A Mononuclear, Nonheme FeII-Piloty's Acid (PhSO2NHOH) Adduct: An Intermediate in the Production of {FeNO}7/8 Complexes from Piloty's Acid

Article abstract of DOI:10.1021/jacs.9b01700

Reaction of the mononuclear nonheme complex [Fe^II(CH₃CN)(N3PyS)]BF₄ (1) with an HNO donor, Piloty's acid (PhSO₂NHOH, P.A.), at low temperature affords a high-spin (S = 2) Fe^II-P.A. intermediate (2), characterized by ⁵⁷Fe M?ssbauer and Fe K-edge X-ray absorption (XAS) spectroscopies, with interpretation of both supported by DFT calculations. The combined methods indicate that P.A. anion binds as the N-deprotonated tautomer (PhSO₂NOH^-) to [Fe^II(N3PyS)]⁺, leading to 2. Complex 2 is the first spectroscopically characterized example, to our knowledge, of P.A. anion bound to a redox-active metal center. Warming of 2 above a'60 °C yields the stable {FeNO}⁷ complex [Fe(NO)(N3PyS)]BF₄ (4), as evidenced by ¹H NMR, ATR-IR, and M?ssbauer spectroscopies. Isotope labeling experiments with ¹⁵N-labeled P.A. confirm that the nitrosyl ligand in 4 derives from P.A. In contrast, addition of a second equivalent of a strong base leads to S-N cleavage and production of an {FeNO}⁸ species, the deprotonated analog of an Fe-HNO complex. This work has implications for the targeted delivery of HNO/NO^-/NO· to nonheme Fe centers in biological and synthetic applications, and suggests a new role for nonheme Fe^II complexes in the assisted degradation of HNO donor molecules.

Full text of DOI:10.1021/jacs.9b01700

Article
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
A Mononuclear, Nonheme FeII−Piloty’s Acid (PhSO2NHOH) Adduct:
An Intermediate in the Production of {FeNO}7/8 Complexes from
Piloty’s Acid
Alex M. Confer,† Avery C. Vilbert,‡ Aniruddha Dey,† Kyle M. Lancaster,*,‡
and David P. Goldberg*,†
†Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, United States
‡Baker Laboratory, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
S
* Supporting Information
ABSTRACT: Reaction of the mononuclear nonheme com-
plex [FeII(CH3CN)(N3PyS)]BF4 (1) with an HNO donor,
Piloty’s acid (PhSO2NHOH, P.A.), at low temperature affords
a high-spin (S = 2) FeII−P.A. intermediate (2), characterized
57
̈
by Fe Mossbauer and Fe K-edge X-ray absorption (XAS)
spectroscopies, with interpretation of both supported by DFT
calculations. The combined methods indicate that P.A. anion
binds as the N-deprotonated tautomer (PhSO2NOH−) to
[FeII(N3PyS)]+, leading to 2. Complex 2 is the first
spectroscopically characterized example, to our knowledge,
of P.A. anion bound to a redox-active metal center. Warming
1
of 2 above −60 °C yields the stable {FeNO}7 complex [Fe(NO)(N3PyS)]BF4 (4), as evidenced by H NMR, ATR-IR, and
Mossbauer spectroscopies. Isotope labeling experiments with 15N-labeled P.A. confirm that the nitrosyl ligand in 4 derives from
̈
P.A. In contrast, addition of a second equivalent of a strong base leads to S−N cleavage and production of an {FeNO}8 species,
the deprotonated analog of an Fe−HNO complex. This work has implications for the targeted delivery of HNO/NO−/NO· to
nonheme Fe centers in biological and synthetic applications, and suggests a new role for nonheme FeII complexes in the assisted
degradation of HNO donor molecules.
rapid (8.0 × 106 M−1 s−1)39 dimerization to give hyponitrous
INTRODUCTION
■
acid, HONNOH, which further decays to yield N2O and
H2O (8.0 × 10−4 s−1).40 To deliver the short-lived HNO in situ
for biochemical studies or medical applications, donor
molecules that release HNO in response to external stimuli
(e.g., pH, temperature, light) must be employed. Sulfohy-
droxamic acids (RSO2NHOH, R = -alkyl, aryl) constitute one
important class of HNO donors that release HNO mainly in
response to pH,41 and in some cases, through photo-
activation.42,43 Piloty’s acid (N-hydroxybenzenesulfonamide;
P.A.), first reported in 1896,44 is the oldest member of this
class, and releases HNO under basic conditions according to
the mechanism shown in Scheme 1. However, donation of
HNO by P.A. is slow (kmax ∼ 10−3 to 10−4 s−1 at 25 °C).41
Given that basic conditions are necessary to produce HNO,
the P.A.− anion is presumed to be the immediate precursor to
HNO release. There are two possible tautomers of
monodeprotonated P.A.−, either PhSO2NOH− (N-deproto-
nated) or PhSO2NHO− (O-deprotonated). Given the
relatively slow rates of HNO release from P.A., we
hypothesized that the P.A.− anion could be an important
Nitroxyl (HNO/NO−, also known as azanone or nitrosyl
hydride) is an important biomolecule whose endogenous
production and pharmacology have yet to be resolved.1−6 The
HNO molecule is a positive cardiac inotrope used in the
treatment of heart failure,7 and is also a potent inhibitor of
aldehyde dehydrogenase,8−10 showing promise as a therapeutic
for alcohol addiction. It is currently thought that HNO
participates in signal transduction in biology by coordinating to
the heme center in soluble guanylate cyclase.11,12 Metal-bound
HNO species (Mn+(HNO)) have been implicated as reactive
intermediates in a number of catalytic cycles that process
NOx,13−15 but only a few examples of Mn+(HNO) complexes
with first row metals have been reported to date.16−26 HNO
has also been used as a surrogate for O2; for example,
substitution of O2 by HNO in reactions with the enzyme
manganese quercetin 2,3-dioxygenase led to substrate nitro-
xygenation (i.e., HNO incorporation).27,28 Furthermore, there
is considerable interest in the reduction/protonation of metal
nitrosyl species (M(NO·)) to produce viable HNO-releasing
agents, as well as to examine the feasibility of HNO production
from metal centers and NO· in vivo.24,25,29−38
In contrast to NO·, which is relatively stable under anaerobic
conditions, HNO has an intrinsically short lifetime due to
Received: February 13, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.9b01700
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Journal of the American Chemical Society
Article
the P.A. ligand in 2 is coordinated through nitrogen. Using a
Scheme 1. Mechanism of HNO Release from Piloty’s Acid
combination of Mossbauer, paramagnetic 1H NMR, and
̈
attenuated total reflectance infrared (ATR-IR) spectroscopies,
we show that the thermal decay of 2 under strictly anaerobic
conditions yields the {FeNO}7 complex 4. In contrast, the
{FeNO}8 complex 3 is produced from adding more base,
consistent with 2 being a precursor to Fe(HNO).
