Metal-assisted oxo atom addition to an fe(III) thiolate

Article abstract of DOI:10.1021/jacs.6b03512

Cysteinate oxygenation is intimately tied to the fonction of both cysteine dioxygenases (CDOs) and nitrile hydratases (NHases), and yet the mechanisms by which sulfurs are oxidized by these enzymes are unknown, in part because intermediates have yet to be

Full text of DOI:10.1021/jacs.6b03512

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Article
Metal-Assisted Oxygen Atom Addition to an Fe(III)-Thiolate.
Gloria Villar-Acevedo, Priscilla Lugo-Mas, Maike Blakely, Abbie S.
Ganas, Erin M. Hanada, Werner Kaminsky, and Julie A. Kovacs
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b03512 • Publication Date (Web): 06 Dec 2016
Downloaded from http://pubs.acs.org on December 6, 2016
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Metal-Assisted Oxygen Atom Addition to an Fe(III)-Thiolate.
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Gloria Villar-Acevedo, Priscilla Lugo-Mas, Maike N. Blakely, Abbie S. Ganas,
Erin M. Hanada, §Werner Kaminsky and *Julie A. Kovacs
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The Department of Chemistry, University of Washington, Box 351700
Seattle, WA 98195-1700
Received date: (to be automatically inserted once manuscript is accepted)
Title running head: Fe(III)-(RS-O) via Addition of PhIO to Fe(III)-SR
Corresponding
author:
J.
Kovacs,
tel.
(206)543-0713,
FAX
(206)685-8665,
kovacs@chem.washington.edu
§ UW staff crystallographer
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Abstract.
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Cysteinate oxygenation is intimately tied to the function of both cysteinate
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dioxygenases (CDO) and nitrile hydratases (NHases), and yet the mechanisms by
which sulfurs are oxidized by these enzymes are unknown, in part because
intermediates have yet to be observed. Herein, we report a five-coordinate bis-thiolate
ligated Fe(III) complex, [FeIII(S2Me2N3(Pr,Pr))]+ (2), that reacts with oxo atom donors
(PhIO and H2O2) to afford a rare example of a singly oxygenated sulfenate, [FeIII(η2-
SMe2O)(SMe2)N3(Pr,Pr)]+ (5), resembling both a proposed intermediate in the CDO
catalytic cycle, as well as the essential NHase Fe–S(O)Cys114 proposed to be intimately
involved in its mechanism. Comparison of the reactivity of 2 with that of a more
electron-rich, crystallographically characterized derivative, [FeIIIS2Me2NMeN2amide(Pr,Pr)]1–
(8), shows that oxo atom donor reactivity correlates with the metal ion’s ability to bind
exogenous ligands. Direct attack at the thiolate sulfurs of 2 is ruled out by DFT
calculations, which show that the average spin-density on the thiolate sulfurs is
approximately the same for 2 and 8, and Mulliken charges on the sulfurs of 8 are
roughly twice that of cationic 2, implying that 8 should be more susceptible to sulfur
oxidation. In contrast to imine-ligated 2, carboxamide-ligated 8 is shown to be
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–
unreactive towards oxo atom donors. Azide (N3 ) is shown to inhibit sulfur oxidation
with 2, and a green intermediate is observed to form, which then slowly converts, with
clear isobestic points, to sulfenate-ligated 5. This suggests that the mechanism of sulfur
oxidation involves initial coordination of the oxo atom donor to the metal ion. Whether
the green intermediate is an oxo atom donor adduct, Fe-O=I-Ph, or an Fe(V)=O remains
to be determined.
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Introduction.
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Cysteinate dioxygenases (CDO) are non-heme Fe-enzymes that catalyze the O2-
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promoted oxygenation of cysteinate (CysS–) to cysteine sulfinic acid (CysSO2 ).1-8
A
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singly oxygenated cysteine sulfenate (CysSO–) is proposed to be involved as an
intermediate. High levels of cysteine can cause neurological disorders such as
Parkinson’s, Alzheimer’s, and rheumatoid arthritis,5,9 and thus having a mechanism for
its degradation is important to human health. The CDO enzyme has also been shown
to suppress tumor growth by combatting its defense against reactive oxygen species.10
An epigenetic event can turn off CDO tumor suppression,11 however, via methylation of
the gene responsible for the biosynthesis of CDO. Methylated CDO genes are found in
~60% of breast cancers, and correlates with the progression of the disease and
outcome.12 The CDO mechanism (Figure 1) is proposed to involve cysteinate binding to
the metal ion, followed by O2 binding, cis to the
•–
cysS–, to afford an FeIII-O2 intermediate (A). The
superoxo radical is then proposed to couple with the
FeII-cysS•↔ FeIII-cysS– sulfur to afford a four
membered ring structure RS-FeII-O-O (B). This
Figure 1.
step is unprecedented, in part because there are
very few reported FeIII-O2•–,8,13,14 but more importantly, because none have RS– ligands
in the coordination sphere. Upon heterolytic cleavage of the O-O bond, a high-valent
iron oxo intermediate with a singly oxygenated sulfenate (RSO)FeIV=O (C) is proposed
to form, which undergoes cis-migration to afford the final doubly oxygenated cysteine
sulfinate.1,5 Although theoretical calculations have indicated that the direct involvement
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of the metal ion of CDO provides a lower energy pathway to sulfur oxygenation,1,5 this
has not been experimentally proven. Intermediates A-C (Figure 1) have yet to be
observed. The lack of spectroscopic data for any of the proposed CDO intermediates
has made it impossible to calibrate theoretical calculations.5
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Dioxygen has been shown to react with small molecule iron thiolate (RS–)
complexes to afford doubly or triply oxygenated thiolates.15-20 For example, cysteinate-
–
ligated TpFeIIScys reacts with O2 to afford cysSO2 (the product of CDO5), however, no
intermediates were observed.21
Iron complexes containing a non-tethered,
monodentate RS-ligand trans to the O2 binding site have been shown to react with O2 to
afford a FeIV=O + RSSR,3,22 in contrast to cis thiolate-ligated complexes, which have
been shown to react with O2 to afford doubly (RSO2-Fe), or triply (RSO3-Fe) oxygenated
derivatives.1-4,22,23 Singly oxygenated RSO-Fe sulfenate intermediates are not observed
in these cases. Whether these reactions are metal-mediated, or involve direct attack of
O2 at sulfur, has not been determined, although the orientation-dependence would
suggest that they are. The mechanism of O2-induced oxygenation of Ni-thiolate
complexes, on the other hand, has been shown to involve direct attack by O2 at the
thiolate sulfurs.24
Oxygenated cysteinates have been shown to play a
Cys109
S
O
Ser113
O
114Cys
O
growing number of diverse roles in cellular processes,
including T-cell activation, redox signaling in mammalian
cells,25 signal transduction, oxygen metabolism, oxidative
III
Fe
N
N
114
S
Cys
S
Cys112
O
O
O
H2O
Scheme 1.
stress response, and transcriptional regulation.25,26 They are also required for enzyme
activity in some cases (e.g, NHase27-31, NADH peroxidase,32 and peroxiredoxins33).
