A Synthetic Model of Enzymatic [Fe4S4]-Alkyl Intermediates

Article abstract of DOI:10.1021/jacs.9b06975

Although alkyl complexes of [Fe₄S₄] clusters have been invoked as intermediates in a number of enzymatic reactions, obtaining a detailed understanding of their reactivity patterns and electronic structures has been difficult owing to

Full text of DOI:10.1021/jacs.9b06975

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A Synthetic Model of Enzymatic [FeS]–Alkyl Intermediates
Mengshan Ye, Niklas B. Thompson, Alexandra C Brown, and Daniel L. M. Suess
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b06975 • Publication Date (Web): 02 Aug 2019
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A Synthetic Model of Enzymatic [Fe4S4]–Alkyl Intermediates
Mengshan Ye, Niklas B. Thompson, Alexandra C. Brown, Daniel L. M. Suess*
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Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
Supporting Information Placeholder
ABSTRACT: Although alkyl complexes of [Fe4S4] clusters have
been invoked as intermediates in a number of enzymatic reac-
tions, obtaining a detailed understanding of their reactivity pat-
terns and electronic structures has been difficult owing to their
transient nature. To address this challenge, we herein report the
Chart 1. Examples of proposed [Fe4S4]–alkyl intermedi-
ates
R1 R2
S
O
OH
A
H2
C
NH2
CR3R4
(H)O
S
O
S
OH
Fe
S
Fe
S
Fe Fe
synthesis and characterization of
a 3:1 site-differentiated
O
S
S
[Fe4S4]2+–alkyl cluster. Whereas [Fe4S4]2+ clusters typically exhibit
pairwise delocalized electronic structures in which each Fe has a
formal valence of 2.5+, Mössbauer spectroscopic and computa-
tional studies suggest that the highly electron-releasing alkyl
group partially localizes the charge distribution within the cubane,
an effect that has not been previously observed in tetrahedrally
coordinated [Fe4S4] clusters.
Fe Fe
Fe
Fe
Cys
Cys
Cys
Cys
Cys
Cys
S
S
Radical SAM enzymes
IspG/GcpE and IspH/LytB
H3C
H3C
CH2
CH2
Fe
S
Fe
S
S
S
S
S
Fe Fe
Fe
L
Fe
Fe
Fe
RS
L
SR
L
RS
S
Iron–sulfur (Fe–S) proteins are found in all kingdoms of life and
perform myriad functions in the cell.1–4 Those that utilize [Fe4S4]
cofactors are the most ubiquitous and have well-documented
roles as electron-transfer and Lewis-acid catalysts.1,2 Recently,
several classes of [Fe4S4] enzymes have been proposed to operate
via organometallic intermediates. In particular, [Fe4S4]–alkyl spe-
cies have been invoked in mechanisms of reductive dehydroxyla-
tion reactions in isoprenoid biosynthesis,5–11 reductive coupling of
CO and CO2 to higher-order hydrocarbons,12 and radical reactions
by the > 100,000 members of the radical S-adenosyl-L-methionine
(SAM) superfamily of enzymes.13–16
S
This work
NifH and synthetic
[Fe4S4] clusters
to alkylated [Fe4S4] clusters. To date, only one alkylated Fe–S clus-
ter has been structurally characterized—an [Fe8S7] cluster in
which the alkyl group is derived from decamethylcobaltacene.21
We herein describe the synthesis of the first [Fe4S4]–alkyl cluster
and investigations into how the alkyl ligand perturbs the electronic
structure of the [Fe4S4] core.
We reasoned that synthetic [Fe4S4]–alkyl clusters could be ac-
cessed by reaction of a nucleophilic alkylating reagent with a 3:1
site-differentiated [Fe4S4]–halide cluster. This route requires that
the clusters be stable and soluble in solvents compatible with al-
kylating reagents, and we therefore pursued clusters with low
overall charge. Inspired by the chelating trithiolate architecture
(LS3) developed by Holm22,23 and adapted by others,24,25 we pre-
pared a structurally analogous ligand, L(N=PTol)3, featuring three
iminophosphorane donors. Iminophosphoranes are strongly basic
and, like thiolates, can serve as both σ- and π-donors due to the
presence of two lone pairs on the nitrogen atom.26 In addition,
iminophosphoranes are neutral (allowing for compounds with low
overall charge and, hence, increased solubility in unreactive sol-
vents), sterically demanding, and tunable at their P-substituents.
