Iron(II)-thiolate S -Oxygenation by O2: Synthetic models of cysteine dioxygenase

Article abstract of DOI:10.1021/ja105591q

The synthesis of structural and functional models of the active site of the nonheme iron enzyme cysteine dioxygenase (CDO) is reported. A bis(imino)pyridine ligand scaffold was employed to synthesize a mononuclear ferrous complex, Fe^II(LN₃S)(OTf) (1), which contains three neutral nitrogen donors and one anionic thiolato donor. Complex 1 is a good structural model of the Cys-bound active site of CDO. Reaction of 1 with O ₂ results in oxygenation of the thiolato sulfur, affording the sulfonato complex Fe^II(LN₃SO₃)(OTf) (2) under mild conditions. Isotope labeling studies show that O₂ is the sole source of O atoms in the product and that the reaction proceeds via a dioxygenase-type mechanism for two out of three O atoms added, analogous to the dioxygenase reaction of CDO. The zinc(II) analog, Zn(LN₃S)(OTf) (4), was prepared and found to be completely unreactive toward O₂, suggesting a critical role for Fe^II in the oxygenation chemistry observed for 1. To our knowledge, S-oxygenation mediated by an Fe ^II-SR complex and O₂ is unprecedented.

Full text of DOI:10.1021/ja105591q

Published on Web 08/16/2010
Iron(II)-Thiolate S-Oxygenation by O2: Synthetic Models of Cysteine Dioxygenase
Yunbo Jiang, Leland R. Widger, Gary D. Kasper, Maxime A. Siegler, and David P. Goldberg*
Department of Chemistry, Johns Hopkins UniVersity, Baltimore, Maryland 21212
Received June 25, 2010; E-mail: dpg@jhu.edu
of a thiolate donor, and (4) include steric protection against the
formation of O- or S-bridged Fe complexes. These criteria were
met with the metal-templated synthesis of LN3S, a bis(imino)py-
Abstract: The synthesis of structural and functional models of the
active site of the nonheme iron enzyme cysteine dioxygenase (CDO)
is reported. A bis(imino)pyridine ligand scaffold was employed to
synthesize a mononuclear ferrous complex, FeII(LN3S)(OTf) (1),
ridine ligand in which a pendant thiolate donor has been incorpo-
rated.2 Herein it is shown that an FeII(LN3S) complex reacts with
which contains three neutral nitrogen donors and one anionic thiolato
O2 via sulfur oxygenation. To our knowledge, S-oxygenation of a
donor. Complex 1 is a good structural model of the Cys-bound active
well-defined FeII-SR species with O2 is unprecedented.
site of CDO. Reaction of 1 with O2 results in oxygenation of the
Reaction of the unsymmetrical ketone 2-(OdCMe)-6-(2,6-(iPr2s
thiolato sulfur, affording the sulfonato complex FeII(LN3SO3)(OTf) (2)
C6H3NdCMe)sC5H3N with 2-aminothiophenol in the presence of
under mild conditions. Isotope labeling studies show that O2 is the
sole source of O atoms in the product and that the reaction proceeds
via a dioxygenase-type mechanism for two out of three O atoms
FeII(OTf)2 and Et3N at 80 °C in ethanol affords the desired dark brown
FeII complex [FeII(LN3S)(OTf)] (1) in good yield (86%) (Scheme 1).
added, analogous to the dioxygenase reaction of CDO. The zinc(II)
analog, Zn(LN3S)(OTf) (4), was prepared and found to be completely
unreactive toward O2, suggesting a critical role for FeII in the oxygenation
chemistry observed for 1. To our knowledge, S-oxygenation mediated
by an FeII-SR complex and O2 is unprecedented.
Scheme 1
The utilization of O2 for the oxidation of organic substrates is a
critical process carried out by metalloenzymes and a highly desirable
one for synthetic chemists to replicate. Cysteine dioxygenase (CDO)
is a mononuclear nonheme iron enzyme that catalyzes the S-
oxygenation of cysteine to cysteine sulfinic acid with O2 as oxidant
(Figure 1).1 Loss of CDO function has been correlated with Alzhe-
imer’s, Parkinson’s, and other neurological disorders. CDO contains
a mononuclear FeII center bound by three His ligands, in contrast to
the 2-His-1-carboxylate “facial triad” that is the canonical motif for
nonheme Fe oxygenases. This unexpected structural variation suggests
that the ligation of three neutral N donors may be important for CDO
The molecular structure of 1 is shown in Figure 2. The FeII ion is
bound by the three neutral N donors and the thiolate S donor of the
LN3S ligand in a distorted square pyramidal geometry (τ ) 0.12), with
the OTf - anion occupying the axial position. The diisopropyl substituents
are projected orthogonal to the pseudoequatorial N3S plane, providing
significant steric protection of the metal center. The Fe-N/S/O distances
are consistent with a high-spin FeII complex.3
function.1h X-ray crystal structures of the native iron(II) CDO,1b
a
Cys-bound complex,1c and an intriguing Cys-persulfenate species1f
have been determined (Figure 1). Little is known regarding the
mechanism of CDO, although the persulfenate structure suggests an
Fe-O2 intermediate may be important.
Figure 2. Displacement ellipsoid plots (50% probability level) of 1 and 3.
The H atoms are omitted for clarity.
Addition of excess O2 to 1 in CH2Cl2 leads to an immediate color
change from black to brown. Analysis of the reaction mixture by laser-
desorption ionization mass spectrometry (LDIMS) shows the complete