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Nitrile hydratases (NHases) are a class of thiolate-ligated non-heme iron enzymes for
which cysteinate oxygenation is intimately tied to function. These enzymes, which
catalyze the enantioselective hydrolysis of nitriles to amides,34-38 contain an Fe(III) (or
Co(III)) active site ligated by three cysteinates, two of which are post-translationally
modified (Scheme 1), one to a sulfenic acid (114Cys–S–OH),31,39,40 and the other to a
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–
sulfinate (112Cys–SO2 ).41 Sulfur K-edge XAS studies have shown that the NHase
–
sulfenic acid residue is protonated,39 whereas the sulfinate (RSO2 ) remains
unprotonated.39 The enzyme becomes inactive when a second oxygen atom is added
to the sulfenic acid, 114Cys–S–OH, implying that it plays a specific role in catalysis.42
This is supported by time-resolved crystallography,43 and crystallographic
characterization of 114Cys–S-bound inhibitors.31 Coupled with theoretical calculations,27
this experimental data provides evidence that the sulfenate oxygen is intimately
involved in the NHase mechanism. The mechanism by which post-translationally
modified NHase sulfenate and sulfinate oxygens are inserted has been proposed to
involve O2–induced formation of a thiolate-ligated high-valent oxo intermediate.22,29
There is no experimental data available to verify this possibility, however.
Singly oxygenated sulfenates are difficult to trap,26,44-47 even when they are coordi-
nated to a metal ion (i.e., M-S(R)O–).24,48,49
There are significantly more examples of
–
doubly oxygenated, metal-sulfinate (RSO2
) complexes.15-20
Examples of singly
Scheme 2
oxygenated RS=O or RS-OH compounds
include, [CoIII(η2-SO)(SO2)N3(Pr,Pr)]+ (Scheme 2),49 [FeIII(ADIT)(ADIT-O)]+ (1),48 and
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[FeIII(ADIT)(ADIT-OH)]2+.48
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In order to understand how thiolates promote O2-activation,50-52 tune reactivity and
the electronic, magnetic, and reactivity properties of peroxo, oxo, and hydroxo
intermediates, we have been exploring the reactivity of coordinatively unsaturated
thiolate-ligated Fe (and Mn) complexes with dioxygen and its reduced derivatives.51,53-59
Although an open coordination site is required for O2 binding, thiolates are known to
intercept open coordination sites by
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forming intermolecular M-SR-M bridges
resulting in oligomerization. Despite this,
we,17,51,54,59-67 and others,2,3,16,18,68-70 have
synthesized a variety of coordinatively
Scheme 3.
unsaturated mononuclear thiolate-ligated iron complexes that are capable of binding
small molecules. For example, thiolate ligated [FeII(N4SMe2(tren))]+,54,71 and bis-thiolate
ligated [FeIII(S2Me2N3(Pr,Pr))] (2)63 (Scheme 3), are five-coordinate, and contain flexible
ligands capable of accommodating a
sixth ligand,62,63,72 as well as a variety of
metal ions, in multiple oxidation
states.59,60,62,63,72-74
Both ligands
constrain the geometry so that added
Scheme 4.
“substrates” are forced to bind cis to a thiolate (e.g., structures 3 and 4, Scheme
–
4).64,65,75,76 For example, superoxide (O2 ) reacts with reduced [FeII(N4SMe2(tren))]+ at
low temperatures in the presence of a proton donor59,75,77 to afford a metastable, low-
spin (S= ½; g⊥ = 2.14; g|| = 1.97) hydroperoxo intermediate [FeIII(N4SMe2(tren))(OOH)]+
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(3; Scheme 4; vO-O= 784 cm-1).54 Bis-thiolate ligated 2 (Scheme 3) was shown to bind
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L= N3 (4) and NO cis to one of the thiolates, and trans to the other (Scheme 4).63,78
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Despite the π-donor properties of RS– ligands, the highly covalent Fe-S bonds of
oxidized [FeIII(N4SMe2(tren))L]+ (L = MeCN, HOO– (3)),54,64,65 [FeIII(S2Me2N3(Pr,Pr))]+ (2)
and [FeIII(S2Me2N3(Pr,Pr))(N3)] (4)63 were all shown to favor a low spin-state (S= ½), due
to the nephalauxetic effect.79 The trans thiolate of 4 was shown to labilize the azide,63
thereby promoting reversible L-binding.80 Herein we examine the reactivity of bis-
thiolate ligated [FeIII(S2Me2N3(Pr,Pr))]+ (2, Scheme 3) with oxo atom donors, in order to
determine how the presence of both a cis and trans thiolate influences electronic
structure and reactivity.
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Experimental Section.
General Methods. All reactions were performed under an atmosphere of dinitrogen in
a glove box, using standard Schlenk techniques, or using a custom–made solution cell
equipped with a threaded glass connector sized to fit a dip probe. Reagents purchased
from commercial vendors were of the highest purity available and used without further
purification. Toluene, tetrahydrofuran (THF), diethyl ether (Et2O), and acetonitrile
(MeCN) were rigorously degassed and purified using solvent purification columns
housed in a custom stainless steel cabinet, dispensed via a stainless steel Schlenk-line
(GlassContour). Methanol (MeOH) and ethanol (EtOH) were distilled from magnesium
methoxide or ethoxide and degassed prior to use. Methylene chloride (DCM) was
distilled from CaH2 and degassed prior to use. 1H NMR spectra were recorded on
Bruker AV 300 or Bruker AV 301 FT-NMR spectrometers and are referenced to an
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external standard of TMS (paramagnetic compounds) or to residual protio-solvent
(diamagnetic compounds). Chemical shifts are reported in ppm and coupling constants
(J) are in Hz. EPR spectra were recorded on a Bruker EPX CW-EPR spectrometer
operating at X-band frequency at 7 K. IR spectra were recorded on a Perkin-Elmer
1700 FT-IR spectrometer as KBr pellets. Cyclic voltammograms were recorded in
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MeCN (100 mM Bun N(PF6) solutions) on a PAR 273 potentiostat utilizing a glassy
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carbon working electrode, platinum auxiliary electrode, and an SCE reference electrode.