Despite the emerging significance of [Fe4S4]–alkyl intermedi-
ates, little is known about how these species form, their reactivity
patterns, or their electronic structures. Moreover, no [Fe4S4]–alkyl
species have been structurally characterized and their identifica-
tion as intermediates (Chart 1) has relied on EPR/ENDOR5–11,13–15
or DFT7,12 studies. And although reliable models for the electronic
structures of [Fe4S4] clusters have been developed,17–19 these
models were derived for clusters ligated by relatively weak-field,
moderately donating ligands (e.g. cysteine thiolates). How binding
of a strong-field, highly electron-releasing alkyl ligand20 perturbs
the electronic structure of [Fe4S4] clusters—to what extent it in-
duces valence localization, how (if at all) it affects Fe–Fe interac-
tions, and how these effects impact the reactivity of the Fe–C
bond—is unknown.
The synthesis of L(N=PTol)3 (3) is shown in Scheme 1. Trianiline 2
was generated via Buchwald-Hartwig coupling between tribro-
mide 1 and Ph2C=NH followed by hydrolysis.27 The iminophospho-
rane groups were then installed in a Kirsanov reaction using
Tol3PCl2 and excess Et3N.
Synthetic chemistry will play an important role in answering
these questions. Synthetic analogues of [Fe4S4]–alkyl intermedi-
ates would, for example, allow for the structures of intermediates
to be elucidated (by linking their structures with their spectro-
scopic features) and for their electronic structures to be interro-
gated. These efforts are hampered by the lack of synthetic access
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Scheme 1. Synthesis of iminophosphorane-ligated [Fe4S4]
a)
1
2
clustersa
3
4
5
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b)
aConditions: (i) a) Ph2C=NH (9 equiv.), Pd2(dba)3 (1.9 mol%), rac-BINAP (2.8
mol%), NaOtBu (10.5 equiv.), toluene, 80 C; b) 2 M HCl, THF; c) excess
NaOH, MeOH; (ii) Tol3PCl2 (3 equiv.), Et3N (7 equiv.), benzene, 80 C; (iii)
NaBPh4 (3 equiv.), [PPh4]2[Fe4S4Cl4] (1.1 equiv.), 1:1 THF/MeCN; (iv) Et2Zn
(1.5 equiv.), THF.
The 3:1 site-differentiated cluster [(L(N=PTol)3)Fe4S4Cl][BPh4] (4)
can be synthesized on a multi-gram scale in 65% yield by reaction
of L(N=PTol)3 with [PPh4]2[Fe4S4Cl4] and 3 equiv NaBPh4 (Scheme 1).
The molecular structure of 4 as determined by single-crystal X-ray
diffraction (XRD) shows the anticipated 3:1 site differentiation
Figure 1. Thermal ellipsoid plots (50%) of (a) 4 and (b) 5. Hy-
drogen atoms, solvent molecules, and anions omitted for
clarity. Fe (red), S (yellow), Cl (purple), N (blue), P (orange), C
(gray).
with three Fe atoms bound by L(N=PTol)3 and the apical Fe (Feapical
)
site bound by Cl (Fig. 1a). The molecule has pseudo-C3 symmetry
with one p-tolyl group of each iminophosphorane aligned with the
pseudo-C3 axis, forming a protective cavity around unique Fe site
(Fig. 1a). The Fe–S distances in 4 are similar to those observed in
the 3:1 site-differentiated cluster [(LS3)Fe4S4Cl]2–.23
each a formal oxidation state (FOS) of 2.5+. Moreover, the similar-
ity between the isomer shifts of 4 and those reported for
[(LS3)Fe4S4Cl]2– (δ = 0.46 mm/s at 80 K)30 and for protein-bound
[Fe4S4]2+ clusters (δ ≈ 0.42 mm/s)31 underscores the utility of the
L(N=PTol)3 ligand in modeling a trithiolate donor set.
