity and Structural Differentiation

Article abstract of DOI:10.1021/jacs.9b01516

The characterization of high-valent iron centers in enzymes has been aided by synthetic model systems that mimic their reactivity or structural and spectral features. For example, the cleavage of dioxygen often produces an iron(IV)-oxo that has been characterized in a number of enzymatic and synthetic systems. In non-heme 2-oxogluterate dependent (iron-2OG) enzymes, the ferryl species abstracts an H-atom from bound substrate to produce the proposed iron(III)-hydroxo and caged substrate radical. Most iron-2OG enzymes perform a radical rebound hydroxylation at the site of the H-atom abstraction (HAA); however, recent reports have shown that certain substrates can be desaturated through the loss of a second H atom at a site adjacent to a heteroatom (N or O) for most native desaturase substrates. One proposed mechanism for the removal of the second H-atom involves a polar-cleavage mechanism (electron transfer-proton transfer) by the iron(III)-hydroxo, as opposed to a second HAA. Herein we report the synthesis and characterization of a series of iron complexes with hydrogen bonding interactions between bound aquo or hydroxo ligands and the secondary coordination sphere in ferrous and ferric complexes. Interconversion among the iron species is accomplished by stepwise proton or electron addition or subtraction, as well as H-atom transfer (HAT). The calculated bond dissociation free energies (BDFEs) of two ferric hydroxo complexes, differentiated by their noncovalent interactions and reactivity, suggest that neither complex is capable of activating even weak C-H bonds, lending further support to the proposed mechanism for desaturation in iron-2OG desaturase enzymes. Additionally, the ferric hydroxo species are differentiated by their reactivity toward performing a radical rebound hydroxylation of triphenylmethylradical. Our findings should encourage further study of the desaturase systems that may contain unique H-bonding motifs proximal to the active site that help bias substrate desaturation over hydroxylation.

Full text of DOI:10.1021/jacs.9b01516

Article
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Radical Rebound Hydroxylation Versus H-Atom Transfer in Non-
Heme Iron(III)-Hydroxo Complexes: Reactivity and Structural
Differentiation
Michael J. Drummond,† Courtney L. Ford,† Danielle L. Gray,† Codrina V. Popescu,‡
and Alison R. Fout*,†
†School of Chemical Sciences, University of Illinois at UrbanaChampaign, 600 South Mathews Avenue, Urbana, Illinois 61801,
United States
‡Department of Chemistry, University of Saint Thomas, 2115 Summit Avenue, Saint Paul, Minnesota 55105, United States
S
* Supporting Information
ABSTRACT: The characterization of high-valent iron
centers in enzymes has been aided by synthetic model
systems that mimic their reactivity or structural and spectral
features. For example, the cleavage of dioxygen often produces
an iron(IV)-oxo that has been characterized in a number of
enzymatic and synthetic systems. In non-heme 2-oxogluterate
dependent (iron-2OG) enzymes, the ferryl species abstracts
an H-atom from bound substrate to produce the proposed
iron(III)-hydroxo and caged substrate radical. Most iron-2OG
enzymes perform a radical rebound hydroxylation at the site
of the H-atom abstraction (HAA); however, recent reports
have shown that certain substrates can be desaturated through
the loss of a second H atom at a site adjacent to a heteroatom (N or O) for most native desaturase substrates. One proposed
mechanism for the removal of the second H-atom involves a polar-cleavage mechanism (electron transfer-proton transfer) by
the iron(III)-hydroxo, as opposed to a second HAA. Herein we report the synthesis and characterization of a series of iron
complexes with hydrogen bonding interactions between bound aquo or hydroxo ligands and the secondary coordination
sphere in ferrous and ferric complexes. Interconversion among the iron species is accomplished by stepwise proton or electron
addition or subtraction, as well as H-atom transfer (HAT). The calculated bond dissociation free energies (BDFEs) of two ferric
hydroxo complexes, differentiated by their noncovalent interactions and reactivity, suggest that neither complex is capable of
activating even weak C−H bonds, lending further support to the proposed mechanism for desaturation in iron-2OG desaturase
enzymes. Additionally, the ferric hydroxo species are differentiated by their reactivity toward performing a radical rebound
hydroxylation of triphenylmethylradical. Our findings should encourage further study of the desaturase systems that may
contain unique H-bonding motifs proximal to the active site that help bias substrate desaturation over hydroxylation.
then perform a radical rebound reaction to hydroxylate the
substrate (Figure 1, right). Computational studies have
estimated an activation barrier of 1−4 kcal/mol for substrate
hydroxylation, with an intermediate lifetime of ∼20 ps,13−17
but attempts to trap an intermediate using a radical clock have
been unsuccessful.
It has recently been shown that the presence of an arginine
residue in proximity of the enzyme active site is necessary to
properly position the oxo moiety and substrate for
hydroxylation.18 Alternatively, the absence of the arginine in
proximity of the active site, among other factors such as the
presence of adjacent heteroatoms (N or O) in substrate, may
favor alternate reactivity, such as substrate desaturation (Figure
1, left),19−21 ring formation,22 decarboxylation,23 or halogen-
INTRODUCTION
■
The characterization of short-lived intermediates in enzymatic
cycles has often been aided by synthetic model systems with
well-defined molecular scaffolds. For example, extensive work
has detailed the spectroscopic properties, structural parame-
ters, and reactivity trends of iron(IV)-oxo species in synthetic
systems.1−5 These findings have been invaluable in assigning
key ferryl intermediates in nonheme 2-oxogluterate dependent
(iron-2OG) enzymes that activate dioxygen.6−8
The ferryl functionality, resulting from the cleavage of
dioxygen, has been identified in enzymatic systems using
stopped-flow UV−visible and Mossbauer spectroscopies.
The proposed subsequent step involves hydrogen atom
abstraction (HAA) from a bound organic substrate to form a
caged substrate radical and an iron(III)-hydroxo species
(Figure 1, center). This ferrichydroxo species, which has not
been observed spectroscopically in enzymatic systems,10,12 can
9−11
̈
Received: February 8, 2019
© XXXX American Chemical Society
A
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Figure 1. Alternative reactivity pathways from a proposed iron(III)-hydroxide intermediate in iron-2OG enzymes.
ation.24 The divergence in reactivity and lack of spectroscopic
information about the iron(III)-hydroxo intermediate leads us
to suggest that noncovalent interactions with the ferric-
hydroxide may dictate some part of the differing reactivity
observed within iron-2OG enzymes.
The few reports of synthetic iron(III)-hydroxo complexes
have mostly required the presence of hydrogen bond donors in
the secondary coordination sphere to stabilize the com-
plex,25−35 and none of these systems have mimicked the C−
O(H) bond formation step common in iron-2OG enzymes.
Goldberg, however, has recently reported iron(III)-methoxide
and iron(IV)-hydroxo complexes that demonstrated radical
rebound-like reactions with the addition of trityl-based C-
centered radicals.36,37
Our group has previously reported the tripodal, tetradentate
ligand, tris(5-cyclohexyliminopyrrol-2-ylmethyl)amine (N-
(piCy)3) (Scheme 1a), that binds first-row transition metal
ions (M = Mn, Fe, Co, Zn), and features a secondary
coordination sphere that interacts with an axial ligand bound
to the metal center.38−40 The arms of the ligand are capable of
tautomerizing from the pyrrole-imine (pi) tautomeric form to
the azafulvene-amine (afa) tautomeric form (Scheme 1c).
Moreover, this tautomerization allows for versatility in the
primary coordination sphere of the ligand (from anionic to
neutral) and the noncovalent interactions imparted by the
secondary coordination sphere (hydrogen bond accepting or
donating).
Inspired by the work of the Chang27 and Goldberg41−43
groups, who have used polypyridyl ligands with a secondary
coordination sphere to study high-valent iron chemistry, we
sought to combine the polypyridyl system with the
tautomerizable pyrrole-imine motif into a single ligand scaffold.
Herein, we report the synthesis of a tetrapodal, pentadentate
ligand, 2,2′,2′-methylbis-pyridyl-6-(2,2′,2′-methylbis-5-cyclo-
hexyliminopyrrol)-pyridine, Py2Py(piCy)2, which features two
of the pyrrole-imine arms previously described (Scheme
1b).44,45 The ligand was complexed with iron(II) salts, and
the synthesis and characterization of five iron complexes are
reported, including two unique iron(III)-hydroxo complexes
with differentiated electronic structures and reactivity analo-
gous to iron-2OG enzymatic systems.
Scheme 1. a) Tripodal Ligand N(piCy)3 Reported in Prior
Studies, b) Tetrapodal Ligand Reported in This Study, and
c) Ligand Arm Tautomerization
RESULTS AND DISCUSSION
■
Synthesis of Py2Py(piCy)2. The synthesis of 1,1-dipyridyl-
ethane was achieved from the reaction of 2-ethylpyridine and
nbutyllithium, followed by the addition of half an equivalent of
2-fluoropyridine. Next, the deprotonated 1,1-dipyridyl was
combined with 2-bromo-6-(1,3-dioxylane)pyridine in tetrahy-
drofuran, with the solvent being refluxed for 36 h to furnish
L1. The deprotection of L1 with aqueous 3 M HCl yielded L2,
which was further functionalized through the installation of
two pyrrole functionalities (L3). Formylation at the 5' position
of the pyrrole moieties was accomplished through a
Vilsmeier−Haack reaction (L4), and the subsequent con-
densation with cyclohexylamine produced the desired ligand,
Py2Py(piCy)2 in good yields (Scheme 2).
Synthesis of Iron(II) Complexes. Py2Py(piCy)2 was
dissolved in tetrahydrofuran to form a pale yellow solution
to which Fe(OTf)2(MeCN)2 (OTf = trifluoromethanesulfo-
nate) was added. An immediate color change to deep red was
observed, followed by the precipitation of a yellow solid. After
1 h of stirring, the suspension was filtered and the yellow
precipitate, [Py2Py(afaCy)2FeIIOTf](OTf) (1), was isolated
(Figure 2). Characterization of this compound by IR
spectroscopy revealed tautomerization of the pyrrole-imine
moieties of the free ligand to the azafulvene-amine tautomeric
form, with a CN stretch at 1635 cm−1 (Figure S11 of the
Supporting Information, SI); the same was reported for the
corresponding iron(II) complex of the tripodal ligand system,
B
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a
Scheme 2. Synthesis of Py2Py(piCy)2
a
n
(i) BuLi (1.6 M in hexanes), THF, 0.5 h, −78 °C (ii) 0.5 equiv 2-fluoropyridine, −20 °C to reflux, 1 h (iii) 0.4 equiv 2-bromo-6-(1,3-
dioxylane)pyridine, 36 h, THF, reflux (iv) 3 M HCl, 4 h, rt (v) excess pyrrole, 3 M HCl, THF, 18 h (vi) 2.1 equiv POCl3, DMF/DCM, 2 h,
NaOAc/H2O, 45 °C, 1 h (vii) 2.1 equiv cyclohexylamine, DCM, 18 h. See the Experimental Section for full details and characterization.