Magnetic moments (solution state) were obtained using the Evans’ method as modified
for super-conducting solenoids.81,82 Temperatures were obtained using Van Geet’s
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method.
Solid state magnetic measurements were obtained with polycrystalline
samples in gel-caps using a Quantum Design MPMS S5 SQUID magnetometer.
Ambient temperature electronic absorption spectra were recorded on a Hewlett-Packard
Model 8450 spectrometer, interfaced to an IBM PC. Low temperature electronic
absorption spectra were recorded using a Varian Cary 50 spectrophotometer equipped
with a fiber optic cable connected to a “dip” ATR probe (C-technologies), with a custom–
built two-neck solution sample holder equipped with a threaded glass connector (sized
to fit the dip probe). Elemental Analyses were performed by Galbraith Labs, Knoxville,
TN and Atlantic Microlabs, Norcross, GA. Thiolate–ligated [FeIII(S2Me2N3(Pr,Pr))](PF6)
(2) was synthesized as previously described.63
Synthesis of [FeIII(η2-SMe2O)(SMe2N3(Pr,Pr)](PF6) (5) via the Addition of PhIO to
2. To a stirred solution of [FeIII(S2Me2N3(Pr,Pr))](PF6) (2) (275 mg, 0.52 mmol) in MeOH
(20 mL) at – 35 °C was added dropwise a 2 mL MeOH solution containing 1.2 equiv of
PhIO (137 mg, 0.62 mmol). The solution was allowed to stir for 1 h at –35 ˚C, and then
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stored in the freezer overnight. After filtration, the volume was reduced to 3 mL, layered
with 25 mL of Et2O, and cooled to – 35 °C overnight to afford 5 (153 mg, 0.28 mmol,
54%) as a pink crystalline solid. Electronic absorption (CH3CN): λmax (ꢀ) = 333(4410),
510(1540) nm; (MeOH): λmax (ꢀ) = 325(4870), 510(1700), 760(248) nm (Figure S-1). IR
(KBr pellet) ν(cm-1): 1625 (C=N); 1024 (S-O). Reduction Potential (MeCN): Ep,c = –
0.960 V (irrev.) vs SCE. Solution magnetic moment (310.2 K; MeOH) µeff = 1.99 BM.
EPR (DCM/toluene glass (1:1), 7 K): g1 = 2.17, g2 = 2.11, g3 = 1.98. ESI-MS calcd for
[FeC16N3S2H31O]+: 401.3; found: 401.2. Anal. calcd for FeC16H31N3OS2PF6: C, 35.2; H,
5.7; N, 7.7. Found: C, 35.39; H, 5.57; N, 7.77.
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Formation of [FeIII(η2-SMe2O)(SMe2N3(Pr,Pr)](PF6) (5) via the Addition of
Hydrogen Peroxide to 2. Oxidized 2 (5 mg, 0.01 mmol) was dissolved in 20 mL of
MeOH and placed in a sealed UV-vis dip-probe cell under a N2 atmosphere. To this, 1.0
equiv of H2O2 (1 µL of a 30 % aqueous solution, 0.01 mmol) was added at room
temperature via syringe to afford a pink air stable compound (λmax= 510(1540) nm; EIS-
MS (M+1): 401). Complex 5 is stable both at room temperature and in air.
Synthesis of (Et4N)[FeIIIS2Me2NMeN2amide(Pr,Pr)] (8). To a stirred solution of
(HSMe)2(NMe)(HNamide)2(Pr,Pr)•HCl (C, see supplemental for synthesis) (100 mg, 0.26
mmol) in MeOH (5 mL) at -35 °C was added dropwise a pre-cooled (-35 °C) solution of
(Et4N)[FeCl4] (85 mg, 0.26 mmol) in MeOH (3 mL). A pre-cooled (-35 °C) solution of
NaOMe (70 mg, 1.3 mmol) in MeOH (5 mL) was then added, and the resulting reaction
mixture was allowed to stir overnight at ambient temperature. The intense olive green
solution was filtered and concentrated to dryness. The solid was re-dissolved in MeCN
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and filtered. The filtrate was concentrated to a minimum amount of MeCN (~2 mL),
layered with 10 mL of Et2O, and cooled to -35 °C overnight to afford 8 (110 mg, 0.21
mmol, 80%) as a dark green crystalline solid. Electronic absorption (CH3CN): λmax (ꢀ) =
352(8210), 428(3590), 581(1280) nm; (MeOH): λmax (ꢀ) = 348(8150), 588(1210) nm;
(H2O): λmax (ꢀ) = 352(8060), 584(1530) nm. IR (KBr pellet) ν(cm-1): 1564 (C=O). E1/2
(MeCN) = -1.51 V vs SCE. Solution magnetic moment (298 K; MeOH) µeff = 3.75 BM.
EPR (MeOH/EtOH glass (9:1), 9 K): g1 = 4.72, g2 = 2.82, g3 = 1.92. ESI-MS calcd for
[FeC13H21N2O2S3]1-: 389.4, found: 389.3. Anal. calcd for FeC23H47N4O2S2: C, 52.0; H,
8.9; N, 10.5. Found: C, 52.3; H, 8.87; N, 10.49.
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Formation of a Green Intermediate, via the Addition of IBX-ester to 2. A 0.238
mM solution of 2 was prepared in 5 mL of MeOH under an inert atmosphere in a drybox.
The resulting solution was transferred via gas-tight syringe to a custom-made two-neck
vial equipped with a septum cap and threaded dip-probe feed-through adaptor that had
previously been purged with argon and contained a stir bar. This solution was cooled in
an acetone/dry ice bath to -73 ˚C. To this, 10 eq. of IBX-ester (isopropyl 2-
iodoxybenzoate) (50 µL of 238 mM solution of IBX-ester in MeOH) was added, resulting
in the formation of a meta-stable green intermediate (λmax= 675 nm).