RESULTS AND DISCUSSION
■
Synthesis and Solution State Properties of
[57FeII(CH3CN)(N3PyS)]BF4 (1-57Fe). The FeII−thiolate start-
ing material, [57FeII(CH3CN)(N3PyS)]BF4 (1-57Fe), was
prepared from the isotopically enriched (57Fe, 95%) FeII−
thioether precursor, [57FeII(CH3CN)(N3PySEtCN)](BF4)2
(5-57Fe) by using the method previously described for the
normal abundance analogue (Scheme 2).29 Treatment of
player in the reactivity with a cationic metal center. Precedent
for this hypothesis comes from a high-resolution crystal
structure of zinc carbonic anhydrase (human isoform II)
inhibited by P.A.45 This structure shows that P.A.− anion binds
to the active site by coordinating to the zinc(II) center through
the sulfonamido N atom, i.e. the N-deprotonated tautomer.
Alkali metal salts of P.A.− anion (e.g., Li+, Na+) exist as the N-
deprotonated tautomer in both the solid state and in dioxane
solution,46,47 and there is computational evidence to suggest
that this tautomer is energetically favored.48 Other data suggest
that it is the O-deprotonated tautomer that leads to HNO
release.49
Scheme 2. Preparation of Isotopically Labeled
[FeII(CH3CN)(N3PyS)]BF4 (1-57Fe)
The interactions of redox-active, biologically relevant metal
ions such as Fe, Mn, or Co, with HNO donors such as P.A.
have been examined previously, and in some cases, were shown
to accelerate the decomposition of P.A. and related
RSO2NHOH compounds.48,50,51 However, there is still
relatively little known about the mechanism of action between
redox-active metal ions and RSO2NHOH compounds. In
particular, direct structural or spectroscopic evidence for
metal−P.A. adducts is lacking. We have set out to examine
nonheme iron complexes and their interaction with P.A. and
other RSO2NHOH donors in order to characterize the
potential binding modes, mechanisms of action, and HNO-
releasing properties of Fe/P.A. systems.
5-57Fe with KOtBu (1 equiv) in dry, deoxygenated CH3CN at
23 °C produces an instantaneous color change from orange-
red to wine-red, corresponding to removal of the base-labile 2-
cyanoethyl protecting group from the sulfur donor. Pure
1-57Fe, a dark red-brown solid, is isolated in 65% crystalline
yield after addition of excess diethyl ether to a concentrated
CH3CN/toluene (1:1 v/v) solution of the product.
The zero-field 57Fe Mossbauer spectra of 5- Fe and 1- Fe,
recorded at 80 K in frozen CH3CN, are shown in Figure 1.
Thioether-ligated 5-57Fe produces a sharp quadrupole doublet
with δ = 0.44 and |ΔEQ| = 0.55 mm s−1, consistent with a low-
spin (ls, S = 0) FeII ground state. Thiolate-ligated 1-57Fe
exhibits parameters (δ = 0.43, |ΔEQ| = 0.31 mm s−1) that are
also consistent with ls FeII and accord well with the data
previously collected at 5.4 K for crystalline 1 (δ = 0.41 and
|ΔEQ| = 0.26 mm s−1).53 This result provides strong evidence
that 1 retains its mononuclear, six-coordinate, solid state
structure upon dissolution in CH3CN. The nearly identical
isomer shifts observed for 5-57Fe and 1-57Fe (0.44 vs 0.43 mm
s−1) make good sense in light of the fact that these complexes
possess very similar first coordination sphere metrics by single
crystal X-ray diffraction. In the solid state, complexes 1 and 5
exhibit average Fe−Npy distances of 1.9594(13) Å and
1.9659(13) and Fe−S distances of 2.2848(4) and 2.3018(4)
Å, respectively.53,54 Despite their similar overall coordination
environments, 5-57Fe and 1-57Fe have different |ΔEQ| values
(0.55 vs 0.31 mm s−1), which likely reflects the difference in
thioether versus thiolate ligation.
57
57
̈
We previously described the reaction of an FeII−solvento
complex, [FeII(CH3CN)(N3PyS)]BF4 (1) with P.A. in the
presence of KOtBu/18-crown-6 (1 equiv) at −40 °C. This
reaction gave a new, thermally unstable species 2, which was
characterized by UV−vis spectroscopy (λmax = 418, 501 nm).29
Reaction of 2 with a further equivalent of base at −40 °C
afforded the {FeNO}8 complex [Fe(NO)(N3PyS)] (3) (λmax
= 520, 720 nm), which was also obtained by one-electron
reduction of an {FeNO}7 precursor, [Fe(NO)(N3PyS)]BF4
(4), with decamethylcobaltocene (CoCp*2). The {FeNO}8
complex 3 exhibited a rare S = 1 ground state,29,52 and was
shown to produce N2O in 54% yield upon standing in solution
at 23 °C. We reasoned that complex 2, the immediate
precursor to the {FeNO}8 species, could be an Fe(HNO) or
Fe−P.A. adduct, but no further spectroscopic characterization
was obtained.
