New Synthetic Routes to Iron-Sulfur Clusters: Deciphering the Repair Chemistry of [2Fe-2S] Clusters from Mononitrosyl Iron Complexes

Article abstract of DOI:10.1021/acs.inorgchem.5b00961

The nitrosylation of inorganic protein cofactors, specifically that of [Fe-S] clusters to form iron nitrosyls, plays a number of important roles in biological systems. In some of these cases, it is expected that a repair process reverts the nitrosylated iron species to intact [Fe-S] clusters. The repair of nitrosylated [2Fe-2S] cluster, primarily in the form of protein-bound dinitrosyl iron complexes (DNICs), has been observed in vitro and in vivo, but the mechanism of this process remains uncertain. The present work expands upon a previous observation (Fitzpatrick et al. J. Am. Chem. Soc. 2014, 136, 7229) of the ability of mononitrosyl iron complexes (MNICs) to be converted into [2Fe-2S] clusters by the addition of nothing other than a cysteine analogue. Herein, each of the critical elementary steps in the cluster repair has been dissected to elucidate the roles of the cysteine analogue. Systematic variations of a cysteine analogue employed in the repair reaction suggest that (i) the bidentate coordination of a cysteine analogue to MNIC promotes NO release from iron, and (ii) deprotonation of the α carbon of the ferric-bound cysteine analogue leads to the C-S cleavage en route to the formation of [2Fe-2S] cluster. The [2Fe-2S] cluster bearing a cysteine analogue has also been synthesized from thiolate-bridged iron dimers of the form [Fe₂(μ-SR)₂(SR)₄]^0/2-, which implies that such species may be present as intermediates in the cluster repair. In addition to MNICs, mononuclear tetrathiolate ferric or ferrous species have been established as another form of iron from which [2Fe-2S] clusters can be generated without need for any other reagent but a cysteine analogue. The results of these experiments bring to light new chemistry of classic coordination complexes and provides further insight into the repair of NO-modified [2Fe-2S] clusters.

Full text of DOI:10.1021/acs.inorgchem.5b00961

This is an open access article published under an ACS AuthorChoice License, which permits
copying and redistribution of the article or any adaptations for non-commercial purposes.
Article
pubs.acs.org/IC
New Synthetic Routes to Iron−Sulfur Clusters: Deciphering the
Repair Chemistry of [2Fe−2S] Clusters from Mononitrosyl Iron
Complexes
Jessica Fitzpatrick and Eunsuk Kim*
Department of Chemistry, Brown University, 324 Brook Street, Providence, Rhode Island 02912, United States
S
* Supporting Information
ABSTRACT: The nitrosylation of inorganic protein cofactors,
specifically that of [Fe−S] clusters to form iron nitrosyls, plays a
number of important roles in biological systems. In some of
these cases, it is expected that a repair process reverts the
nitrosylated iron species to intact [Fe−S] clusters. The repair of
nitrosylated [2Fe−2S] cluster, primarily in the form of protein-
bound dinitrosyl iron complexes (DNICs), has been observed in
vitro and in vivo, but the mechanism of this process remains
uncertain. The present work expands upon a previous
observation (Fitzpatrick et al. J. Am. Chem. Soc. 2014, 136,
7229) of the ability of mononitrosyl iron complexes (MNICs) to
be converted into [2Fe−2S] clusters by the addition of nothing
other than a cysteine analogue. Herein, each of the critical elementary steps in the cluster repair has been dissected to elucidate
the roles of the cysteine analogue. Systematic variations of a cysteine analogue employed in the repair reaction suggest that (i)
the bidentate coordination of a cysteine analogue to MNIC promotes NO release from iron, and (ii) deprotonation of the α
carbon of the ferric-bound cysteine analogue leads to the C−S cleavage en route to the formation of [2Fe−2S] cluster. The
[2Fe−2S] cluster bearing a cysteine analogue has also been synthesized from thiolate-bridged iron dimers of the form [Fe2(μ-
SR)2(SR)4]0/2−, which implies that such species may be present as intermediates in the cluster repair. In addition to MNICs,
mononuclear tetrathiolate ferric or ferrous species have been established as another form of iron from which [2Fe−2S] clusters
can be generated without need for any other reagent but a cysteine analogue. The results of these experiments bring to light new
chemistry of classic coordination complexes and provides further insight into the repair of NO-modified [2Fe−2S] clusters.
NO.19 Formation of the DNIC activates this protein to
function as a transcription activator for the soxS gene, another
1. INTRODUCTION
Nitric oxide (NO) plays important roles in many biological
processes1 including, but not limited to, vasoregulation,2
transcription factor whose product promotes the expression of
immunity,3 and neurotransmission.4 Many of these functions
a large group of defensive proteins that increase the ability of
the microorganism to survive oxidative stress.19 Other systems
arise from the interaction of NO with metal centers in
biological systems, such as the cofactors of metalloproteins. The
study of NO as a ligand in coordination chemistry has a long
in which the conversion of [Fe−S] clusters to DNICs has been
proposed to play a functional role include the bacterial NO
sensor-regulator NsrR,20−22 and the WhiB-like (Wbl) family of
history, albeit one complicated by the noninnocence that it
regulatory proteins found in actinomycetes.23,24 Conversely, the
often exhibits.5,6 A class of complexes that exemplifies the
effects of this noninnocence on structure and bonding
comprises the dinitrosyl iron complexes (DNICs). As a result
of their electronic structures, these molecules display character-
interaction of NO with aconitases can be destructive rather
than functional because nitrosylation of the active-site [4Fe−
4S] cluster abrogates enzymatic activity and interrupts a vital
metabolic process.25,26 This metabolic disruption may be an
important mechanism by which the NO released by macro-
phages at sites of infection kills pathogenic microorganisms.
For cells to employ NO as an effective signaling agent or to
better survive immunological attacks, it is logical to propose
that a DNIC-to-[Fe−S] cluster transformation may exist
(Scheme 1). Evidence for such repair was observed in whole-
cell EPR spectroscopic experiments performed on E. coli
istic EPR signals.7−9 This spectroscopic handle led to a series of
discoveries that revealed the biological relevance of DNICs of
the form [Fe(NO)2X2]−/0, where the X ligand can be a free
thiolate, the thiolates of cysteine residues of a protein, or the
imidazoles of histidine residues of a protein.10−16 One source of
protein-bound DNICs is the interaction of NO with [Fe−S]
cluster cofactors of certain metalloproteins.8,17,18 The con-
version of [Fe−S] cluster to DNIC can bring about significant
changes in protein function. For example, the SoxR protein
found in Escherichia coli bears a [2Fe−2S] cluster that
decomposes to form protein-bound DNIC upon exposure to
Received: April 28, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.5b00961
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Scheme 1. [Fe−S] Clusters and Protein-Bound DNICs Can
Interconvert
Scheme 2. Conversion of MNIC to [2Fe−2S] Cluster Upon
Addition of the Cysteine Analogue, MMP
overexpressing SoxR.19 Exposure of the cells to NO gas resulted
in disappearance of the EPR signal from the mixed-valent
[2Fe−2S] cluster of SoxR and its replacement by a character-
istic DNIC signal. Continued incubation of the cells in the
absence of NO resulted in conversion of the DNIC EPR signal
back to that of the intact [Fe−S] cluster. Related in vitro
experiments with [2Fe−2S] ferredoxin revealed that isolated
protein-bound DNIC could be converted to protein with intact
[2Fe−2S] cluster upon aerobic incubation with cysteine and
cysteine desulfurase.27,28 Importantly, no additional iron was
added to the system, indicating that the iron present in the
DNIC was recycled to form the [Fe−S] cluster. The detailed
mechanism by which these transformations occur, however,
remains unknown.
Scheme 3. Previously Proposed Mechanism of the MNIC-to-
[2Fe−2S] Cluster Transformation
One approach used to gain insight into these biochemical
transformations is to investigate the reactivity of small-molecule
models of these bioinorganic species. Investigation of the
interaction of NO with [Fe−S] clusters has benefitted greatly
from the extensive work carried out by many researchers,
perhaps most notably those of the Holm group, to investigate
the inorganic syntheses of small-molecule [Fe−S] clusters.29,30
The reactivity of these complexes has provided significant
insight into the biosynthesis of their protein-bound counter-
parts and the reactions that such metalloproteins undergo.