Formation of a Green Intermediate, via the Addition of PhIO to 2. A 0.238 mM
solution of 2 was prepared in 5 mL of MeOH or THF under an inert atmosphere in a
drybox. The resulting solution was transferred via gas-tight syringe to a custom-made
two-neck vial equipped with a septum cap and threaded dip-probe feed-through adaptor
that had previously been purged with argon and contained a stir bar. This solution was
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cooled in an acetone/dry ice bath to -73 ˚C. To this, 1-4 eq. of PhIO (50-200 µL of 23.8
mM solution in MeOH) was added, resulting in the formation of a meta-stable
intermediate (λmax= 675 nm).
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DFT Calculations. All geometry optimizations were performed utilizing the ORCA
v.3.0 quantum chemistry package84 and originated from X-ray crystallographic
coordinates. A BP86 functional,85,86 with the resolution of identity approximation (RI),87
dispersion correction (D3BJ),88 and zeroth-order regular approximation for relativistic
effects (ZORA),89 was employed, using a dense integration grid (Grid4), def2-TZVP
basis set,90 and def2-TZVP/J auxiliary basis set. In addition, the conductor-like
screening model (COSMO) using acetonitrile (ε =37.5) as the solvent,91 was employed.
All optimized geometries were visualized using Avogadro.
X-ray Crystallographic Structure Determination. A red crystal plate 0.24 x
0.24 x 0.05 mm of 5 was mounted on a glass capillary with oil. Data was collected at -
143 °C. The crystal-to-detector distance was set to 30 mm and exposure time was 30
seconds per degree for all data sets with a scan width of 1.4o. The data collection was
89.1% complete to 28.34o and 96.1% complete to 25o in ꢀ. A total of 63733 partial and
complete reflections were collected covering the indices, h = -16 to 16, k = -18 to 20, l =
-19 to 19. 5215 reflections were symmetry independent and the Rint = 0.0698 indicated
that the data was of average quality (0.07). Indexing and unit cell refinements indicated
a monoclinic P lattice in the space group P 21/c (No.14).
A black crystal prism size 0.48 x 0.44 x 0.36 mm of 8 was mounted on a glass
capillary with oil. Data was collected at -143 °C. The crystal-to-detector distance was
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set to 40 mm and exposure time was 20 seconds per degree for all sets with a scan
width of 1.5o. The data collection was 94.5% complete to 28.63o and 99.8% complete
to 25o in ꢀ. A total of 41494 partial and complete reflections were collected covering the
indices, h = -23 to 23, k = -11 to 11, l = -23 to 23. 6353 reflections were symmetry
independent and the Rint = 0.063 indicated that the data was of average quality (0.07).
Indexing and unit cell refinement indicated an orthorhombic P lattice in the space group
P n a 21 (No.33).
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The data for both 5 and 8 was integrated and scaled using Denzo-hkl-
SCALEPACK, and an absorption correction was performed using SORTAV. Scattering
factors were obtained from Waasmair and Kirfel.92 Solution by direct methods (SIR97)
produced a complete heavy atom phasing model consistent with the proposed
structures. All non-hydrogen atoms were refined anisotropically by full-matrix least-
squares methods, while all hydrogen atoms were then located using a riding model.
Crystal data for 5 and 8 is presented in Table 1 (below). Selected bond distances and
angles are assembled in Table 2.
Table 1. Crystal data for [FeIII(η2-SMe2O)(SMe2N3(Pr,Pr)](PF6)
(5), and (Et4N)[FeIIIS2Me2NMeN2amide(Pr,Pr)] (8).
5
8
formula
C16H31F6FeN3OPS2 C23H47FeN4O2S2
MW (g/mol) 546.38
531.62
130(2)
Orthorhombic
P n a 21
17.9580(3)
8.5610(6)
18.1560(7)
90
90
90
2791.3(2)
4
1.265
temp (K)
unit cella
space group
a (Å)
b (Å)
c (Å)
130(2)
Monoclinic
P 21/c
12.3550(5)
15.3070(8)
14.8020(7)
90
123.147(3)
90
2343.79(19)
4
α (°)
β (°)
γ (°)
V (Å3)
Z
σcalc (mg/m3) 1.548
Rb
Rw
0.0721
0.2040
1.001
0.0477
0.1219
0.940
GOF
a In all cases: Mo Kα(λ = 0.71070 Å) radiation.b R = Σ Fo - Fc /Σ Fo
; Rw = [Σw( Fo - Fc )2/ΣwFo2]1/2, where w-1 = [σ2count + (0.05F2)2]/4F2.
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Results and Discussion.
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Addition of 1.2 equiv of PhIO to five-coordinate [FeIII(S2Me2N3(Pr,Pr))]+ (2)63 at
ambient temperatures induces a color change from orange to magenta, and causes
the S!Fe CT band in the
electronic
absorption
spectrum, at 415(4200)
nm, to disappear, and a
new band to grow in at
510(1540) nm (Figure 2).
A red-shifted absorption
band would be consistent
with an increase in metal
Figure 2. Using electronic absorption spectroscopy to monitor the
reaction between [FeIII(S2Me2N3(Pr,Pr))]+ (2) and 1.2 equiv of PhIO,
added in 0.15 equiv aliquots, in MeOH at 298 K.
ion Lewis acidity, as a consequence of oxo atom addition. The ESI mass spec of this
magenta species, 5, shows a peak at m/z = 401 corresponding to the parent ion M + 16
(Figure S-2), consistent with the addition of a single oxygen atom. Addition of one equiv
of H2O2 (aq) to 2 in MeOH (Figure S-3) affords an identical magenta-colored species (5,
λmax= 510(1540) nm), with an identical ESI-MS indicative of the addition of a single
oxygen atom. Single oxo atom addition to 2 would be consistent with the formation of
either a high-valent iron oxo, or an iron-sulfenate (FeS(R)–O–) species. The low
temperature (7 K), perpendicular-mode EPR spectrum of 5 (Figure 3) reproducibly
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displays an intense rhombic signal with g–values of 2.16, 2.10 and 1.97 indicative of an
S= ½ ground-state. The precursor to 5, [FeIII(S2Me2N3(Pr,Pr))]+ (2), is also low-spin
S= ½, but has distinctly
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different g-values (g = 2.20,
2.15, 2.00).63
temperature, MeOH solution
magnetic moment of 5 (µeff
The ambient
=
1.99 BM), is also consistent
Figure 3. Low temperature (7 K) X-band EPR spectrum of 5 in
CH2Cl2/Toluene (1:1) glass.
with an S= ½ spin- state, and
indicates that there are no thermally accessible higher spin-states in the range 7-300 K.