Figure 2. Complexation of Py2Py(piCy)2 and interconversion between iron complexes. All pKa and reduction potential values are reported in
acetonitrile.
N(afaCy)3FeIIOTf2).38 Stretches observed at 3215 and 3285
cm−1 were assigned as N−H stretches of the amines. Crystals
suitable for X-ray diffraction were grown from vapor diffusion
of diethyl ether into a concentrated solution of 1 in
acetonitrile. Refinement of the data revealed an octahedral
iron(II) center coordinated to three pyridyl nitrogen atoms,
two azafulvene nitrogen atoms, and one oxygen atom of a
triflate anion (Figure 3). Both of the amine moieties of the
secondary coordination sphere were pointed away from the
iron center, with one amine engaged in hydrogen bonding to
the outer-sphere triflate.
To determine if the ligand arms were able to hydrogen bond
with a bound axial ligand, 1 was reacted with KOH in
acetonitrile, turning the yellow solution bright red over the
course of 1 h. After the removal of solvent in vacuo, the residue
was treated with dichloromethane to solubilize the desired
metal species. Analysis of the red product, [Py2Py-
(afaCy)2FeIIOH]OTf (2), by IR spectroscopy showed a C
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Figure 3. Structural characterization of complexes 1−5. Complexes 3 and 4 contained half of the target molecule in the unit cell with the other half
being symmetry generated. Non-hydrogen bonding hydrogen atoms, anions, and solvent molecules have been omitted for clarity.
Table 1. Selected Bond Distances (Å), Angles (°), and IR Data of Iron(II) and Iron(III) Complexes, 1−5
1
2
3
4
5
Fe−O1
Fe−N1
Fe−Nafa
Fe−Nafa
2.2035(18)
2.2078(19)
2.1313(19)
2.1313(19)
2.0020(12)
2.2531(14)
2.1498(15)
2.1639(14)
2.0954(17)
2.218(2)
1.861(8)
2.211(10)
2.074(7)
1.8755(18)
2.245(2)
2.106(2)
Fe−Npi
Fe−py(4/5)(avg)
2.1394(14)
2.2095(14)
2.066(2)
2.201(2)
2.670(3)
2.747(3)
103.66
2.186(2)
2.2238(14)
2.6853
2.183(7)
2.769
N
afa(H)---O (avg)
Npi---(H)O (avg)
2.7490(16)
99.77
1615
Fe−O−N6/7(avg)
102.72
1656
3640
104.72
1660
3585
νC=N(cm−1
)
1635
3215, 3285
1616, 1658
νO−Hor νN−H(cm−1
)
N stretch at 1656 cm−1, consistent with the azafulvene−amine
tautomeric form of the ligand, and a feature at 3640 cm−1,
assigned as an O−H stretch (Figure S13). Crystals suitable for
X-ray diffraction were grown from the vapor diffusion of
diethyl ether into a concentrated solution of 2 in acetonitrile.
Analysis of the data showed a hydroxo ligand bound to the iron
center (Figure 3). Both azafulvene-amine arms of the
secondary coordination sphere were engaged in hydrogen
bonding to the hydroxo ligand with distances of 2.6666(18)
and 2.7040(19) Å between the O and Nafa hydrogen bond
donor (Table 1). The Fe−O distance of 2.0020(12) Å is
similar to reported iron(II)-hydroxo complexes.26,27,38,46−49
The solution magnetic moment (μeff) of 5.21 μB was
determined by Evans’ method, corroborating the assignment
of 2 as a high spin S = 2 iron(II) species.
It was determined that 2 could be converted back to 1
through the addition of 2-aminobenzimidazolium triflate (pKa
= 16.1 in acetonitrile)50 to form what we tentatively assign as
an iron(II)-aquo species (Figure S25); subsequent release of
water reforms 1 upon removal of volatiles or attempts at
crystallization (Figure 2). Alternatively, the addition of
benzylammonium triflate (pKa = 16.9 in acetonitrile) showed
1
no reactivity toward 2, as assayed by H NMR spectroscopy
(Figure S26). The observed reactivity led to the assignment of
a lower limit pKb value of 16.1 for 2 (the exact pKb may be
slightly different due to ligand exchange energetics between the
loss of water and triflate binding to the iron).
We were also interested in investigating both the acidity of 2
and the possibility of anionic coordination of the ligand to the
iron center. The addition of 1 equiv of KH to 2 in acetonitrile
led to the formation of a bright pink precipitate identified as
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Py2Py(piCy)2FeIIOH2 (3). The pKa of 2 was determined
through the addition of bases with reported pKa values in
acetonitrile50 and monitoring the solution for the precipitation
of 3. It was determined that triethylamine (pKa = 18.82) could
not deprotonate 2 (Figure S27), while addition of pyrrolidine
(pKa = 19.56) led to the precipitation of 3, establishing a lower
bound for the pKa for 2. Complex 3 could also be protonated
with 2,6-lutidinium triflate (LuHOTf) to reform 2 (Figure 2).
Further characterization of 3 was achieved with IR
spectroscopy, displaying a single CN stretch in the IR
spectrum at 1615 cm−1 (Figure S14), consistent with
previously reported values of anionic coordination of the
pyrrole-imine tautomeric form.38 Crystals suitable for X-ray
diffraction were grown from vapor diffusion of diethyl ether
into a concentrated solution of 3 in a 5:1 mixture of
dimethylacetamide and dichloromethane. The resulting
structure showed an iron(II)-aquo complex, with an Fe−O
bond length of 2.0954(17) Å (Figure 3).
Furthermore, the interconversion of the ferric hydroxide
complexes was achieved by the addition of LuHOTf to 5 or
KH to 4. The pKa of 4 was determined through the addition of
bases with reported pKa values in acetonitrile50 and monitoring
1
the solution by H NMR spectroscopy for the formation of 5.
It was determined that benzylamine (pKa = 16.91) could
deprotonate 4 while 2-aminobenzimidazole (pKa = 16.08)
could not (Figure S28 and S29), giving an estimated pKa of
16.5 ( 0.4) (Figure 2).
Characterization of Electronic Structure. The elec-
tronic structures of the iron complexes were investigated using
̈
̈
Mossbauer spectroscopy. The 6 K Mossbauer spectra of
polycrystalline powders of compounds 1−3 show quadrupole
doublets (Figure 4) with isomer shifts between 1.10 and 1.15
Synthesis of Iron(III) Complexes. With the iron(II)
complexes in hand, the oxidations of 2 and 3 to their
corresponding ferric species were investigated (Figure 2).
Silver triflate (AgOTf) was added to a solution of 2 in
dichloromethane in the dark, turning the bright red solution
dark red-brown immediately. Filtration of the solution revealed
the presence of Ag0, and upon evaporation of the filtrate, a
brown residue was obtained. Analysis of the product by IR
spectroscopy showed a CN stretching frequency at 1660
cm−1 (Figure S16), confirming the azafulvene-amine tauto-
meric form of the ligand. There was also a broad stretch
observed at 3585 cm−1 that we tentatively assign as an O−H
stretch. Crystals suitable for X-ray diffraction were grown from
the vapor diffusion of diethyl ether into a concentrated
solution of the metal complex in acetonitrile. Refinement of
the data revealed an iron(III)-hydroxo species, [Py2Py-
(afaCy)2FeIIIOH](OTf2) (4), isostructural to 2, with a
contraction of the Fe−O bond to 1.861(8) Å and the presence
of a second outer-sphere triflate anion (Figure 3). The solution
magnetic moment of 4 was determined by the Evans method
to be 6.11(26) μB, consistent with a high spinS = 5/2 iron(III)
center.26,32,51,52
Similarly, a pink suspension of 3 in dichloromethane was
oxidized in the presence of AgOTf, forming a red-brown
1
solution. Analysis of the product by H NMR spectroscopy
yielded a similar spectrum to 4 (Figure S17); however, a new
resonance at 22 ppm and changes in the broad resonances
between 60 and 120 ppm suggested the formation of a new
species. IR spectroscopy showcased the presence of both the
pyrrole-imine and azafulvene-amine tautomers, with CN
stretches at 1616 and 1658 cm−1, respectively (Figure S18).
Crystals suitable for X-ray diffraction were grown from the
vapor diffusion of diethyl ether into a concentrated solution of
the metal complex in dichloromethane. Refinement of the data
revealed another iron(III)-hydroxo complex, [Py2Py(afaCy)-
(piCy)FeIIIOH]OTf (5), that contained one outer-sphere
triflate anion and the presence of a hydroxo ligand bound to
the iron. The hydroxo proton was acting as a hydrogen bond
donor to a pyrrole-imine ligand arm, with the other ligand arm
in the azafulvene-amine tautomer donating a hydrogen bond to
the hydroxo moiety. (Figure 3). The Fe−O bond length of 5
(1.8755(18) Å) is similar to 4, which was also assigned as an
iron(III)-hydroxo and contracted relative to 2. The solution
magnetic moment of 5 was 5.97(14) μB, supporting its
formulation as a high spin S = 5/2 iron(III) center.
̈
Figure 4. Mossbauer spectra of complexes 1−3 at 6 K and 70 mT for
(1) and (2) and in 0 T (3). Hash marks are raw data, red lines are
spectral simulations with the parameters shown. Full simulation
parameters are located in the SI.
mm/s and large quadrupole splittings (ΔEQ > 1.90 mm/s)
typical for high spin ferrous complexes with N/O coordina-
tion.53 As is typical for molecular complexes in polycrystalline
̈
samples, the electronic spin relaxes quickly on the Mossbauer
time-scale, therefore no hyperfine structure is observed. The
asymmetric broadening of doublets 1 and 2 is consistent with
anisotropic spin relaxation effects and it is not observed at
higher temperatures (>100 K) or in zero field (spectrum of
complex 3). In contrast to 1−3, complexes 4 and 5 exhibit
paramagnetic spectra at 6 K. The isomer shifts of 0.5 mm/s
and small quadrupole splittings are typical for S = 5/2 ground
states, as are the spectral patterns (Table S1).
