Influence of the Second Coordination Sphere on O₂ Activation by a Nonheme Iron(II) Thiolate Complex

Abstract

The synthesis and characterization of a new 1-(bis(pyridin-2-ylmethyl) amino)-2-methylpropane-2-thiolate-ligated nonheme iron complex, Fe^II(BPA^Me2S)Br (1), is reported. Reaction of 1 with O₂ at −20 °C generates a high-spin iron(III)-hydroxide complex, [Fe^III(OH)(BPA^Me2S)(Br)] (2), that was characterized by UV-vis, ⁵⁷Fe Mössbauer, and electron paramagnetic resonance (EPR) spectroscopies, and electrospray ionization mass spectrometry (ESI-MS). Density functional theory (DFT) calculations were employed to support the spectroscopic assignments. In a previous report (J. Am. Chem. Soc. 2024, 146, 7915 – 7921), the related bis((6-(neopentylamino) pyridinyl) methyl)amino)-2-methylpropane-2-thiolate-ligated iron(II) complex, Fe^II(BNPA^Me2S)Br, was reported and shown to react with O₂ at low temperature to give a rare (η²-superoxo)-iron(III) intermediate, which then converts to an S-oxygenated sulfinate as seen for the nonheme iron thiol dioxygenases. This complex includes two hydrogen bonding neopentylamino groups in the second coordination sphere. Complex 1 does not include these H-bonding groups, and its reactivity with O₂ does not yield a stabilized Fe/O₂ intermediate or S-oxygenated products, although the data suggest an inner-sphere mechanism and formation of an iron-oxygen species that is capable of abstracting hydrogen atoms from solvent or weak C-H bond substrates. This study indicates that the H-bond donors are critical for stabilizing the Fe^III(O₂^−•) intermediate with the BNPA^Me2S⁻ ligand, which in turn leads to S-oxygenation, as opposed to H-atom abstraction, following O₂ activation by the nonheme iron center.

Graphical Abstract

1. Introduction

Dioxygen activation at nonheme iron centers is crucial for the function of a wide array of enzymatic and synthetic catalysts.^1–4 An archetypal example is seen in the alpha-ketoglutarate (α-KG) dependent nonheme iron enzyme taurine dioxygenase (TauD). The mechanism of TauD involves the binding of O₂ to Fe^II, followed by O−O bond cleavage to give a ferryl (Fe^IV(O)) intermediate. The Fe^IV(O) species then abstracts hydrogen from a substrate C−H bond to give an Fe^III(OH) species and carbon radical (R•), which rapidly recombines to give hydroxylated (ROH) product.^5–7 A related subset of nonheme iron enzymes activates O₂ for sulfur oxidation,^8–10 leading to C-S^11–14 or O-S^15–25 bond formation. However, the mechanisms of these sulfur-ligated enzymes are much less well understood than TauD.^26–28 One family of these enzymes are the thiol dioxygenases (TDOs), which utilize O₂ for the S-oxygenation of iron-bound thiolate substrates.^{17, 20, 29–30} For example, cysteine dioxygenase (CDO) catalyzes cysteine oxygenation to cysteine sulfinic acid (CSA) at a nonheme iron center bound by three histidines, rather than the 2-His/1-carboxylate motif seen in TauD and other nonheme iron enzymes. A proposed mechanism for the TDOs, including CDO, begins with formation of an Fe^III(superoxo) intermediate from Fe^II + O₂, followed by two sequential mono-oxygenation steps, first giving an Fe^IV(O)(sulfenate) intermediate, followed by intramolecular O-atom transfer to give an Fe^II(sulfinate) product.^{25, 31–33} This mechanism shares some commonalities with α-KG-dependent enzymes such as TauD.²⁶ Other mechanisms rely on the direct attack of O₂ on sulfur as seen with nickel thiolate complexes,³⁴ or possibly formation of a persulfenate-iron(II) intermediate from iron(III)(superoxide), followed by O-O cleavage to give the Fe^II(sulfinate) and avoiding formation of an Fe^IV(O) intermediate.³⁵ A recent study involving cobalt(II)-substituted cysteamine dioxygenase (ADO) supports this pathway.³⁶