Herein we provide new characterization of 2 by 57Fe
Reactivity of 1 with Piloty’s Acid. It was shown
previously that complex 1 reacted with Piloty’s acid (P.A.)
and KOtBu/18-crown-6 in CH3CN at −40 °C to give a new,
thermally unstable intermediate 2, which was characterized by
UV−vis spectroscopy (λmax = 418, 501 nm).29 This species is
not observed when the reaction is run at 23 °C (vide infra).
̈
Mossbauer and Fe K-edge X-ray absorption spectroscopies,
which show that 2 is in fact a high-spin (S = 2) FeII−P.A.
adduct. Density functional theory (DFT) calculations,
̈
including predicted Mossbauer parameters for N-bound Fe−
P.A. adducts, support the spectroscopic results and suggest that
B
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57
̈
Figure 2. Fe Mossbauer spectrum (80 K, C3H7CN/THF, 9:1 v/v)
of 1-57Fe + P.A. (1 equiv) + KOtBu/18-crown-6 (1 equiv) reacted at
−60 °C for 2 h according to Scheme 3. Experimental data (black
dots); best fit (red line); majority hs FeII species 2-57Fe (dashed blue
line, 75%; Voigt line shape); minor ls FeII species (solid orange line,
7%; Lorentzian line shape) corresponding to 1-57Fe; minor species
(solid gray line, 18%) corresponding to an unidentified product.
57
̈
Figure 1. Fe Mossbauer spectra (80 K in frozen CH3CN) showing
the conversion of 5-57Fe to 1-57Fe according to Scheme 2.
Experimental data are plotted as black dots while best fits are
overlaid as solid lines. (a) Spectrum of 5-57Fe fit to a single doublet
with δ = 0.44, |ΔEQ| = 0.55, ΓL=R = 0.29 mm s−1. (b) Spectrum of
1-57Fe fit to a single doublet with δ = 0.43, |ΔEQ| = 0.31, ΓL=R = 0.25
mm s−1.
with a hs FeII species (δ = 1.05, |ΔEQ| = 2.93 mm s−1; dashed
blue line). We assign this doublet to 2-57Fe. A minor
component corresponds to starting 1-57Fe (δ = 0.42, |ΔEQ|
= 0.33 mm s−1; solid orange line, 7% of fit). A third, broad
component with δ = 0.36 and |ΔEQ| = 0.61 mm s−1 (18%) is
required to fit the remaining area and corresponds to an
unidentified product. Longer reaction times (3−6 h), or lower
temperature (−80 °C), did not significantly increase the
spectroscopic yield of 2-57Fe.
We hypothesized that this intermediate might be either an
Fe(HNO) or Fe(P.A.) adduct, but no further spectroscopic
characterization was obtained. With 1-57Fe in hand,
57
̈
intermediate 2- Fe could be examined by Mossbauer
spectroscopy. To increase the stability of 2-57Fe, we changed
to a mixed solvent system of n-butyronitrile/THF, which
allowed for lower temperatures to be employed and for the
solubility of KOtBu/18-crown-6 to be maintained.
Our group has shown that an analogous change from low-
spin to high-spin FeII occurs when 1 is dissolved in
methanol.53,55 This hs FeII species was assigned as
The generation of 2-57Fe at ca. −60 °C in C3H7CN/THF is
outlined in Scheme 3. Anaerobic, dropwise addition of a THF
II
+
̈
[Fe (CH3OH)(N3PyS)] (1-CH3OH). The 80 K Mossbauer
spectrum for 1-57Fe dissolved in methanol contains two
quadrupole doublets, one arising from the acetonitrile-bound
species (60%), and the other arising from the proposed
methanol adduct (δ = 1.05, |ΔEQ| = 2.77 mm s−1; 40%)
(Figure S1). At room temperature, the CH3CN in 1 is
completely displaced by CH3OH as evidenced by Evans
Scheme 3. Reaction of [FeII(CH3CN)(N3PyS)]BF4 and
Piloty’s Acid at −60 °C
method measurements,53 and the Mossbauer data show that
̈
upon freezing the rebinding of CH3CN is greatly favored. In
contrast, the P.A.-derived ligand readily outcompetes either
CH3CN or the related C3H7CN solvent for the Fe binding site.
Notably, the isomer shifts for 1-57Fe in CH3OH and 2-57Fe are
identical, which indicates that their overall coordination
environments are similar. The combined observations suggest
that binding of the reactive P.A. species to Fe is favored over
CH3CN, and maintains the [FeII(N3PyS)]+ core structure.
As shown in Scheme 1, HNO release from Piloty’s acid is
accompanied by generation of benzenesulfinate anion
solution of KOtBu/18-crown-6 (1 equiv) to a precooled
mixture of 1-57Fe (5 mM) and P.A. (1 equiv) in C3H7CN
produces a gradual color change from deep wine red to bright
pink red over 2 h. This color change is due to loss of the
intense charge-transfer bands for diamagnetic 1 (ε ∼ 4000−
5000 M−1 cm−1)53 as intermediate 2 (ε ∼ 1000 M−1 cm−1) is
formed, as shown previously. An aliquot of the cold,
deoxygenated reaction mixture can be quickly transferred to
(PhSO2 ), a possible ligand for [FeII(N3PyS)]+. To test
−
whether 2-57Fe is an FeII−PhSO2− adduct rather than a P.A. or
HNO adduct, 1-57Fe was reacted with Na+PhSO2 according
−
̈
a Mossbauer sample holder using air-free techniques.
to Scheme 4. This reaction yielded a new, thermally stable hs
FeII species (6-57Fe) with δ = 1.07 and |ΔEQ| = 3.11 mm s−1,
which we assign to a coordinated FeII(O2SPh) adduct.