Small-molecule [2Fe−2S] clusters are typically prepared by
reaction of ferric or ferrous tetrathiolate complexes with
elemental sulfur.30 An alternative strategy involves the reaction
of ferric chloride, thiolate, hydrosulfide, and methoxide.30
Reaction of such synthetic [2Fe−2S] clusters with NO typically
affords DNICs.9,31 The opposing reaction, in which a DNIC is
converted into a [2Fe−2S] cluster, has also been inves-
tigated.32,33 For example, the DNIC [Fe(NO)2(S5)]− could be
irradiated with UV light to photolytically release NO, which
was trapped by an exogenous Fe-based NO-acceptor.32 If the
photolysis is carried out in the presence of elemental sulfur,
then the corresponding [2Fe−2S] cluster re-forms.32 In light of
the known NO reactivity of tetrathiolatoiron(II) complexes in
which 1 equiv produces a mononitrosyl iron complex (MNIC),
whereas 2 equiv produces a DNIC,34 we were intrigued by the
possibility that MNICs could be intermediates in the
conversion of DNICs to [2Fe−2S] clusters.
analysis because no intermediates build up in the course of the
reaction. In this current work, we provide a detailed
characterization of each of the elementary steps by which
thiolate-bridged ferrous dimers can form [2Fe−2S] clusters
upon addition of a cysteine analogue or both a cysteine
analogue and an exogenous oxidant. We have also further
explored the scope of this reactivity to probe the mechanism by
which the MNIC-to-[2Fe−2S] cluster transformation occurs
and to uncover which molecular features of the cysteine
analogues permit this reactivity. The results of these experi-
ments are consistent with those of our previously proposed
model of the conversion of MNIC to the [2Fe−2S] cluster and
allowed us to uncover a reactivity of these classic complexes
that has remained unappreciated until now. In this way, these
results not only add to the repertoire of fascinating chemistry
available to iron thiolate compounds, but also corroborate our
hypothesis that such species may play key roles in the
bioinorganic chemistry of iron nitrosyl complexes.
2. RESULTS AND DISCUSSION
2.1. Coordination of a Cysteine Analogue to Fe
Assists NO Release from Mononitrosyl Iron Complexes
(MNICs). The MNIC-to-[2Fe−2S] cluster transformation
involves release of NO from the iron center. Previous
computational studies35 on an MMP-ligated MNIC suggested
that bidentate coordination of iron by MMP positions the
sulfur atom of MMP trans to NO, which promotes the release
of NO• (Scheme 4). To further validate this idea, we carried
out reactions of [Fe(NO)(StBu)3]− (1a) with various types of
thiols including several cysteine analogues (Table 1).
We recently reported that MNICs with alkylthiolate ligands
are converted into [2Fe−2S] clusters following addition of
nothing other than the cysteine analogue methyl 3-
mercaptopropionate (MMP) (Scheme 2).35 Subsequent experi-
ments revealed that the sulfur atoms of the bridging sulfides of
the [2Fe−2S] cluster originate from MMP. Careful analysis of
the other reaction products revealed that a DNIC and the
thioether S(CH2CH2C(O)OMe)2 were also generated at the
end of the reaction, Scheme 3.
In our previous report, we proposed that a thiolate-bridged
Fe(II) dimer of the form [Fe(μ-SR)(SR)2]2 (5) might be an
intermediate in the transformation (Scheme 3). Our initial
proposal of 5 as an intermediate was largely based on product
Addition of simple thiols or thiolates such as alkyl or aryl
thiol derivatives to 1a or 1b did not lead to the formation of
[2Fe−2S] clusters. In fact, the {Fe(NO)}7 unit in the MNIC is
quite robust and persists in the presence of a wide range of
2−
B
DOI: 10.1021/acs.inorgchem.5b00961
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Stripping these reaction mixtures of volatile components,
washing with a 1:1 mixture of THF and diethyl ether, and
dissolving the residue in acetonitrile afforded solutions with
electronic absorption and FTIR spectra consistent with the
formation of the corresponding [2Fe−2S] clusters (Supporting
Information Figures S1−S4). The THF−diethyl ether wash
removes the DNIC byproduct that is formed, and the excess
thiol is removed under vacuum.
Scheme 4. Participation of 5-Coordinate Isomer in the
Formation of [2Fe−2S] Cluster
The data collected to date suggest that two features of the
thiol added to the MNIC are required for release of NO en
route to [2Fe−2S] cluster formation. The first is that the thiol
must be able to substitute for the thiolate ligands initially bound
to the iron center. This property can be related to the pKa of
the thiol. Indeed, MMP (pKaSH = 9.33 in 50% water−50%
DMSO)39 is unable to produce the cluster from PhS-bound
MNIC, [Fe(NO)(SPh)3]− (pKaSH = 6.6 in water).40,41 Once
substitution at the metal center has occurred, pKa appears to no
longer be a decisive factor in cluster formation. For instance,
EtSH, MMP, and HSCH2CH2C(O)Me are expected to have
very similar pKa values and yet only the latter two, cysteine
analogues, can form cluster. The conspicuous difference
between the thiols/thiolates that can effect formation of
[2Fe−2S] cluster and those that cannot is the presence or
absence, respectively, of the carbonyl moiety. These results are
consistent with our previous proposal that the 5-coordinate
isomer (Scheme 4) is the key species to remove NO from iron
to eventually generate the [2Fe−2S] cluster. The NO releasing
chemistry observed here is in line with the known ability of
thiolate ligands of heme and nonheme iron complexes to
labilize trans ligand including NO•.42,43 The NO released from
MNIC can then interact with another equivalent of MNIC
generating a DNIC and a thiolate-bridged ferrous dimer
(Scheme 3).
Table 1. Ability of Different Thiols To Mediate Formation of
[2Fe−2S] Cluster
a
The maximum theoretical yield of cluster in these reactions is 50%
given the reaction stoichiometry shown in Scheme 3.
2.2. [2Fe−2S] Clusters Can Be Generated from a Bis(μ-
thiolato) Differous Precursor. Once NO is released from
{Fe(NO)}7 MNIC, the resulting ferrous moiety is presumed to
form a thiolate-bridged species, 5, Scheme 3. To probe the
possibility that such a thiolate-bridged ferrous dimer, 5, can
function as a viable reaction intermediate, we have
independently synthesized a known44 ethyl analogue
(PPN)2[Fe2(μ-SEt)2(SEt)4] ((PPN)2-5a) and investigated the
reaction of 5a with (SCH2CH2C(O)OMe)2 (Scheme 5).
thiols or thiolates. Many of these thiols and thiolates can,
however, substitute for the thiolate ligands of MNIC. For
example, addition of a thiol that is more acidic than tert-butyl
thiol to the starting 1a leads to thiolate ligand exchange,
analogous to the well-known ligand exchange chemistry of
[Fe−S] clusters.30 Literature precedent suggests that removal
of NO from an iron nitrosyl by a thiolate would be feasible via
nucleophilic attack on the iron-bound nitrosyl when an NO
ligand is present with NO+ character.36,37 Therefore, the lack of
success in removing NO from 1a by addition of simple thiol/
thiolate is perhaps unsurprising because 1a has already been
well-established as an {Fe(III)(NO−)}7 complex34 rather than
an {Fe(I)(NO+)}7 species.