Our research group has made significant efforts toward synthesizing inorganic analogs of the TDOs.^37–42 A significant part of these efforts have involved identifying the general structural and electronic requirements for synthesizing mononuclear Fe^II complexes that facilitate O₂ activation. Recently, we reported a thiolate-ligated nonheme iron complex as a functional model for TDOs.⁴² In this work, a new tetradentate, monoanionic ligand, (bis((6-(neopentylamino) pyridinyl) methyl)amino)-2-methylpropane-2-thiol) (BNPA^Me2SH) was prepared, which contains two pivaloylamino groups in the second coordination sphere as potential hydrogen bond donors. The corresponding complex Fe^II(BNPA^Me2S)(Br), reacts with O₂ at low temperature (-40 °C) to give an iron(III)-superoxide (Fe^III(O₂^.-)) species,^43–45 which was characterized by UV-vis, ⁵⁷Fe Mössbauer, and ATR-FTIR spectroscopies, as well as cold-spray ionization mass spectrometry (CSIMS). A “side-on”, as opposed to “end-on” binding mode, was implicated for the superoxide ligand, as shown in Scheme 1. Upon warming from −40 to 25 °C, this species converts to an iron(II) sulfinate product as well as an iron(III) hydroxide complex. Mechanistic probes indicate that a singly oxygenated iron sulfenate species is formed along the pathway to the ferrous sulfinate product, as shown in Scheme 1. This mechanism is analogous to the proposed mechanism for the TDOs involving an (Fe^III(O₂^•-)) adduct. Although the mechanism of formation of the hydroxide complex has not been determined, this species forms either following the thermal decay of the low temperature Fe^III(O₂^•-) intermediate, or upon reaction of the iron(II) complex with O₂ at room temperature.

Scheme 1.

Dioxygen reactivity of the H-bonded analog Fe^II(BNPA^Me2S)(Br) system

Herein we report a new tetradentate ligand, 1-(bis(pyridin-2-ylmethyl) amino)-2-methylpropane-2-thiol (BPA^Me2SH) and its iron(II) derivative, Fe^II(BPA^Me2S)Br (1), which was characterized by single crystal X-ray diffraction. The new ligand is an analog of BNPA^Me2S⁻, in which the pivaloylamino groups have been removed from the second coordination sphere. Our hypothesis was that the hydrogen bonding groups were critical to the stabilization of the side-on bound superoxide species in Scheme 1, and by removing these groups in the BPA^Me2S⁻ ligand, we could test this hypothesis. Reaction of 1 with O₂ at low temperature was examined, and although inner-sphere O₂ activation is implicated, the formation of a side-on bound superoxide is not observed, and S-oxygenation does not occur in this case. Our observations are consistent with an end-on superoxide species forming as the initial intermediate, which then leads to a putative Fe^IV(O) species, followed by H-atom abstraction to give a final Fe^III(OH) complex. Isotope labeling experiments show that the OH group is derived from O₂, and a hydrogen atom is abstracted from either the solvent or a weak C-H bond substrate.

2. Results and Discussion

The new tetradenate ligand 1-(bis(pyridin-2-ylmethyl) amino)-2-methylpropane-2-thiol (BPA^Me2SH) was prepared by mixing 2,2’-dipicolylamine and 2,2-dimethylthiirane in CH₃CN, followed by heating at 75 °C for 48 h.⁴⁶ The crude product was partially purified by chromatography on neutral alumina as a mixture of disulfide and polysulfide compounds. Reduction with lithium aluminum hydride followed by organic workup and purification on neutral alumina gave the target ligand, L as a pure yellow oil. The iron(II) complex 1 is synthesized by deprotonation of L with NaH in THF, followed by mixing with FeBr₂ ^● 2 THF dissolved in CH₃CN at −35 °C. An immediate color change from yellow to dark orange is noted, and this mixture is then warmed to room temperature and stirred for 4 h to ensure complete metalation (Scheme 2). Complex 1 was obtained as an orange powder, which was then recrystallized from THF/pentane to afford orange block crystals over 12 h. Analysis of these crystals by single crystal X-ray diffraction led to the structure shown in Figure 1. As expected, 1 is a five-coordinate, iron(II) complex with a bromide ligand occupying the fifth site. The geometry is close to trigonal bipyramidal (tbp) based on a tau value of 0.81.⁴⁷ The geometry for the previously reported hydrogen-bonded BNPA derivative is closer to square pyramidal (τ = 0.53), likely a result of the strain imposed by the neopentyl amino groups through H-bonding to the Br ligand and steric crowding around the metal. The Fe-N and Fe-S bond distances for 1 are in the typical range for high-spin (hs) (S = 2) Fe^II complexes and are similar to the BNPA derivative.