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Figure 5. Preference in reactivities between complexes 4 and 5. Complex 4 performs radical rebound hydroxylation at a faster rate then H atom
transfer for selected substrates. Complex 5 will not perform radical rebound hydroxylation unless in the presence of opportunistic water, while
readily performing an H atom transfer reaction.
Complexes 4 and 5 were further differentiated using X-band
EPR spectroscopy at 10 K. The spectra are complex and not
easily simulated, but they illustrate the differentiated electronic
structures for each ferric hydroxo species (Figure S24). Further
characterization of the electronic structures of this series of
iron complexes is ongoing due to the complexities of the
BDFE may be slightly different due to the ligand exchange
energetics going between complexes). These data show that 4
and 5 have similar driving forces for the oxidation of substrates
with BDFEs falling between the iron(IV)-oxo (87 kcal/mol)
and iron(III)-oxo (66 kcal/mol) complexes reported by
Borovik.51,56
̈
Mossbauer and EPR spectra.
BDFE(O−H)
=
23.06E1/2 + 1.37pKa + C
Reduction of Iron(III) Complexes. The reversibility of
the iron redox events was examined using cyclic voltammetry
(Figure S23) and chemical reductants. The cyclic voltammo-
grams showed the reversible reduction of 4 to 2 and 5 to 3
with the reduction potential of 5 nearly 300 mV more positive
than that of 4 (Figure 2). The chemical reductions of the
iron(III)-hydroxo complexes, 4 and 5 in acetonitrile, were also
investigated. Addition of cobaltocene (Cp2Co, E0 = −1.33 V vs
Fc0/+) to 4 and 5 produced the corresponding iron(II)
products, 3 from 5, in good yields (99% and 78%,
respectively).
C = 54.9 kcal/mol in acetonitrile
(1)
To assay the calculated BDFEs experimentally, 1,2-
diphenylhydrazine (DPH, BDFE = 69 kcal/mol) was used as
an H-atom transfer source. The addition of DPH to 5 in
acetonitrile resulted in a color change from dark brown-red to
bright red over the course of 1 h (Figure 5, top right). The
formation of azobenzene was noted and 2 was identified as the
only product by 1H NMR spectroscopy (Figure S35).
Similarly, the addition of DPH to 4 resulted in the formation
of the proposed iron(II)-aquo intermediate, before loss of
water resulted in the formation of 1 (Figure 5, top left). The
rates of the reactions were also surveyed under pseudo-first
order conditions using time-resolved UV−visible spectroscopy
(Figures S44 and S45).
Interestingly, the addition of weak C−H bonds such as 1,4-
cyclohexadiene or dihydroanthracene (BDFEs ∼77 kcal/mol
in acetonitrile) resulted in no reaction for either complex
(Figures S37 and S38). Since complexes 4 and 5 are unable to
activate even weak C−H bonds, these species may provide
important mechanistic insights concerningiron-2OG desatur-
ase enzymes.
H-Atom Transfer to Iron(III)-Hydroxo Complexes. One
other synthetic iron(III)-hydroxo has been reported to
perform HAA using 1-hydroxy-2,2,6,6-tetramethylpiperidine
(TEMPO-H, BDFE = 66.5 kcal/mol) to form a ferrous-aquo
species, analogous to dehydration reactions observed in some
nonheme iron enzymes.54 Determination of the iron(II)/
iron(III) redox couples and the pKa values (Figure 2) allowed
for an estimation of the bond dissociation free energies
(BDFEs) of the iron complexes using eq 1.55 Due to the
complicating nonequilibrium effects in the pKa determination
of 2 (precipitation of 3 in acetonitrile), the reduction potential
between 2 and 4 and the pKa between 4 and 5 were used in the
BDFE calculation of 2, which was determined to be
approximately 71 kcal/mol. Similarly, a BDFE of ∼70 kcal/
mol was determined for 1 using the reduction potential
between 2 and 4 and the pKa between 2 and 1 (the exact
The desaturation of C−C bonds is most commonly
observed in heme and di-iron enzymes that maintain an
iron(IV) species after the first HAA from substrate.57 By
comparison, iron-2OG enzymes do not seem as well-equipped
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to perform a second HAA with the formation of an iron(III)-
hydroxo, instead of a more oxidatively potent iron(IV) species.
However, multiple iron-2OG enzymes have demonstrated the
installation of olefins, with most studies invoking a mechanism
of two subsequent HAAs.22,58,59 A recent mechanistic
investigation for one of these desaturases highlighted the
importance a heteroatom (N or O) adjacent to the
desaturation site for most native substrates in iron-2OG
enzymes.20 The heteroatom is proposed to assist in the
removal of the second H-atom through a polar cleavage
mechanism (electron transfer-proton transfer), as opposed to a
concerted HAA by the iron(III)-hydroxo.
Attempts to determine if the hydroxyl ligand is concertedly
transferred to substrate through the use of para-substituted
trityl derivatives were inconclusive due to incompatible
solvents for the production of the monomeric trityl radical
derivatives and solubility of 4. A future study modifying the
ligand scaffold, as has previously been demonstrated,49 should
allow for discrimination between a concerted hydroxyl transfer
mechanism or an electron-transfer cation-transfer (ET-CT)
pathway.
To contrast the differences between the two ferric-hydroxide
complexes, Gomberg’s dimer was added to 5 to determine if it
was also capable of performing the hydroxylation reaction that
is dominant in most iron-2OG enzymes. Over the course of 24
h the reaction mixture turned from dark red-brown to bright
red. Analysis of the reaction by 1H NMR spectroscopy showed
the formation of 2, with partial consumption of Gomberg’s
dimer to form triphenylmethanol over 24 h (Figure S34). The
sluggish rate of the reaction and the formation of 2 (a net H-
atom transfer) suggest that the hydroxylation of Gomberg’s
dimer cannot be accomplished by 5 alone.
The same study, and others60,61 have shown that substrates
without the presence of a heteroatom can also be desaturated
by iron-2OG enzymes. Not all reports of iron-2OG
desaturation enzymes have detailed product profiles, so it is
difficult to know if substrate hydroxylation overrides or is in
close competition with desaturation in all iron-2OG enzymes.
However, the inability of 4 and 5 to react with weak C−H
bonds supports the observation that a second HAA may not be
operative in all desaturation mechanisms for iron-2OG
desaturase enzymes due to the iron(III)-hydroxo intermedi-
ate’s lack of oxidizing power. It is possible that the C−H bond
involved in the second H atom removal (proximal to the C·
from the first HAA) is significantly weakened to the point
where the iron(III)-hydroxo could abstract an H atom, but we
are not aware of studies that identify the extent to which that
C−H bond is weakened.
We hypothesized that adventitious water interacts with 5
and the triphenylmethylradical to perform the small amount of
hydroxylation that is observed (Figure 5, bottom right). To
test this, the reaction of 5 and Gomberg’s dimer was performed
in solvent spiked with H218O. The conversion of 5 to 2 was
observed over the course of 1 h, and analysis of the
triphenylmethanol by EI−MS showed 72% incorporation of
the 18O label into the substrate (Figure S46, Table S3), leading
to the proposed intermediate shown in Figure 5. Alternatively,
an 18O isotopologue of 5 was synthesized from H218O and
reacted in dry solvent for 24 h. Not enough triphenylmethanol
was formed from the reaction to determine the amount of
isotope label that was incorporated, showing the need for
adventitious water for the hydroxylation reaction to occur.
Hydroxylation of Gomberg’s Dimer. In addition to
exploring the oxidative potency of 4 and 5, we sought to
determine if a radical rebound hydroxylation could be
accomplished by the iron(III)-hydroxide complexes.
The addition of 0.5 equiv of Gomberg’s dimer to 4 at room
temperature led to a rapid color change from dark brown-red
to yellow. Analysis of the organic species in the reaction
showed production of triphenylmethanol in high yield (99% by
GC−MS with mesitylene internal standard, Figure S31). The
hydroxylation of the triphenylmethylradical species was
concomitant with the formation of 1 (83% crystalline yield,
Figure 5, bottom left). This reaction represents a rare example
of radical rebound hydroxylation with an iron(III)-hydroxo.
The rate of hydroxylation was investigated further using
UV−visible spectroscopy. The stoichiometric reaction of 4
reacting with Gomberg’s dimer was completed within a minute
with isosbestic conversion to 1 (Figure S40). Under pseudo-
first order conditions with 10 equiv of Gomberg’s dimer, the
hydroyxlation was complete within approximately 10 s at 25
°C (Figure S41). Additionally, an 18O isotopologue of 4 was
synthesized from 1 with the use of H218O, and incorporation
up to 71% was observed for the 18O isotopologue of 4 (Figure
S46, Table S2). We hypothesize the actual isotope
incorporation is higher due to a larger percentage incorpo-
ration of the 18O label into triphenylmethanol (82%, Figure
S49, Table S5) from the reaction of the 18O isotopologue
of 4 with triphenylmethyl radical. The observed differences
suggest there may be some exchange with adventitious water
and the hydroxo ligand of 4 prior to analysis of the metal
complexes by ESI−MS. The exchange of water and the
hydroxo ligand was corroborated by stirring unlabeled 4 in
acetonitrile spiked with H218O for 1 h, leading to ∼50%
incorporation of 18O label in complex 4 (Figure S48, Table
S4).
1
Monitoring the H NMR spectrum of 5 showed no change
in the paramagnetic resonances upon addition of H2O;
however, upon addition of Gomberg’s dimer to this solution,
conversion to 2 was observed (Figure S39). Similarly, the
reaction of 5 and Gomberg’s dimer in wet solvent under
pseudo-first order conditions was monitored by UV−visible
spectroscopy, and showed the reaction proceeding to
completion within 10 min (Figure S43). Monitoring the
reaction in dry solvent did show some consumption of the
triphenylmethylradical, but only minimal conversion was
observed (Figure S42).
We propose that the noncovalent interactions from the
ligand have a profound effect on regulating the hydroxylation
reactivity of complexes 4 and 5 and could be representative of
noncovalent interactions observed in iron-2OG enzymes. Since
the proposed iron(III)-hydroxo intermediate has not been
observed in iron-2OG enzymes, an analysis of the residues
surrounding the iron active site, and subsequent mutagenesis
studies, may lend insight into how desaturase enzymes bias
their reactivity from hydroxylation to desaturation. Our data
suggest the engagement of the hydroxo ligand with a hydrogen
bond acceptor in the secondary coordination sphere may play
a role in deterring the rebound hydroxylation pathway
observed in most iron-2OG enzymes. Further kinetic studies
will need to be conducted on our system to determine the
effect of reduction potential on hydroxylation reactivity, as well
as assigning a concerted hydroxylation mechanism or an ET-
CT mechanism.