Scheme 5. Thiolate-Bridged Fe(II) Dimer as a Precursor to
[2Fe−2S] Cluster
The stability of the {Fe(NO)}7 unit of 1a disappears when
cysteine analogues are added. For instance, when a THF or
acetonitrile solution of (PPN)[Fe(NO)(StBu)3] (PPN-1a),
where PPN = bis(triphenylphosphine)iminium, is allowed to
react with an excess of a cysteine analogue, such as MMP,
HSCH2CH2C(O)Me, or N-acetylcysteine methyl ester (Table
1), the red-brown solution turns brown and IR spectra of the
reaction mixture reveal that no MNIC remains. If the reaction
with MMP is carried out in THF, the final [2Fe−2S] cluster,
(PPN)2[Fe2S2(SCH2CH2C(O)OMe)4] ((PPN)2-2), can be
readily isolated from the reaction mixture.38 An alternative
method of isolating (PPN)2-2 involving elaborate serial
recrystallizations has been reported earlier.35 In contrast to
the reaction with MMP, all of the products of the reactions
involving other cysteine analogues, HSCH2CH2C(O)Me and
N-acetylcysteine methyl ester, with 1a or 1b are readily soluble
in THF, precluding facile isolation as was possible with MMP.
Addition of this disulfide to an acetonitrile solution of 5a
induced a rapid change in the color of the solution. The near
colorless solution of 5a immediately acquired a deep red-brown
color (λmax = 470 nm) and then more slowly changed to a final
true brown color (Figure 1). The spectrum of the final solution,
with absorption maxima at 330, 425, and 450 nm, corresponds
to that of the [2Fe−2S] cluster that we have previously
reported, [Fe2(μ-S)2(SCH2CH2C(O)OMe)4]2− (2).35
C
DOI: 10.1021/acs.inorgchem.5b00961
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Figure 1. UV−vis spectral changes associated with the reaction of
[Fe2(μ-SEt)2(SEt)4]2− (5a) (dashed trace) and an excess of
(SCH2CH2C(O)OMe)2 to yield the rapid formation of a red-brown
species with λmax = 470 nm (black trace) followed by a slow formation
of [Fe2(μ-S)2(SCH2CH2C(O)OMe)4]2− (2) (red trace) in acetonitrile
at room temperature over the course of 6 h.
Figure 2. UV−vis spectral changes associated with the reaction of
[Fe2(μ-SEt)2(SEt)4]2− (5a) (dashed trace) and 2 equiv of FcPF6 to
form a putative [Fe2(μ-SEt)2(SEt)4] (6) (green trace), followed by
addition of excess MMP and DBN to yield another ferric intermediate
species (blue trace) which converts to [Fe2(μ-S)2(SCH2CH2C(O)-
OMe)4]2− (2) (red trace) in acetonitrile at room temperature. Total
reaction time was 20 min.
In addition to small-scale experiments designed to
spectroscopically monitor the progress of this reaction, the
conversion of (PPN)2[Fe2(μ-SEt)2(SEt)4] ((PPN)2-5a) to
(PPN)2[Fe2(μ-S)2(SCH2CH2C(O)OMe)4] ((PPN)2-2) was
also carried out on a preparative scale. Similarly to the method
employed to isolate 2 from the reaction of MMP and
[Fe(NO)(tBuS)3]− (1) as described in section 2.1, pure
product 2 from the reaction of 5a with (SCH2CH2C(O)OMe)2
was obtained by exploiting differential solubility of the starting
materials and product. If 20 equiv of (SCH2CH2C(O)OMe)2
are added to a THF solution of 5a, then the [2Fe−2S] cluster 2
simply precipitates from solution over the course of 4 h.
Starting from a THF solution of (PPN)2[Fe2(μ-SEt)2(SEt)4]
(5a), analytically pure [2Fe−2S] cluster can be obtained in 76%
yield simply by filtering the reaction mixture. The spectroscopic
features of this material are consistent with those obtained in
the monitoring experiments or preparative reactions in
acetonitrile.
In contrast to the evident reaction that occurs upon addition
of a disulfide, (SCH2CH2C(O)OMe)2, addition of a related
thiol, MMP, to a solution of [Fe2(μ-SEt)2(SEt)4]2− (5a) results
in no cluster formation. In order to probe whether the
formation of the [2Fe−2S] cluster from 5a depends on the
ability of (SCH2CH2C(O)OMe)2 to act as an oxidant, we
combined 5a with 2 equiv of ferrocenium (Fc+) followed by an
excess of −SCH2CH2C(O)OMe, generated in situ by
combining MMP and the base 1,5-diazabicyclo[4.3.0]non-5-
ene (DBN), Scheme 5. Optical absorption spectroscopic
measurements were used to follow the progress of this stepwise
reaction (Figure 2). Addition of the 2 equiv of ferrocenium
(Fc+) to 5a results in the formation of a green species with
absorption maxima at 320, 390, and 635 nm. We propose that
this green complex is the corresponding thiolate-bridged ferric
dimer, [Fe2(μ-SEt)2(SEt)4] (6), Scheme 5. Addition of MMP
and DBN to this solution results in an immediate color change
with the appearance of new absorption maxima at 355 and 490
nm, which is reminiscent of tetrathiolatoiron(III) species,
[Fe(SCH2CH2C(O)OMe)4]−. This species subsequently trans-
forms to [Fe2(μ-S)2(SCH2CH2C(O)OMe)4]2− (2) over the
next 20 min (Figure 2) and can be isolated in a 45% yield. We
note briefly that dioxygen instead of Fc+ can also be effectively
used as the oxidant in these reactions, affording the product
with a 56% isolated yield. The justification for using the thiolate
form of MMP will be provided below, but here we highlight
that if the thiol is not deprotonated, and then the reaction stops
at the formation of the green intermediate, 6, and does not
proceed to give cluster. Regrettably, we have been unable to
isolate the proposed diferric intermediate, [Fe2(μ-SEt)2(SEt)4]
(6), because the complex appears to be unstable. However, the
Holm group has reported a series of analogous green
compounds with the empirical formula [Fe(SR)3]n, where R
is Me, Et, CH2Ph, or CH2C6H11 although the exact structures
have not been elucidated.45 The formation of such Fe(SR)3
species was also reported by the Henkel group.46 We assign
absorption features that give the intermediate solution a green
color to a diferric species as opposed to a mixed-valent complex
because addition of only 1 equiv of ferrocenium to the diferrous
complex produces absorption features with half the intensity
and addition of the second equivalent of ferrocenium produces
the full intensity peaks (Supporting Information Figure S5). We
note that, at each stage of this two-step titration, the absorbance
from the added ferrocenium is completely extinguished,
indicating full consumption of the oxidant.
Although the putative diferric thiolate-bridged intermediate,
[Fe2(μ-SEt)2(SEt)4] (6), observed during the reaction of Fc+
and the ferrous dimer could not be isolated, the corresponding
monomeric tetrathiolatoiron(III) complexes are readily pre-
pared. If (PPN)[Fe(SEt)4] (PPN-7) is combined with MMP,
no reaction other than ligand substitution occurs. If added to
D
DOI: 10.1021/acs.inorgchem.5b00961
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
MMP and DBN, however, clean conversion to the [2Fe−2S]
cluster takes place (Figure 3), and the product can be isolated
in an 85% yield. The significance of this reaction condition is
described in the following section.
Scheme 6. Deprotonation of Fe-Bound Thiolate Drives C−S
Bond Cleavage and Formation of the [2Fe−2S] Cluster
To test the hypothesis that deprotonation of the α-carbon of
MMP is crucial for the formation of the [2Fe−2S] cluster from
both ferrous and ferric precursors, [Fe2(μ-SEt)2(SEt)4]2− (5a)
and [Fe(SEt)4]− (7), respectively, analogues of (SCH2CH2C-
(O)OMe)2 and MMP were used that differ from these
compounds in key respects with regard to proton position
and acidity. If the orientation of the ester linkage is reversed, as
in the case of HSCH2CH2OC(O)Me, the CH2 unit highlighted
in bold has lowered acidity47,48 and cannot be readily
deprotonated. Accordingly, addition of the disulfide form of
HSCH2CH2OC(O)Me to a solution of 5a results in no cluster
formation. If, alternatively, the carbon chain between the sulfur
atom and the carbonyl functional group is lengthened by one
methylene unit, as in (SCH2CH2CH2C(O)OMe)2, again no
cluster was observed upon addition to 5a. The lack of cluster
formation in this latter case arises from the fact that the acidic
proton is no longer properly positioned for carbon−sulfur bond
breaking. We have found, however, that analogues of MMP in
which the protons of the α-carbon are appropriately acidic47
and properly positioned do lead to cluster formation. For
example, HSCH2CH2C(O)Me is a ketone analogue of MMP
that forms the corresponding [2Fe−2S] cluster when added to
7 in the presence of DBN (Supporting Information Figure S7).