Scheme 2.

Synthesis of L and 1

Figure 1.

Displacement ellipsoid plot (50% probability level) for 1 at 110(2) K. Hydrogen atoms have been omitted for the sake of clarity.

The ¹H nuclear magnetic resonance (NMR) spectrum of 1 in CD₃CN reveals paramagnetically shifted peaks from +140 to −20 ppm (Figure 2), indicative of a high-spin (hs) Fe^II complex. The spectrum of 1 is similar to the spectrum of the BNPA analog iron(II) complex, with the exception of two additional peaks at 76.7 ppm and 135.9 ppm. Based on assignments in the literature, the peak at 76.7 ppm can be assigned to the α-pyridine proton resonance. The assignment of the new peak at 135.9 ppm is not clear at this time. The Mössbauer spectrum of 1 in THF at 80 K shows a sharp quadrupole doublet with δ = 0.93 and |ΔE_Q| = 3.29 mm s⁻¹ (Figure 3b), typical of a hs-Fe^II center and similar to the BNPA^Me2S⁻ analog (δ = 0.96 and |ΔE_Q| = 2.80 mm s⁻¹).

Figure 2.

¹H NMR spectra in CD₃CN of a) 1 and b) Fe^II(BNPA^Me2S)(Br).

Figure 3.

a) UV−vis spectra showing the conversion of 1 (0.6 mM, black solid line) to 2 (red dotted line) after O₂ addition in THF at −20 °C, and b) Mössbauer spectra of 1 (top) and 1 + O₂ (bottom) in THF at 80 K; isomer shift (δ) and quadrupole splitting (|ΔE_Q|) values are given in mm s⁻¹; solid lines represent the best fits.

3. Dioxygen Reactivity

Complex 1 is orange in THF and exhibits a UV−vis absorption band at 365 nm (ε = 1460 M⁻¹ cm⁻¹) and 450 nm (ε = 1050 M⁻¹ cm⁻¹). Addition of O₂ to a solution of 1 in THF, 2-MeTHF, or CH₃CN at 23 °C shows no reaction. However, upon cooling the solution to −20 °C, a color change from orange to brown-green is observed. Addition of O₂ to 1 in THF results in the appearance of an absorption band at 580 nm (ε = 1340 M⁻¹ cm⁻¹), corresponding to a new species, 2. Complex 2 is stable at −20 °C. Vigorously bubbling Ar(g) through a solution of 2 at −20 °C or warming the solution to 20 °C results in no spectral change, and the formation of 2 cannot be reversed by repeated vacuum/purge cycles. These data indicate that 2 is not a reversible O₂ adduct of iron(II).

Analysis of the reaction mixture for 1 + O₂ in THF by Mössbauer spectroscopy at 80 K reveals a broad quadruple doublet with parameters δ = 0.48 mm s⁻¹ and |ΔE_Q| = 1.41 mm s⁻¹, accounting for ≥ 80% of the total area (Figure 3c). These parameters are consistent with a hs-Fe^III species, and are very similar to those of other high-spin, Fe^III(OH) complexes prepared with the H-bonded BNPA analogue.^{42, 48–51} A minor quadruple doublet is also observed (δ = 1.14 mm s⁻¹; |ΔE_Q| = 3.09 mm s⁻¹) which accounts for ≤ 20% of the total area, and corresponds to a hs-Fe^II species different from the starting material. The experimental parameters also do not match the calculated Mössbauer parameters (DFT) for either sulfur mono- or di-oxygenated iron(II) complexes (Table S2). The X-band EPR spectrum (20 K) of the same reaction mixture reveals peaks at g = 8.92, 4.31 (Figure S2), consistent with a hs-Fe^III (S = 5/2) complex, and matches the EPR spectra for the other Fe(III)(OH) complexes with either BNPA^Me2S⁻ or BNPA^Ph2O⁻ supporting ligands.^{42, 48, 50}

