Iron(II)-Thiolate S-Oxygenation by O₂: Synthetic Models of Cysteine Dioxygenase

Abstract

The synthesis of structural and functional models of the active site of the non-heme 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 3 neutral nitrogen and one anionic thiolato donor. Complex 1 is a good structural model of the Cys-bound active site of CDO. Reaction of 1 with O₂ 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.

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 non-heme 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 Alzheimer’s, Parkinson’s, and other neurological disorders. CDO contains a mononuclear Fe^II center bound by 3 His ligands, in contrast to the 2-His-1-carboxylate “facial triad” that is the canonical motif for non-heme Fe oxygenases. This unexpected structural variation suggests that the ligation of three neutral N donors may be important for CDO function.^1h X-ray crystal structures of the native iron(II) CDO,^1b a Cys-bound complex,^1c and an intriguing Cys-persulfenate species^1f 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 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 3 neutral N donors, 2) stabilize Fe^II, 3) allow for the facile incorporation 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)pyridine ligand in which a pendant thiolate donor has been incorporated.² Herein it is shown that an Fe^II(LN₃S) complex reacts with O₂ via sulfur oxygenation. To our knowledge, S-oxygenation of a well-defined Fe^II-SR species with O₂ is unprecedented.

Reaction of the unsymmetrical ketone 2-(O=CMe)-6-(2,6-(ⁱPr₂-C₆H₃N=CMe)-C₅H₃N with 2-aminothiophenol in the presence of Fe^II(OTf)₂ and Et₃N at 80 °C in ethanol affords the desired dark brown Fe^II complex [Fe^II(LN₃S)(OTf)] (1) in good yield (86%) (Scheme 1). The molecular structure of 1 is shown in Figure 2. The Fe^II 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 pseudo-equatorial N₃S plane, providing significant steric protection of the metal center. The Fe-N/S/O distances are consistent with a high-spin Fe^II complex.³

Scheme 1.

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 [Fe^II(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 ([Fe^II(LN₃S)]⁺, m/z 484.2) and 2, together with a smaller peak at m/z 516.1, corresponding to a doubly-oxygenated product which disappears as the reaction proceeds. The peak at 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 reverse-phase HPLC (H₂O/CH₃CN 95/5, 0.1% TFA) shows 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 Fe^III (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-phenanthroline yields a total Fe^II content of 91% after O₂ addition (see Supporting Information).

Further support for the identity of 2 comes from the synthesis of a close analog. A template reaction with Fe^IICl₂, unsymmetrical ketone 2-(O=CMe)-6-(2,6-(ⁱPr₂-C₆H₃N=CMe)-C₅H₃N, 2-H₂N-C₆H₄SO₃H and Et₃N followed by re-crystallization from CH₃CN/iPr₂O affords [Fe^II(LN₃SO₃)(Cl)] (3) (Figure 2). The sulfonato group coordinates as expected to the Fe^II 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.

Isotopic labeling studies provide important mechanistic information regarding the oxygenation reaction. Addition of ¹⁸O₂ (98%) to 1 results in fully labeled [Fe^II(LN₃S¹⁸O₃)]⁺ (Figure 3). In contrast, no ¹⁸O incorporation is observed when the reaction is run in the presence of excess H₂ ¹⁸O. 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).

Figure 3.

Oxygen isotope studies using LDIMS. ¹⁸O₂/¹⁶O₂ (~ 49/51) mixture (left) and ¹⁸O₂ (98%) (right). Exptl (black), simulation (red).

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 (Figure 3 and Figure 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.

(1)

The role of the Fe^II ion in the S-oxygenation of 1 is not yet known, and mechanisms that involve both redox and non-redox pathways can be envisioned.^1e However, synthesis of the redox-inert Zn^II 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 Fe^III-SR complexes.^6,7 However, prior to the present study, the reaction of O₂ with Fe^II-SR complexes has led only to the formation of Fe^III-O-Fe^III complexes, in lieu of S-oxygenates.⁸ Interestingly, Darensbourg observed that the site of O-capture (Fe vs S) in the reaction of Fe^II-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.