G
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Article
path length and capped with a rubber septum. Elemental analyses
were performed by the University of Illinois at Urbana−Champaign
School of Chemical Sciences Microanalysis Laboratory in Urbana, IL.
Samples submitted for elemental analyses were dried under vacuum
for a minimum of 12 h; solvates were confirmed by 1H NMR
spectroscopy. Mass spectra were recorded by the University of Illinois
mass spectroscopy laboratory.
Electrochemical experiments were carried out using a CH
Instruments CHI410C Electrochemical Workstation. The supporting
electrolyte was 0.1 M [nBu4N][PF6] in acetonitrile. A glassy carbon
working electrode, a platinum wire counter electrode, and a silver wire
pseudoreference electrode were used. The concentration of each
analyte was 1 mM. Experiments were performed at a scan rate of 100
mV/s. Each scan was referenced to internal Fc0/+. EPR samples were
prepared in an MBraun glovebox under a dinitrogen atmosphere. The
sample concentration was 5 mM in 1:1 acetonitrile/dichloromethane.
EPR spectra were recorded on a Varian E-line 12′′ Century series X-
band CW spectrometer, and the spectra were simulated using the
program SIMPOW6.3. Analysis by Gas Chromatography Mass
Spectrometry (GC−MS) was performed using a Shimadzu GC-
2010 Plus Gas Chromatograph equipped with a Shimadzu GCMS-
QP2010 SE mass spectrometer using electron impact ionization (EI)
after traveling through a SH-RxiTM-5 ms 30 m × 0.32 mm × 0.25 μm
column with helium carrier gas. Zero and low-field (0.07 T), variable-
CONCLUSIONS
■
The synthesis and characterization of five iron complexes,
including two differentiated ferric hydroxo complexes, was
accomplished. Interestingly, both iron(III)-hydroxo complexes
exhibited BDFEs incapable of abstracting an H atom from even
weak C−H bonds. These data support recent observations in
iron-2OG desaturase enzymes that go through a polar-cleavage
mechanism instead of a second HAA to desaturate substrates.
The two ferric hydroxo complexes showed differing reactivity
toward the hydroxylation of radical substrate, with 5 not
performing hydroxylation without the presence of water. We
attribute the differences in reactivity to the presence of an H-
bond acceptor interacting with the hydroxo ligand and the
lesser oxidizing reduction potential of 5. It is plausible that
iron-2OG desaturase enzymes are also able to tune the metal
reduction potential or employ H-bond acceptors proximal to
the metal active site to help bias the enzyme toward substrate
desaturation and away from hydroxylation.
EXPERIMENTAL SECTION
■
General Considerations. All manipulations of air- and moisture-
sensitive metal compounds were carried out in the absence of water
and dioxygen using a MBraun inert atmosphere drybox under a
dinitrogen atmosphere. All glassware was oven-dried for a minimum
of 8 h and cooled in an evacuated antechamber prior to use in the
drybox. Solvents were dried and deoxygenated on a Glass Contour
System (SG Water U.S.A., Nashua, NH) and stored over 4 Å
molecular sieves (3 Å in the case of acetonitrile) purchased from
Strem prior to use. Chloroform-d1, dichloromethane-d2, and
acetonitrile-d3 were purchased from Cambridge Isotope Laboratories
and stored over 4 Å molecular sieves prior to use. 2,6-
dibromopyridine (Oakwood Chemical), nbutyllithium (1.6 M in
hexanes) (Sigma-Aldrich), dimethylacetamide (Sigma-Aldrich), ethyl-
ene glycol (Macron), p-TSA·H2O (Sigma-Aldrich), 2-fluoropyridine
(Oakwood Chemical), pyrrole (Sigma-Aldrich), POCl3 (Sigma-
Aldrich), cyclohexylamine (Oakwood Chemical), lithium oxide
(Sigma-Aldrich), silver triflate (Strem), potassium hydride (Sigma-
Aldrich), triflic acid (Sigma-Aldrich), potassium hydroxide (Fischer
Scientific), 2,6-lutidine (Sigma-Aldrich), triethylamine (Sigma-Al-
drich), 2-aminobenzimidazole (Sigma-Aldrich), pyrrolidine (Sigma-
Aldrich), benzylamine (Sigma-Aldrich), 1,8-diazabicyclo[5.4.0]undec-
7-ene (Sigma-Aldrich), and H218O (97% enrichment, Sigma-Aldrich)
were purchased from the vendor listed and used as received.
Diphenylhydrazine (Sigma-Aldrich) was recrystallized from a mixture
̈
temperature (5−200 K) Mossbauer spectra were recorded on a
closed-cycle refrigerator spectrometer, model CCR4K (SeeCo, Edina,
MN) equipped with a 0.07 T permanent magnet, maintaining
temperatures between 5 and 300 K. The samples consisted of solid
powders (or crystalline material) suspended in Icosane placed in
Delrin 1.00 mL cups, and frozen in liquid nitrogen. The isomer shifts
are quoted at 5 K with respect to iron metal standard at 298 K.
̈
Mossbauer spectra were analyzed using the software WMOSS4 (Ion
Prisecaru, www.wmoss.org).
2,2′,2′-Methylbis-pyridyl-6-(2-methyl-1,3-dioxolan-2-yl)-
pyridine (L1). L1 was synthesized using a modified literature
procedure.65 To a 250 mL three neck Schlenk flask outfitted with a
gas inlet, reflux condenser topped with a rubber septum, and septum,
were added 2-ethylpyridine (3.0 g, 28.0 mmol) and 50 mL of
anhydrous tetrahydrofuran. N2 was flowed through the reaction vessel
for 5 min. The flask was cooled to −78 °C (dry ice/acetone) for 10
min prior to adding nButyllithium in hexanes (17.5 mL of 1.6 M, 28.0
mmol) within 5 min, turning the yellow solution deep red. After 30
min, the reaction vessel temperature was raised to −20 °C, and 2-
fluoropyridine (1.35 g, 14 mmol, 0.5 equiv) was added dropwise to
the reaction. The solution was brought to room temperature over 20
min, then refluxed for 1 h. The solution was subsequently cooled to
room temperature and 2-bromo-6-(1,3-dioxylane)pyridine (2.5 g,
10.2 mmol) was added in 10 mL of THF. The solution was then
returned to reflux for 36 h. The flask was then cooled to room
temperature and quenched with 25 mL of H2O. The biphasic mixture
was extracted with three 25 mL portions of ethyl acetate, with organic
fractions combined and dried over Na2SO4. Volatiles were removed
under reduced pressure leaving a brown oil that was used without
62
of diethyl ether and hexanes at −35 °C. Fe(OTf)2(MeCN)2,
LuHOTf,63 and Gomberg’s37 dimer were prepared according to
modified literature procedures. Ligand precursors 6-bromo-2-
acetylpyridine64 and 2-bromo-6-(1,3-dioxylane)pyridine64 were syn-
1
thesized according to literature procedures and identified by their H
NMR spectra. Celite 545 (J.T. Baker) and tetrabutylammonium
hexafluorophosphate ([nBu4N][PF6]) (Sigma- Aldrich) were dried in
Schlenk flasks for 24 h under a dynamic vacuum while heating to at
least 150 °C prior to use in a drybox.
1
further purification. H NMR (CDCl3, 500 MHz, 21 °C): δ 1.64 (s,
3H, −CH3), 2.34 (s, 3H, −CH3), 3.90−3.85 (m, 2H, −CH2), 4.06−
4.01 (m, 2H, −CH2), 7.14−7.03 (m, 5H, py−CH), 7.35 (d, J = 7.7
Hz, 1H, py−CH), 7.62−7.52 (m, 3H, py−CH), 8.56 (ddd, J = 6.0,
3.6, 2.5 Hz, 2H py−CH). 13C NMR (CDCl3, 126 MHz, 21 °C): δ
24.28, 27.10, 60.19, 64.99, 108.84, 116.85, 121.09, 122.55, 123.63,
135.77, 136.37, 148.63, 159.33, 164.79, 166.20 ESI−MS: calculated
[C21H22N3O2]+: 348.1712, found: 348.1699.
Physical Measurements. NMR spectra for ligand precursors
were recorded on a Varian spectrometer operating at 400 MHz (1H
NMR) or a Bruker spectrometer operating at 500 MHz (1H NMR) or
126 MHz (13C NMR). NMR spectra of metal complexes were
recorded on a Varian spectrometer at 500 MHz (1H NMR) or 377
MHz (19F NMR). All 1H and 13C chemical shifts (ppm) are reported
relative to the resonance of the residual solvent; 19F chemical shifts
are reported relative to an external standard (1% CFCl3 in CDCl3).
Solid-state infrared spectra were recorded using a PerkinElmer
Frontier FT−IR spectrophotometer equipped with a KRS5 thallium
bromide/iodide universal attenuated total reflectance accessory. UV−
visible spectra were recorded on an Agilent 8453 Spectrophotometer
with accompanying software. All samples were prepared in a drybox
containing a dinitrogen atmosphere in quartz cuvettes with a 1 cm
2,2′,2′-Methyl-bis-pyridyl-6-acyl-pyridine (L2). To a 20 mL
scintillation vial was added L1 (2.8 g, 8.0 mol) and 10 mL of 3 M
HCl. The reaction was allowed to stir for 4 h at room temperature.
The solution was then transferred to a separatory funnel and
neutralized with 20 mL of a saturated aqueous solution of NaHCO3,
followed by 10 mL of distilled H2O. The aqueous layer was extracted
with 3 portions of 25 mL of dichloromethane, with the organic layers
combined and dried over Na2SO4. After removing the volatiles under
reduced pressure, the product was obtained as a brown oil that was
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used without further purification (Yield, 2 steps: 2.4 g, 8.0 mmol,
78%) H NMR (CDCl3, 400 MHz, 21 °C): δ 2.37 (s, 3H, −CH3),
2H, py−CH), 9.67 (s, 2H, pyrrole−NH). 13C NMR (CDCl3, 126
MHz, 21 °C): 25.12, 25.83, 27.65, 29.00, 29.86, 34.83, 46.33, 60.42,
69.51, 108.12, 112.86, 118.62, 121.30, 123.74, 130.50, 136.24, 137.38,
139.81, 148.83, 148.92, 163.11, 164.75, 166.30. IR νmax: 1635 cm−1
(CN) ESI−MS: calculated [C41H48N7]+: 638.3971, found:
638.3957.