Importantly, these parameters are significantly different from
those seen for 2-57Fe. Complex 6-57Fe was also produced in
Alternatively, the reaction mixture can be rapidly frozen by
pouring into liquid N2, giving frozen pellets which then can be
further pulverized to give a fine powder that is loaded into the
̈
Mossbauer sample holder.
A representative Mossbauer spectrum for the reaction
mixture is shown in Figure 2. The major species, which
accounts for 75% of the fitted area, has parameters consistent
̈
̈
good yield as seen by Mossbauer spectroscopy (84%) when
1-57Fe was treated with a previously reacted 1:1 mixture of
P.A. and KOtBu/18-crown-6 at 23 °C (Scheme 4 and Figure
C
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Scheme 4. Reaction of [FeII(CH3CN)(N3PyS)]BF4 and
Benzenesulfinate Anion (PhSO2 ) Sources at 23 °C
complex 2 is shown in Figure 4, overlaid with the spectrum of
starting 1 for comparison. The energies of the rising edge
−
Figure 4. Normalized Fe K-edge XANES of the hs FeII−P.A. complex
2 (solid red line) and the FeII−solvento precursor 1 (solid blue line)
(frozen C3H7CN/THF, 9:1 v/v).
inflection points increase upon going from 1 (7120.7 eV) to 2
(7121.5 eV), consistent with back-donation of electron density
from FeII to the N−O π* of P.A.
The extended X-ray absorption fine structure (EXAFS) of 2
is shown in Figure 5a (solid green line), along with an overlaid
3). The latter result confirms that a 1:1 mixture of P.A. and
strong base in organic solvent produces PhSO2 , as it does in
−
57
̈
Figure 3. Fe Mossbauer spectrum (80 K, C3H7CN/THF, 9:1 v/v)
for the reaction of 1-57Fe with a previously reacted 1:1 mixture of P.A
and KOtBu/18-crown-6 at 23 °C. Experimental data (black dots);
−
best fit (red line); FeII(PhSO2 ) adduct 6-57Fe (dashed green line,
84%); 1-57Fe (solid orange line, 12%); minor species (solid gray line,
4%) corresponding to an unidentified product.
water.41 Taken together, these experiments show that
metastable 2 is not an FeII(PhSO2 ) adduct.
−
Next, we considered the possibility that 2 may be an Fe−
HNO adduct ({Fe(HNO)}8 in the Enemark−Feltham
̈
notation). The only Mossbauer data on a proposed {Fe-
(HNO)}8 species comes from cryoreduction of the {FeNO}7
Figure 5. (a) Fe K-edge EXAFS of 2 plotted from k = 2 to 12 Å−1
(solid green line) overlaid with the corresponding best fit (dashed
black line). (b) Fourier transforms of the EXAFS data, fit, and residual
(data minus fit; solid gray line).
adduct of taurine dioxygenase (TauD).56 This species could
̈
only be generated in low yield (17%), but gave Mossbauer
parameters of δ = 0.80 and |ΔEQ| = 1.64 mm s−1 that were
consistent with DFT calculations for a quintet (S = 2)
{Fe(HNO)}8 structure. We turned to DFT methods to predict
line of best fit (dashed black line). The corresponding Fourier
transforms of the EXAFS data and best fit are shown in Figure
5b. Relevant fitting parameters are provided in Table 1, and
result from sequentially improved fits to the data (see Table
S1). The best fit for complex 2 includes four N/O scatterers at
2.17 Å and one S scatterer at 2.66 Å, which correspond to the
Fe(N3PyS) core. A fifth N/O scatterer at 1.95 Å, an Fe−O
scatterer at 2.51 Å, and a second S scatterer at 3.25 Å can be
isolated from the data. These structural features strongly
support the assignment of 2 as a hs FeII−Piloty’s acid adduct.
DFT-Calculated Structures for the FeII−Piloty’s Acid
Adduct (2). Given the availability of EXAFS data for 2, a
constrained geometry optimization approach was employed.
+
̈
the Mossbauer parameters for [Fe(HNO)(N3PyS)] . Opti-
mized geometries of the S = 0, 1, and 2 spin states were
obtained and employed for the Mossbauer calculations (see
̈
Supporting Information). The calculated isomer shifts range
from 0.27 to 0.55 mm s−1 (estimated maximum errors of 0.1
mm s−1),57 which are clearly inconsistent with the
experimental data for 2-57Fe (δ = 1.05 mm s−1). The
calculated quadrupole splittings (estimated maximum errors
of 0.5 mm s−1)57 are also in poor agreement with 2-57Fe. We
conclude that 2 is not an Fe−HNO adduct.
Fe K-Edge X-ray Absorption Spectroscopy (XAS). The
normalized X-ray absorption near-edge structure (XANES) of
D
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̈
S3), the calculated Mossbauer parameters were δ = 1.06 and
|ΔEQ| = 2.84 mm s−1. An increase in the average Fe−
N(N3PyS) distance for 5B (2.28 Å) translates to an increase in
isomer shift of ∼0.1 mm s−1, but has no impact on the
calculated quadrupole splitting. Thus, EXAFS-derived struc-
Table 1. Best Fit to Fe K-edge EXAFS of Complex 2 (F =
32.97%)
a b c
, ,
CN
R (Å)
σ2
Fe−S
Fe−N
Fe−N(O)
Fe−S
Fe−C
1
4
1
1
8
2.66
2.17
1.95
3.25
4.74
3.84
2.51
0.01
0.01
0.01
0.01
0.01
0.01
0.02
0.005
0.010
0.005
0.005
0.004
0.002
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.002
̈
tures for 2 lead to calculated Mossbauer parameters that are
fully consistent with the experimental data.