Treatment of 4 with Et2Zn generates the [Fe4S4]–alkyl complex
[(L(N=PTol)3)Fe4S4Et][BPh4] (5), which can be isolated as brown sol-
ids in 85% yield. The structure of 5 (Fig. 1b) was confirmed by sin-
gle-crystal XRD and is similar to that of 4. The Feapical–C bond length
of 5 (2.05 Å) is shorter than that in the [Fe8S7]–decamethylcobalto-
cenyl cluster (2.12 Å)21 and comparable to that in a tris(thi-
oether)borate-ligated Fe2+–Me complex (2.03 Å).28 In solution, 5
slowly decomposes to unidentified products; further reactivity
studies of 5 are underway.
Complexes 4 and 5 show similar ligand-derived resonances in
their room-temperature H and 31P NMR spectra. Both exhibit C3
1
symmetry in solution as indicated by splitting of the diastereotopic
Ar–CH2–Ar and Ar–CH2–CH3 resonances. Their 31P NMR reso-
nances (at 102.1 and 96.0 ppm for 4 and 5, respectively) are
shifted downfield from that of the free ligand 3 (−0.4 ppm), re-
flecting both the expected downfield-shift upon binding a Lewis-
acidic metal center32,33 and the population of paramagnetic ex-
cited states as is commonly observed in [Fe4S4]2+ clusters;23,34–38
their room-temperature solution magnetic moments (μeff = 2.7
and 2.8 μB, respectively) are consistent with this interpretation
The 57Fe Mössbauer spectrum of solid 4 at 90 K (Fig. 2a) was
simulated as three quadrupole doublets in a 2:1:1 ratio with iden-
tical isomer shifts of 0.47 mm/s (Fig. 2a, Table 1). This simulation
is in accordance with the canonical electronic structure of an
[Fe4S4]2+ cluster: an S = 0 ground state arising from antiferromag-
and typical of [Fe4S4]2+ clusters.35,39–41 The H NMR signals corre-
1
sponding to the -CH3 and -CH2- protons of the ethyl ligand in 5 are
observed at −4.6 and 70.0 ppm, respectively, which also indicates
the population of paramagnetic excited states.23,34–38 Clusters 4
and 5 exhibit reversible, one-electron reduction events in their cy-
clic voltammagrams at –1.48 and –1.78 V vs. Fc/Fc+, respectively
(see SI), reflecting the greater electron-donating ability of the
ethyl group compared with that of the chloride.
As above, the 57Fe Mössbauer spectrum of solid 5 (Fig. 2b) at 90
K was simulated with three quadrupole doublets in a 2:1:1 ratio.
This approach produces two reasonable models (Table 1); our
9
netic coupling of two S = /2 [Fe2S2]+ rhombs, each of which con-
sists of spin-aligned, high-spin Fe2.5+ ions engaged in a double-ex-
change interaction.29 Thus, we assign the doublet comprising 50%
of the spectral area to the [Fe2S2]+ rhomb bound by two imino-
phosphorane donors and the second pair of doublets to the Cl-
ligated Feapical center and its ferromagnetically aligned, iminophos-
phorane-ligated partner (Fig. 2c). That identical isomer shifts are
observed for all sites in this spectrum indicates that each Fe site
possesses similar core-charge density, and hence we can assign to
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Table 1. Experimental (90 K) and computed Mössbauer parameters for 4 and 5
1
2
3
4
5
6
7
8
9
4 (X = Cl)
5 (X = Et)
Disfavored
simulation
Favored
simulation
Simulation
|∆EQ|
Calculation
Calculation
Doublet
Site
δ
δcalc
δ
|∆EQ|
δ
|∆EQ|
δcalc
FOSa
FOSa
(mm/s) (mm/s) (mm/s)
(mm/s) (mm/s) (mm/s) (mm/s) (mm/s)
0.45
2.50+
2.59+
2.54+
2.54+
0.46
2.58+
2.51+
2.31+
2.79+
1
Fe–L
0.47
0.48
0.46
0.59
0.46
0.58
0.46
0.45
0.50
0.47
0.52
0.22
2
3
Fe–Lb
Fe–Xb
0.47
0.47
0.75
1.06
0.52
0.30
1.09
1.18
0.44
0.39
0.93
1.36
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aFOSs are determined from a population analysis of the LMOs of each BS determinant. See SI for details. bSites coupled via double exchange.