In contrast, 2 has an ambient temperature magnetic moment of µeff = 3.5 BM reflecting
the thermal accessibility of an S=3/2 excited state.63 The S= ½ ground state of 5 would
be consistent with either an Fe(V)93-96 or an Fe(III) oxidation state,48,54,59-61,63,66,79,97 but
inconsistent with an even-spin, S= 1 or S= 2 system, thereby ruling out an Fe(IV)=O.
The redox properties of the 5 also differ from those of 2. Whereas five-coordinate
2 is reversibly reduced at a potential of E1/2= –
425 mV vs SCE (Figure 4), 5 is irreversibly
reduced at a significantly more negative
potential (Epc= –958 mV, Epa= -690 mV vs
SCE; Figure S-4).
The latter is quasi-
reversible. Oxygen atom addition therefore
increases the stability of the Fe+3 oxidation
state. Oxygenation at sulfur would be
Figure 4. Cyclic voltammogram of five-coordinate
[FeIII(S2Me2N3(Pr,Pr))]+ (2) in MeCN at 298 K (0.1 M
(Bu4N)PF6, glassy carbon electrode, 150 mV/sec
scan rate). Peak potentials versus SCE indicated.
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expected to shift the potential in the opposite direction, given that it would decrease the
electron donating properties of the sulfur, unless, of course, the unmodified thiolate
overcompensates for the increase in Lewis acidity by forming a more covalent
unmodified Fe-SR bond.48 If the latter were the case, then the S!Fe CT band should
blue shift,48 as opposed to red-shift as shown in Figure 2. On the other hand, ferric
ions are more stable in a six-coordinate environment, relative to a five-coordinate
environment, perhaps suggesting that oxo atom addition involves the metal ion.
Iodosyl benzene (PhIO) typically promotes 2-electron chemistry, and has been
shown to convert Fe(II) compounds to Fe(IV)=O compounds.98-101 There are fewer
examples of Fe(III) compounds that are reactive towards oxo atom donors, such as
PhIO (Figure 2).102-104 One example of the latter involves the addition of PhIO to
cytochrome P450 in its Fe(III) resting state, to afford (por/cysS)Fe(V)=O (cmpd I).105 The
oxidizing equivalents of P450 cmpd I have been shown to be delocalized over both the
redox active porphyrin (por) and redox active thiolate ligands.106-111 Hydrogen peroxide
(H2O2) has also been shown to convert Fe(III) compounds to Fe(V)=O species,112,113 or
its redox equivalent.107,109,111 In contrast to Fe(IV)=O compounds,52,98,114-123 however,
very few synthetic non-heme Fe(V)=O species have been observed.93,95,96,104,113,124 The
most thoroughly characterized example, [Fe(V)(TAML)(O)]–, incorporates an electron
donating tetra-anionic carboxamide ligand (vide infra), TAML4-.93,95,104,125,126
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The IR spectrum of 5 contains a stretch at ꢀ= 1024 cm-1 (Figure S-5) that is absent
in the IR spectrum of 2. This stretching frequency would be extremely high for an
Fe(V)=O (ꢀFe=O = 798-828 cm-1)96,127 and is somewhat above the usual range (ꢀS=O
=
970-900 cm-1)24,35,46,128-130 for a metal-sulfenate complex. Sulfenate ꢀS=O stretches have,
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however, previously been shown to shift out of the usual range if the RS=O is η2–
coordinated to a metal ion.49,131 For example, sulfenate/sufinate-ligated [CoIII(η2-
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5
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9
SO)(SO2)N3(Pr,Pr)]+
1066 cm-1.49
(Scheme
2)
has
a
sulfenate
ꢀS=O
stretch
at
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X-ray Structure of [FeIII(η2-SMe2O)(SMe2)N3(Pr,Pr)]PF6 (5). The identity of the
product, 5, formed in the reaction between 2 and PhI, (or H2O2), was ultimately
determined by X-ray crystallography. Single crystals of 5 were grown at -30 ˚C by
layering Et2O onto a MeCN solution. As shown
in the ORTEP diagram of Figure 5, the single
oxygen atom O(1) adds ~trans (O(1)-Fe-S(2)=
152.3(2)˚) to one of the thiolate sulfurs (S(2)),
and forms a bond to the cis-thiolate S(1),
affording an η2-coordinated sulfenate [FeIII(η2-
SMe2O)(SMe2)N3(Pr,Pr)]+ (5). A similar side-on
sulfenate
observed
(η2-RSO–)
previously
binding-mode
was
Figure 5. ORTEP of the cation of [FeIII(η2-
SMe2O)(SMe2)N3(Pr,Pr)](PF6) (5) showing atom
labeling scheme. All hydrogen atoms have
been removed for clarity.
in
[CoIII(η2-
SO)(SO2)N3(Pr,Pr)]+ (Scheme 2).49 As
mentioned earlier, singly oxygenated metal sulfenates (RSO–) are rare24,48,49 since they
tend to be more reactive than their thiolate precursor.26,46,47,49 There are significantly
more examples of doubly oxygenated, structurally–rearranged oxygen–bound, metal-
–
sulfinate (RSO2 ) complexes.16-20
Comparison of structure 5 (Figure 4) with that of its [FeIII(S2Me2N3(Pr,Pr))]+ (2)
precursor (Table 2) shows that the singly oxygenated Fe-S(1) bond of 5 elongates only
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slightly as a result of oxygen atom addition (from 2.133(2) Å in 2 to 2.142(2) Å in 5).
One would have expected a more dramatic change in bond length. The Fe-S(2) bond,
on the other hand, decreases in length from 2.161(2) Å in 2 to 2.148(2) Å in 5, indicating
that the unmodified thiolate compensates for the shifting of electron density away from
the metal ion towards the oxygen atom. This compensatory effect was observed
previously with [FeIII(ADIT)(ADIT-O)]+ (1; Scheme 2).48 The sulfenate S(1)-O(1) bond in
5 (1.447 Å; Table 2) is slightly shorter than the few known sulfenate complexes (range:
1.50–1.60 Å),24,46,131 including that of [CoIII((η2–SO)(SO2)N3(Pr,Pr))]+ (Scheme 2) (S(1)-
O(1), 1.548(3) Å).49
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Table 2. Comparison of Selected bond distances (Å) and
angles
(deg)
for
Imine-Ligated
[FeIII(S2Me2N3
(Pr,Pr))]+ (2),63, its Singly Oxygenated Derivative [FeIII(η2-
SMe2O)(SMe2N3(Pr,Pr)]+ (5), and Carboxamide-Ligated
[FeIIIS2Me2NMeN2amide(Pr,Pr)]1– (8).