Moreover, the greater acidity of the α-carbon of HSCH2CH2C-
(O)Me, as compared to MMP, is reflected in the greater rate at
which the starting material is consumed in the former case
(Supporting Information Figure S8). Similarly, N-acetylcysteine
methyl ester forms a [2Fe−2S] cluster upon reaction with 7
and DBN, but appears to also participate in a side reaction, as
evidenced by the lack of clear isosbestic points (Supporting
Information Figure S9), precluding a comparison of the rate of
this reaction with those of the other cysteine analogues. As
described in section 2.1, these two cysteine analogues are also
able to generate [2Fe−2S] cluster upon reaction with MNIC.
Formation of [2Fe−2S] cluster from tetrathiolatoiron(III) and
the cysteine analogues could be followed by electronic
absorption monitoring experiments. The spectra of the final
solutions match those of the products obtained from the
reactions with MNIC (vide supra) and from independently
prepared samples of the clusters synthesized by ligand
substitution of the (PPN)2[Fe2(μ-S)2(StBu)4] with the
corresponding thiol (Supporting Information Figures S7−S8).
These clusters have proven to be highly unstable. Although
they persist in solution long enough to permit spectral
characterization, and in the solid state at −35 °C for ca. 12
h, we have yet been unable to further characterize these
clusters.
Figure 3. UV−vis spectral changes associated with the reaction of
[Fe(SEt)4]− (7) (blue trace), 50 equiv of MMP, and 50 equiv of DBN
in acetonitrile at room temperature to form the corresponding [2Fe−
2S] cluster, [Fe2(μ-S)2(SCH2CH2C(O)OMe)4]2− (2) (red trace).
The total reaction time was 35 min.
2.3. Deprotonation of Cysteine Analogue Leads to
the C−S Cleavage To Form [2Fe−2S] Cluster. As described
in section 2.1, the transformation of MNIC to [2Fe−2S] cluster
by cysteine analogues depends on the ability of these molecules
to form 5-coordinate isomers of the MNIC via coordination of
the carbonyl oxygen atom. Two other results from the product
analyses indicate that some other characteristic features of the
cysteine analogue are essential for the transformation in
question. The first is the observation that the bridging sulfides
in the [2Fe−2S] cluster originated from MMP.35 The second is
the quantitative formation of the thioether analogue of MMP,
S(CH2CH2C(O)OMe)2 during the reaction (Scheme 3).35
These observations imply that the C−S bond of MMP is
cleaved in the reaction of MNIC with MMP. The analogous
reaction of [Fe(NO)(StBu)3]− (1a) with EtSH produces no
cluster and the reaction of [Fe2(μ-SEt)2(SEt)4]2− (5a) with
EtSSEt produces only [Fe(SEt)4]− (7) (Scheme 5, Supporting
Information Figure S6). Given the similarities in the pKaSH
values and redox potentials of EtSH and MMP, some other
feature of MMP must be giving rise to the observed reactivity.
We suggest that this feature is the acidity of the proton on the
α-carbon.
We propose that, in the transformation of [Fe(SEt)4]− (7) to
[Fe2(μ-S)2(SCH2CH2C(O)OMe)4]2− (2) as described in the
previous section (Figure 3), a base (DBN or the thiolate form
of MMP) deprotonates the α-carbon of the Fe-coordinated
MMP (Scheme 6). Consequent heterolytic cleavage of the C−S
bond provides an Fe-coordinated sulfide (S2−) which is
required in the final [2Fe−2S] cluster product, as well as an
equivalent of methyl acrylate (Scheme 6). Reaction of MMP
with methyl acrylate results in the quantitative formation of the
thioether form of MMP, S(CH2CH2C(O)OMe)2. We have
independently confirmed that addition of MMP to a solution of
methyl acrylate produces S(CH2CH2C(O)OMe)2.
With the insight gained from the reactions with monomeric
tetrathiolatoiron(III) complexes, we can revisit the working
model for the MNIC-to-[2Fe−2S] cluster transformation
E
DOI: 10.1021/acs.inorgchem.5b00961
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
depicted in Scheme 3 and propose a mechanism for the final
step of the reaction (Scheme 7). Reaction of [Fe2(μ-
Scheme 8. Mechanism for the Transformation of
Tetrathiolatoiron(II) to [2Fe−2S] Cluster
Scheme 7. Role of Deprotonation in the [2Fe−2S] Cluster
Repair
3. SUMMARY AND CONCLUSIONS
[2Fe−2S] clusters can be formed by the reaction of cysteine
analogues with mononitrosyl iron complexes (MNICs). An
investigation of the mechanism of this reaction has led to the
discovery that different Fe(II) and Fe(III) thiolate complexes
are also competent to carry out this cysteine-analogue-mediated
transformation. The various reactions by which [2Fe−2S]
clusters have been observed to form from Fe nitrosyl and iron
thiolate species as a result of reaction with a cysteine analogue
(or its disulfide) are summarized in Scheme 9.
SR)2(SR)4]2− (5) with (SCH2CH2C(O)OMe)2 oxidizes 5 to
the diferric state, generating free thiolate. The released thiolate
deprotonates the α-carbon of the bridging thiolate of 6
triggering C−S bond cleavage and formation of the [2Fe−2S]
cluster.
The data obtained thus far have revealed the ability of
cysteine analogues to facilitate the conversion of MNICs and
[Fe(SR4)]− complexes to [2Fe−2S] clusters via a mechanism
that relies on the deprotonation of the α-carbon and
consequent C−S bond heterolysis. Starting from either
MNICs, tetrathiolatoiron(III) complexes, or thiolate-bridged
iron(II) dimers, it is possible to form [2Fe−2S] clusters from
cysteine analogues, provided that an acidic proton is properly
positioned so as to facilitate C−S bond scission.
Scheme 9. Summary of the Reactions by Which [2Fe−2S]
Clusters Were Formed in This Paper
2 . 4 . C y s t e i n e A n a l o g u e s C a n C o n v e r t
Tetrathiolatoiron(II) Monomers to [2Fe−2S] Clusters.
As described above, our observation of the reactivity of Fe(III)
dimers with (SCH2CH2C(O)OMe)2 led us to investigate the
reactivity of Fe(III) monomers. Tetrathiolatoiron(III) mono-
mers were consequently found to be competent for [2Fe−2S]
cluster formation, provided that the thiolate form of the
cysteine analogue is present. Given this pattern of reactivity, we
reasoned that tetrathiolatoiron(II) monomers may be con-
verted to [2Fe−2S] clusters by (SCH2CH2C(O)OMe)2. The
oxidation of the Fe(II) center by disulfide generates the
equivalent of base (i.e., thiolate) needed to facilitate the C−S
cleavage, and no extra base should be needed to generate the
[2Fe−2S] cluster.
Indeed, this was found to be the case. UV−vis spectroscopic
monitoring experiments demonstrate that addition of
(SCH2CH2C(O)OMe)2 to a solution of [Fe(SEt)4]2− (8b)
leads to the development of the characteristic spectroscopic
features of [Fe2(μ-S)2(SCH2CH2C(O)OMe)4]2− (2) (Scheme
8, Supporting Information Figure S10). The reaction could also
be carried out on a preparative scale, and the cluster isolated in
85% yield. The use of [Fe(StBu)4]2− (8a) instead of the ethyl
analogue did not influence the reactivity, and the [2Fe−2S]
cluster was isolated in 50% yield. In further analogy to the
experiments described above, if 8b is treated with EtSSEt
instead of (SCH2CH2C(O)OCH3)2, the tetrathiolatoiron(III)
species is formed, but the reaction does not proceed to cluster.
Finally, if 8b is allowed to react with 1 equiv of ferrocenium,
excess MMP, and excess DBN, then the cluster is formed in a
68% yield (Supporting Information Figure S11).