Unconstrained geometry optimizations for quintet FeII(κ1-
N-PhSO2NOH)(N3PyS)] produce shorter Fe−S(N3PyS)
distances of 2.4−2.5 Å, depending on the choice of functional
or basis set. These distances are not consistent with the EXAFS
results. We considered the possibility that the N3PyS sulfur
donor might be protonated, which could lead to elongation of
the Fe−S bond. Accordingly, we investigated [FeII(κ1-N-
PhSO2NO)(N3PySH)], a tautomer of [FeII(κ1-N-
PhSO2NOH)(N3PyS)] that could be obtained by internal
proton transfer from P.A. to N3PyS. Although this structure
exhibits an Fe−S(N3PySH) distance of 2.60 Å, which is similar
to the EXAFS-derived distance, it is ∼20 kcal mol−1 higher in
energy than S-deprotonated structures obtained through fully
Fe−C
Fe−O
16
1
a
b
See Table S1 for a list of sequentially improved fits. EXAFS data
were fit with EXAFSPAK using paths calculated by FEFF7.
Coordination numbers were held constant, whereas distances (R)
and Debye−Waller factors (σ2) were allowed to float. Goodness of fit
w a s m e a s u r e d w i t h F , w h i c h w a s d e fi n e d a s
Ä
Å
Å
Å
Å
Å
É
1/2
Ñ
Ñ
Ñ
2
)
n
i
3
i
∑ [k (EXAFS − EXAFScalc)i] /n . E0 for the best fit was
Ñ
(
obs
Ñ
Å
Ç
Ñ
Ö
c
+3.64 eV. Tabulated errors ( ) correspond to fitting errors.
Expected errors for R are 0.02 Å; expected errors in CN are ca.
20−25% (ref 58).
5
relaxed optimizations. Thus, we conclude that A is the most
Our computational studies commenced with quintet [FeII(κ1-
N-PhSO2NOH)(N3PyS)] (5A, Figure 6 and Table S3) which
reasonable structure for 2. However, we cannot exclude the
possibility that hydrogen bonding or other electrostatic effects
(such as an N3PyS···K+ interaction) could contribute to
elongation of the Fe−S bond in solution.
Generation of an {FeNO}7 Complex from 2. A rapid
color change from bright red to dark brown is observed when
samples of 2 are warmed to 23 °C. These changes correspond
to loss of the UV−vis bands for 2 and the appearance of a
species with λmax = 350, 450, and 550 nm (Figure 7). This
Figure 6. DFT structure of [FeII(κ1-N-PhSO2NOH)(N3PyS)] (5A)
5
Figure 7. UV−vis spectra (CH3CN, 0.1 mM Fe, −40 °C) comparing
FeII−solvento starting material 1 (solid black line), FeII−P.A. complex
2 (dashed red line), and the thermal decay product(s) of 2 (solid
brown line).
obtained by optimizing with constraints from the EXAFS data for 2
(Table 1). Calculated 57Fe Mossbauer parameters are shown in green.
̈
C and H atoms are depicted as brown and white spheres, respectively.
contains an N-bound, monodeprotonated P.A. ligand in the
sixth coordination site. From EXAFS, the Fe−S(N3PyS), Fe−
S(P.A.), Fe−N(O), and Fe···O(N) distances were fixed at
2.66, 3.25, 1.95, and 2.51 Å, respectively. The four Fe−
N(N3PyS) distances, which typically have a large degree of
asymmetry, were chosen such that their average would equal
2.17 Å (i.e., the EXAFS value for four Fe−N scatterers), while
all remaining distances were allowed to vary. Using this
spectroscopically constrained structure led to the optimized
geometry shown in Figure 6. Calculation of the 57Fe
spectrum is consistent with Fe(NO)(N3PyS)]BF4 (4), an
{FeNO}7 complex previously synthesized.59 A representative
1H NMR spectrum of final reaction mixtures in CD3CN is
shown in Figure 8 (top), and contains a number of well-
separated, paramagnetically shifted peaks between +35 and −5
ppm. These signals match the spectrum seen for 4 (Figure 8,
bottom). No other paramagnetically shifted 1H resonances are
observed across the acquisition window (+130 to −50 ppm).
The generation of 4 is further evidenced by attenuated total
reflectance infrared (ATR-IR) spectroscopy with 15N-labeled
Piloty’s acid, which was synthesized according to Scheme 5.
Samples of 2-57Fe were prepared with 14N−P.A. and 15N−P.A.
according to the standard protocol, yielding identical
̈
Mossbauer parameters for this geometry gave δ = 0.96 and
|ΔEQ| = 2.81 mm s−1, both of which accord well with the
experimental data for 2-57Fe (δ = 1.05 and |ΔEQ| = 2.93 mm
s−1). Similarly, when the four Fe−N(N3PyS) distances were
5
̈
allowed to optimize without constraints (structure B, Table
Mossbauer spectra which established the same purity level
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line).55,59 No other 14N/15N shifts are evident in the ATR-IR
data. Importantly, these results establish that the nitrosyl ligand
in 4 derives from Piloty’s acid.
57
̈
Mossbauer data for the thermal decay of 2- Fe is presented
in Figure 10. Given that the spectrum of 4-57Fe is well-resolved
1
Figure 8. Paramagnetic H NMR spectra (CD3CN, 23 °C) showing
the thermal decay product of 2 (top) and an authentic sample of the
{FeNO}7 complex 4 (bottom).