2
5
8
Fe-S(1)
Fe-S(2)
Fe-N(1)
Fe-N(2)
Fe-N(3)
Fe-O(1)
S(1)-O(1)
2.133(2) 2.142(2)
2.161(2) 2.148(2)
1.967(4) 1.976(4)
2.049(4) 2.044(5)
1.954(4) 1.954(4)
2.231(1)
2.210(1)
1.934(3)
2.212(3)
1.924(3)
N/A
N/A
N/A
2.115(4)
1.447(6)
N/A
N(1)-Fe-N(3)
S(1)-Fe-S(2)
S(1)-Fe-N(2)
S(2)-Fe-N(2)
178.1(2) 178.0(2)
177.8(1)
121.0(1) 112.62(8) 144.40(5)
132.3(1) 134.1(1)
106.5(1) 113.2(2)
107.14(9)
108.43(9)
S(1)-Fe-N(1)
S(2)-Fe-N(1)
S(1)-Fe-O(1)
O(1)-Fe-S(2)
86.7(1)
95.2(1)
N/A
86.1(1)
91.9(1)
39.7(2)
152.3(2)
87.6(2)
94.3(2)
94.4(2)
N/A
85.37(9)
93.8(1)
N/A**
N/A**
N/A**
N/A**
N/A**
0.56
N/A
O(1)-Fe-N(1) N/A
O(1)-Fe-N(2) N/A
O(1)-Fe-N(3) N/A
ꢀ
0.76
**In this structure the only oxygens are associated with the
carboxamide, and are not coordinated to the metal.
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Synthesis of a More Electron-Rich Derivative of 2. In order to provide an even
more electron-rich environment, analogous to that of the TAML4– ligand shown
previously to stabilize an Fe(V)=O,93 we synthesized (Scheme S-1) the tetra-anionic
carboxamide ligand [(Pr,Pr)(NMeNamide2SMe2)]4– (Figure S-6–S-9), and its corresponding
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Fe-complex (Scheme 5). As mentioned earlier, the
O
1–
N
preferred 2e- chemistry of PhIO makes it conceivable that
Me
III
S
Fe
N
oxo atom addition to Fe(III)-2 results in the formation of an
unobserved metastable Fe(V)=O intermediate along the
reaction pathway to 5. The highly covalent Fe–S bonds
would help to delocalize the oxidizing equivalents, thereby
S
N
O
8
Scheme 5.
making the higher-valent state accessible. The placement of a nucleophilic thiolate cis
to an electrophilic oxo would facilitate rapid intramolecular trapping of the oxo via the
formation of an S-O bond. A similar reaction sequence was recently observed to
convert a sulfur-bound substrate analogue of isopenicillin-N-synthase to an FeII((η2–
SO),132 and may be the mechanism by which the catalytically important sulfenate is
inserted into the Fe-NHase active site.27
Bis-thiolate/carboxamide-ligated [FeIIIS2Me2NMeN2amide(Pr,Pr)]1- (8) displays a ꢀC=O
stretch at 1564 cm-1 (Figure S-11), and parent ion peak in the negative-mode ESI-MS
(Figure S-10). This would be consistent with the structure shown in Scheme 5, which
incorporates anionic carboxamides in place of the neutral imines of 2. X-ray quality
crystals of (Et4N)[FeIIIS2Me2NMe N2amide(Pr,Pr)] (8) were obtained via slow vapor diffusion
of Et2O into a MeCN solution at –35 ˚C. As shown in the ORTEP diagram of Figure 6,
the Fe3+ ion of 8 maintains a five- coordinate structure, despite being crystallized from a
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coordinating solvent (MeCN), and resides in a
5
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8
highly distorted N3S2 environment (ꢀ = 0.56)
halfway between trigonal bipyramidal and square
pyramidal. The Fe3+ ion is ligated by two cis-
thiolate sulfurs and a tertiary amine (N(2)) in the
equatorial plane, and two trans- carboxamide
nitrogens (N(1), N(3)) in apical positions. Key
bond distances and angles are compared with
those of its cationic imine analogue 2 in Table 2.
The incorporation of anionic carboxamides in 8, in
place of the neutral imines of 2, causes the mean
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Figure 6. ORTEP of the anion of
(Et4N)[FeIIIS2Me2NMeN2amide(Pr,Pr)]
(8)
showing atom labeling scheme. All
hydrogen atoms have been removed for
clarity.
Fe–S bond distance to significantly elongate (from 2.147 Å in 2 to 2.22 Å in 8). A similar
increase in Fe–S bond lengths (from 2.189 Å to 2.219 Å) is seen upon replacement of
the neutral imines in [FeIII(tame-N3)
S2Me2)]+, with anionic carboxamides in
[FeIII((tame–N2S)S2Me2)]2–.17 The S–
Fe–S angle (144˚) in 8 is significantly
wider than that of 2 (121˚), as well as
imine-ligated
(105˚).61
[FeIII(S2Me2N3(Et,Pr))]+
Figure 7. Electronic absorption spectrum of
(Et4N)[FeIIIS2Me2NMeN2amide(Pr,Pr)] (8) in H2O at 298
K.
Carboxamide-ligated 8 is soluble
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in a variety solvents,including H2O, forms intense olive green-colored solutions, and
5
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8
displays a solvent-independent electronic absorption spectrum with bands at λmax
352(8060), 450(sh), and 584(1530) nm, indicating that solvents (MeCN, MeOH, H2O
(Figure 7)) do not coordinate to the
=
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metal ion.
The redox potential of
anionic 8 (–1.51V vs SCE; Figure 8) is
shifted by over 1 V, relative to that of
cationic imine-ligated 2 (E1/2 = –425 mV
vs SCE, Figure 4), demonstrating that
carboxamide ligands provide significant
stability to the Fe+3 oxidation state.133
Figure
8.