Examination of complex 1 in CH₃CN using electrospray ionization mass spectrometry (ESI-MS) indicates an intense cluster centered at 423.73 m/z (Figure S3) which matches the theoretical m/z value for [Fe(BPA^Me2S)(Br)]+H⁺. The cluster also exhibits the isotopic pattern associated with natural abundance ^79/81Br. Reaction of 1 with O₂ at −40 °C followed by direct injection into the ESI-MS instrument reveals that the cluster for 1 has disappeared, and a new, intense cluster appears centered at 359.01 m/z. The mass and isotopic pattern of this cluster matches that for the loss of the bromine atom in 1 and addition of an OH group, i.e. [Fe(BPA^Me2S)(OH)]⁺. Addition of isotopically pure ¹⁸O₂ (98% ¹⁸O) to 1 lead to a 2 unit shift of the cluster centered at 361.01 m/z. Fitting of the isotopic pattern indicates an incorporation of 78 – 83% of the ¹⁸O isotope (Figure 4). In contrast, the addition of H₂¹⁸O (99% ¹⁸O) to 1 followed by reaction with O₂ does not lead to any ¹⁸O label in the final product as seen by ESI-MS. These data, together with the Mössbauer and EPR spectra, provide strong evidence that the major product from the reaction of 1 and O₂ is the Fe^III(OH) complex 2 shown in Scheme 3, in which the OH ligand is derived from O₂.

Figure 4.

ESI-MS data for 2(¹⁶O₂) (a) and 2(¹⁸O₂) (b). Calculated isotopic cluster for 2(¹⁶O₂) (c) and for 2(¹⁸O₂) (d) with 80% ¹⁸O incorporation.

Scheme 3.

Reaction of 1 with O₂.

The density functional theory (DFT) optimized geometry for 1 matches the crystal structure and was employed as the starting point for calculations on 2. Geometry optimizations by DFT of 2 in the possible spin states (S = 1/2, 3/2, and 5/2) were carried out with the CAM-B3LYP/def2-TZVP/6–31g*(C, H) functional/basis set combination. The same level of theory was used to obtain an optimized structure for the starting material 1 in the quintet state, and this structure matched well with the structure from XRD, helping to validate the theoretical methods. The optimized geometry for 2 converged to the expected 6-coordinate structure shown in Figure 5a, and gave metal−ligand (Fe-N,S,O) bond distances that were slightly contracted by 0.02 – 0.03 Å as compared to the H-bonded Fe(BNPA^Me2S)(OH)(Br) complex. Calculation of the Mössbauer parameters for 2 showed that the S = 5/2 ground state gives δ = 0.48 mm s⁻¹ and |ΔE_Q| = 1.11 mm s⁻¹, which is a good match with the experiment (Figure 5a), while the parameters for the other spin states (S = 3/2, 1/2) deviate significantly. We also considered the possibility of formation of a di-ferric, oxo-bridged species in the reaction of 1 + O₂, and an optimized geometry is shown in Figure 5b. The calculated Mössbauer parameters for this di-ferric, μ-oxo complex in both the fully antiferromagnetically and ferromagnetically coupled spin states do not match the experimental data, as is the case with other possible intermediate spin states (Table S3). These calculations bolster the assignment of 2 as a 6-coordinate, Fe^III(BPA^Me2S)(OH)(Br) complex.

Figure 5.

Optimized geometries and Mössbauer parameters (δ, |ΔE_Q| (mm s⁻¹)) for the different spin states from DFT calculations for a) 2, and b) [Fe^III₂(BPA^Me2S)₂(μ-O)(Br)₂].