Scheme 5. Synthesis of 15N-Labeled Piloty’s Acid
for both samples (Figures S3). Warming of these samples to 23
°C similarly yielded identical 1H NMR spectra consistent with
the {FeNO}7 complex 4. These samples gave the ATR-IR
57
̈
Figure 10. Fe Mossbauer spectrum (80 K in frozen CH3CN)
showing the thermal decay of 2-57Fe. Experimental data (black dots);
best fit (red line); majority {FeNO}7 product 4-57Fe (dashed purple
line, 78%); minor hs FeII coproduct (solid green line, 22%).
at 80 K in CH3CN (but not in mixtures of C3H7CN/THF),
final reaction mixtures in C3H7CN/THF (9:1 v/v) were
concentrated to dryness under high vacuum before adding
CH3CN. The major species with δ = 0.31 and |ΔEQ| = 0.57
mm s−1 (78% of fit) corresponds to 4-57Fe. A minor species
with δ = 1.09 and |ΔEQ| = 3.12 mm s−1 (22% of fit) is also
observed, which we tentatively attribute to coordination of the
−
PhSO2 byproduct at Fe. Thus, the combined data
̈
(Mossbauer, NMR, ATR-IR, and UV−vis) confirm that the
major product obtained from warming 2 at −60 to +23 °C is
the {FeNO}7 complex 4.
Mechanism. A mechanism for the formation and decay of
Fe−P.A. complex 2 is shown in Scheme 6. Deprotonation of
P.A. with KOtBu/18-crown-6 (1 equiv) at −60 °C affords the
P.A. monoanion (PhSO2NOH−). Our data suggest that this
species displaces CH3CN from 1, yielding P.A.-bound 2,
̈
before HNO release can occur. The combined Mossbauer and
XAS data, supported by DFT calculations, indicate that the
P.A. ligand in 2 is coordinated via nitrogen. Addition of a
second equivalent of KOtBu/18-crown-6 leads to the {FeNO}8
complex 3. In the absence of excess base, thermal decay of 2
from −60 to +23 °C induces S−N bond cleavage, leading to
the {FeNO}7 complex 4 (78%). There is precedent for the
production of {FeNO}7 complexes from Piloty’s acid and
related RSO2NHOH compounds;60,61 however, these com-
plexes were obtained from iron(III) precursors via HNO/NO−
transfer. The production of an {FeNO}7 complex from Piloty’s
acid, base, and an iron(II) precursor, as described here, is
consistent with formation of a highly reactive {Fe(HNO)}8
species, which is then oxidized by an as yet unidentified
pathway to give a stable {FeNO}7 complex. Efforts are ongoing
to trap the elusive nonheme {Fe(HNO)}8 species.
Figure 9. ATR-IR spectra following the thermal decay of 2. Top:
authentic 4-14N (green line). Middle: thermal decay of 2-14N (black
line) and 2-15N (red line), overlaid and normalized relative to the
1467 and 1444 cm−1 N3PyS ligand vibrations (denoted by *). The
1351 cm−1 mode originates from 18-crown-6. Bottom: difference
spectrum (15N minus 14N, blue line) showing isotopically sensitive
modes at 1735 and 1642 cm−1 corresponding to 4.
spectra shown in Figure 9 (14N, black line; 15N, red line). The
15N minus 14N difference spectrum (blue line) contains a
prominent band at 1642 cm−1 (positive intensity) that shifts to
1613 cm−1 (negative intensity) upon 15N substitution. This
band is assigned to the ν(N−O) mode of 4 and exhibits a
downshift of 29 cm−1 that is in excellent agreement with the
harmonic oscillator prediction for an isolated N−O stretch
(−30 cm−1). A second isotopically sensitive band is observed
at 1735 cm−1 (15N: 1703 cm−1), also arising from 4. These two
ν(N−O) modes belong to the S = 1/2 and S = 3/2 spin states
of 4, which are both populated at 23 °C (Figure 9, green
CONCLUSIONS
■
This work describes the characterization of a rare mono-
nuclear, nonheme FeII−Piloty’s acid adduct by UV−vis and
57
̈
Fe Mossbauer spectroscopy, Fe K-edge XAS, and DFT. This
F
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Scheme 6. Possible Mechanism of Formation of the FeII−Piloty’s Acid Adduct and Reaction Pathways Leading to {FeNO}7
and {FeNO}8 Products
from a purple sodium/benzophenone ketyl radical solution.
Butyronitrile was distilled from Na2CO3/KMnO4 according to a
literature procedure.63 Diethyl ether was obtained from a PureSolv
solvent purification system (SPS) and used without further
purification. All solvents were degassed by a minimum of three
freeze−pump−thaw cycles and stored over freshly activated 3 Å
molecular sieves in the drybox.
adduct can be formed at low temperatures from an iron(II)
precursor, P.A., and one equivalent of a strong alkoxide base.
Piloty’s acid and related RSO2NHOH compounds are
synthetic precursors to HNO/NO−, and are known to react
with iron(III) centers to give {FeNO}7 species.60,61 Much less
is known about the reactions of RSO2NHOH donors with
iron(II) complexes, where the anticipated products are less
stable, one-electron reduced {FeNO}8 or {Fe(HNO)}8
species. In this regard, we have provided strong evidence for
a reaction where the P.A. donor, rather than free HNO, binds
to a nonheme iron(II) complex, which then ultimately
facilitates nitroxyl production on the metal. Our results
indicate that binding of monoanionic P.A. to the FeII center
is favored over spontaneous S−N bond cleavage to give HNO
Instrumentation. UV−visible spectra were recorded on a Varian
Cary 50 Bio spectrophotometer integrated with a Unisoku USP-203
1
cryostat. H NMR spectra were recorded on a Bruker Avance 400
MHz FT-NMR spectrometer at 298 K and referenced against residual
solvent proton signals. Attenuated total reflectance (ATR) infrared
spectra were obtained with a Golden Gate Reflectance diamond cell
57
̈
in a Nexus 670 Thermo-Nicolet FTIR spectrometer. Fe Mossbauer
spectra were collected on a spectrometer from SEE Co. (Science
Engineering & Education Co., MN) integrated with a Janis SVT-400-
MOSS LHe/LN2 cryostat. The spectrometer was operated in the
constant acceleration mode in a transmission geometry.