1
2.52 (s, 3H, −CH3), 7.12−7.06 (m, 2H, py−CH), 7.14 (ddd, J = 7.5,
4.8, 1.1 Hz, 2H, py−CH), 7.40 (dd, J = 7.9, 1.0 Hz, 1H, py−CH),
7.59 (tt, J = 7.6, 2.0 Hz, 3H, py−CH), 7.72 (t, J = 7.8 Hz, 1H, py−
CH), 7.87 (dd, J = 7.7, 1.0 Hz, 1H, py−CH), 8.59 (ddd, J = 4.9, 1.9,
0.9 Hz, py−2H). 13C NMR (CDCl3, 126 MHz, 21 °C): δ 25.72,
27.21, 60.27, 119.07, 121.44, 123.47, 127.66, 136.08, 136.69, 148.91,
152.21, 165.05, 165.76, 200.80. IR νmax: 1650 cm−1, 1694 cm−1 (C
O). ESI−MS: calculated [C19H18N3O]+: 304.1450, found: 304.1459.
2,2′,2′-Methyl-bis-pyridyl-6-(2,2′,2′-methylbis-pyrrolyl)-
pyridine (L3). L2 (2.5 g, 8.2 mmol) was added to a 20 mL
scintillation vial, along with 5 mL of pyrrole, 5 mL of 3 M HCl, and 2
mL of tetrahydrofuran. The solution was left to stir at room
temperature for 18 h. Upon neutralization with a saturated aqueous
solution of NaHCO3, the biphasic mixture was extracted with
dichloromethane (3 × 25 mL). Upon removal of the volatiles, diethyl
ether was added to the orange oil to form yellow suspension. After
stirring for 2 h, the suspension was filtered and the off-white solid, L3,
[Py2Py(afaCy2)FeIIOTf]OTf (1). To a 20 mL scintillation vial were
added Py2Py(piCy)2 (0.032 g, 0.050 mmol), a stir bar, and 4 mL of
tetrahydrofuran. After dissolution, Fe(OTf)2(MeCN)2 (21.8 mg,
0.050 mmol) was added to the solution. An immediate color change
to red was observed, followed by the formation of a yellow precipitate
over the course of 1 h. The reaction was filtered over diatomaceous
earth, and the filtrate was discarded. The yellow solid collected by
filtration was eluted with five, 1 mL portions of acetonitrile (or until
no visible yellow solid remained on filter). Volatiles were removed
under reduced pressure to give a yellow powder (0.040 g, 0.40 mmol,
81%). Crystals suitable for X-ray analysis were grown from the vapor
diffusion of diethyl ether into a concentrated solution of the target
molecule in acetonitrile at room temperature. Analysis for
C43H47F6FeN7O6S2·MeCN (calcd., found:): C (52.33, 52.10), H
(4.88, 4.68), N (10.85, 10.40). The poor solubility of the complex
precluded its characterization by 1H NMR spectroscopy and the
determination of its solution magnetic moment by the Evans’
Method. IR νmax: 1635 cm−1 (CN), 3215, 3283 cm−1 (N−H).
[Py2Py(afaCy2)FeIIOH]OTf (2) from 1. To a 20 mL scintillation
vial was added 1 (0.032 g, 0.32 mmol), a stir bar, and 4 mL of
acetonitrile. KOH (0.0030 g, 0.54 mmol) was added to the yellow
suspension, and a red solution formed over 1 h. After the removal of
volatiles under reduced pressure, the red residue was dissolved in
dichloromethane and filtered over a pad of diatomaceous earth. The
volatiles were again removed under reduced pressure, yielding a red
powder (0.0265 g, 0.031 mmol, 95%). Crystals suitable for X-ray
analysis were grown from vapor diffusion of diethyl ether into a
concentrated solution of the target molecule in acetonitrile at room
temperature. (0.0225 g, 0.026 mmol, 81%). 1H NMR (d3-CD3CN, 21
°C): −5.7, −4.9, −3.8, −0.6, 0.2, 0..4, 0.7, 1.1, 1.9, 2.7, 2.9, 6.6, 24.0,
24.8, 34.3 (d), 51.2, 61.0, 65,0, 82.1 Analysis for C42H48F3FeN7O4S:
(calcd., found) C (58.67, 58.33), H (5.63, 5.73), N (11.40, 11.34). IR
νmax: 1656 cm−1 (CN), 3637 cm−1 (O−H). μeff = 5.21(9) μB.
Py2Py(piCy2)FeIIOH2 (3) from 1. To a 20 mL scintillation vial was
added 1 (0.041 g, 0.041 mmol), a stir bar, and 4 mL of acetonitrile.
Li2O (0.005 mg, 0.16 mmol) was added to the solution. The solution
changed from yellow to red over the course of 1 h, followed by the
precipitation of a bright pink solid over the course of 18 h. The
suspension was filtered, and the precipitate was washed with 1 mL of
acetonitrile. The solid was eluted with dimethylacetamide (DMA).
Crystals suitable for X-ray analysis were grown from the vapor
diffusion of diethyl ether into a concentrated solution of the target
complex in dimethylacetamide and dichloromethane at room
temperature (0.0275 g, 92%). Analysis for C41H47FeN7O·1DMA·
0.5CH2Cl2 (calcd., found): C (65.11, 65.28), H (6.85, 6.49), and N
(13.35, 12.93). The poor solubility of the complex precluded its
1
was collected (1.86 g 4.4 mmol, 54%). H NMR (CDCl3, 400 MHz,
21 °C): δ 2.02 (s, 3H, −CH3), 2.35 (s, 3H, −CH3), 5.93 (ddd, J = 3.3,
2.5, 1.6 Hz, 2H, pyrrole−CH), 6.04−6.00 (m, 2H, pyrrole−CH), 6.48
(td, J = 2.6, 1.5 Hz, 2H, pyrrole−CH), 6.96 (dd, J = 7.9, 0.8 Hz, 1H,
py−CH), 7.12 (dt, J = 8.1, 1.0 Hz, 2H, py−CH), 7.22−7.16 (m, 3H,
py−CH), 7.65−7.54 (m, 3H, py−CH), 8.65 (ddd, J = 4.9, 1.9, 1.0 Hz,
2H, py−CH), 9.03 (b, 2H, pyrrole N−H). 13C NMR (CDCl3, 126
MHz, 21 °C): δ 27.34, 44.94, 59.96, 104.87, 107.37, 116.81, 117.98,
20.32, 21.62, 123.73, 136.35, 136.92, 137.85, 149.02, 164.16, 164.90,
165.87 ESI−MS: calculated [C27H26N5]+: 420.2188, found: 420.2203.
2,2′,2′-Methyl-bis-pyridyl-6-(2,2′,2′-methylbis-5-formyl-
pyrrol)-pyridine (L4). To a 250 mL round-bottom flask were added
L3 (1.30 g, 3.1 mmol), 5 mL of dimethylformamide, and 30 mL of
dichloromethane, forming a yellow solution. A solution of POCl3 (1.0
g, 6.5 mmol, 2.1 equiv) in 10 mL of dichloromethane was added to
the solution dropwise forming a pink solution that was heated to 40
°C for 2 h. An aqueous solution of sodium acetate was prepared (2.5 g
in 50 mL distilled water) and added to the stirred solution and heated
at 45 °C for 1 h. The biphasic mixture was then cooled and
neutralized using solid Na2CO3, and extracted with dichloromethane
(3 × 20 mL). Organic fractions were combined and dried over
Na2SO4, followed by the removal of volatiles under reduced pressure
to give a pink powder, L4, (1.6 g, 3.1 mmol, 99%). 1H NMR (CDCl3,
400 MHz, 21 °C): δ 2.03 (s, 3H, −CH3), 2.39 (s, 3H, −CH3), 6.04
(dd, J = 3.9, 2.4 Hz, 2H, pyrrole−CH), 6.80 (dd, J = 3.9, 2.4 Hz, 2H,
pyrrole−CH), 7.03 (d, J = 7.8 Hz, 1H, py−CH), 7.12 (ddd, J = 7.5,
4.8, 1.1 Hz, 2H, py−CH), 7.15 (dt, J = 8.0, 1.1 Hz, 2H, py−CH), 7.57
(td, J = 7.7, 1.9 Hz, 2H, py−CH), 7.63 (t, J = 7.9 Hz, 1H, py−CH),
8.57 (ddd, J = 4.9, 2.0, 1.0 Hz, 2H, py−CH), 9.37 (s, 2H, −CHO),
9.86 (b, 2H-pyrrole−NH). 13C NMR (CDCl3, 126 MHz, 21 °C): δ
27.16, 27.28, 46.26, 60.07, 109.62, 118.32, 121.46, 122.20, 123.36,
128.31, 129.11, 132.61, 136.33, 137.67, 144.72, 148.94, 160.63,
165.56, 178.68. IR νmax: 1650 cm−1 (C = O). ESI-MS: calculated
[C29H26N5O2]+: 476.2087, found: 476.2065.
1
characterization by H NMR spectroscopy and the determination of
2,2′,2′-Methyl-bis-pyridyl-6-(2,2′,2′-methylbis-5-cyclohexy-
liminopyrrol)-pyridine (Py2Py(piCy)2). In a 20 mL scintillation vial
L4 (1.55 g, 3.0 mmol, 1 equiv) was dissolved in 10 mL of
dichloromethane, followed by the addition of cyclohexylamine (0.65
g, 6.5 mmol, 2.2 equiv). The reaction was stirred at room temperature
for 18 h followed by the removal of volatiles under reduced pressure,
yielding a brown powder. The brown powder was taken into a dry
glovebox, dissolved in dichloromethane, and stored over 4 Å
molecular sieves overnight. Removal of volatiles under reduced
pressure furnished a tan powder, Py2Py(piCy)2, (1.7 g, 2.7 mmol, 88%)
1H NMR (CDCl3, 400 MHz, 21 °C): 1.15−1.77 (m, 20H,
cyclohexyl−CH2), 1.90 (s, 3H, −CH3), 2.47 (s, 3H, −CH3), 3.00
(tt, J = 10.5, 4.0 Hz, 2H, cyclohexyl−CH), 5.91 (d, J = 3.6 Hz, 2H,
pyrrole−CH), 6.30 (d, J = 3.6 Hz, 2H, pyrrole−CH), 6.86 (d, J = 7.8
Hz, 1H, py−CH), 7.09 (ddd, J = 7.5, 4.8, 1.1 Hz, 2H, py−CH), 7.16
(d, J = 7.9 Hz, 1H, py−CH), 7.20 (dt, J = 8.1, 1.1 Hz, 2H, py−CH),
7.58−7.48 (m, 3H, py−CH), 7.96 (s, 2H, imine−CH), 8.60−8.57 (m,
its solution magnetic moment by the Evans’ Method. IR νmax: 1615
cm−1 (CN).