4. Mechanism

As shown in Scheme 1, reaction of the H-bonded analog Fe^II(BNPA^Me2S)Br with O₂ at −40 °C led to a thermally metastable, side-on bound, iron(III)(superoxide) adduct in which the Br⁻ anion is no longer coordinated. This species, upon warming, converts to give a singly oxygenated sulfenate (RS(O)⁻) group as implicated by chemical trapping experiments, as well as a putative Fe^IV(O) species. O-atom transfer from the proposed ferryl to the sulfenate gives an Fe^II(sulfinate) product. Removal of the H-bonding groups in the BPA^Me2S⁻ ligand leads to a dramatically different O₂ activation mechanism. There are no spectroscopic signals observed for any Fe/O₂ intermediates in the reaction of 1 with O₂ at low temperature. However, reaction of 1 with O₂ at −20 °C does lead to a ferric hydroxide complex, 2, as the final product. Analysis by ESI-MS in combination with ¹⁸O labeling indicates that reaction of O₂ with 1 proceeds by an inner-sphere mechanism, where the OH⁻ in 2 is derived from O₂. The lack of reactivity of 1 with O₂ at room temperature is readily explained by a lack of binding of O₂ to the metal under these conditions.

A reasonable mechanism for the binding and activation of O₂ by 1 at low temperature is proposed in Scheme 4. It is based on proposed mechanisms of O₂ activation for other nonheme and heme iron complexes.^52–53 Initial binding of O₂ to 1 leads to a 6-coordinate Fe^III(O₂^−•) species, with the superoxide coordinated in an end-on fashion. This species reacts rapidly with another equivalent of iron(II) to give the peroxo-bridged di-ferric intermediate, which then undergoes homolytic O-O bond cleavage to produce the Fe^IV(O) species. This species then rapidly decays via H-atom abstraction from solvent to give the final Fe^III(OH) product.

Scheme 4.

Proposed mechanism for the formation of 2.

The final step involving solvent oxidation in the proposed mechanism in Scheme 4 was examined by running the reaction in deuterated THF. Exposure of 1 to excess O₂ in THF-d₈ followed by ESI-MS showed a clear shift of +1 in the major ion, from 359.06 to 360.07 m/z (Figure 6). This ion corresponds to [Fe^III(BPA^Me2S)(OD)]⁺, confirming that the source of the H atom in the final hydroxide product is derived from the solvent. These results are fully consistent with 2 being formed from H-atom abstraction by a ferryl intermediate in the final step in Scheme 4.

Figure 6.

ESI-MS data for 2 in THF in the presence of DHA (a), 2 in THF in the presence of d₄-DHA (b), 2 in THF-d₈ (c), 2 in THF-d₈ in the presence of DHA (d).

Additional evidence for the participation of an Fe^IV(O) species comes from interception by addition of an exogenous H-atom donor. Reaction of 1 + O₂ in the presence of 9,10-dihydroanthracene (9,10-DHA) led to formation of the Fe^III(OH) product and the dehydrogenated anthracene in ~60% yield. Replacement of 9,10-DHA with the deuterated version (9,10-DHA-d₄) led to the same increase of +1 m/z in the ESI-MS spectrum as seen for THF-d₈. The cross-over experiment, in which 1 + O₂ is carried out with excess 9,10-DHA in THF-d₈, gives only the proteo analog [Fe^III(BPA^Me2S)(OH)]⁺ (Figure 6), with no sign of deuteration. These results confirm that 9,10-DHA is the source of the hydrogen (deuterium) atom in the final product. These results are fully consistent with a short-lived, Fe^IV(O) intermediate being intercepted by 9,10-DHA, which is considerably more reactive than the solvent because of its relatively weak C-H bond (BDE = 76 kcal/mol). Further efforts were made to characterize the putative ferryl species and any other iron/oxygen intermediates (e.g. Fe^III(O₂^−•)) by examining the reaction of 1 + O₂ at temperatures as low as −95 °C, but only the slow formation of the final Fe^III(OH) species 2 was observed.