loss of starting material after 24 h and the appearance of a prominent
ion at m/z 532.1, consistent with the triply oxygenated cation
[FeII(LN3SO3)]+ of 2 (Scheme 1). The reaction is solvent independent,
giving the same product in CH3CN or THF. Reaction mixtures at earlier
times (e.g., 5-180 min) contain starting material 1 ([FeII(LN3S)]+,
m/z 484.2) and 2, together with a smaller peak at m/z 516.1,
Figure 1. Depiction of the active sites of CDO derived from X-ray
crystallography for (a) the iron(II) resting state, (b) the Cys-bound form,
and (c) a trapped persulfenate complex; (d) CDO reaction scheme.
Herein we describe the first structural and functional synthetic
models of CDO. To obtain biologically relevant models, we targeted
polydentate ligand platforms that would (1) provide three neutral
N donors, (2) stabilize FeII, (3) allow for the facile incorporation
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C O M M U N I C A T I O N S
corresponding to a doubly oxygenated product which disappears as
the reaction proceeds. The peak at m/z 516.1 is consistent with either
a sulfinato (RSO2-) complex or a persulfenate species analogous to
that seen for CDO. A sulfenato (RSO-) complex is not observed.
Attempts to crystallographically characterize 2 after O2 addition were
unsuccessful. However, demetalation and acid hydrolysis (1 M HCl),
followed by quantitative reversed-phase HPLC (H2O/CH3CN 95/5,
0.1% TFA), show that the expected oxygenated organic fragment
2-H2N-C6H4SO3H is formed in good yield (60%). These data confirm
that S-oxygenation occurs upon reaction of O2 with 1. EPR spectra at
15 K of mixtures of 1 + O2 reveal a signal for high-spin FeIII (g 4.3),
but double integration shows this signal accounts for less than 5 (
2% of the total iron content. The lack of a significant EPR signal
indicates a +2 oxidation state for 2. Quantitation with 1,10-phenan-
throline yields a total FeII content of 91% after O2 addition (see
Supporting Information).
pathways can be envisioned.1e However, synthesis of the redox-
inert ZnII analog [Zn(LN3S)(OTf)] (4) provides some initial
insights.5 Exposure of 4 to O2 for up to 7 d at 25 °C (eq 1) gives
no reaction as determined by 1H NMR and LDIMS. Thus the
requirement for iron(II), the native metal in CDO, appears to be
critical for the S-oxygenation of 1.
There are only a few reports of O2-mediated S-oxygenation of
FeIII-SR complexes.6,7
However, prior to the present study, the
reaction of O2 with FeII-SR complexes has led only to the formation
of FeIII-O-FeIII complexes, in lieu of S-oxygenates.8
Interestingly,
Darensbourg observed that the site of O-capture (Fe vs S) in the
reaction of FeII-SR + O2 resulted in the exclusive selection of Fe
over S.8a
Our findings establish that an Fe
II-SR complex, in the
appropriate ligand environment, can selectively react with O2 to yield
S-oxygenates. Further examination of 1 and related complexes should
provide new, general insights regarding Fe/S/O2 reactivity.
Further support for the identity of 2 comes from the synthesis
of a close analog. A template reaction with FeIICl2, the unsym-
metrical ketone 2-H2NsC6H4SO3H, and Et3N followed by recrys-
tallization from CH3CN/iPr2O affords [FeII(LN3SO3)(Cl)] (3) (Figure
2). The sulfonato group coordinates as expected to the FeII center,
completing a distorted square pyramidal geometry (τ ) 0.33) with
the N and Cl donors. Thus complex 3 is a reasonable structural analog
of the sulfonato product 2 proposed in Scheme 1.
Acknowledgment. The NIH (GM62309) is gratefully acknowl-
edged for financial support. We thank Prof. S. Michel, S. J. Lee,
and J. Michalek for assistance with HPLC.
Supporting Information Available: Experimental details, spectra,
and crystallographic data for complexes 1, 3, and 4. This material is
available free of charge via the Internet at http://pubs.acs.org.
Isotopic labeling studies provide important mechanistic information
regarding the oxygenation reaction. Addition of 18O2 (98%) to 1 results
in fully labeled [FeII(LN3S18O3)]+ (Figure 3). In contrast, no 18O
incorporation is observed when the reaction is run in the presence of
excess H218O. Thus O2 is the source of S-oxygenation in 2, which
parallels the results obtained from 18O-labeling studies with CDO.1f
Two mechanistic possibilities for the formation of complex 2 are (1)
incorporation of an intact molecule of O2 before or after the addition
of a single O atom (2 + 1 case) or (2) single O atom addition for all
three sulfonato oxygens (1 + 1 + 1 case). Reaction of 1 with a mixture
of 18O2/16O2 (∼49:51), followed by LDIMS and statistical simulation
of the isotopic distribution pattern in 2, provides a means for
distinguishing these two possibilities.4 Simulations of the isotopic
envelope show that the 2 + 1 mechanism is the dominant pathway
(Figures 3 and S1). This pathway indicates that a dioxygenase-type
reaction is occurring, as seen for CDO. The failure to detect a singly
oxygenated sulfenato complex at earlier reaction times suggests that
the third O atom is incorporated after dioxygenation, not before.
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