−
and benzenesulfinate anion (PhSO2 ).
Our findings also have implications for the chemistry of
HNO with nonheme iron−thiolate metalloenzymes. The FeII
precursor used in this study, [FeII(CH3CN)(N3PyS)]BF4, was
previously shown to be a structural and functional mimic of the
thiol dioxygenases.53,59 It is well-known that HNO reacts with
free thiols to give sulfinamide (RS(O)NH2) and (RSSR)
products,12 but considerably less is known about the reaction
of HNO with metal-bound thiols/thiolates. In the present
example, we have shown that coordination of P.A. to Fe can
lead to either sulfur-ligated {FeNO}8 or {FeNO}7 species,
while the sulfur donor remains unmodified. Thus, formation of
a metal-bound adduct might protect sulfur sites from
irreversible modification by preventing formation of free
HNO from P.A. or similar HNO donors.
[57FeII(CH3CN)(N3PySEtCN)](BF4)2 (5-57Fe). The title compound
was prepared as described previously29 with a 57Fe enrichment level of
approximately 80%. A three-necked, 100 mL round-bottom flask
equipped with a stir bar was fitted with a reflux condenser. Amounts
of 57Fe powder (33 mg, 0.58 mmol) and 56Fe powder (11 mg, 0.20
mmol) were added. A solution of HBF4 (48 wt % in H2O, 0.3 mL, 2.9
equiv) was added via syringe, and the mixture was refluxed for 45 min
until no Fe powder remained. The resulting aqueous solution of 57Fe-
enriched 57Fe(BF4)2 was cooled to 23 °C and then diluted with
CH3CN (5 mL). The N3PySEtCN ligand (355.1 mg, 0.786 mmol)
was dissolved in CH3CN (10 mL), and the 57Fe(BF4)2 solution was
added at 23 °C, producing an immediate color change from pale
orange to dark red-orange. After 2 h, excess Et2O (20 mL) was added,
resulting in precipitation of a brown residue. The supernatant was
decanted by cannula, and the residue was dried in vacuo over P4O10.
Slow vapor diffusion of Et2O into a concentrated CH3CN solution of
the product furnished large red crystals of 5-57Fe after 24 h (413 mg,
73% yield).
EXPERIMENTAL SECTION
■
General Procedures. All syntheses and manipulations were
conducted in an N2-filled drybox (vacuum atmospheres, O2 < 0.2
ppm, H2O < 0.5 ppm) or using standard Schlenk techniques under an
atmosphere of dry Ar unless otherwise noted. 57Fe powder (95.5 atom
%; 98% purity) was purchased from Cambridge Isotope Laboratories.
Piloty’s acid (N-hydroxybenzenesulfonamide) and 15N-labeled
Piloty’s acid were synthesized according to a literature procedure.62
Potassium tert-butoxide (KOtBu, sublimed grade, 99.99% trace metals
basis) was purchased from Sigma-Aldrich and stored in the drybox.
18-crown-6 was purchased from Sigma-Aldrich and purified by
recrystallization from hot acetonitrile. The 18-crown-6-acetonitrile
solvate thus obtained was dried under high-vacuum at 35 °C for
several hours to afford pure 18-crown-6. Acetonitrile, acetonitrile-d3,
and toluene were distilled from CaH2. Tetrahydrofuran was distilled
[57FeII(CH3CN)(N3PyS)]BF4 (1-57Fe). The title compound was
prepared as described previously29 with a 57Fe enrichment level of
approximately 80%. An amount of crystalline 5-57Fe (81 mg, 0.11
mmol) was dissolved with vigorous stirring in CH3CN (4 mL). A
slurry of KOtBu (13 mg, 0.11 mmol, 1 equiv) in CH3CN (2 mL) was
prepared and added dropwise at 23 °C to the solution of 5-57Fe,
resulting in an immediate color change from dark red-orange to dark
wine red. The mixture was stirred for 2.5 h, filtered through a short
plug of Celite, and concentrated under reduced pressure. The dark
red-brown residue was triturated with Et2O (3 × 5 mL) and dried to
yield crude 1-57Fe. The crude product was dissolved in a mixture of
CH3CN and toluene (1:1 v/v, 4 mL) and filtered through Celite.
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Excess Et2O (12 mL) was added, and the turbid mixture was placed in
a −35 °C freezer overnight, resulting in precipitation of a red-brown
microcrystalline solid. Removal of the yellow-brown supernatant and
further drying under reduced pressure yielded pure 1-57Fe (42 mg,
65% yield).
rapidly bleached within ∼5 s. Stirring was continued for 48 h at 23 °C,
at which point the mixture appeared colorless and slightly turbid.
An amount of 1-57Fe (2 mg, 3 μmol) was dissolved in C3H7CN
(600 μL). An aliquot of the previously reacted P.A./KOtBu/18-
crown-6 mixture (80 μL; 3 μmol, 1 equiv per Fe based on initial P.A.)
was added dropwise to 1-57Fe, resulting in an immediate color change
57
57
̈
̈
Fe Mossbauer Spectroscopy. Fe Mossbauer spectra were
̈
from dark wine-red to bright pink-red. For Mossbauer spectroscopy,
collected on frozen solution samples at 80 K in the absence of an
applied magnetic field. 57Fe-enriched samples were prepared in
custom Delrin cups (10.0 mm × 12.4 mm OD) equipped with tight-
fitting Delrin inserts (5.0 mm × 11.0 mm). All samples were stored
an aliquot of the reaction mixture (400 μL) was transferred to a
sample cup and frozen in liquid N2.