5. Conclusion

In summary, we have successfully synthesized a new thiolate-ligated, tetradentate ligand, BPA^Me2SH. This ligand was designed as a direct analog of the previously reported BNPA^Me2SH ligand, but without the hydrogen bonding amine groups in the second coordination sphere. The new Fe^II complex Fe^II(BPA^Me2S)(Br) (1) was reacted with O₂ at −20 °C and gave the high-spin, iron(III)-hydroxide complex (2), similar to one of the products seen in the reaction of Fe(BNPA^Me2S)(Br) and O₂. However, no spectroscopic evidence for an Fe^III(O₂^−•) intermediate or other Fe/O₂ species was observed, and there was no indication of S-oxygenation for the [Fe(BPA^Me2S)]⁺ complex. The O₂ reactivity for 1 is clearly different from that of Fe(BNPA^Me2S)(Br), which leads to the observation of a side-on bound superoxide complex [Fe(BNPA^Me2S)(O₂^−•)]⁺ at low temperature, and an Fe^II(sulfinate) product. The combined data indicate that 1 activates O₂ via an inner-sphere mechanism (Scheme 4), but this mechanism differs significantly from the mechanism shown in Scheme 1. We suggest that the H-bonding groups in [Fe(BNPA^Me2S)]⁺ may be critical for stabilizing a side-on, as opposed to end-on, superoxide intermediate, which then leads to the S-oxygenation pathway in Scheme 1. In the absence of the H-bonding and steric protection afforded by the second coordination sphere neopentylamino substituents in [Fe(BNPA^Me2S)]⁺, we speculate that O₂ may bind through an end-on superoxide species, Fe^III(BPA^Me2S)(O₂^−•), which then leads to facile formation of a dinuclear, peroxo-bridged intermediate. The deuteration experiments with THF and the exogenous donor 9,10-DHA provide strong evidence for an O₂-derived intermediate that must abstract hydrogen from the C-H bonds of either the solvent or the substrate immediately prior to Fe^III(OH) formation. An Fe^IV(O) species is perhaps the most likely intermediate to abstract hydrogen from C-H bonds to give the Fe^III(OH) product, and follows nicely from O-O cleavage of a peroxo-bridged differic precursor. Further work is underway in our laboratory to examine the factors that control O₂ activation at nonheme iron centers and the requirements for S-oxygenation, C-H activation, and other oxidative transformations.

5.1. Experimental section

All syntheses and manipulations were conducted in an N₂-filled drybox (Vacuum Atmospheres, O₂ < 0.2 ppm, H₂O < 0.5 ppm) or using standard Schlenk techniques under an atmosphere of Ar unless otherwise noted. Bis(2-pyridylmethyl)amine was purchased from TCI America. ⁵⁷Fe metal (99.9%) and ¹⁸O₂ (98 atom %) were purchased from Sigma-Aldrich. All other reagents were purchased from commercial vendors and used without further purification. Acetonitrile and acetonitrile-d₃ were distilled using CaH₂. Tetrahydrofuran was dried over Na/benzophenone and subsequently distilled. Diethyl ether was obtained from a PureSolv solvent purification system (SPS). All solvents were degassed by a minimum of three freeze−pump−thaw cycles and stored over freshly activated 4 Å molecular sieves in the drybox following distillation.

The ¹H, ¹³C NMR spectra were measured on a Bruker 300 MHz and 400 MHz spectrometer. UV−vis experiments were carried out on a Cary 60 UV−vis spectrophotometer equipped with a Unisoku USP-203A cryostat using a 1 cm modified Schlenk cuvette. Electrospray ionization (ESI) mass spectra were acquired with an AccuTOF^™ LC-Express Time-of-Flight Mass Spectrometer. Theoretical isotopic clusters were generated using msAxel isotopic pattern simulator (version 2.1 (2.1.1.1)). The experimental data was simulated by manually combining different relative percentages of each pure isotopic cluster, and then the best overall simulation was selected by comparing the theoretical ratio of intensities for peaks at 361 and 359 m/z versus the ratio of intensities in the experimental data. Mössbauer spectra were recorded on a spectrometer from SEE Co. (Edina, MN) operating in constant acceleration mode in a transmission geometry. The sample was kept in an SVT-400 cryostat from Janis Research Co. (Wilmington, MA), using liquid N₂ as a cryogen for 80 K measurements. Isomer shifts were determined relative to the centroid of the spectrum of a metallic foil of α-Fe collected at room temperature. Data analysis was performed using version F of the program WMOSS, and quadrupole doublets were fit to Lorentzian line shapes. Electron paramagnetic resonance (EPR) spectra were recorded with a Bruker EMX spectrometer equipped with a Bruker ER041 X G microwave bridge and a continuous-flow liquid helium cryostat (ESR900) coupled to an Oxford Instruments TC503 temperature controller for low temperature data collection.