more negative.42,43 The major difference between these two mod-
a)
els is in the magnitude of this effect (δ = 0.30 vs. 0.39 mm/s) and
whether the remaining iminophosphorane-bound Fe site, which is
coupled to the alkylated site via a double-exchange interaction,
possesses an isomer shift that is greater or less than that of the
other ligand-bound pair (δ = 0.52 vs. 0.44 mm/s, compared with
0.46 mm/s for the other ligand-bound pair).
In order to distinguish between these two models, we turned to
broken-symmetry density functional theory (BS DFT) calculations
(see SI for details). The isomer shifts calculated for 4 (Table 1) are
in good agreement with the experimental values. To make the
connection between δ and the formal oxidation state (FOS) of the
Fe sites, we further analyzed the BS determinants in terms of lo-
b)
calized molecular orbitals (LMOs).44 For each site, the LMOs natu-
rally partition into a set of five spin-up (or down) Fe 3d orbitals,
plus an extra spin down (or up) 3d-derived orbital that is delocal-
ized over a single additional Fe site. This picture corresponds to
the canonical electronic structure of the [Fe4S4]2+ cluster18,19 in
which the double-exchange interaction is mediated by the extra
delocalized orbital (Fig. 3). Through a population analysis, we char-
acterized the tendency of the itinerant electron to localize on ei-
ther of the two Fe sites engaging in double exchange and thereby
assigned FOSs. In the case of 4, the itinerant electrons are fully
delocalized, leading to FOS assignments of ~2.5+ for each site (Ta-
ble 1 and Figure 3), consistent with the experimentally observed
isomer shifts.
c)
For 5, the calculated isomer shift of the alkylated site, 0.22
mm/s, is most consistent with the simulated value of 0.30 mm/s.
Moreover, the calculation predicts that the isomer shift of the
iminophosphorane-bound Fe that is coupled to the alkylated site
via double exchange increases relative to those of the remaining
Fe sites—precisely what is observed in the simulation presented
in Table 1. While the low isomer shift of the alkylated site might
be expected on the basis of the properties of the alkyl ligand (vide
supra), the compensatory increase in the isomer shift of the spin-
aligned Fe is unusual and suggests partial charge localization
within this double-exchange-coupled pair. Indeed, the calculated
FOS of 2.31+ for this site in 5 suggests increased ferrous character,
and this charge localization is coupled to an increase in the FOS of
the alkylated site to 2.79+ (Table 1).
Figure 2. Zero-field 57Fe-Mössbauer spectra of solid (a) 4 and
(b) 5 at 90 K. Black circles represent experimental data, solid
lines are simulations. (c) Assignments of doublets to individ-
ual Fe sites. Isomer shifts indicated in mm/s and double-ex-
change interactions indicated by orange arrows.