The MNIC-to-[2Fe−2S] cluster transformation relies on the
ability of the cysteine analogue to substitute for the initially
bound thiolate ligands, release nitric oxide, and undergo base-
dependent heterolytic C−S bond cleavage. In addition to
MMP, related thiols, such as HSCH2CH2C(O)Me and N-
acetylcysteine methyl ester, were also able to convert MNICs
into [2Fe−2S] clusters. Thiolatoiron(II) complexes, either
monomeric tetrathiolates or dimers with bridging thiolates,
could be converted to [2Fe−2S] clusters using the disulfide
forms of these thiolates or sequential addition of an external
oxidant and the thiolate. Additionally, monomeric Fe(III)
tetrathiolate complexes could be converted to [2Fe−2S]
clusters using the thiolate form of cysteine analogues.
These reactions add to the palette of reactivity that is
available to chemists exploring iron thiolate complexes and
[Fe−S] clusters. More importantly, they also provide valuable
insight into the mechanisms of the biochemical transformations
that analogous metallocofactors may undergo in biological
systems. Importantly, in the long history of investigation into
the chemistry of [Fe−S] clusters, the source of the bridging
sulfide has typically been elemental S or a sulfide salt such as
NaSH. This work shows that a cysteine analogue is sufficient to
promote [2Fe−2S] cluster assembly. Moreover, the results
presented here provide support for the putative role of MNICs
in the transformation of DNICs to [2Fe−2S] clusters. The
simplicity of these reactants contrasts sharply with the
complexity of the cascade reactions that unfold, but careful
F
DOI: 10.1021/acs.inorgchem.5b00961
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
of 15 mL of Et2O followed by cooling to −38 °C for 12 h induced
precipitation of a dark brown powder. This precipitate was filtered and
washed with cold Et2O to afford 3 mg (1.8 μmol, 40%) of
(PPN)2[Fe2(μ-S)2(SCH2CH2C(O)CH3)4]. For method 2, a solution
of (PPN)2[Fe2(μ-S)2(StBu)4] (22 mg, 0.014 mmol) in 2.0 mL of
MeCN was transferred to a 10 mL Schlenk flask, to which
HSCH2CH2C(O)CH3 (72 μL, 0.7 mmol) was added at −15 °C.
The reaction was allowed to proceed for 2 h. Addition of 15 mL of
Et2O followed by cooling to −38 °C for 12 h induced precipitation of
a dark brown powder. This precipitate was filtered and washed with
cold Et2O to afford 5 mg (0.0027 mmol, 20%) of (PPN)2[Fe2(μ-
S)2(SCH2CH2C(O)CH3)4]. UV−vis in MeCN, λ in nm (ε in M−1
cm−1): 320 (25 540), 420 (18 810), 450 sh (16 880). IR (KBr, cm−1):
1702 (s, νCO), 1264 (s), 1115 (s), 724 (s), 693 (s), 533 (s).
Reaction of (PPN)2[Fe2(μ-SEt)2(SEt)4] ((PPN)2-5a) and (SCH2CH2C-
(O)OMe)2. An alternative, more laborious procedure for the reaction
monitoring and isolation of the reaction products was previously
reported.35 A solution of (PPN)2[Fe2(μ-SEt)2(SEt)4] ((PPN)2-5a)
(15 mg, 9.6 μmol) in 2.0 mL of THF was transferred to a 10 mL
Schlenk flask, to which (SCH2CH2C(O)OCH3)2 (40 μL, 192 μmol)
was injected at room temperature. The reaction was allowed to
proceed for 5 h over which time a dark-brown powder precipitated.
The solid was collected by filtration, washed with THF (5 × 1 mL),
and then dissolved in 1 mL of MeCN. Addition of 15 mL of Et2O
followed by cooling to −38 °C for 12 h induced precipitation of a dark
brown microcrystalline solid. This precipitate was filtered and washed
with cold Et2O to afford 12 mg (6.8 μmol, 70%) of (PPN)2[Fe2(μ-
S)2(SCH2CH2C(O)OCH3)4] ((PPN)2-2). Physical characterization
(UV−vis and IR spectroscopy) was in agreement with that previously
reported for ((PPN)2-2).35
Reaction of (PPN)2[Fe2(μ-SEt)2(SEt)4] ((PPN)2-5a), FcPF6, MMP,
and DBN. A solution of (PPN)2[Fe2(μ-SEt)2(SEt)4] ((PPN)2-5a) (21
mg, 13 μmol) in 2.0 mL of THF was transferred to a 10 mL Schlenk
flask, to which FcPF6 (8.9 mg, 26 μmol) was added. After the addition,
the solution took on a dark green color. To this solution was injected
HSCH2CH2C(O)OMe (59 μL, 520 μmol), followed by DBN (33 μL,
260 μmol). The reaction was allowed to proceed for 5 h at room
temperature over which time a dark-brown powder precipitated. The
solid was collected by filtration, washed with THF (5 × 1 mL), and
then dissolved in 1 mL of MeCN. Addition of 15 mL of Et2O followed
by cooling to −38 °C for 12 h induced precipitation of a dark brown
microcrystalline solid. This precipitate was filtered and washed with
cold Et2O to afford 11 mg (6.2 μmol, 46%) of (PPN)2[Fe2(μ-
S)2(SCH2CH2C(O)OCH3)4] ((PPN)2-2). Physical characterization
(UV−vis and IR spectroscopy) was in agreement with that previously
reported for ((PPN)2-2).35
analysis of these reactions has provided deeper insight into the
inorganic chemistry of these molecules as well as the
biochemistry in which they might participate.
4. EXPERIMENTAL SECTION
4.1. General Considerations. Unless otherwise specified, all
reactions and manipulations were carried out under an inert
atmosphere of nitrogen using an MBraun glovebox (<0.1 ppm of
O2, < 0.1 ppm of H2O). Fe(II) chloride, Fe(III) chloride, PPNCl,
methyl 3-mercaptopropionate (MMP), N-acetyl cysteine methyl ester,
methyl vinyl ketone, sodium sulfide nonahydrate, 1,5-
diazabicyclo[4.3.0]non-5-ene (DBN), diethyl disulfide (EtSSEt),
(SCH2CH2OC(O)CH3)2, and ferrocenium hexafluorophosphate
(FcPF6) were purchased from Aldrich and used as received.
(SCH2CH2C(O)OCH3)2 was purchased from TCI Chemicals and
used as received. Solvents were purified by passage over an alumina
column under an Ar atmosphere and stored over activated molecular
sieves (4 Å). Diethyl ether and tetrahydrofuran were additionally dried
over sodium prior to use. Nitric oxide (Matheson, 99%) was purified
following a literature method,49 in which the NO gas stream is passed
through an Ascarite column, and then distilled at −80 °C.
4.2. Physical Methods. Unless otherwise specified, samples for
spectroscopic analysis were prepared inside a nitrogen glovebox.
Infrared spectra were recorded on a Bruker Tensor 27 FTIR
Spectrometer. UV−vis spectra were recorded on a Varian Cary 50
Bio spectrometer.
4.3. Synthesis and Reactivity Studies. The compounds
(PPN)2[Fe(StBu)4],50 (PPN)2[Fe(SEt)4],51 (PPN)2[Fe(SPh)4],52
(PPN)2[Fe2(μ-SEt)2(SEt)4],44 (PPN)2[Fe2(μ-S)2(StBu)4],53 (PPN)-
[Fe(SEt)4],54 (PPN)[Fe(StBu)3(NO)],34 (PPN)[Fe(SEt)3(NO)],55
57
(SCH2CH2CH2C(O)CH3)2,56 and HSCH2CH2C(O)CH3 were
prepared according to published procedures or with slight
modifications.