5.2. Crystallographic data collection and structure refinement

All reflection intensities were measured at 110(2) K using a SuperNova diffractometer (equipped with Atlas detector) with Mo Kα radiation (λ = 0.71073 Å) under the program CrysAlisPro (Version CrysAlisPro 1.171.42.49, Rigaku OD, 2022). The same program was used to refine the cell dimensions and for data reduction. The structure was solved with the program SHELXS-2018/3 (Sheldrick, 2018) and was refined on F² with SHELXL-2018/3 (Sheldrick, 2018). Numerical absorption correction based on gaussian integration over a multifaceted crystal model was performed using CrysAlisPro. The temperature of the data collection was controlled using the system Cryojet (manufactured by Oxford Instruments). The H atoms were placed at calculated positions using the instructions AFIX 23, AFIX 43 or AFIX 137 with isotropic displacement parameters having values 1.2 or 1.5 U_eq of the attached C atoms. The structure is ordered.

5.3. Theoretical calculations

All calculations were performed with the ORCA-5.0.2 program package.⁵⁴ Starting geometry was derived from the single crystal X-ray structure of 1. For geometry optimizations, the CAM-B3LYP functional was employed in conjunction with the 6–31g* basis set on C and H atoms and the def2- TZVP basis set on all other atoms (Fe, S, N, O). The RIJCOSX approximation was implemented to reduce computation time. All optimizations utilized the CPCM solvation model for THF. Frequency calculations at the same level of theory confirmed that all optimizations had converged to true minima on the potential energy surface (i.e., no imaginary frequencies). The optimized structures obtained using the CAM-B3LYP functional were used for all Mössbauer parameter calculations. Mössbauer parameters were computed using the B3LYP functional and basis sets CP(PPP)⁵⁵ for Fe and def2-TZVP^56–57 for all other atoms. The isomer shift was obtained from the electron density at the Fe nucleus, using a linear fit function previously reported: δ = α(ρ(0)– c)+β. For the methodology described here, α = −0.44024 mm s⁻¹a.u., β= 2.1042 mm s⁻¹, and C = 11813 au. The calibrated quadrupole splitting was obtained from a linear fit function: |ΔE_Q|_calibrated = η(|ΔE_Q|_calc) − B₀ with η = 0.84003 and B₀ = −0.0019275 mm s⁻¹.⁴⁰

5.4. Synthesis of BPA^Me2SH

The starting material bis(2-pyridylmethyl)amine (996 mg, 4.98 mmol) was added in CH₃CN to a schlenck flask under Ar. An amount of isobutylene sulfide (661 mg, 7.50 mmol) was added, and the reaction mixture was heated at 75 °C for 12 h. The reaction mixture was cooled to 23 °C which was then dried under vacuo, giving a yellow oil. The product was purified by neutral alumina column using EtOAc/hexanes as eluent to give a yellow oil. The ¹H NMR spectrum showed a mixture of thiol and disulfide product, which was used in the next step without further purification.

Lithium aluminum hydride (110 mg, 2.95 mmol) was added to anhydrous diethyl ether (50 mL) at 0 °C and stirred for 10 min. An amount of crude mixture of BPA^Me2SH and disulfide (211 mg, 0.37 mmol) was added, and the suspension was stirred for an additional 30 min. The reaction mixture was warmed to 23 °C and stirred for 18 h, during which time a yellow solution was formed. The reaction mixture was quenched by the addition of a few drops of distilled water, and then after addition of ethyl acetate, the reaction was washed with brine, and dried over Na₂SO₄. The volatiles were removed under vacuo to obtain a yellow oil, which was purified by silica gel chromatography using CH₂Cl₂ as eluent. The pure product was isolated as a pale-yellow oil; 87 mg (82%). ¹H NMR (CD₃CN, 400 MHz): δ 8.50 (d, 2H), 7.67 (t, 2H), 7.48 (d, 2H), 7.18(m, 2H), 3.93 (s, 4H), 2.91 (s, 2H), 2.24 (s,1H), 1.22 (s, 6H) ppm.