[Py2Py(afaCy2)FeIIIOH]OTf2 (4) from 2. To a 20 mL scintillation
vial wrapped in black electrical tape were added 2 (0.0190 g, 0.022
mmol), 4 mL of dichloromethane, and a stir bar. AgOTf (0.0057 g,
0.022 mmol) was added to the solution and stirring was continued for
1 h. The mixture was filtered over diatomaceous earth, and the filtrate
was dried under reduced pressure, producing a red-brown powder
(0.0209 g, 0.021 mmol, 93%). Crystals suitable for X-ray analysis were
grown from vapor diffusion of diethyl ether into a concentrated
solution of dichloromethane and acetonitrile (1:1) at room
temperature. Bulk purification was achieved by vapor diffusion of
diethyl ether into a concentrated solution of dichloromethane (0.019
g, 0.019 mmol, 87%). Analysis for C43H48F6FeN7O7S2 (calcd., found):
C (49.03, 49.27), H (4.68, 4.66), N (9.10, 9.08). 1H NMR (d3-
CD3CN, 500 MHz, 21 °C): 2.6, 8.2, 13.9, 19.0, 64.1, 72.6, 88.8, 109.0.
IR νmax: 1660 cm−1 (CN), 3585 cm−1 (O−H), μeff = 6.11(26) μB.
I
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Journal of the American Chemical Society
Article
[Py2Py(afaCy)(piCy)FeIIIOH]OTf (5) from 3. To a 20 mL
scintillation vial wrapped in black electrical tape was added 3
(0.0270 g, 0.031 mmol, 0.038 mmol), 4 mL of dichloromethane, and
a stir bar. AgOTf (0.0098 g, 0.038 mmol) was added to the solution,
which was stirred for 1 h. The mixture was filtered over diatomaceous
earth, and the filtrate was dried under reduced pressure, producing a
red-brown powder (0.0320 g, 0.037 mmol, 94%). Crystals suitable for
X-ray analysis and bulk purification were grown from vapor diffusion
of diethyl ether into a concentrated solution of dichloromethane and
acetonitrile (1:1) at room temperature (0.0237 g, 0.028 mmol, 70%).
Analysis for C42H47F3FeN7O4S·CH2Cl2 (calcd., found): C (54.73,
amount of AgOTf in acetonitrile spiked with H218O, followed by
filtration and recrystallization from the acetonitrile solution by vapor
diffusion of diethyl ether.
Synthesis of 18O Isotopologue of 5. Complex 1 (40 mg, 0.04
mmol) was dissolved in 2 mL of acetonitrile spiked with H218O (∼5
mg/mL) forming an orange solution. One drop of triethylamine (∼10
mg, 0.1 mmol) was added to the solution turning it bright red
immediately. The solution was left to stir for 1 h and then
concentrated to ∼1 mL volume, and the 18O isotopologue of 2 was
recrystallized from vapor diffusion of diethyl ether into acetonitrile.
The recrystallized product was then reacted with 1,8-
Diazabicyclo[5.4.0]undec-7-ene (DBU) to form the 18O isotopologue
of 3 as a precipitate from acetonitrile. Filtration of the suspension over
diatomaceous earth and addition of a stoichiometric amount of
AgOTf to the filter pad, followed by addition of 1 mL of acetonitrile
spiked with H218O to the filter formed a solution of the 18O
isotopologue of 5 which was recrystallized from the acetonitrile
solution by vapor diffusion of diethyl ether.
1
54.82) H (5.23, 5.23) N (10.39, 9.90). H NMR (d2-CD2Cl2, 500
MHz, 21 °C): 13.4, 20.0, 31.5, 65.6, 75.0, 82.2, 90.6. IRνmax: 1616,
1658 cm−1 (CN), μeff = 5.97(14) μB.
Alternative Synthesis of 3 from 2. To a 20 mL scintillation vial
were added 2 (0.0245 g, 0.30 mmmol), a stir bar, 4 mL of acetonitrile,
and KH (0.0014 g, 0.035 mmol). The solution was stirred for 18 h,
and formation of a pink precipitate was observed. Upon filtration, 3
was isolated (0.0151 g, 0.021 mmol, 76%).
Rebound Hydroxylation using Gomberg’s dimer and 4. To
a 20 mL scintillation vial was added 4 (0.0202 g, 0.020 mmol), a stir
bar, and 2 mL of acetonitrile. In a separate vial Gomberg’s dimer
(0.0057 g, 0.010 mmol, 0.5 equiv) was dissolved in 2 mL of
tetrahydrofuran and added to the vial containing 4. The reaction
quickly turned from a dark red-brown solution to a bright yellow
solution. After 0.5 h of stirring, volatiles were removed under reduced
pressure and a yellow powder was triturated with diethyl ether. The
ether-soluble products were separated and dried. The resulting white
powder was dissolved in 2 mL of tetrahydrofuran containing a known
amount of mesitylene as an internal standard. The formation of
Alternative Synthesis of 2 from 3. To a 20 mL scintillation vial
were added 3 (0.0104 g, 0.015 mmol), a stir bar, 4 mL of
tetrahydrofuran, and 2,6-lutidinium triflate (LuHOTf, 0.0038 g, 0.015
mmol). The solution was stirred for 1 h, changing from a pink
solution to a bright red suspension. The solution was filtered, and the
red solid was eluted with acetonitrile. Subsequent removal of volatiles
under reduced pressure yielded a red powder, 2 (0.0069 g, 0.008
mmol, 55%).
Alternative Synthesis of 5 from 4. To a 20 mL scintillation vial
were added 4 (0.0255 g, 0.026 mmol), a stir bar, 2 mL of acetonitrile,
and potassium hydride (0.0011 g, 0.028 mmol). The solution was
stirred for 1 h, with the color remaining dark red-brown. Volatiles
were removed under reduced pressure and the product, 5, was
recrystallized from dichloromethane with slow diffusion of diethyl
ether (0.0173 g, 0.020 mmol, 77%).
1
triphenylmethanol was observed by GC−MS and H NMR analyses
(Yield: 0.0052 g, 0.020 mmol, 99%). Quantification of triphenylme-
thanol by GC−MS was achieved through the use of a standard curve
containing mesitylene as a standard.
Alternative Synthesis of 4 from 5. To a 20 mL scintillation vial
were added 5 (0.0118 g, 0.014 mmol) , a stir bar, 4 mL of
dichloromethane, and 2,6-lutidinium triflate (LuHOTf, 0.0036 g,
0.014 mmol). The solution was stirred for 1 h, remaining dark red-
brown in color. Volatiles were removed under reduced pressure, and
the product was recrystallized from acetonitrile with slow diffusion of
diethyl ether (0.0102 g, 0.010 mmol, 73%).
Alternative Synthesis of 2 from 4. To a 20 mL scintillation vial
were added 4 (0.0123 g, 0.012 mmol), a stir bar, and 4 mL of
acetonitrile. Cobaltocene (0.0024 g, 0.012 mmol) was added to the
stirred solution and an immediate color change was noted from dark
red-brown to bright red. Volatiles were removed under reduced
pressure and the product was recrystallized from acetonitrile with slow
diffusion of diethyl ether (0.0105 g, 0.012 mmol, > 99%).
Alternative Synthesis of 3 from 5. To a 20 mL scintillation vial
were added 5 (0.0183 g, 0.021 mmol), a stir bar, and 4 mL of
acetonitrile. Cobaltocene (0.0041 g, 0.021 mmol) was added to the
stirred solution and the appearance of a pink precipitate over the
course of 1 h was noted. Filtration of the suspension yielded the
product, 3 (0.0117 g, 0.016 mmol, 78%).
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.9b01516.
Experimental spectra and crystallographic information
(CIF)
Experimental spectra and crystallographic information
(CIF)
Experimental spectra and crystallographic information
(CIF)
Experimental spectra and crystallographic information
(CIF)
Experimental spectra and crystallographic information
(CIF)
Experimental data (PDF)
Alternative Synthesis of 2 from 5. To a 20 mL scintillation vial
were added 0.0129 g (0.015 mmol) of 5, 4 mL of acetonitrile, and
0.0028 mg (0.015 mmol) of 1,2-diphenylhydrazine. The solution was
stirred for 1 h, producing a bright red solution. Volatiles were
removed under reduced pressure, and the resulting powder was
triturated with diethyl ether. The product, 2, was recrystallized from
acetonitrile with vapor diffusion of diethyl ether (0.0079 g, 0.009
mmol, 61%).
AUTHOR INFORMATION
■
Corresponding Author
*fout@illinois.edu.
ORCID
Michael J. Drummond: 0000-0002-1912-5116
Courtney L. Ford: 0000-0002-1509-4850
Danielle L. Gray: 0000-0003-0059-2096
Codrina V. Popescu: 0000-0003-2369-3383
Alison R. Fout: 0000-0002-4669-5835
Synthesis of 18O Isotopologue of 4. Complex 1 (40 mg, 0.04
mmol) was dissolved in 2 mL of acetonitrile spiked with H218O (∼5
mg/mL) forming an orange solution. One drop of triethylamine (∼10
mg, 0.1 mmol) was added to the solution turning it bright red
immediately. The solution was left to stir for 1 h and then
concentrated to ∼1 mL volume and the 18O isotopologue of 2 was
recrystallized from vapor diffusion of diethyl ether into acetonitrile.
The recrystallized product was then oxidized with a stoichiometric
Notes
The authors declare no competing financial interest.
J
DOI: 10.1021/jacs.9b01516
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(17) Mai, B. K.; Kim, Y. Is It Fe (III)-Oxyl Radical That Abstracts
Hydrogen in the C−H Activation of TauD? A Theoretical Study
Based on the DFT Potential Energy Surfaces. Inorg. Chem. 2016, 55,
3844−3852.
(18) Mitchell, A. J.; Dunham, N. P.; Martinie, R. J.; Bergman, J. A.;
Pollock, C. J.; Hu, K.; Allen, B. D.; Chang, W.; Silakov, A.; Bollinger,
J. M.; Krebs, C.; Boal, A. K. Visualizing the Reaction Cycle in an
Iron(II) and 2-(Oxo)-Glutarate- Dependent Hydroxylase. J. Am.
Chem. Soc. 2017, 139, 13830−13836.