Synthesis of (PPN)2[Fe2(μ-S)2(SCH2CH(NHAc)C(O)OMe)4]. For
method 1, a solution of (PPN)[Fe(StBu)3(NO)] ((PPN)-1a) (32
mg, 36 μmol) in 2.0 mL of acetonitrile was transferred to a 10 mL
Schlenk flask covered in Al-foil, to which N-acetylcysteine-methyl ester
(127 mg, 717 μmol) was added at −15 °C. The reaction was allowed
to proceed for 2 h over which time the color of the solution changed
from red-brown to brown. The volatiles were removed in vacuo, and
the resulting residue was washed with Et2O (3 × 1 mL), and 10% v/v
MeOH in Et2O (3 × 1 mL). The residue was then dissolved in 1 mL
of cold MeCN. Addition of 15 mL of Et2O followed by cooling to −38
°C for 12 h induced precipitation of a dark brown powder. This
precipitate was filtered and washed with cold Et2O to afford 13 mg
(6.6 μmol, 37%) of (PPN)2[Fe2(μ-S)2(SCH2CH(NHAc)C(O)-
OMe)4]. For method 2, a solution of (PPN)2[Fe2(μ-S)2(StBu)4] (47
mg, 0.029 mmol) in 3.0 mL of THF was transferred to a 10 mL
Schlenk flask, to which N-acetylcysteine-methyl ester (250 mg, 1.46
mmol) was added at room temperature. The reaction was allowed to
proceed for 3 h. The volatiles were removed in vacuo, and the residue
was washed with Et2O:MeOH (10:1, 3 × 1 mL). The residue was then
dissolved in 1 mL of MeCN. Addition of 15 mL of Et2O followed by
cooling to −38 °C for 12 h induced precipitation of a dark brown
powder. This precipitate was filtered and washed with cold Et2O to
afford 33 mg (0.017 mmol, 65%) of (PPN)2[Fe2(μ-S)2(SCH2CH-
(NHAc)C(O)OMe)4]. UV−vis in MeCN, λ in nm (ε in M−1 cm−1):
320 (14 990), 420 (9817), 450 sh (8154). IR (KBr, cm−1): 1745 (s,
νCO), 1669 (s, νCO), 1513 (s, νCO), 1115 (w), 724 (w), 693 (w), 533
(w), 499 (s).
Reaction of (PPN)2[Fe2(μ-SEt)2(SEt)4] ((PPN)2-5a), O2, MMP, and
DBN. A solution of (PPN)2[Fe2(μ-SEt)2(SEt)4] ((PPN)2-5a) (20 mg,
13 μmol) in 2.0 mL of THF was transferred to a 10 mL Schlenk flask,
to which O2 (26 μmol) was injected. After 1 min, HSCH2CH2C(O)-
OCH3 (57 μL, 520 μmol), followed by DBN (32 μL, 260 μmol), was
injected to the solution. The reaction was allowed to proceed for 12 h
at room temperature over which time a dark-brown powder
precipitated. The solid was collected by filtration, washed with THF
(5 × 1 mL), and then dissolved in 1 mL of MeCN. Addition of 15 mL
of Et2O followed by cooling to −38 °C for 12 h induced precipitation
of a dark brown microcrystalline solid. This precipitate was filtered and
washed with cold Et2O to afford 12 mg (6.9 μmol, 53%) of
(PPN)2[Fe2(μ-S)2(SCH2CH2C(O)OCH3)4]. Physical characteriza-
tion (UV−vis and IR spectroscopy) was in agreement with that
previously reported for (PPN)2-2.35
Synthesis of (PPN)2[Fe2(μ-S)2(SCH2CH2C(O)CH3)4]. For method 1, a
solution of (PPN)[Fe(StBu)3(NO)] ((PPN)-1a) (8 mg, 9.0 μmol) in
1.0 mL of acetonitrile was transferred to a 10 mL Schlenk flask covered
in Al-foil, to which HSCH2CH2C(O)CH3 (19 μL, 179 μmol) was
injected at −15 °C. The reaction was allowed to proceed for 2 h over
which time the color of the solution changed from red-brown to
brown. The volatiles were removed in vacuo, and the resulting residue
was washed with Et2O (3 × 1 mL), and 10% v/v THF in Et2O (3 × 1
mL). The residue was then dissolved in 1 mL of cold MeCN. Addition
UV−Vis Monitoring of the Reactions of (PPN)2[Fe2(μ-SEt)2(SEt)4]
((PPN)2-5a) and RSSR. In a typical experiment, 0.5−0.9 mM solutions
of (PPN)2[Fe2(μ-SEt)2(SEt)4] ((PPN)2-5a) in MeCN were prepared
in a 2 mm Schlenk cuvette equipped with a rubber septum. The
disulfides were injected via syringe and the reactions monitored by
their UV−vis spectra at room temperature.
a. (SCH2CH2C(O)OMe)2. To a 700 μL solution of (PPN)2-5a (0.7
mM) was added (SCH2CH2C(O)OMe)2 (5 μL, 25 μmol). The total
G
DOI: 10.1021/acs.inorgchem.5b00961
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
reaction time was 6 h. The product is consistent with formation of
(PPN)2[Fe2(μ-S)2(SCH2CH2C(O)OCH3)4] ((PPN)2-2).35
b. EtSSEt. To a 700 μL solution of (PPN)2-5a (0.9 mM) was added
EtSSEt (4 μL, 32 μmol). The total reaction time was 4 h. The product
is consistent with formation of (PPN)[Fe(SEt)4] (7).54
c. (SCH2CH2CH2C(O)OMe)2. To a 700 μL solution of (PPN)2-5a
(0.7 mM) was added (SCH2CH2CH2C(O)OMe)2 (6.5 mg, 24.5
μmol). The total reaction time was 5 h. No cluster formation was
observed.
Physical characterization (UV−vis and IR spectroscopy) was in
agreement with that previously reported for (PPN)2-2.35
t
For R = Bu, as described for R = Et, a solution of (PPN)2[Fe-
(StBu)4] ((PPN)2-8a) (28 mg, 18.8 μmol) in 2.0 mL of THF and
(SCH2CH2C(O)OCH3)2 (73 μL, 0.66 mmol) yielded 8 mg (4.6
μmol, 50%) of (PPN)2-2.
Reaction of (PPN)2[Fe(SEt)4] ((PPN)2-8b), FcPF6, MMP, and DBN.
A solution of (PPN)2[Fe(SEt)4] ((PPN)2-8b) (18.8 mg, 13.6 μmol) in
1.0 mL of THF was transferred to a 10 mL Schlenk flask, to which
FcPF6 (4.5 mg, 14 μmol) was added. After the addition, the solution
took on a dark red-brown color. To this solution was injected
HSCH2CH2C(O)OCH3 (75 μL, 0.70 mmol), followed by DBN (84
μL, 0.70 mmol). The reaction was allowed to proceed for 4 h at room
temperature over which time a dark-brown powder precipitated. The
solid was collected by filtration, washed with THF (5 × 1 mL), and
then dissolved in 1 mL of MeCN. Addition of 15 mL of Et2O followed
by cooling to −38 °C for 12 h induced precipitation of a dark brown
microcrystalline solid. This precipitate was filtered and washed with
cold Et2O to afford 8.1 mg (4.7 μmol, 68%) of (PPN)2[Fe2(μ-
S)2(SCH2CH2C(O)OCH3)4] ((PPN)2-2). Physical characterization
(UV−vis and IR spectroscopy) was in agreement with that previously
reported for (PPN)2-2.35
UV−Vis Monitoring of the Reaction of (PPN)2[Fe(SEt)4] ((PPN)2-
8b) and (SCH2CH2C(O)OMe)2. A 700 μL solution of (PPN)2[Fe-
(SEt)4] ((PPN)2-8b) (0.6 mM) in MeCN was prepared in a 2 mm
Schlenk cuvette equipped with a rubber septum. To this solution was
injected a 1.0 M MeCN solution of (SCH2CH2C(O)OMe)2 (14 μL,
13 μmol) and the reaction monitored by their UV−vis spectra at room
temperature. The total reaction time was 4 h. The product is
consistent with formation of (PPN)2-2.35
d. (SCH2CH2OC(O)Me)2. To a 700 μL solution of (PPN)2-5a (0.7
mM) was added (SCH2CH2OC(O)Me)2 (5 μL, 30 μmol). The total
reaction time was 2.5 h. No cluster formation was observed.