preferred model (vide infra) is shown in Fig. 2b/c, and alternatives
are discussed in the SI. Both models feature a doublet comprising
50% of the total area with parameters that are nearly identical to
those found for the iminophosphorane-bound Fe2.5+ sites in 4 (Ta-
ble 1); this doublet is therefore assigned to the analogous Fe2.5+
sites in 5. The quadrupole doublet with the lowest isomer shift is
assigned to the alkylated Fe site because the stronger electron-
donating ability (vide supra) and increased covalency of the ethyl
group relative to L(N=PTol)3 are expected to enhance the charge
density at the 57Fe nucleus and drive the isomer shift of this site
Physically, this partial charge localization can be understood in
terms of localized ligand field effects about the Fe sites engaging
in double-exchange delocalization.17,45 Assuming that double ex-
change occurs through a single orbital interaction, then two Fe
sites with very similar ligand fields will share the itinerant electron
equally, producing an effective valence of 2.5+; this appears to be
the case, both experimentally and computationally, for all sites in
4 (Fig. 3). The symmetry of the double-exchange interaction is re-
moved by alkylation in 5 whereby the electron-releasing alkyl
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Mengshan Ye: 0000-0003-2709-8135
Cl
L
R
L
4
5
1
2
3
4
5
6
Niklas B. Thompson: 0000-0003-2745-4945
Alexandra C. Brown: 0000-0002-2410-9217
Daniel L. M. Suess: 0000-0002-0916-1973
Notes
FeA
FeB
FeA
FeB
7
8
9
The authors declare no competing financial interests.
partially
localized
ACKNOWLEDGMENT
delocalized
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We thank Prof. Theodore Betley (Harvard) for use of his Möss-
bauer spectrometer, Kevin Anderton (Harvard) for assistance with
acquiring Mössbauer data, and Drs. Peter Müller and Charlene
Tsay (MIT) for assistance with X-ray crystallographic experiments.
This work was supported by the MIT Skoltech Program.
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Figure 3. Double-exchange interactions in the beta manifold
of the [Fe2S2]+ rhombs in 4 and 5. (Top) Molecular orbital dia-
gram showing polarization of the double-exchange interac-
tion upon introduction of an electron-rich alkyl ligand. (Bot-
tom) Isosurface plots (0.05 a.u.) of the double-exchange inter-
action orbitals with Fe-based Löwdin population analysis.
ligand raises the average energy of the local Fe 3d manifold (Fig.
3). As a result, the itinerant electron will tend to localize on the
site to which the alkylated Fe is coupled, as observed. Similar elec-
tronic desymmetrization may alternatively be induced by differ-
ences in coordination number between double-exchange-coupled
Fe sites.46 Although a two-orbital model of double exchange is
likely too simplistic, these arguments should hold in the case of a
more complex multi-orbital picture.18,47
In conclusion, we have reported the synthesis and characteriza-
tion of 3:1 site-differentiated [Fe4S4]2+–Cl and [Fe4S4]2+–Et clusters
that are supported by a chelating triiminophosphorane ligand.
NMR and Mössbauer spectroscopic data indicate that although
both clusters have typical diamagnetic ground states, the
[Fe4S4]2+–Et cluster exhibits a polarized Fe–Fe double-exchange in-
teraction, partially localizing ferric character at the alkylated Fe
site and ferrous character at its ferromagnetically aligned partner.
Based on these results, we anticipate that enzymatic [Fe4S4]–alkyl
intermediates may exhibit partial or even complete localization of
Fe3+ at their alkylated sites. Further investigations into the reactiv-
ity of [Fe4S4]2+–alkyl clusters and efforts to access [Fe4S4]–alkyl
clusters in other redox states and coordination numbers are cur-
rently underway in our laboratory.
ASSOCIATED CONTENT
Supporting Information
Supporting Information containing experimental procedures,
spectra, and computational details is available free of charge on
the ACS Publications websites at DOI: xxx
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Caranto, J. D.; Dzikovski, B.; Sharma, A.; Lancaster, K. M.; Freed, J. H.;
Ealick, S. E.; Hoffman, B. M.; Lin, H. Organometallic and Radical
Intermediates Reveal Mechanism of Diphthamide Biosynthesis. Science
2018, 359, 1247–1250.
(15) Byer, A. S.; Yang, H.; McDaniel, E. C.; Kathiresan, V.; Impano, S.;
Pagnier, A.; Watts, H.; Denler, C.; Vagstad, A. L.; Piel, J.; Duschene, K. S.;
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Broderick, W. E.; Hoffman, B. M.; Broderick, J. B. Paradigm Shift for Radical
S-Adenosyl-l-Methionine Reactions: The Organometallic Intermediate ω Is
AUTHOR INFORMATION
Corresponding Author
*Email: suess@mit.edu
ORCID
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