(19) Topf, M.; Sandala, G. M.; Smith, D. M.; Schofield, C. J.;
Easton, C. J.; Radom, L. The Unusual Bifunctional Catalysis of
Epimerization and Desaturation by Carbapenem Synthase. J. Am.
Chem. Soc. 2004, 126, 9932−9933.
(20) Dunham, N. P.; Chang, W.; Mitchell, A. J.; Martinie, R. J.;
Zhang, B.; Bergman, J. A.; Rajakovich, L. J.; Wang, B.; Silakov, A.;
Krebs, C.; Boal, A. K.; Bollinger, M. J. Two Distinct Mechanisms for
C−C Desaturation by Iron(II)- and 2-(Oxo)Glutarate-Dependent
Oxygenases: Importance of α-Heteroatom Assistance. J. Am. Chem.
Soc. 2018, 140, 7116−7126.
(21) Dunham, N. P.; Mitchell, A. J.; Del Río Pantoja, J. M.; Krebs,
C.; Bollinger, M. J.; Boal, A. K. α-Amine Desaturation of D-Arginine
by the Iron(II)- and 2-(Oxo)Gluterate-Dependent L-Arginine 3-
Hydroxylase, VioC. Biochemistry 2018, 57, 6479−6488.
(22) Zhou, J.; Kelly, W. L.; Bachmann, B. O.; Gunsior, M.;
Townsend, C. A.; Solomon, E. I. Spectroscopic Studies of Substrate
Interactions with Clavaminate Synthase 2, a Multifunctional α-KG-
Dependent Non-Heme Iron Enzyme: Correlation with Mechanisms
and Reactivities. J. Am. Chem. Soc. 2001, 123, 7388−7398.
(23) Huang, J.; Tang, Y.; Yu, C.; Sanyal, D.; Jia, X.; Liu, X.; Guo, Y.;
Chang, W. Mechanistic Investigation of Oxidative Decarboxylation
Catalyzed by Two Iron(II)- and 2-Oxoglutarate-Dependent Enzymes.
Biochemistry 2018, 57, 1838−1841.
(24) Blasiak, L. C.; Drennan, C. L. Structural Perspective on
Enzymatic Halogenation. Acc. Chem. Res. 2009, 42, 147−155.
(25) Ogo, S.; Wada, S.; Watanabe, Y.; Iwase, M.; Wada, A.; Harata,
M.; Jitsukawa, K.; Masuda, H.; Einaga, H. Synthesis, Structure, and
Spectroscopic Properties of [Fe(III)(Tnpa)(OH)(PhCOO)ClO4: A
Model Complex for an Active Form of Soybean Lipoxygenase-1.
Angew. Chem., Int. Ed. 1998, 37, 2102−2104.
(26) MacBeth, C. E.; Golombek, A. P.; Young, V. G.; Yang, C.;
Kuczera, K.; Hendrich, M. P.; Borovik, A. S. O2 Activation by
Nonheme Iron Complexes: A Monomeric Fe(III)- Oxo Complex
Derived from O2. Science 2000, 289, 938−941.
(27) Soo, H. S.; Komor, A. C.; Iavarone, A. T.; Chang, C. J. A
Hydrogen-Bond Facilitated Cycle for Oxygen Reduction by an Acid-
and Base-Compatible Iron Platform. Inorg. Chem. 2009, 48, 10024−
10035.
(28) Cook, S. A.; Ziller, J. W.; Borovik, A. S. Iron(II) Complexes
Supported by Sulfonamido Tripodal Ligands: Endogenous versus
Exogenous Substrate Oxidation. Inorg. Chem. 2014, 53, 11029−
11035.
(29) Yeh, C. Y.; Chang, C. J.; Nocera, D. G. Hangman” Porphyrins
for the Assembly of a Model Heme Water Channel. J. Am. Chem. Soc.
2001, 123, 1513−1514.
(30) MacBeth, C. E.; Hammes, B. S.; Young, V. G.; Borovik, A. S.
Hydrogen-Bonding Cavities about Metal Ions: Synthesis, Structure,
and Physical Properties for a Series of Monomeric M-OH Complexes
Derived from Water. Inorg. Chem. 2001, 40, 4733−4741.
(31) Ogo, S.; Yamahara, R.; Roach, M.; Suenobu, T.; Aki, M.;
Ogura, T.; Kitagawa, T.; Masuda, H.; Fukuzumi, S.; Watanabe, Y.
Structural and Spectroscopic Features of a Cis (Hydroxo)-FeIII-
(Carboxylato) Configuration as an Active Site Model for Lip-
oxygenases. Inorg. Chem. 2002, 41, 5513−5520.
(32) MacBeth, C. E.; Gupta, R.; Mitchell-Koch, K. R.; Young, V. G.;
Lushington, G. H.; Thompson, W. H.; Hendrich, M. P.; Borovik, A. S.
Utilization of Hydrogen Bonds to Stabilize M-O(H) Units: Synthesis
and Properties of Monomeric Iron and Manganese Complexes with
Terminal Oxo and Hydroxo Ligands. J. Am. Chem. Soc. 2004, 126,
2556−2567.
ACKNOWLEDGMENTS
■
This work was supported by the U.S. Department of Energy,
Office of Science, Office of Basic Energy Sciences, Chemical
Sciences, Geosciences and Biosciences Division under award
̈
DE-SC-0016026. The Mossbauer study is supported by start-
up funds from The College of Arts and Sciences at The
University of St. Thomas to CVP. The authors would like to
thank M. J. Nilges for assistance with EPR spectroscopy and T.
B. Rauchfuss for useful discussions and feedback. A.R.F. is a
Sloan Research Fellow and a Camille Dreyfus Teacher-Scholar.
REFERENCES
■
(1) Rohde, J.-U.; In, J.-H.; Lim, M. H.; Brennessel, W. W.; Bukauski,
M. R.; Stubna, A.; Mu
Spectroscopic Characterization of a Nonheme Fe(IV)O Complex.
̈
nck, E.; Nam, W.; Que, L. Crystallographic and
Science 2003, 299, 1037−1039.
(2) Nam, W. Synthetic Mononuclear Nonheme Iron-Oxygen
Intermediates. Acc. Chem. Res. 2015, 48, 2415−2423.
(3) Puri, M.; Que, L. Toward the Synthesis of More Reactive S = 2
Non-Heme Oxoiron(IV) Complexes. Acc. Chem. Res. 2015, 48,
2443−2452.
(4) Nam, W.; Lee, Y. M.; Fukuzumi, S. Tuning Reactivity and
Mechanism in Oxidation Reactions by Mononuclear Nonheme
Iron(IV)-Oxo Complexes. Acc. Chem. Res. 2014, 47, 1146−1154.
(5) Sahu, S.; Goldberg, D. P. Activation of Dioxygen by Iron and
Manganese Complexes: A Heme and Nonheme Perspective. J. Am.
Chem. Soc. 2016, 138, 11410−11428.
(6) Price, J. C.; Barr, E. W.; Tirupati, B.; Bollinger, J. M.; Krebs, C.
The First Direct Characterization of a High-Valent Iron Intermediate
in the Reaction of an α-Ketoglutarate-Dependent Dioxygenase: A
High-Spin Fe(IV) Complex in Taurine/α-Ketoglutarate Dioxygenase
(TauD) from Escherichia Coli. Biochemistry 2003, 42, 7497−7508.
(7) Krebs, C.; Fujimori, D. G.; Walsh, C. T.; Bollinger, J. M. Non-
Heme Fe (IV)− Oxo Intermediates. Acc. Chem. Res. 2007, 40, 484−
492.
(8) Meunier, B.; de Visser, S. P.; Shaik, S. Mechanism of Oxidation
Reactions Catalyzed by Cytochrome P450 Enzymes. Chem. Rev. 2004,
104, 3947−3980.
(9) Bollinger, J. M.; Price, J. C.; Hoffart, L. M.; Barr, E. W.; Krebs, C.
Mechanism of Taurine: α-Ketoglutarate Dioxygenase (TauD) from
Escherichia Coli. Eur. J. Inorg. Chem. 2005, 21, 4245−4254.
(10) Martinez, S.; Hausinger, R. P. Catalytic Mechanisms of Fe (II)
and 2-Oxoglutarate-Dependent Oxygenases. J. Biol. Chem.2015, 290,
20702−20711.
(11) Price, J. C.; Barr, E. W.; Glass, T. E.; Krebs, C.; Bollinger, J. M.
Evidence for Hydrogen Abstraction from C1 of Taurine by the High-
Spin Fe (IV) Intermediate Detected during Oxygen Activation by
Taurine: α-Ketoglutarate Dioxygenase (TauD). J. Am. Chem. Soc.
2003, 125, 13008−13009.
(12) Proshlyakov, D. A.; Mccracken, J.; Hausinger, R. P.
Spectroscopic Analyses of 2-oxoglutarate-Dependent Oxygenases:
TauD as a Case Study. J. Biol. Inorg. Chem.2017, 22, 367−379.
(13) de Visser, S. P. Propene Activation by the Oxo-Iron Active
Species of Taurine/α-Ketoglutarate Dioxygenase (TauD) Enzyme.
How Does the Catalysis Compare to Heme-Enzymes? J. Am. Chem.
Soc. 2006, 128, 9813−9824.
(14) de Visser, S. P. Differences in and Comparison of the Catalytic
Properties of Heme and Non-Heme Enzymes with a Central Oxo−
Iron Group. Angew. Chem., Int. Ed. 2006, 45, 1790−1793.
(15) Latifi, R.; Bagherzadeh, M.; de Visser, S. P. Origin of the
Correlation of the Rate Constant of Substrate Hydroxylation by
Nonheme Iron (IV)− Oxo Complexes with the Bond-Dissociation
Energy of the C-H Bond of the Substrate. Chem. - Eur. J. 2009, 15,
6651−6662.
(16) Ye, S.; Neese, F. Nonheme Oxo-Iron (IV) Intermediates Form
an Oxyl Radical upon Approaching the C−H Bond Activation
Transition State. Proc. Natl. Acad. Sci. U. S. A.2011, 108, 1228−1233.
K
DOI: 10.1021/jacs.9b01516
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
(33) Harman, W. H.; Chang, C. J. N2O Activation and Oxidation
Reactivity from a Non-Heme Iron Pyrrole Platform. J. Am. Chem. Soc.
2007, 129, 15128−15129.
Potential in Nonheme Fe(II)-Hydroxo Complexes through Primary
and Secondary Coordination Sphere Modifications. Inorg. Chem.