UV−vis monitoring of the reactions of (PPN)2[Fe2(μ-SEt)2(SEt)4]
((PPN)2-5a) and Fc+, MMP, DBN. To a 700 μL MeCN solution of
(PPN)2-5a (0.7 mM) was added 108 μL of an MeCN solution of
FcPF6 (9.1 mM, 0.98 μmol) at room temperature. The cuvette was
gently shaken and the changes in the UV−vis spectra were monitored
for 1 min. MMP (2.7 μL, 25 μmol), DBN (3.0 μL, 25 μmol) were then
added via syringe and UV−vis spectra were recorded for 20 min. The
product is consistent with formation of (PPN)2-2.35
Reaction of (PPN)[Fe(SEt)4] ((PPN)-7), HSCH2CH2C(O)OMe, and
DBN. A solution of (PPN)[Fe(SEt)4] ((PPN)-7) (21.6 mg, 25.7
μmol) in 1.5 mL of THF was transferred to a 10 mL Schlenk flask, to
which MMP (114 μL, 1.03 mmol) and DBN (64 μL, 0.52 mmol) were
added. After the addition, the solution immediately took on a red-
purple color, and then more slowly turned brown. The reaction was
allowed to proceed for 4 h at room temperature over which time a
dark-brown powder precipitated. The solid was collected by filtration,
washed with THF (5 × 1 mL), and then dissolved in 1 mL of MeCN.
Addition of 15 mL of Et2O followed by cooling to −38 °C for 12 h
induced precipitation of a dark brown microcrystalline solid. This
precipitate was filtered and washed with cold Et2O to afford 19.7 mg
(11.4 μmol, 89%) of (PPN)2[Fe2(μ-S)2(SCH2CH2C(O)OCH3)4]
((PPN)2-2). Physical characterization (UV−vis and IR spectroscopy)
was in agreement with that previously reported for (PPN)2-2.35
UV−Vis Monitoring of the Reactions of (PPN)[Fe(SEt)4] ((PPN)-7)
and RSH. In a typical experiment, 0.5−0.7 mM solutions of
(PPN)[Fe(SEt)4] (PPN)-7 in MeCN were prepared in a 2 mm
Schlenk cuvette equipped with a rubber septum. The thiols and DBN
were injected via syringe and the reactions monitored by their UV−vis
spectra at room temperature.
UV−Vis Monitoring of the Reaction of (PPN)2[Fe(SEt)4] ((PPN)2-
8b), FcPF6, MMP, and DBN. To a 700 μL MeCN solution of
(PPN)2[Fe(SEt)4] ((PPN)2-8b) (0.7 mM) was added 41 μL of an
MeCN solution of FcPF6 (12.1 mM, 0.49 μmol) at room temperature.
The cuvette was gently shaken, and the changes in the UV−vis spectra
were monitored for 1 min. MMP (2.7 μL, 25 μmol) and DBN (3.0 μL,
25 μmol) were then added via syringe, and UV−vis spectra were
recorded for 30 min. The product is consistent with formation of
(PPN)2-2.35
a. HSCH2CH2C(O)OMe. To a 700 μL solution of (PPN)-7 (0.7
mM) was added HSCH2CH2C(O)OMe (2.7 μL, 25 μmol), and DBN
(3.0 μL, 25 μmol). The total reaction time was 35 min. The product is
consistent with formation of (PPN)2-2.35
b. HSCH2CH2C(O)Me. To a 700 μL solution of (PPN)-7 (0.7 mM)
was added HSCH2CH2C(O)Me (2.5 μL, 25 μmol), and DBN (3.0 μL,
25 μmol). The total reaction time was 4 min, after which time
decomposition occurred. The spectral characteristics of the product
that formed prior to decomposition are consistent with formation of
(PPN)2[Fe2(μ-S)2(SCH2CH2C(O)CH3)4] (Supporting Information
Figure S7).
ASSOCIATED CONTENT
■
S
* Supporting Information
UV−vis and IR spectra of [2Fe−2S] clusters bearing N-
acetylcysteine methyl ester and 4-mercaptobutan-2-one. UV−
vis titration of [Fe2(μ-SEt)2(SEt)4]2− (5a) and FcPF6. UV−vis
monitoring of a series of reactions including: (i) 5a and EtSSEt,
(ii) [Fe(SEt)4]− (7) and HSCH2CH2C(O)Me with DBN, (iii)
7 and N-acetylcysteine methyl ester with DBN, (iv) [Fe-
(SEt)4]2− (8b) and (SCH2CH2C(O)OMe)2, and (v) 8b and
FcPF6 followed by addition of HSCH2CH2C(O)OMe with
DBN. The Supporting Information is available free of charge on
the ACS Publications website at DOI: 10.1021/acs.inorg-
chem.5b00961.
c. N-Acetylcysteine Methyl Ester. To a 700 μL solution of (PPN)-7
(0.5 mM) was added N-acetylcysteine methyl ester (3.1 mg, 18 μmol),
and DBN (2.2 μL, 18 μmol). The total reaction time was 30 min. The
spectral characteristics of the product are consistent with formation of
(PPN)2[Fe2(μ-S)2(SCH2CH(NHAc)C(O)OMe)4] (Supporting In-
formation Figure S9).
t
Reaction of (PPN)2[Fe(SR)4] (R=Et ((PPN)2-8b), Bu ((PPN)2-8a))
AUTHOR INFORMATION
■
and (SCH2CH2C(O)OMe)2. For R = Et, a solution of (PPN)2[Fe-
(SEt)4] ((PPN)2-8b) (22.4 mg, 16.4 μmol) in 2.0 mL of THF was
transferred to a 10 mL Schlenk flask, to which (SCH2CH2C(O)-
OCH3)2 (63 μL, 0.33 mmol) was injected at room temperature. The
reaction was allowed to proceed for 5 h over which time a dark-brown
powder precipitated. The solid was collected by filtration, washed with
THF (5 × 1 mL), and then dissolved in 1 mL of MeCN. Addition of
15 mL of Et2O followed by cooling to −38 °C for 12 h induced
precipitation of a dark brown microcrystalline solid. This precipitate
was filtered and washed with cold Et2O to afford 12 mg (6.7 μmol,
85%) of (PPN)2[Fe2(μ-S)2(SCH2CH2C(O)OCH3)4] ((PPN)2-2).
Corresponding Author
*E-mail: Eunsuk_Kim@brown.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the Vince Wernig Fellowship (J.F.) and the
NSF (E.K., CHE 1254733) for financial support.
H
DOI: 10.1021/acs.inorgchem.5b00961
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(33) Lu, T. T.; Huang, H. W.; Liaw, W. F. Inorg. Chem. 2009, 48,
9027−9035.
REFERENCES
■
(1) Butler, A. R.; Nicholson, R. Life, Death and Nitric Oxide; The
Royal Society of Chemistry: Cambridge, U.K., 2003.
(2) Ignarro, L. J. Annu. Rev. Pharmacol. Toxicol. 1990, 30, 535−560.
(3) MacMicking, J.; Xie, Q. W.; Nathan, C. Annu. Rev. Immunol.
1997, 15, 323−350.
(34) Harrop, T. C.; Song, D.; Lippard, S. J. J. Am. Chem. Soc. 2006,
128, 3528−3529.
(35) Fitzpatrick, J.; Kalyvas, H.; Filipovic, M. R.; Ivanovic-
Burmazovic, I.; MacDonald, J. C.; Shearer, J.; Kim, E. J. Am. Chem.
Soc. 2014, 136, 7229−7232.
(36) Souza, M. L.; Roveda, A. C., Jr.; Pereira, J. C. M.; Franco, D. W.
Coord. Chem. Rev. 2015, DOI: 10.1016/j.ccr.2015.03.008.
(37) Roncaroli, F.; Videla, M.; Slep, L. D.; Olabe, J. A. Coord. Chem.
Rev. 2007, 251, 1903−1930.
(38) Complex 1 was completely consumed upon addition of a single
equivalent of MMP to yield unstable nitrosyl species. However, we
were not able to characterize it.
(39) Bernasconi, C. F.; Killion, R. B. J. Am. Chem. Soc. 1988, 110,
7506−7512.
(4) Brenman, J. E.; Bredt, D. S. Methods. Enzymol. 1996, 269, 119−
129.
(5) Ford, P. C.; Lorkovic, I. M. Chem. Rev. 2002, 102, 993−1018.
(6) Berto, T. C.; Speelman, A. L.; Zheng, S.; Lehnert, N. Coord.
Chem. Rev. 2013, 257, 244−259.
(7) McDonald, C. C.; Phillips, W. D.; Mower, H. F. J. Am. Chem. Soc.
1965, 87, 3319−3326.
(8) Lewandowska, H.; Kalinowska, M.; Brzoska, K.; Wojciuk, K.;
Wojciuk, G.; Kruszewski, M. Dalton Trans. 2011, 40, 8273−8289.
(9) Tsai, M. L.; Tsou, C. C.; Liaw, W. F. Acc. Chem. Res. 2015, 48,
1184−1193.
(10) Mallard, J. R.; Kent, M. Nature 1964, 204, 1192.
(11) Vithayathil, A. J.; Ternberg, J. L.; Commoner, B. Nature 1965,
207, 1246−1249.
(12) Vanin, A. F.; Nalbandian, R. M. Biofizika 1965, 10, 167−168.
(13) Cesareo, E.; Parker, L. J.; Pedersen, J. Z.; Nuccetelli, M.;
Mazzetti, A. P.; Pastore, A.; Federici, G.; Caccuri, A. M.; Ricci, G.;
Adams, J. J.; Parker, M. W.; Lo Bello, M. J. Biol. Chem. 2005, 280,
42172−42180.
(14) D’Autreaux, B.; Horner, O.; Oddou, J. L.; Jeandey, C.;
Gambarelli, S.; Berthomieu, C.; Latour, J. M.; Michaud-Soret, I. J.
Am. Chem. Soc. 2004, 126, 6005−6016.
(15) Kennedy, M. C.; Antholine, W. E.; Beinert, H. J. Biol. Chem.
1997, 272, 20340−20347.
(16) Lee, M.; Arosio, P.; Cozzi, A.; Chasteen, N. D. Biochemistry
1994, 33, 3679−3687.
(17) Hyduke, D. R.; Jarboe, L. R.; Tran, L. M.; Chou, K. J.; Liao, J. C.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8484−8489.
(18) Crack, J. C.; Green, J.; Thomson, A. J.; Le Brun, N. E. Curr.
Opin. Chem. Biol. 2012, 16, 35−44.
(19) Ding, H.; Demple, B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
5146−5150.
(20) Tucker, N. P.; Hicks, M. G.; Clarke, T. A.; Crack, J. C.;
Chandra, G.; Le Brun, N. E.; Dixon, R.; Hutchings, M. I. PloS One
2008, 3, e3623.
(40) Serjeant, E. P.; Dempsey, B. Ionization Constants of Organic Acids
in Aqueous Solution; Permagon: Oxford, U.K., 1979.
(41) Warren, J. J.; Tronic, T. A.; Mayer, J. M. Chem. Rev. 2010, 110,
6961−7001.
(42) Praneeth, V. K. K.; Haupt, E.; Lehnert, N. J. Inorg. Biochem.
2005, 99, 940−948.
(43) Shearer, J.; Kung, I. Y.; Lovell, S.; Kaminsky, W.; Kovacs, J. A. J.
Am. Chem. Soc. 2001, 123, 463−468.
(44) Hagen, K. S.; Holm, R. H. J. Am. Chem. Soc. 1982, 104, 5496−
5497.
(45) Averill, B. A.; Herskovi, T.; Holm, R. H.; Ibers, J. A. J. Am. Chem.
Soc. 1973, 95, 3523−3534.
(46) Strasdeit, H.; Krebs, B.; Henkel, G. Inorg. Chem. 1984, 23,
1816−1825.
(47) The estimated48 pKa’s of the following protons in boldface type
are: 24.6 for HSCH2CH2C(O)OMe, 36.4 for HSCH2CH2OC(O)Me,
25.3 for HSCH2CH2CH2C(O)OMe, 19.6 for HSCH2CH2C(O)Me,
and 24.6 for HSCH2CH(NHAc)C(O)Me.
(48) Szegezdi, J.; Csizmadia, F. Abstr. Pap. Am. Chem. Soc. 2007, 233.
The software of the pKa calculator is commercially available from
ChemAxon (Budapest, Hungary).
(49) Schopfer, M. P.; Mondal, B.; Lee, D. H.; Sarjeant, A. A.; Karlin,
K. D. J. Am. Chem. Soc. 2009, 131, 11304−11305.
(50) Ueyama, N.; Sun, W. Y.; Nakamura, A. Inorg. Chem. 1992, 31,
4053−4059.
(51) Hagen, K. S.; Watson, A. D.; Holm, R. H. J. Am. Chem. Soc.
1983, 105, 3905−3913.
(21) Yukl, E. T.; Elbaz, M. A.; Nakano, M. M.; Moenne-Loccoz, P.
(52) Hagen, K. S.; Reynolds, J. G.; Holm, R. H. J. Am. Chem. Soc.
1981, 103, 4054−4063.
Biochemistry 2008, 47, 13084−13092.
(22) Kommineni, S.; Yukl, E.; Hayashi, T.; Delepine, J.; Geng, H.;
Moenne-Loccoz, P.; Nakano, M. M. Mol. Microbiol. 2010, 78, 1280−
1293.
(53) Ueno, S.; Ueyama, N.; Nakamura, A.; Tukihara, T. Inorg. Chem.
1986, 25, 1000−1005.
(54) Maelia, L. E.; Millar, M.; Koch, S. A. Inorg. Chem. 1992, 31,
4594−4600.
(23) Smith, L. J.; Stapleton, M. R.; Fullstone, G. J.; Crack, J. C.;
Thomson, A. J.; Le Brun, N. E.; Hunt, D. M.; Harvey, E.; Adinolfi, S.;
Buxton, R. S.; Green, J. Biochem. J. 2010, 432, 417−427.
(24) Crack, J. C.; Smith, L. J.; Stapleton, M. R.; Peck, J.; Watmough,
N. J.; Buttner, M. J.; Buxton, R. S.; Green, J.; Oganesyan, V. S.;
Thomson, A. J.; Le Brun, N. E. J. Am. Chem. Soc. 2011, 133, 1112−
1121.
(55) Lu, T. T.; Chiou, S. J.; Chen, C. Y.; Liaw, W. F. Inorg. Chem.
2006, 45, 8799−8806.
(56) Andrews, C. G.; Langler, R. F.; Branch, D. R. J. Sulfur Chem.
2009, 30, 22−28.
(57) Ross, N. C.; Levine, R. J. Org. Chem. 1964, 29, 2346−2350.
(25) Drapier, J. C.; Hibbs, J. B., Jr. J. Clin. Invest. 1986, 78, 790−797.
(26) Drapier, J. C.; Pellat, C.; Henry, Y. J. Biol. Chem. 1991, 266,
10162−10167.
(27) Yang, W.; Rogers, P. A.; Ding, H. J. Biol. Chem. 2002, 277,
12868−12873.
(28) Yang, J.; Duan, X.; Landry, A. P.; Ding, H. Free Radical Biol.
Med. 2010, 49, 268−274.
(29) Beinert, H.; Holm, R. H.; Munck, E. Science 1997, 277, 653−
659.
(30) Rao, P. V.; Holm, R. H. Chem. Rev. 2004, 104, 527−559.
(31) Tran, C. T.; Skodje, K. M.; Kim, E. Prog. Inorg. Chem. 2014, 59,
339−379.
(32) Tsai, M. L.; Chen, C. C.; Hsu, I. J.; Ke, S. C.; Hsieh, C. H.;
Chiang, K. A.; Lee, G. H.; Wang, Y.; Chen, J. M.; Lee, J. F.; Liaw, W. F.
Inorg. Chem. 2004, 43, 5159−5167.
I
DOI: 10.1021/acs.inorgchem.5b00961
Inorg. Chem. XXXX, XXX, XXX−XXX