Cyclic
voltammogram
of
(Et4N)[FeIIIS2Me2NMeN2amide(Pr,Pr)] (8) in MeCN at 298 K
(0.1 M (Bu4N)PF6, glassy carbon electrode, 150 mV/sec
scan rate). Peak potentials versus SCE indicated.
As shown by fits to the inverse magnetic susceptibility (1/χm) versus temperature plot of
Figure 9, and low temperature (7 K) perpendicular-mode EPR (g =4.72, 2.82, 1.92;
Figure S-12) bis-thiolate ligated 8 has
an S= 3/2 ground state, and maintains
this spin-state over a wide temperature
range in the solid state and frozen
solution.
The ambient temperature
MeCN solution magnetic moment of µeff
= 3.75 µB indicates that 8 maintains this
spin-state in solution. In contrast,
Figure 9. Inverse molar magnetic susceptibility 1/χm
vs
temperature
(T)
plot
for
(Et4N)[FeIIIS2Me2NMeN2amide(Pr,Pr)] (8) fit to an S =
3/2 spin-state.
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imine-ligated 2 has an S=1/2 ground state, with a thermally accessible S= 3/2 state that
is ~23% populated at ambient temperature, and 11% populated at T= -80 ˚C. Spin-
states have been shown to influence reactivity in profound ways.134,135
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Comparison of the Reactivity Properties of Carboxamide-Ligated 8 versus
Imine-Ligated 2. In contrast to 2, carboxamide-ligated 8 does not react with oxo atom
donors PhIO or H2O2, under any conditions, even with a huge excess of oxidant, and
over a wide temperature range (-78 ˚C – 25 ˚C). One would have anticipated that the
thiolates of 8 would be more readily oxygenated, given the anionic molecular charge,
and expected increase in electron density on the thiolate sulfurs. Given the similarity of
its ligand environment to that of [Fe(V)(TAML)(O)]–, one would have also anticipated
that an Fe(V)=O might be stabilized, possibly to the point where it might be observable,
prior to intramolecular trapping, and S-O bond formation. If, on the other hand, sulfur
oxidation involves direct attack at the thiolate sulfurs, then one would also expect
carboxamide-ligated 8 to be more reactive towards oxo atom donors than 2, because
the thiolate sulfurs should carry more negative charge. In order to determine whether
the latter is indeed the case, and quantify this difference if it exists, we turned to
theoretical calculations. Density functional theory (DFT) calculations were performed
using the ORCA package, a BP86 functional and def2-TZVP basis set. For both 2 and
8, metrical parameters of the DFT-optimized geometry are within ≤ 1.96 % of the
crystallographically-determined bond lengths (Table S-1). Based on these calculations,
the Mulliken charges on the sulfurs of anionic 8 (–0.484 for S(1), –0.488 for S(2)) were
found to be roughly twice that of cationic 2 (–0.288 for S(1),–0.245 for S(2)). The longer
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Fe–S bond distances in 8, relative to 2, would be consistent with this. The calculated
average spin-density on the thiolate sulfurs is approximately the same for 2 (0.12) and 8
(0.15). Theoretical calculations therefore suggest that if oxo atom donors were to
directly attack the thiolate sulfur in a 2e– process, then carboxamide-ligated 8 would be
more reactive than imine-ligated 2, when in fact the opposite is observed. If a 1e–
radical process, involving direct attack at the sulfurs, were involved, then the two
complexes 2 and 8 should be equally reactive. Oxo atom donor reactivity does, on the
other hand, correlate with ligand binding properties. Despite its open coordination site,
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anionic 8 does not bind neutral (pyridine, MeCN) or anionic (N3 , CN–) ligands, even
when added in excess (>100 eq) at low temperatures (–40 ˚C to –78 ˚C), as
determined using electronic absorption spectroscopy. This is likely due to the fact that a
spin-state change would be required in order for 5-coordinate 8 to convert to a 6-
coordinate structure. Whereas 8 is S=3/2, six-coordinate, thiolate-ligated ferric
complexes are predominantly low-spin S=1/2.39,59,79,97,136 As one would expect,
calculated unpaired spin-density on the iron of 8 (2.59) is significantly larger than that of
2 (0.83), reflecting the S= ½ ground state of 2. A Me-group on N(2) of 8, in place of the
N(2)-H proton of 2, also likely inhibits ligands from binding to 8, since it would sterically
clash with S(1) when the S(1)-Fe-N(2) bond angle decreased as a consequence of a
the concomitant geometry change (~trigonal bipyriamidal! ~octahedral). Imine-ligated
–
2, on the other hand, readily binds both N3 and NO to its open coordination site,63,78 in
addition to reacting with oxo atom donors.
Thus, both correlations between sulfur oxidation and the molecule’s affinity for a
sixth ligand and ability to undergo a geometry change, and theoretical calculations,
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suggest that the mechanism of sulfur oxidation with 2 involves initial coordination of the
5
6
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oxo atom donor to the metal ion. Consistent with this, azide (N3 ) is found to inhibit
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sulfur oxidation with 2. If 1.2 equiv of N3 is added to 2 in THF at -73 ˚C, then the
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intense band 415(4200) nm characteristic of 2, is replaced by a band at 708(1600) nm
(Figure 10) characteristic of coordinatively
saturated,
azide-bound
[FeIII(S2Me2N3
(Pr,Pr))(N3)] (4).63
If, following the
formation of azide-bound 4 in THF, 1.2
equiv of PhIO is added, no reaction is
observed, even after prolonged reaction
times (2 hr). If, under the same conditions
Figure
10.
Monitoring
the
addition
of
iodosylbenzene (PhIO) to [FeIII(S2Me2N3(Pr,Pr))N3]
(4) in MeOH at -73 ˚C via electronic absorption
(-73 ˚C in THF) 1.2 equiv of PhIO is added spectroscopy showing that no reaction occurs.
–
to 2, in the absence of N3 , then a metastable intermediate that converts to singly
oxygenated [FeIII(η2-SMe2O)(SMe2N3(Pr,Pr)]+ (5) (λmax= 510(1540)) forms within minutes.
Thus, azide inhibits sulfur oxidation with 2, presumably by preventing the oxo atom
donor from binding to the metal ion. Consistent with this, we observe an intermediate in
the oxo atom donor reaction in the absence of azide.