5.5. Synthesis of 1

The ligand BPA^Me2SH (46.3 mg, 0.16 mmol) was dissolved in THF (2 mL) and a suspension of NaH (3.8 mg, 0.16 mmol) in THF was added. An amount of FeBr₂•2THF (57.9 mg, 0.16 mmol) was dissolved in acetonitrile (1.5 mL) and added dropwise to the ligand/NaH mixture. An immediate color change from pale yellow to dark orange was noted, and the reaction mixture was stirred for 6 h. The resulting orange reaction mixture was evaporated to dryness under vacuum giving an orange solid. The orange solid was re-dissolved in THF, filtered through celite, and the solution was left to stand with slow vapor diffusion of pentane at room temperature. Orange crystals: 43.7 mg (65 %) suitable for X-ray structure determination were obtained after 48 h. ¹H NMR (CD₃CN, 400 MHz): δ 135.91, 96.58, 76.74, 57.52, 40.51, 7.29, 6.35, 4.35, 2.31, −9.6 ppm. UV−vis (THF): λ_max = 365 nm (ε = 1460 M⁻¹cm⁻¹), 450 nm (ε = 1050 M⁻¹cm⁻¹). HRMS (ESI) m/z: calcd for [M]⁺= 420.99069, observed mass: 420.9907.

5.6. Preparation of samples for Mössbauer spectroscopy

A solution of ⁵⁷Fe-enriched 1(⁵⁷Fe) (5.2 mM, 250 μL) in THF was transferred to a Delrin Mössbauer cup and cooled to −20 °C. Excess O₂ was bubbled through the solution of 1(⁵⁷Fe), resulting in the formation of dark green solution of 2. The solution of 2 was frozen in liquid nitrogen and stored at 77 K until it was loaded into the Mössbauer spectrometer.

5.7. Preparation of samples for EPR spectroscopy

A stock solution of Fe(BPA^Me2S)Br (1) was prepared in 2-MeTHF (2.1 mM). An aliquot of the stock solution (300 μL) of 1 was transferred into a 4 mm EPR tube and sealed with a septum in the glovebox. The tube was removed from the glove box and cooled to −20 °C. Excess O₂ was then bubbled directly through the solution of 1 resulting in a color change from orange to green, and the reaction was allowed to proceed for 15 min with frequent manual mixing. The sample was then slowly annealed in liquid nitrogen and stored at 77 K until needed.

5.8. Preparation of samples for ESI-MS for reaction of 1 and O₂ in the presence of excess 9,10-DHA

Crystalline 1 (6 mg, 0.014 mmol) was dissolved in THF and an amount of 9,10-DHA (50.5 mg, 0.28 mmol, 20 equiv) was added. Excess, dry O₂ was bubbled through the solution at −20 °C, and the color of the solution changed from orange to the green of 2. The reaction mixture was allowed to stir for 30 min at −20 °C and then was analyzed by ESI-MS (+ ion mode).

Supplementary Material

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jinorgbio.2024.112776.

Scheme 5.

Reaction of 1 + O₂ in the presence of the H-atom donor 9,10-DHA.

Abbreviations

TauD

taurine dioxygenase

TDOs

thiol dioxygenases

CDO

cysteine dioxygenase

BPA^Me2SH

1-(bis(pyridin-2-ylmethyl)amino)-2-methylpropane-2-thiol

BNPA^Me2SH

bis((6-(neopentylamino)pyridinyl) methyl)amino)-2-methylpropane-2-thiol

ESI-MS

electrospray ionization mass spectrometry

EPR

electron paramagnetic resonance

DFT

density functional theory

XRD

x-ray diffraction

9,10-DHA

9,10-dihydroanthracene