2017, 56, 4852−4863.
(50) Kaljurand, I.; Kutt, A.; Soovali, L.; Rodima, T; Maemets, V.;
Leito, I.; Koppel, I. A. Extension of the Self-Consistent Spectrophoto-
metric Basicity Scale in Acetonitrile to a Full Span of 28 p K a Units:
Unification of Different Basicity Scales. J. Org. Chem. 2005, 70, 1019−
1028.
(34) Çelenligil-Çetin, R.; Paraskevopoulou, P.; Dinda, R.; Staples, R.
J.; Sinn, E.; Rath, N. P.; Stavropoulos, P. Synthesis, Characterization,
and Reactivity of Iron Trisamidoamine Complexes That Undergo
Both Metal- and Ligand-Centered Oxidative Transformations. Inorg.
Chem. 2008, 47, 1165−1172.
(35) Mukherjee, J.; Lucas, R. L.; Zart, M. K.; Powell, D. R.; Day, V.
W.; Borovik, A. S. Synthesis, Structure, and Physical Properties for a
Series of Monomeric Iron(III) Hydroxo Complexes with Varying
Hydrogen-Bond Networks. Inorg. Chem. 2008, 47, 5780−5786.
(36) Zaragoza, J. P. T.; Yosca, T. H.; Siegler, M. A.; Moenne-Loccoz,
P.; Green, M. T.; Goldberg, D. P. Direct Observation of Oxygen
Rebound with an Iron-Hydroxide Complex. J. Am. Chem. Soc. 2017,
139, 13640−13643.
(37) Pangia, T. M.; Davies, C. G.; Prendergast, J. R.; Gordon, J. B.;
Siegler, M. A.; Jameson, G. N. L.; Goldberg, D. P. Observation of
Radical Rebound in a Mononuclear Nonheme Iron Model Complex.
J. Am. Chem. Soc. 2018, 140, 4191−4194.
(38) Matson, E. M.; Bertke, J. A.; Fout, A. R. Isolation of Iron(II)
Aqua and Hydroxyl Complexes Featuring a Tripodal H-Bond Donor
and Acceptor Ligand. Inorg. Chem. 2014, 53, 4450−4458.
(39) Matson, E. M.; Park, Y. J.; Bertke, J. A.; Fout, A. R. Synthesis
and Characterization of M(II) (M = Mn, Fe and Co) Azafulvene
Complexes and Their X3− Derivatives. Dalt. Trans. 2015, 44, 10377−
10384.
(51) Gupta, R.; Borovik, A. S. Monomeric MnIII/II and FeIII/II
Complexes with Terminal Hydroxo and Oxo Ligands: Probing
Reactivity via O-H Bond Dissociation Energies. J. Am. Chem. Soc.
2003, 125, 13234−13242.
(52) Matson, E. M.; Park, Y. J.; Fout, A. R. Facile Nitrite Reduction
in a Non-Heme Iron System: Formation of an Iron(III)-Oxo. J. Am.
Chem. Soc. 2014, 136, 17398−17401.
(53) Gutlich, P.; Bill, E.; Trautwein, A. X. Mossbauer Spectroscopy
̈
̈
and Transition Metal Chemistry Fundamentals and Application;
Springer-Verlag: Berlin/Heidelberg, 2011.
(54) Ching, W.; Zhou, A.; Klein, J. E. M. N.; Fan, R.; Knizia, G.;
Cramer, C. J.; Guo, Y.; Que, L. Characterization of the Fleeting
Hydroxoiron (III) Complex of the Pentadentate TMC-Py Ligand.
Inorg. Chem. 2017, 56, 11129−11140.
(55) Warren, J. J.; Tronic, T. A.; Mayer, J. M. Thermochemistry of
Proton-Coupled Electron Transfer Reagents and Its Implications.
Chem. Rev. 2010, 110, 6961−7001.
(56) Usharani, D.; Lacy, D. C.; Borovik, A. S.; Shaik, S.
Dichotomous Hydrogen Atom Transfer vs Proton-Coupled Electron
Transfer During Activation of X−H Bonds (X = C, N, O) by
Nonheme Iron−Oxo Complexes of Variable Basicity. J. Am. Chem.
Soc. 2013, 135, 17090−17104.
(57) Guengerich, F. P. Mechanisms of Cytochrome P450-Catalyzed
Oxidations. ACS Catal. 2018, 8, 10964−10976.
(40) Park, Y. J.; Matson, E. M.; Nilges, M. J.; Fout, A. R. Exploring
Mn−O Bonding in the Context of an Electronically Flexible
Secondary Coordination Sphere: Synthesis of a Mn(III)−Oxo.
Chem. Commun. 2015, 51, 5310−5313.
(41) Sahu, S.; Widger, L. R.; Quesne, M. G.; De Visser, S. P.;
(58) Ishikawa, N.; Tanaka, H.; Koyama, F.; Noguchi, H.; Wang, C.
C. C.; Hotta, K.; Watanabe, K. Non-Heme Dioxygenase Catalyzes
Atypical Oxidations of 6,7-Bicyclic Systems To Form the 6, 6-
Quinolone Core of Viridicatin-Type Fungal Alkaloids. Angew. Chem.,
Int. Ed. 2014, 53, 12880−12884.
̈
Matsumura, H.; Moenne-Loccoz, P.; Siegler, M. A.; Goldberg, D. P.
Secondary Coordination Sphere Influence on the Reactivity of
Nonheme Iron(II) Complexes: An Experimental and DFT Approach.
J. Am. Chem. Soc. 2013, 135, 10590−10593.
(42) Widger, L. R.; Davies, C. G.; Yang, T.; Siegler, M. A.;
̈
(59) Brauer, A.; Beck, P.; Hintermann, L.; Groll, M. Structure of the
́
́
Troeppner, O.; Jameson, G. N. L.; Ivanovic-Burmazovic, I.; Goldberg,
D. P. Dramatically Accelerated Selective Oxygen-Atom Transfer by a
Nonheme Iron(IV)-Oxo Complex: Tuning of the First and Second
Coordination Spheres. J. Am. Chem. Soc. 2014, 136, 2699−2702.
(43) Widger, L. R.; Jiang, Y.; McQuilken, A. C.; Yang, T.; Siegler, M.
Dioxygenase AsqJ: Mechanistic Insights into a One- Pot Multistep
Quinolone Antibiotic Biosynthesis. Angew. Chem., Int. Ed. 2016, 55,
422−426.
(60) Nakashima, Y.; Mori, T.; Nakamura, H.; Awakawa, T.;
Hoshino, S.; Senda, M.; Senda, T.; Abe, I. Structure Function and
Engineering of Multifunctional Non-Heme Iron Dependent Oxy-
genases Oxygenases in Fungal Meroterpenoid Biosynthesis. Nat.
Commun. 2018, 9, 1−10.
̈
A.; Matsumura, H.; Moenne-Loccoz, P.; Kumar, D.; de Visser, S. P.;
Goldberg, D. P. Thioether-Ligated Iron(II) and Iron(III)-Hydro-
peroxo/Alkylperoxo Complexes with an H-Bond Donor in the Second
Coordination Sphere. Dalt. Trans. 2014, 43, 7522.
(61) Meng, S.; Han, W.; Zhao, J.; Jian, X.; Pan, H.; Tang, G. A Six-
Oxidase Cascade for Tandem C-H Bond Activation Revealed by
Reconstitution of Bicyclomycin Biosynthesis Communications
Angewandte. Angew. Chem., Int. Ed. 2018, 57, 719−723.
(62) Hagadorn, J. R.; Que, L.; Tolman, W. B. N-Donor Effects on
Carboxylate Binding in Mononuclear Iron (II) Complexes of a
Sterically Hindered Benzoate Ligand. Inorg. Chem. 2000, 39, 6086−
6090.
(44) Hart, J. S.; Nichol, G. S.; Love, J. B. Directed Secondary
Interactions in Transition Metal Complexes of Tripodal Pyrrole Imine
and Amide Ligands. Dalt. Trans. 2012, 41, 5785−5788.
(45) Peters, G. M.; Winegrad, J. B.; Gau, M. R.; Imler, G. H.; Xu, B.;
Ren, S.; Wayland, B. B.; Zdilla, M. J. Synthesis and Structure of 2,5-
Bis[N-(2,6-Mesityl)Iminomethyl]Pyrrolylcobalt(II): Evidence for
One-Electron- Oxidized, Redox Noninnocent Ligand Behavior.
Inorg. Chem. 2017, 56, 3377−3385.
(63) Curley, J. J.; Bergman, G.; Tilley, T. D. Preparation and
Physical Properties of Early-Late Heterobimetallic Compounds. Dalt.
Trans. 2012, 41, 192−200.
(46) Hikichi, S.; Ogihara, T.; Fujisawa, K.; Kitajima, N.; Akita, M.;
Moro-oka, Y. Synthesis and Characterization of the Benzoylformato
Ferrous Complexes with the Hindered Tris(Pyrazolyl)Borate Ligand
as a Structural Model for Mononuclear Non-Heme Iron Enzymes.
Inorg. Chem. 1997, 36, 4539−4547.
(64) Zong, R.; Wang, D.; Hammitt, R.; Thummel, R. P. Synthetic
Approaches to Polypyridyl Bridging Ligands with Proximal Multi-
dentate Binding Sites. J. Org. Chem. 2006, 71, 167−175.
́
(47) Benisvy, L.; Halut, S.; Donnadieu, B.; Tuchagues, J. P.;
̈
(65) Unal, E. A.; Wiedemann, D.; Seiffert, J.; Boyd, J. P.; Grohmann,
Chottard, J. C.; Li, Y. Monomeric Iron(II) Hydroxo and Iron(III)
Dihydroxo Complexes Stabilized by Intermolecular Hydrogen
Bonding. Inorg. Chem. 2006, 45, 2403−2405.
A. Efficient Synthesis of Pentakis- and Tris(Pyridine) Ligands.
Tetrahedron Lett. 2012, 53, 54−55.
(48) Kendall, A. J.; Zakharov, L. N.; Gilbertson, J. D. Synthesis and
Stabilization of a Monomeric Iron(II) Hydroxo Complex via Intra
Molecular Hydrogen Bonding in the Secondary Coordination Sphere.
Inorg. Chem. 2010, 49, 8656−8658.
(49) Gordon, Z.; Drummond, M. J.; Matson, E. M.; Bogart, J. A.;
Schelter, E. J.; Lord, R. L.; Fout, A. R. Tuning the Fe(II/III) Redox
L
DOI: 10.1021/jacs.9b01516
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX