Iron acyl thiolato carbonyls: Structural models for the active site of the [Fe]-hydrogenase (Hmd)

Article abstract of DOI:10.1021/ja1072228

Phosphine-modified thioester derivatives are shown to serve as efficient precursors to phosphine-stabilized ferrous acyl thiolato carbonyls, which replicate key structural features of the active site of the hydrogenase Hmd. The reaction of Ph₂P

Full text of DOI:10.1021/ja1072228

Published on Web 11/09/2010
Iron Acyl Thiolato Carbonyls: Structural Models for the Active
Site of the [Fe]-Hydrogenase (Hmd)
Aaron M. Royer,† Marco Salomone-Stagni,‡ Thomas B. Rauchfuss,*,† and
Wolfram Meyer-Klaucke*,‡
School of Chemical Sciences, UniVersity of Illinois at Urbana-Champaign, Urbana,
Illinois 61801, United States, and EMBL Outstation c/o DESY, Notkestrasse 85,
D-22603 Hamburg, Germany
Received August 19, 2010; E-mail: rauchfuz@uiuc.edu; wolfram@embl-hamburg.de
Abstract: Phosphine-modified thioester derivatives are shown to serve as efficient precursors to
phosphine-stabilized ferrous acyl thiolato carbonyls, which replicate key structural features of the active
site of the hydrogenase Hmd. The reaction of Ph2PC6H4C(O)SPh and sources of Fe(0) generates
both Fe(SPh)(Ph2PC6H4CO)(CO)3 (1) and the diferrous diacyl Fe2(SPh)2(CO)3(Ph2PC6H4CO)2, which
carbonylates to give 1. For the extremely bulky arylthioester Ph2PC6H4C(O)SC6H3-2,6-(2,4,6-
trimethylphenyl)2, oxidative addition is arrested and the Fe(0) adduct of the phosphine is obtained.
Complex 1 reacts with cyanide to give Et4N[Fe(SPh)(Ph2PC6H4CO)(CN)(CO)2] (Et4N[2]). 13C and 31P
NMR spectra indicate that substitution is stereospecific and cis to P. The IR spectrum of [2]- in νCN
and νCO regions very closely matches that for HmdCN. XANES and EXAFS measurements also indicate
close structural and electronic similarity of Et4N[2] to the active site of wild-type Hmd. Complex 1 also
stereospecifically forms a derivative with TsCH2NC, but the adduct is more labile than Et4N[2].
Tricarbonyl 1 was found to reversibly protonate to give a thermally labile derivative, IR measurements
of which indicate that the acyl and thiolate ligands are probably not protonated in Hmd.
Introduction
terin dehydrogenase (Mtd, PDB 3IQF). Hmd was originally
thought to be free of metals because catalysis was found initially
The conversion of carbon dioxide to methane is accomplished
on a massive scale biologically, as indicated by the world’s
natural gas reserves (6254 trillion cubic feet).1 The principal
means by which microorganisms carry out this conversion has
been delineated over the previous few decades.2 This reduction
involves a sequence of enzyme-catalyzed steps that utilize
several unusual cofactors. For example, the biochemical cycle
starts with the conversion of CO2 to a formamide using a
methanofuran cofactor and ends with the hydrogenolysis of a
CH3-S bond in coenzyme M. As a source on new ideas on
catalysis, this collection of cofactors represent potentially
rewarding targets. The biosynthesis and mode of action are areas
ripe for discovery, and perhaps applications.
The most recently elucidated step in the archaeal methano-
genic cycle is the reduction of a stabilized carbocation to the
corresponding methylene derivative.3 Normally, this conversion
is effected by a [NiFe]-hydrogenase in the hydrogenotrophic
methanogens, but under conditions where nickel is insufficiently
bioavailable, the organism up-regulates backup enzymes that
catalyze the same conversion.4 These enzymes are called H2-
forming methylenetetrahydromethanopterin dehydrogenase (Hmd,
PDB 3F47) and F420-dependent methylene tetrahydromethanop-
to be insensitive to the presence of CO. Later work showed
that Hmd requires iron and is inhibited by CO.5 Over the course
of the preceding five years, the structure of the active site has
been elucidated using both the native protein as well as
mutants.6-10 The protein harbors an active site consisting of
the third example of a iron thiolato carbonyl center found in
biology.11,12 The fact that these species catalyze reactions
involving H2 is an example of convergent evolution and an
indicator of the deep significance of the Fe-SR-CO system.11
The environment of the Fe center in Hmd is Fe(SR)
(acyl)L(CO)2X, where L is an N-bonded ligand that is a derivative
(4) Afting, C.; Hochheimer, A.; Thauer, R. K. Arch. Microbiol. 1998,
169, 206–210. Afting, C.; Kremmer, E.; Brucker, C.; A., H.; Thauer,
R. K. Arch. Microbiol. 2000, 174, 225–232.
(5) Lyon, E. J.; Shima, S.; Buurman, G.; Chowdhuri, S.; Batschauer, A.;
Steinbach, K.; Thauer, R. K. Eur. J. Biochem. 2004, 271, 195–204.
(6) Shima, S.; Pilak, O.; Vogt, S.; Schick, M.; Stagni, M. S.; Meyer-
Klaucke, W.; Warkentin, E.; Thauer, R. K.; Ermler, U. Science 2008,
321, 572–575.
(7) Hiromoto, T.; Ataka, K.; Pilak, O.; Vogt, S.; Stagni, M. S.; Meyer-
Klaucke, W.; Warkentin, E.; Thauer, R. K.; Shima, S.; Ermler, U.
FEBS Lett. 2009, 583, 585–590.
(8) Hiromoto, T.; Warkentin, E.; Moll, J.; Ermler, U.; Shima, S. Angew.
Chem., Int. Ed. 2009, 48, 6457–6460.
(9) Lyon, E. J.; Shima, S.; Boecher, R.; Thauer, R. K.; Grevels, F.-W.;
Bill, E.; Roseboom, W.; Albracht, S. P. J. J. Am. Chem. Soc. 2004,
126, 14239–14248.
† University of Illinois at Urbana-Champaign.
‡ EMBL Outstation.
(1) Energy Information Administration. World Proved Reserves of Oil
and Natural Gas, Most Recent Estimates, http://www.eia.doe.gov/
emeu/international/reserves.html, March 3, 2009.
(2) Thauer, R. K. Microbiology 1998, 144, 2377–2406.
(3) Shima, S.; Thauer, R. K. Chem. Rec. 2007, 7, 37–46.
(10) Korbas, M.; Vogt, S.; Meyer-Klaucke, W.; Bill, E.; Lyon, E. J.; Thauer,
R. K.; Shima, S. J. Biol. Chem. 2006, 281, 30804–30813.
(11) Armstrong, F. A.; Fontecilla-Camps, J. C. Science 2008, 321, 498–
499.
(12) Heinekey, D. M. J. Organomet. Chem. 2009, 694, 2671–2680.
9
10.1021/ja1072228  2010 American Chemical Society
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A R T I C L E S
Royer et al.
been described for complexes of Rh(I)20 and Pt(0)21 and is of
continuing interest for applications in organic synthesis.22,23
Thioester-iron interactions have been implicated in transforma-
tions relevant to the origin of life.24
We had previously described the oxidative addition of
thioester-modified phosphines to Fe(0) reagents to give diiron
µ-thiolato species of the type Fe2(SPh)2(Ph2PC6H4CO)2(CO)3.25
This diferrous species carbonylates to afford the metastable
monomer fac-Fe(SPh)(Ph2PC6H4CO)(CO)3 (1). This tricarbonyl
was found to undergo monosubstitution to give derivatives
mimicking other ligand-inhibited forms of Hmd, abbreviated
HmdL. In our preliminary report, we had not crystallized
monomeric models of known inhibited states, but this problem
has now been solved. Herein we describe the structural
characterization of 1 and its cyanide derivative, which constitute
structural models for the active site of Hmd.
Following the discovery of the Fe-acyl bond in Hmd, several
models have appeared. Hu and co-workers originally generated
iron acyls stabilized by 2-mercaptopicoline derivatives.26 More
recently both the Hu and Pickett groups have developed acyl-
or carbamoylpyridine derivatives of ferrous carbonyls.27 The
mechanism, including the role of the 2-pyridinol cofactor, has
also attracted much interest from computational chemists.28
Figure 1. Structure of the active site of the jHmd C176A mutant with
dithiothreitol (DTT) replacing cys176 (PDB 3H65) (Methanococcus jan-
naschii).7 One OH of the DTT occupies the site trans to acyl. Color scheme:
brown, Fe; blue, N; red, O; purple, P; yellow, S; gray, C.
of either 2-hydroxypyridine or 2-pyridonate and X occupies an
apparently labile site. In the crystal structure X has been modeled
as oxygen (e.g., of water),7 which is in line with the EXAFS
analysis.13 In the mutant protein, the oxygenic ligand is provided
by an alcohol (Figure 1). Inhibited forms of the protein have
been prepared where X is cyanide and CO. The cyanide
derivative HmdCN is stable, but its formation reverses at high
dilution, whereas the CO-inhibited form HmdCO is highly labile,
in accord with the weak inhibiting effect of this ligand.3 These
inhibited forms, especially HmdCO and HmdCN, represent suitable
synthetic targets, since they are coordinatively saturated.
Furthermore, models for HmdCO could serve as precursors to
catalytically active states.
From the structural perspective, the presence of the acyl ligand
is striking. Fe-acyls are common in synthetic organometallic
chemistry,14 forexample, (C5H5)(CO)2FeC(O)Meand[(CO)4FeC-
(O)Me]-, but are unusual in biology. Acyl nickel intermediates
have been invoked in acetogenesis,15 which is catalyzed by the
enzyme acetyl Co-A synthase.16 In Hmd, the acyl ligand may
function as a trans directing group, stereoselectively labilizing
the site that binds H2. Normally, acyl ligands are cis labilizing
because of the facility of the η1- to η2-acyl conversion,17 but if
constrained in a chelate ring as in Hmd’s active site, then we
speculate that an acyl can exert a trans influence comparable to
that of an aryl group.18
Results
Phosphine Thioesters. Thioester phosphines containing a variety
of aryl and alkylthio substituents can be prepared via carbodiimide
coupling of 2-diphenylphosphinobenzoic acid with a range of thiols
(Scheme 1). The new compounds are air-stable pale yellow to
colorless crystalline solids. Such thioesters have been previously
investigated as peptide coupling reagents.29 Isomeric thioesters of
the type Ph2PC6H4SC(O)R are also known.30
Scheme 1
For first-generation models of Hmd, we sought to incorporate
the most distinctive ligand, the acyl. Prior to our work, acyl
thiolato monoiron complexes were unknown, although diiron
complexes of the type Fe2(SR)(acyl)(CO)6 had been described.
Specifically, Seyferth and co-workers prepared a series of diiron
acyl thiolates via the oxidative addition of alkyl and arythioesters
to Fe(0) reagents (eq 1).19 The results presented in this paper
Fe(II) Acyl Thiolates. We found that several of the phosphine
thioesters react with iron carbonyls to afford iron(II) acyl thiolato
derivatives. A useful iron(0) source was Fe(bda)(CO)3 (bda )
(18) Hartwig, J. F. Organotransition Metal Chemistry, from Bonding to
Catalysis; University Science Books: New York, 2010.
(19) Seyferth, D.; Womack, G. B.; Archer, C. M.; Dewan, J. C. Organo-
metallics 1989, 8, 430–442.
(20) Shaver, A.; Uhm, H. L.; Singleton, E.; Liles, D. C. Inorg. Chem. 1989,
28, 847–851.
suggest that these diiron compounds arise via monoiron acylthi-
olate complexes. The oxidative addition of thioesters has also
(21) Minami, Y.; Kato, T.; Kuniyasu, H.; Terao, J.; Kambe, N. Organo-
metallics 2006, 25, 2949–2959.
(22) Tokuyama, H.; Yokoshima, S.; Yamashita, T.; Lin, S.-C.; Li, L.;
Fukuyama, T. J. Braz. Chem. Soc. 1998, 9, 381–387. Fukuyama, T.;
Lin, S.-C.; Li, L. J. Am. Chem. Soc. 1990, 112, 7050–7051. Mori, Y.;
Seki, M. Org. Synth. 2007, 84, 285–294. Liebeskind, L. S.; Srogl, J.
J. Am. Chem. Soc. 2000, 122, 11260–11261.
(13) Salomone-Stagni, M.; Stellato, F.; Whaley, C. M.; Vogt, S.; Morante,
S.; Shima, S.; Rauchfuss, T. B.; Meyer-Klaucke, W. Dalton Trans.
2010, 39, 3057–3064.
(14) Plietker, B., Ed. Iron Catalysis in Organic Chemistry; Wiley-VCH:
Weinheim, 2008.
(23) Looman, S. D.; Giese, S.; Arif, A. M.; Richmond, T. G. Polyhedron
1996, 15, 2809–2811.
(15) Tucci, G. C.; Holm, R. H. J. Am. Chem. Soc. 1995, 117, 6489–6496.
(16) Lindahl, P. A.; Graham, D. E. Met. Ions Life Sci. 2007, 2, 357–415.
Fontecilla-Camps, J. C.; Amara, P.; Cavazza, C.; Nicolet, Y.; Volbeda,
A. Nature 2009, 460, 814–822.
(24) Huber, C.; Wa¨chtersha¨user, G. Science 1997, 276, 245–247. Wa¨cht-
ersha¨user, G. Philos. Trans R. Soc. 2006, 361, 1787–1806.
(25) Royer, A. M.; Rauchfuss, T. B.; Gray, D. L. Organometallics 2009,
28, 3618–3620.
(17) Ford, P. C.; Rokicki, A. AdV. Organometal. Chem. 1988, 28, 139–
206.
(26) Chen, D.; Scopelliti, R.; Hu, X. J. Am. Chem. Soc. 2009, 132, 928–
929.
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Iron Acyl Thiolato Carbonyls To Model Hmd
A R T I C L E S
Scheme 2. Isomers Proposed for Fe2(SPh)2(Ph2PC6H4CO)2(CO)3
Figure 2. Energies of νCN and νCO for selected model compounds and
HmdCO and HmdCN
.
benzylideneacetone), but Fe2(CO)9 was also effective. Spectro-
scopic analysis of the reaction of Ph2PC6H4-2-C(O)SPh (La) with
the highly labile cyclooctene complex Fe(CO)3(C8H14)231 indi-
cated displacement of only one cyclooctene ligand to give a
monophosphine adduct. Thioesters derived from EtSH and tert-
BuSH gave diiron compounds that were spectroscopically
similar to the PhS derivative (see below), but these alkylthiolato
diiron compounds resisted carbonylation to the monoiron
complexes (see below).
Table 1. Selected IR Data for Model Compounds and for Hmd
from M. marburgensis9
samplea
νCO (cm-1
)
HmdCO from M. marburgensis9
Fe(SPh)(Ph2PC6H4CO)(CO)3 (1)
(1:1 CH2Cl2:MeOH)
2074, 2020, 1981
2075, 2025, 2001
(2077, 2028, 2005)
2090 (νCN), 2020, 1956
2093 (νCN), 2013, 1954
HmdCN from M. marburgensis9
Et4N[Fe(SPh)(Ph2PC6H4CO)(CN)(CO)2]
(Et4N[2])
The simple thioester La gave the cleanest and highest yielding
reaction and became the main focus for our efforts. Treatment
of a hot THF suspension of Fe2(CO)9 with La was found to
give diiron dithiolato complexes with the formula Fe2(SPh)2-
(CO)3[Ph2PC6H4C(O)]2 (eq 2).25
(1:1 CH2Cl2:MeOH)
(2093 (br, νCN), 2026, 1973)
Et4N[Fe(SPh)(Ph2PC6H4CO)(13CN)(CO)2] 2050 (νCN), 2012, 1954
Fe(SPh)(Ph2PC6H4CO)(CNCH2Ts)(CO)2
2154 (νCN), 2042, 1988
a Models in CH2Cl2 solution unless otherwise indicated; enzyme in
aqueous solution.
Fe(SPh)(Ph2PC6H4CO)(CO)3. Compound 1 was obtained by
high-pressure carbonylation of a warm solution of the afore-
mentioned diiron derivative. Solutions of the monomer 1 were
found to decarbonylate at room temperature, returning to the
diiron complex, but solutions of 1 were stable for days at < -10
°C. Crystalline samples of 1 proved stable at room temperature
in air for weeks. Exposure of a solution of complex 1 to 1
atm of 13CO at room temperature resulted in rapid (<5 min)
exchange of all sites to give Fe(SPh)(Ph2PC6H4CO)(13CO)3.
The 31P NMR spectrum of this species confirmed the
arrangement of the CO ligands, since three separate 13C-31P
couplings are observed with J ) 58, 21, and 16 Hz. In
Fe(CO)3(PMe3)(η2-Me3SiCCSiMe3), the 13CO-31P coupling
constants are 59 (trans) and 35 Hz (cis).34
This diiron species exists as a mixture of isomers that are
proposed to differ with respect to the orientation of the µ-SPh
groups (Scheme 2).32 Other aryl thioesters (Ar ) C6H4-2-OMe,
2,4,6-iPr3C6H2) also gave derivatives of the type Fe2(SAr)2-
(Ph2PC6H4CO)2(CO)3, but these conversions were less efficient.
Using an extremely bulky arylthioester, we were able to arrest
the oxidative addition. Thus, treatment of Fe2(CO)9 with
Ph2PC6H4C(O)SC6H4-2,6-(Ar*)2 (Ar* ) 2,4,6-trimethylphenyl)
gave the monophosphine adduct of Fe(CO)4. The IR spectrum
of this product matches that for known derivatives of the type
Fe(CO)4(PR3).33
Thioester Derivatives of Nitrogen-Based Heterocycles. We
also examined the reaction of thioester-functionalized N-
heterocycles with Fe(0) reagents (see Scheme 1). The thiophe-
nolate esters of quinoline-8-carboxylic acid20 and 2-hydroxy-
4-methylpyridine-6-acetic acid were synthesized by the usual
coupling methods (Scheme 1). Both derivatives were reactive
toward Fe2(CO)9 and (bda)Fe(CO)3, but Fe2(SPh)2(CO)6 was
the only Fe carbonyl product detected.
[Fe(SPh)(Ph2PC6H4CO)(CN)(CO)2]-.Complex1reactssmoothly
with cyanide to afford the anion [Fe(SPh)(Ph2PC6H4CO)-
(CN)(CO)2]- ([2]-), isolated as its Et4N+ salt. The 13C and 31
P
NMR spectra for Et4N[2] and its 13CN-labeled derivative indicate
that substitution is stereospecific. The value of J(31P,13C)
indicates that cyanide is located cis to the phosphine ligand.35
Unlike 1, solutions of [2]- are stable with respect to loss of
CO, as also seen for HmdCN.9 The IR spectrum of [2]- in CH2Cl2
solution also closely matches the cyanide-inhibited form of Hmd,
wherein cyanation also proceeds stereoselectively9 (Figure 2,
Table 1).
The cyanide derivative was further characterized by X-ray
absorption spectroscopy. The XANES spectrum (Figure 3),
indicative of the electronic structure of the iron site, closely
(27) Chen, D.; Scopelliti, R.; Hu, X. Angew. Chem., Int. Ed. 2010, 49,
7512–7515. Turrell, P. J.; Wright, J. A.; Peck, J. N. T.; Oganesyan,
V. S.; Pickett, C. J. Angew. Chem., Int. Ed. 2010, 49, 7508–7511.
(28) Yang, X.; Hall, M. B. J. Am. Chem. Soc. 2009, 131, 10901–10908.
Dey, A. J. Am. Chem. Soc. 2010, 132, 13892–13901. Stiebritz, M. T.;
Reiher, M. Inorg. Chem. 2010, 49, 5818–5823.
(29) Myers, E. L.; Raines, R. T. Angew. Chem., Int. Ed. 2009, 48, 2359–
2363.
10
matches that of the iron binding site in wild type mHmdCN
.
The intense pre-edge peak for the new complex is resolved as
a doublet, assigned tentatively to the 1s-d/4p transition(s).
[Note: The high resolution in the K-edge region was achieved
with the Si(220) monochromator with an intrinsic resolution of
0.4 eV vs 0.9 eV for Si(111).] The XANES spectra for both
(30) Zhang, J.; Wang, H.; Xian, M. J. Am. Chem. Soc. 2009, 131, 3854–
3855.
(31) Fleckner, H.; Grevels, F. W.; Hess, D. J. Am. Chem. Soc. 1984, 106,
2027–2032.
(34) Dennett, J. N. L.; Ferguson, M. J.; McDonald, R.; Takats, J. Can.
J. Chem. 2005, 83, 862–868.
(32) King, R. B. J. Am. Chem. Soc. 1962, 84, 2460.
(33) Conder, H. L.; Darensbourg, M. Y. J. Organomet. Chem. 1974, 67,
93–97.
(35) Rocchini, E.; Rigo, P.; Mezzetti, A.; Stephan, T.; Morris, R. H.; Lough,
A. J.; Forde, C. E.; Fong, T. P.; Drouin, S. D. J. Chem. Soc., Dalton
Trans. 2000, 3591–3602.
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A R T I C L E S
Royer et al.
Figure 4. Structure of 1, drawn with 35% probability ellipsoids. Hydrogen
atoms were omitted for clarity.
Table 3. Selected Bond Lengths (Å) for 1 and mHmdCO
bond
1
HmdCO M. jannaschii
Fe1-CO1
Fe1-CO2
Fe1-CO3
Fe1-Cacyl4
Fe1-N
1.7844 (0.0018)
1.8229 (0.0019)
1.8395 (0.0018)
2.0202 (0.0016)
-
1.8260 (0.0081)
1.8260 (0.0081)
1.8260 (0.0081)
1.927 (0.044)
2.0194 (0.076)
2.3162 (0.0056)
Figure 3. (Top) XANES spectrum of Et4N[Fe(SPh)(Ph2PC6H4CO)(CN)(CO)2]
(left) and wild-type Hmd from M. marburgensis, the isolated Fe guanylyl-
derived cofactor, as well as the cyanide-inhibited protein (right).10 (Bottom)
EXAFS spectrum and its Fourier transform with fits (red) for the model
Fe(CO)3(CN)P(S) using the parameters in Table 2.
Fe1-S1
2.3457 (0.0005)
Table 2. Results of EXAFS Refinements for
Et4N[Fe(SPh)(Ph2PC6H4CO)(CN)(CO)2] (Et4N [2])a
r (Å)b
2σ2 (Å2)b
Protonation. Tricarbonyl 1 was found to undergo protonation
ligand
N
at low temperatures to give unstable derivatives. In CH2Cl2
CO
CO
CN
CN
C
2
2
1
1
1
1
1
1.81 ( 0.01
2.93 ( 0.01
2.07 ( 0.03c
3.19 ( 0.03
2.07 ( 0.03c
2.32 ( 0.02
2.18 ( 0.06
0.016 ( 0.003
0.012 ( 0.002a
0.012 ( 0.004b
0.012 ( 0.002a
0.012 ( 0.004b
0.008 ( 0.004
0.02 ( 0.01
solution at -30 °C, protonation with H(OEt2)2BArF (ArF )
4
3,5-(CF3)2C6H3) afforded a single product [1H]+, which lacks
1
apparent hydride signals in its H NMR spectrum. The IR
spectrum (-26 °C) showed νCO bands (2090, 2041, and 2024
cm-1), which are shifted by an average of 18 cm-1 to higher
energies vs those for 1. Addition of Et3N to this cold solution
of [1H]+ cleanly returned 1 (IR analysis). The IR data are thus
consistent with protonation at a ligand, probably the thiolate36
or the acyl group.37 The corresponding S- and O-protonation
of the ferrous thiolate (C5H5)Fe(CO)2SPh and the ferrous acyl
S
P
a Energy range, 10-750 eV; EF ) -6.3 ( 0.3 eV; fit index, 0.954.
b Indices a, b, and c indicate parameters that were refined jointly in
order to lower the number of free parameters.
the CN-inhibited enzyme and Et4N[2] show a mild shoulder in
their rising edge, followed by a sharp resonance at about 7130
eV and a broad resonance at about 7150 eV. The position of
the subsequent minimum at 7170 eV is mainly attributed to the
distance of those ligands dominating the EXAFS pattern (Figure
3, Table 2). The high similarity of its position indicates strong
similarities in the overall geometry of the active sites.
Adducts with TsCH2NC. Reminiscent of its reactivity toward
cyanide, complex 1 was found to undergo substitution by
TsCH2NC under mild conditions. 31P NMR spectroscopic
measurements show that substitution occurs in seconds at about
-10 °C. The formation of a single derivative (δ70.7 vs 72.5
for 1; see Table 1 for νCO and νCN). The TsCH2NC-substituted
complex, 3, is stable in solution at temperatures below -10
°C, but 31P NMR measurements indicate that over the course
of several minutes at -5 °C. The initial species converts to a
mixture of three additional complexes, which are assumed to
be isomers or the result of decarbonylation. At room temper-
ature, this mixture simplifies over the course of a few minutes,
as indicated by the appearance of two 31P NMR signals in a 1:1
ratio, assigned to metastable diiron derivatives of the type
Fe2(SPh)2(Ph2PC6H4CO)2(CO)(RNC)2.
(C5H5)Fe(CO)(PPh3)C(O)Me with HBF4 and HBr, respectively,
36,37
induces shifts in νCO by 40 and 32 cm-1
.
Upon warming
its solution to 20 °C, [1H]+ was found to convert to a new
product, which decomposed over the course of a few minutes.
Structure of Fe(SPh)(Ph2PC6H4CO)(CO)3. The tricarbonyl
monomer 1 was further characterized by X-ray crystallography
(Figure 4). The structure of 1 compares well with the recent
structure of the C176A mutant of Hmd, which has been
characterized at 2.15 Å resolution.7 In this mutant, one cysteinyl
ligand is replaced by one thiolate of dithiothreitol, which also
provides an alcohol ligand in the coordination site trans to the
acyl (see Figure 1). Selected bond lengths for 1 and mHmdCO
and for 2 and mHmdCN are presented in Tables 3 and 4,
respectively. The agreement between the bond lengths is
excellent except for the Fe-acyl bond,25,27 which for unknown
reasons is 5-10% longer in models than in the protein.
Conclusions
Thioester derivatives of 2-diphenylphosphinobenzoic acid are
versatile multifunctional reagents that enable easy access to
phosphine-stabilized metal acyl thiolato carbonyls. The PhS
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Iron Acyl Thiolato Carbonyls To Model Hmd
A R T I C L E S
Table 4. Selected Bond Lengths (Å) for 2 and mHmdCN
derivative was examined in detail and shown to adopt a structure
very similar to the one modeled for the Fe site in HmdCO.8 The
major difference is the presence of the phosphine ligand vs the
pyridyl group of the organic cofactor, but the phosphorus center
offers the advantage of enabling 31P NMR analysis of reaction
mixtures. The pathway for the oxidative addition of the thioester
to Fe(0) is illuminated by the finding that an extremely
bulky phosphine thioester gave an adduct of the type
Fe(Ph2PC6H4COSR)(CO)4. The isolation of this adduct is
consistent with phosphine coordination preceding the chelate-
assisted oxidative addition38 of the thioester group. The oxidative
addition of the thioester can then be envisioned to proceed via
coordination of the thioether-like sulfur center23 (eq 3). In
contrast, oxidative addition of simple thioesters to Fe(0) reagents
proceeds in low yields to give [Fe(I)]2 derivatives of the type
Fe2(SR)2(CO)6 or Fe2(SR)(C(O)R′)(CO)6.19
bond
2
HmdCN M. jannaschii
Fe1-CO1
Fe1-CO2
Fe1-CN3
Fe1-Cacyl4
Fe1-N
Fe1-S1
Fe1-P1
1.81 ( 0.01
1.81 ( 0.01
2.07 ( 0.03
2.07 ( 0.03
-
2.32 ( 0.02
2.18 ( 0.06
1.776 ( 0.006
1.776 ( 0.006
2.06 ( 0.01
1.89 ( 0.01
1.97 ( 0.01
2.342 ( 0.006
-
of both the electronic and geometric structure of the active site.
This close match provides compelling evidence for the presence
of a ferrous center in Hmd. The oxidation state of the Fe center
in Hmd has been of recurring interest,40 but the issue should
now be considered settled in light of this and related modeling
work on acyl-containing models.26,27
The present results highlight the anomalous effect of
cyanide on the IR spectrum of Hmd. In HmdCN, two νCO
bands are shifted to higher energies by 9 and 12 cm-1. In
this conversion, CN- is assumed to displace a labile ligand
such as water. Nonetheless, it is extremely rare that νCO bands
shift to higher energy upon installing a cyanide ligand. For
example, the average of the two νCO bands is 50 cm-1
lower in [Fe(SPh)(Ph2PC6H4CO)(CN)(CO)2]- than in
Fe(SPh)(Ph2PC6H4CO)(CO)3.9 One possible explanation for
this anomaly is that CN- affects the second coordination
sphere of the ferrous center in Hmd, such as the protonation
state of the pyridone ligand.
Our tricarbonyl model was also susceptible to reversible
protonation, but only with strong acids. The νCO bands for the
protonated tricarbonyl occur at 20-30 cm-1 above those seen
for HmdCO. Furthermore, the protonated derivative is unstable
at temperatures above -30 °C.
Whereas the phosphine facilitates oxidative addition of the
thioester, the low affinity of Fe(0) for pyridines39 prevented
incorporation of the more biomimetic N-heterocyclic ligand on
the Fe(CO)3 center. Reactivity studies reinforce the electronic
similarity between our model and the active site. Hmd stereo-
selectively binds 13CO and CN-.9 31P NMR data show that 1
undergoes stereoselective substitution by both CN- and
TsCH2CN, but CO is so labile in our model that stereoselective
binding of 13CO was not observed (eq 4). The lability of the
site trans to acyl combined with the bridging tendency of thiolate
ligands explains the facile conversion of 1 into the related
Fe2(SR)2 derivative.25 Our results suggest that the Fe-GP
cofactor might be expected to degrade via dimerization.
Experimental Section
General Considerations. Unless otherwise indicated, reactions
were conducted using standard Schlenk techniques (N2) at room
temperature with stirring. All solvents were dried and degassed prior
to use. Literature procedures afforded the following reagents:
2-diphenylphosphinobenzoic acid,41 2,6-dimesitylphenylthiol, 2,4,6-
triisopropylthiol,42 2-hydroxy-4-methylpyridine-6-acetic acid,43
[H(Et2O)2]BArF ,44 and Fe(bda)(CO)3.45 Benzenethiol, 2-methyl-
4
2-propanethiol, ethanethiol, TsCH2NC, and Et4NCN were purchased
from Sigma-Aldrich. DCC, 2-mercaptopyridine, and 4-dimethy-
laminopyridine were obtained from Fluka Analytical. EDAC (1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) was
(36) McGuire, D. G.; Khan, M. A.; Ashby, M. T. Inorg. Chem. 2002, 41,
2202–2208.
(37) Green, M. L. H.; Hurley, C. R. J. Organomet. Chem. 1967, 10, 188–
190.
(38) Landvatter, E. F.; Rauchfuss, T. B. Organometallics 1982, 1, 506.
(39) Boxhoorn, G.; Cerfontain, M. B.; Stufkens, D. J.; Oskam, A. J. Chem.
Soc., Dalton Trans. 1980, 1336–1341.
(40) Wang, X.; Li, Z.; Zeng, X.; Luo, Q.; Evans, D. J.; Pickett, C. J.; Liu,
X. Chem. Commun. 2008, 3555–3557. Guo, Y.; Wang, H.; Xiao, Y.;
Vogt, S.; Thauer, R. K.; Shima, S.; Volkers, P. I.; Rauchfuss, T. B.;
Pelmenschikov, V.; Case, D. A.; Alp, E. E.; Sturhahn, W.; Yoda, Y.;
Cramer, S. P. Inorg. Chem. 2008, 47, 3969–3977.
(41) Hoots, J. E.; Rauchfuss, T. B.; Wrobleski, D. A. Inorg. Synth. 1982,
21, 175.
(42) Blower, P. J.; Bishop, P. T.; Dilworth, J. R.; Hsieh, T. C.; Hutchinson,
J.; Nicholson, T.; Zubieta, J. Inorg. Chim. Acta 1985, 101, 63–65.
(43) Royer, A. M.; Rauchfuss, T. B.; Wilson, S. R. Inorg. Chem. 2008,
47, 395–397.
(44) Brookhart, M.; Grant, B.; Volpe, A. F. Organometallics 1992, 11,
3920–3922.
(45) Alcock, N. W.; Richards, C. J.; Thomas, S. E. Organometallics 1991,
10, 231–238.
As indicated by comparisons of the XANES, EXAFS, and
IR spectra for Hmd and HmdCN (M. marburgensis),
[Fe(SPh)(Ph2PC6H4CO)(CN)(CO)2]- replicates the major details
9
J. AM. CHEM. SOC. VOL. 132, NO. 47, 2010 17001

A R T I C L E S
Royer et al.
purchased from Chem-Impex International. MgSO4 and NaHCO3
were purchased from Fisher Chemicals. Fe2(CO)9 was purchased
from Strem Chemicals. Quinoline-8-carboxylic acid was purchased
from Karl Industries. 2-Methoxythiophenol was obtained from
SAFC Supply Solutions (St. Louis, MO). K13CN was purchased
from Isotec. The silica gel used was 230-400 mesh Siliaflash P60
from Silicycle. Electrospray ionization mass spectra (ESI-MS) were
acquired using a Micromass Quattro QHQ quadrupole-hexapole-
quadrupole instrument. 1H and 31P NMR spectra were acquired on
Varian UNITY INOVA TM 500NB and UNITY 500 NB instru-
ments. Elemental analyses were performed by the School of
Chemical Sciences Microanalysis Laboratory utilizing a model CE
440 CHN analyzer. In situ IR spectroscopic measurements were
obtained using a ReactIR 4000 (Mettler-Toledo) instrument.
temperature. The mixture was evaporated to dryness under vacuum,
and the solid was rinsed with ∼15 mL of hexanes. The solid was
recrystallized from 15 mL of Et2O/30 mL of hexanes. Yield: 90
mg (76%). 31P NMR (202 MHz, CD2Cl2): δ72.0 (s). IR (THF,
cm-1): νCO ) 2048, 1969, 1950, 1933. IR spectroscopic measure-
ments indicated that a solution of the compound in refluxing THF
remained unchanged for 24 h.
Fe(SPh)(Ph2PC6H4CO)(CO)3, 1. Under a stream of N2, a solution
of Fe(bda)(CO)3 (445 mg, 1.55 mmol) and Ph2PC6H4C(O)SPh (652
mg, 1.63 mmol) in 20 mL of benzene was heated to reflux for 4 h.
The solution was evaporated under vacuum and washed with a few
milliliters of Et2O. The brown crystalline solid was dried overnight to
give 743 mg of diiron dithiolato complexes. A solution of this mixture
(985 mg, 0.992 mmol) in 6 mL of CH2Cl2 was stirred under 1600 psi
of CO at 60 °C for 24 h to give a near-quantitative conversion to 1.
Pure samples of 1 could be obtained by slow crystallization at -30
°C (see below). For such carbonylations, the solution is first pressurized
at 100-500 psi followed by careful venting. This gas-exchange
procedure is repeated twice more. The bomb is then pressurized to
1400-1800 psi, with cooling of the bomb as needed to achieve the
final pressure. 31P NMR (202 MHz, CD2Cl2): δ 72.5. IR (CH2Cl2,
cm-1): νCO ) 2075, 2025, 2001,1629 (acyl). Single crystals of 1
suitable for X-ray diffraction were obtained by layering hexanes over
a solution of 450 mg of 1 in 5 mL of CH2Cl2 at -30 °C for 96 h.
Orange crystals of 1 were manually separated from a brown unidenti-
fied powder.
The preparation and purification of thioesters was found to be
slightly less cumbersome utilizing the water-soluble reagent EDAC
instead of DCC.
Ph2PC6H4-2-C(O)SPh, La. To a stirred solution of PhSH (870
µL, 8.475 mmol) in CH2Cl2 (20 mL) were added 2-diphenylphos-
phinobenzoic acid (2.36 g, 7.705 mmol) and DCC (1.75 g, 8.475
mmol). The reaction mixture was stirred 1 h, and the precipitated
1,3-dicyclohexylurea was filtered off. The yellow filtrate was
concentrated under vacuum, and the residue was purified by column
chromatography on silica gel, eluting with 50:1 hexane:ethyl acetate.
The light yellow fraction was evaporated to give a crystalline solid
that was dried under vacuum. Yield: 2.5 g (82%). 31P NMR (202
MHz, CD2Cl2): δ -5.53. FDMS m/z: 398.2 (Calcd for M+: 398.1).
IR (CH2Cl2, cm-1): νCO ) 1677 (acyl). Anal. Calcd for C25H19OPS
(found): C, 75.36 (75.08); H, 4.81 (4.82); N, 0.00 (0.51).
Ph2PC6H4-2-C(O)SC6H3-2,6-(C6H2-2,4,6-Me3)2. To a stirred
solution of 2,6-dimesitylphenylthiol (1000 mg, 2.89 mmol) in
CH2Cl2 (20 mL) were added 2-diphenylphosphinobenzoic acid (884
mg, 2.89 mmol), 4-dimethylaminopyridine (35 mg, 0.29 mmol),
and EDAC ·HCl (830 mg, 4.33 mmol) successively. The reaction
solution was stirred for 3.5 h, and then washed three times with 1
N HCl (30 mL), followed by two times with saturated aqueous
NaHCO3 (30 mL), and once with water (30 mL). After drying over
MgSO4, the solution was evaporated. The residue was extracted
into hexanes (8 mL) and precipitated a white solid within 15 min.
The solid was collected by filtration, washed with 5 mL of hexanes,
and dried under vacuum. Yield: 1.32 g (74%). 1H NMR (500 MHz,
CD2Cl2): δ 1.96 (s, 12H, mesityl-2,6-CH3), 2.27 (s, 6H, mesityl-
4-CH3), 6.86 (s, 5H, aryl-H), 6.98-7.35 (15 H, aryl-H), 7.54 (t,
1H, aryl-H). 31P NMR (202 MHz, CD2Cl2): δ -8.39. ESI-MS m/z:
635.6 (Calcd for MH+: 635.3). IR (CH2Cl2, cm-1): νCO ) 1674
(acyl). Anal. Calcd for C43H39OPS (found): C, 81.36 (80.89); H,
6.19 (6.32); N, 0.00 (0.46).
Fe(SPh)(Ph2PC6H4CO)(13CO)3, 113CO. A solution of a mixture
of 1 and 2 (8.5 mg, 0.009 mmol, ∼9:1 in favor of 1) in 1 mL of
CH2Cl2 in a J-Young NMR tube was frozen, and the tube was
evacuated under vacuum. An atmosphere of 0.8 atm of 13CO was
introduced, and the tube was sealed. The solution was thawed and
analyzed by 31P NMR spectroscopy within 5 min. IR data were
obtained within 25 min. 31P NMR (202 MHz, CD2Cl2): δ72.0 (d
of d of d, 2JCPtrans ) 53, 2JCPcis ) 21, 2JCPcis ) 16 Hz). IR (CH2Cl2,
cm-1): νCO ) 2027, 1980, 1957, 1629 (acyl).
Et4N[Fe(SPh)(Ph2PC6H4CO)(CN)(CO)2], Et4N[2]. A solution
of Et4NCN (51.6 mg, 0.330 mmol) in 5 mL of CH2Cl2 was added
to a solution of 1 (164 mg, 0.3304 mmol) in 20 mL of CH2Cl2.
The solution was stirred 10 min and then concentrated to 2 mL.
An oil precipitated upon the addition of 10 mL of Et2O. The oil
was dissolved in THF and reprecipitated by the addition of ether.
The resulting oily solid was recrystallized from THF/Et2O twice
more to give an orange tacky solid that converted to an orange
powder upon vacuum drying. Yield: 115 mg (52%). 31P NMR (202
MHz, CD2Cl2): δ66.73 (s). ESI-MS (negative mode, m/z): 536.1
(Calcd for C28H19FeNO3PS: 536.0). IR (CH2Cl2, cm-1): νCN/CO
)
2094 (CN), 2013, 1954, 1597 (acyl). Anal. Calcd for
C36H39FeN2O3PS. Found: C, 64.87 (64.14); H, 5.90 (5.99); N 4.20
(4.33).
2-HO-4-Me-6-[CH2C(O)SPh]-C5H2N. To a stirred solution of
PhSH (61.4 µL, 0.5982 mmol) in THF (50 mL) were added
2-hydroxy-4-methylpyridine-6-acetic acid (100 mg, 0.5982 mmol)
and DCC (123.4 mg, 0.5982 mmol) successively. The reaction
mixture was stirred 4 days. Solvent was removed under vacuum,
and CH2Cl2 (20 mL) was added. The precipitated 1,3-dicyclohexy-
lurea was filtered off. The filtrate was concentrated under vacuum,
and the yellow residue was washed with hexanes. The solid was
extracted into ∼15 mL of EtOAc, and this extract was filtered
through a ∼5-cm plug of silica gel. Solvent was removed by
vacuum, and the residue was recrystallized from CH2Cl2 by the
addition of hexanes, giving a white powder. Yield: 62 mg (40%).
1H NMR (500 MHz, CD2Cl2): δ 2.23 (s, 3H, 4-CH3), 3.89 (s, 2H,
CH2C(O)S) 6.15 (s, 1H, pyridyl-H), 6.34 (s, 1H, pyridyl-H), 7.43
(m, 5H, SC6H5). ESI-MS m/z: 260.3 (Calcd for MH+: 260.1). IR
(CH2Cl2, cm-1): νCO ) 1657 (acyl). Anal. Calcd for C14H13NO2S
(found): C, 64.84 (64.96); H, 5.05 (5.61); N, 5.40 (6.14).
Et4N[Fe(SPh)(Ph2PC6H4CO)(13CN)(CO)2].
A solution of
Et4N13CN (17 mg, 0.108 mmol) was generated by K13CN and
Et4NCl in MeOH followed by filtration to remove KCl. Solvent
was removed by vacuum, and the residue was dissolved in 3 mL
of CH2Cl2 and added to a solution of 1 (54.0 mg, 0.108 mmol) in
5 mL of CH2Cl2. The solution was stirred 10 min and evaporated
under vacuum. Upon slurrying in Et2O, the product converted to
an oily orange powder that was dried under vacuum. 31P NMR (202
MHz, CD2Cl2): δ66.7, doublet, 2JCP ) 24 Hz. IR (CH2Cl2, cm-1):
νCO ) 2050, 2012, 1954, 1597 (acyl).
Fe(SPh)(Ph2PC6H4CO)(CO)2(NCCH2Ts), 3. A solution of 1
(99.6 mg, 0.185 mmol) in 3 mL of CH2Cl2 was cooled to -30 °C.
A background IR spectrum was recorded in situ. A solution of
TsCH2NC (36.5 mg, 0.185 mmol) in 1 mL of CH2Cl2 was added,
and IR spectra were collected every minute as the solution was
allowed to warm. The CO region of the IR spectra changed cleanly
between -10 and -6 °C over the course of ∼30 min. 31P NMR
(202 MHz, CD2Cl2): δ70.7 (s). IR (CH2Cl2, cm-1): νCN/CO ) 2153
(CN), 2038, 1983, 1615 (acyl). Upon warming the sample above
-6 °C, three new 31P NMR signals at δ73.2, 72.6, and 72.2 were
Fe(Ph2PC6H4C(O)SC6H3-2,6-Ar*2(CO)4 (Ar* ) 2,4,6-trimeth-
ylphenyl). A solution of Ph2PC6H4C(O)SC6H3-2,6-Ar*2 (94.2 mg,
0.148 mmol) in 20 mL of CH2Cl2 was transferred to a mixture 54
mg (0.148 mmol) of Fe2(CO)9 in 10 mL of CH2Cl2 at 0 °C. The
mixture was stirred for 15 min and then allowed to warm to room
9
17002 J. AM. CHEM. SOC. VOL. 132, NO. 47, 2010

Iron Acyl Thiolato Carbonyls To Model Hmd
A R T I C L E S
initially observed before many signals appeared at further times.
Upon prolonged standing at room temperature, loss of CO was
observed.
XAS Data Analysis. The extended X-ray absorption fine
structure (EXAFS) oscillations were analyzed with EXCURV9.2,48
and the σ3-weighted spectra were used. Different integer coordina-
tion numbers were considered for the ligands CO, CN, C, O, N, P,
and S. The fit index was used as a measure of the goodness of the
fits. Ab initio theoretical phase and amplitude functions were
generated within EXCURV. The experimental spectra are compared
with the theoretical simulations based on small atom theory. No
Fourier filtering was applied. The best model is described in Table
3, and the fit is shown in Figure 3.
Protonation of 1. A solution of 1 (30 mg, 0.056 mmol) in 3
mL of CH2Cl2 solution cooled to -72 °C was examined by FT-IR
spectroscopy to confirm its integrity. A solution of [H(Et2O)2]BArF
4
(56.4 mg, 0.056 mmol) in 1 mL of CH2Cl2 was added with stirring.
Within 5 min the IR spectrum confirmed complete conversion to a
new product (IR: 2090, 2041, 2024 cm-1). Addition of Et3N (10
µL, 0.072 mmol, -72 °C) gave back 1. The protonated product
isomerizes slowly at -30 °C. Protonation was also conducted in a
J.Young NMR tube by adding 15.3 mg (0.028 mmol) of 1 and
Acknowledgment. Research at Illinois was supported by the
U.S. Department of Energy - Office of Basic Energy Sciences, grant
DE FG01-90er14146 and in part the Petroleum Research Fund. We
thank Dr. S. Shima (MPI-Marburg) for helpful advice. The authors
are grateful to SSRL for providing beamtime and thank Erik Nelson
and colleagues for their excellent support during the beamtime.
Portions of this research were carried out at the Stanford Synchro-
tron Radiation Lightsource, a national user facility operated by
Stanford University on behalf of the U.S. Department of Energy,
Office of Basic Energy Sciences. The SSRL Structural Molecular
Biology Program is supported by the Department of Energy, Office
of Biological and Environmental Research, and by the National
Institutes of Health, National Center for Research Resources,
Biomedical Technology Program.
30.3 mg (0.028 mmol) of [H(Et2O)2]BArF , followed by vacuum
4
transfer of 0.8 mL of CD2Cl2. The sample was warmed to -78 °C
and inserted into the NMR probe that had been precooled to -50
°C. The sample was slowly warmed to -30 °C, at which
temperature the 31P NMR signal for starting material disappeared
and a new signal appeared at δ68.1. The spectrum remained
unchanged over the course of 30 min. Upon allowing the sample
to warm to 0 °C, no change was noted, but at 20 °C, we observed
rapid growth of a new singlet in the 31P NMR spectrum (δ76.7).
After 5 min, the signal at δ68.1 had completely disappeared. At
longer times at room temperature, many new 31P NMR signals were
observed. The resulting solution had an odor of thiol.
XAS Data Collection. In a glovebox, the powder sample (25
µL in volume) was transferred into plastic sample holders covered
with polyimide windows, frozen in liquid nitrogen, and kept at 4
K during the experiment. X-ray absorption spectra at the Fe K-edge
were recorded in transmission mode at Wiggler station 7-3 (SSRL,
Menlo Park, CA) equipped with a Si(220) double-crystal mono-
chromator, a focusing mirror, and a 30-element Ge solid-state
fluorescence detector (Canberra). Energy axis of each scan was
calibrated by a reference sample (Fe foil). Scan averaging was done
with Athena,46 and normalization and data reduction were per-
formed with the EXPROG47 using E0(Fe) ) 7120 eV.
Supporting Information Available: Crystallographic analysis
of 1; additional spectroscopic and preparative details (including
protonation data); reanalysis of EXAFS data for mHMDCN and
mHMDCO. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA1072228
(46) Ravel, B.; Newville, M. J. Synchrotron Radiat. 2005, 12, 537–541.
(47) Nolting, H.-F.; Hermes, C., EXPROG, EMBL EXAFS data analysis
and evaluation program package; 1992.
(48) Binsted, N.; Strange, R. W.; Hasnain, S. S. Biochemistry 1992, 31,
12117–12125.
9
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