Reaction of 1-57Fe with NaPhSO2 at 23 °C. An amount of
1-57Fe (2 mg, 3 μmol) was dissolved in a mixture of C3H7CN (500
μL) and THF (50 μL). Sodium benzenesulfinate (11 mg, 66 μmol)
and 18-crown-6 (18 mg, 70 μmol) were combined with C3H7CN (1.0
mL), and the resulting slurry was vigorously stirred for 1 h. An
̈
under LN2 prior to analysis. The zero velocity of each Mossbauer
spectrum refers to the centroid of a 25 μm metallic iron (α-Fe) foil
collected at room temperature. The WMOSS program (SEE Co.,
formerly WEB Research Co., Edina, MN) was used to analyze the
−
amount of the slurry (60 μL; 4 μmol Na+PhSO2 ) was added
̈
Mossbauer data. Data were fit to quadrupole doublets with Lorentzian
dropwise to 1-57Fe with continued stirring, producing an immediate
or Voigt line shapes as specified.
̈
color change from dark wine-red to bright pink-red. For Mossbauer
Fe K-Edge X-ray Absorption Spectroscopy. Fe K-edge XAS
and extended X-ray absorption fine structure (EXAFS) data were
collected at the 16-pole, 2-T wiggler Beamline 9-3 at the Stanford
Synchrotron Radiation Lightsource (SSRL) under ring conditions of
500 mA and 3 GeV. All samples were 57Fe-enriched in order to assess
spectroscopy, an aliquot of the reaction mixture (400 μL) was
transferred to a sample cup and frozen in liquid N2.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
■
̈
purity by Mossbauer spectroscopy, and were prepared in custom
S
̈
screw-top Delrin Mossbauer cups. The base of each cup contained a
slit (1 × 4 mm) that was covered in 38 μM Kapton tape to provide an
X-ray transparent window, thus allowing for sequential Mossbauer
ACS Publications website at DOI: 10.1021/jacs.9b01700.
̈
57
and XAS measurements. Samples were maintained at 10 K in an
Oxford liquid He flow cryostat. A Si(220) double-crystal mono-
chromator was used for energy selection. A Rh-coated mirror (set to
an energy cutoff of 9 keV) was used for harmonic rejection. Internal
incident energy calibration was achieved by assigning the first
inflection points of a Fe foil placed downstream of the sample to
7111.2 eV. Data were collected in fluorescence mode using a
Canberra 30-element Ge array detector windowed on Fe Kα
emission. Elastic scatter into the detector was attenuated using a
Soller slit with an upstream Co filter. Multiple scans were measured
and averaged with SIXPACK64 software package. No spectral changes
due to photodamage were observed after multiple scans for these
complexes. Data were normalized to postedge jumps of 1.0 at 7130 eV
in SIXPACK by applying a Gaussian normalization for the pre-edge
and a three-region cubic spline to model the smooth background
above the edge. EXAFS data were fitted using the OPT module of
EXAFSPAK65 using initial scattering paths calculated using
FEFF7.66,67
̈
Fe Mossbauer data/fits, Fe K-edge EXAFS data/fits,
and DFT calculations (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: dpg@jhu.edu
*E-mail: kml236@cornell.edu
ORCID
Alex M. Confer: 0000-0002-9922-894X
Aniruddha Dey: 0000-0001-7162-0483
Kyle M. Lancaster: 0000-0001-7296-128X
David P. Goldberg: 0000-0003-4645-1045
Notes
The authors declare no competing financial interest.
Generation of 2-57Fe. A dark red solution of 1-57Fe (5 mg, 9
μmol) in C3H7CN (1.6 mL) was combined with a stir bar in a 10 mL
Schlenk flask. The flask was cooled to −60 °C in a slurry of dry ice
and chloroform. A stock solution of Piloty’s acid (9 mg, 53 μmol) in
C3H7CN (1.15 mL) was prepared, and an aliquot (200 μL, 9 μmol, 1
equiv per Fe) was loaded into a gastight syringe. A stock solution of
KOtBu (6 mg, 50 μmol) and 18-crown-6 (13 mg, 50 μmol) in THF
(1.1 mL) was prepared, and an aliquot (200 μL, 9 μmol, 1 equiv per
Fe) was loaded into a separate gastight syringe. The Piloty’s acid
solution was added dropwise over 10 min to the solution of 1-57Fe at
−60 °C, and stirring was continued for an additional 5 min. Next, the
KOtBu/18-crown-6 solution was added dropwise over 10 min.
Stirring was continued for 2 h, over which time the color of the
reaction mixture turned from dark wine red to bright pink red. For
ACKNOWLEDGMENTS
■
The NSF (CHE1566007 to D.P.G.) and NIH
(R35GM124908 to K.M.L.) are gratefully acknowledged for
financial support. A.M.C. thanks the Harry and Cleio Greer
Fellowship for support. XAS data were obtained at the
Stanford Synchrotron Radiation Lightsoure (SSRL). Use of
SSRL, SLAC National Accelerator Laboratory, is supported by
the U.S. Department of Energy, Office of Science, Office of
Basic Energy Sciences under Contract No. DE-AC02-
76SF00515. The SSRL Structural Molecular Biology Program
is supported by the DOE Office of Biological and Environ-
mental Research, and by the National Institutes of Health,
National Institute of General Medical Sciences (including
P41GM103393). The Kirin cluster at JHU KSAS is thanked
for CPU time to A.M.C.
̈
Mossbauer spectroscopy, the reaction mixture was poured into liquid
N2, and the resulting frozen powder was packed into a sample cup
under liquid N2. Alternatively, the reaction mixture was further cooled
to −98 °C, and transferred by precooled syringe to a precooled
sample cup. Samples for Fe K-edge X-ray absorption spectroscopy
were prepared in an identical manner.
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combined and dissolved in THF (1.0 mL). The solution of KOtBu/
18-crown-6 was added to the solution of Piloty’s acid with stirring,
resulting in a dark yellow mixture. The color of the reaction mixture
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