Observation of an Intermediate in the Reaction Between Imine-Ligated 2 and
Oxo Atom Donors PhIO and IBX-ester. If the reaction between 10 equiv of PhIO and
five-coordinate 2 is monitored by electronic absorption spectroscopy at low tempera-
tures (-73 ˚C) in MeOH, a green intermediate with a λmax= 675 nm is observed (Figure
S-13), which then slowly (t=360 min, [2]= 0.238 mM) converts to sulfenate-ligated 5
(Figure S-14). As shown in Figure 11, an ~identical intermediate (λmax= 677 nm) is
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formed when isopropyl 2-iodoxybenzoate (IBX-ester) is used in place of PhIO, however
its rate of conversion to sulfenate-ligated 5 (36 min, [2]= 0.238 mM) is significantly
faster. IBX-ester contains an IV(=O)2 moiety,137 whereas PhIO contains an IIII=O moiety,
providing a possible explanation for the differences in rates. The observation of
intermediates in these reactions provides evidence that sulfur oxidation is assisted by
the metal ion, and involves the initial
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binding of oxo-atom donors to Fe.
Clear isobestic points visible during
these reactions show that the new
intermediate
is
stoichiometrically
related to complex 2 and converts
directly to the sulfenate complex 5,
thus eliminating the possibility of direct
attack at sulfur. Whether the green
Figure 11. Detection of a green metastable intermediate in the
reaction between (Pr,Pr)Fe(III) (2) and 10 eq. of IBX-ester at
–73˚C in MeOH to form sulfenate 5 over the course of 36
minutes.
intermediate is an oxo atom donor adduct, Fe-O=I-Ph, or an Fe(V)=O remains to be
determined. Given how rare Fe-O=I-Ph138,139 and Fe(V)=O93,113,124 species are, both
possibilities are intriguing. The λmax of our green intermediate (677 nm) is in the
reported range for both Fe(III)-O=I-Ph (λmax = 660 nm)139 and Fe(V)=O (λmax = 630(5400
M-1cm-1) nm)93,104 consistent with either possibility. Iodosylarene adducts have been
shown to be competent oxidants in OAT reactions.138-142 Spectroscopic characterization
of the green intermediate, along with the kinetics of both its formation and conversion to
sulfenate-ligated 5, will be the topic of a separate manuscript. Possible mechanisms for
sulfur oxygenation would involve initial formation of an oxo atom donor adduct Fe-O=I-
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Ph that is either directly attacked by the adjacent nucleophilic sulfur, or which converts
first to an Fe(V)=O, which is then rapidly and irreversibly trapped by the adjacent sulfur.
The observation of one intermediate, as opposed to two, is consistent with either (a)
rate-determining cis-migration of a thiolate sulfur to the oxo of a coordinated Fe-O=I-Ph,
or (b) rate-determining cleavage of the I–O bond to afford an Fe(V)=O. The virtually
identical electronic spectra associated with the green intermediate, regardless of the
nature of the oxo atom donor is a bit puzzling, given that one would expect an Fe-O=IV-
Ph species to have electronic properties that differ from that of an Fe-O=IIII-Ph species.
Inhibition studies, involving PhI, should help to distinguish mechanisms (a) and (b). The
anticipated rapid rate of intramolecular trapping of an Fe(V)=O, relative to the likely rate
of its formation, would suggest that it would be unobservable. This remains to be
determined, however.
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Summary and Conclusions. Cysteinate oxygenation is intimately tied to the
function of both cysteinate dioxygenases (CDO)1-8 and nitrile hydratases
(NHases).31,39-41 However, the mechanisms by which sulfurs are oxidized by these
enzymes is unknown, in part because intermediates have yet to be observed. Herein
we show that oxo atom donors, PhIO and H2O2, react with coordinatively unsaturated
[FeIII(S2Me2N3(Pr,Pr))]+ (2) to afford a rare example of a singly oxygenated sulfenate,
[FeIII(η2-SMe2O)(SMe2)N3(Pr,Pr)]+ (5). Sulfenate-ligated 5 is low-spin (S= ½; g= 2.17,
2.11, 1.98), and resembles both an intermediate proposed to form during CDO
catalyzed cysteine oxidation, as well as the catalytically essential NHase Fe–SCys114
–
O, proposed to be intimately involved in its mechanism.27,31,42,43 In contrast to imine-
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ligated 2, carboxamide-ligated [FeIIIS2Me2NMeN2amide(Pr,Pr)]1– (8) was shown to be
unreactive towards oxo atom donors PhIO or H2O2, under any conditions. DFT-
calculated Mulliken charges on the sulfurs of anionic 8 were found to be roughly twice
that of cationic 2, indicating that if oxo atom donors were to directly attack the thiolate
sulfur, then carboxamide-ligated 8 would be more reactive than imine-ligated 2.
Reactivity was shown to correlate with the metal ion’s ability to bind exogenous
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–
ligands, and azide (N3 ) was found to inhibit sulfur oxidation with 2, suggesting that the
mechanism of sulfur oxidation involves initial coordination of the oxo atom donor to the
metal ion. Consistent with this, a green intermediate is observed to form, which then
slowly converts to sulfenate-ligated 5. Whether the green intermediate is an oxo atom
donor adduct, Fe-O=I-Ph, or an Fe(V)=O remains to be determined. The placement of
a nucleophilic thiolate cis to an electrophilic oxo would facilitate rapid intramolecular
trapping of a high-valent oxo via the formation of an S-O bond. Although the direct
involvement of the metal ion has been theoretically calculated to provide a lower
energy pathway to sulfur oxygenation by CDO,5 this has not been experimentally
proven.7
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Acknowledgements. This work was supported by NIH (GM45881). P. L. -M.
gratefully acknowledges support by an NIH pre-doctoral minority fellowship (F31
GM73583-01). We also gratefully acknowledge Steven J. Lippard, and an anonymous
donor for financial support.
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Supporting Information. Contains experimental details regarding the synthesis and
characterization of the [(Pr,Pr)(NMeHNamide Me(SH)2)]•HCl ligand. ESI mass spec, IR,
2
and cyclic voltammogram of 5. ESI mass spec, IR, cyclic voltammogram, aqueous
electronic absorption and EPR spectra of 8, and electronic spectral evidence for the
formation of 5, via a green metastable intermediate. Crystallographic tables for 5 and 8,
and metrical parameters for DFT optimized structures are also included. This material
is available free of charge via the Internet at http://pubs.acs.org.
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