Characterized cis-FeV(O)(OH) intermediate mimics enzymatic oxidations in the gas phase

FeV(O)(OH) species have long been proposed to play a key role in a wide range of biomimetic and enzymatic oxidations, including as intermediates in arene dihydroxylation catalyzed by Rieske oxygenases. However, the inability to accumulate these intermediates in solution has thus far prevented their spectroscopic and chemical characterization. Thus, we use gas-phase ion spectroscopy and reactivity analysis to characterize the highly reactive [FeV(O)(OH)(5tips3tpa)]2+ (32+) complex. The results show that 32+ hydroxylates C–H bonds via a rebound mechanism involving two different ligands at the Fe center and dihydroxylates olefins and arenes. Hence, this study provides a direct evidence of FeV(O)(OH) species in non-heme iron catalysis. Furthermore, the reactivity of 32+ accounts for the unique behavior of Rieske oxygenases. The use of gas-phase ion characterization allows us to address issues related to highly reactive intermediates that other methods are unable to solve in the context of catalysis and enzymology.

H igh-valent iron species are highly reactive molecules involved in numerous oxidative processes of synthetic and biological relevance 1,2 . In particular, Fe(V) intermediates have been proposed as the oxidation agents in key organic synthesis reactions, such as C-H, C=C and arene oxidations, and in energy-related transformations, including water oxidation 3 . Moreover, iron-dependent enzymes such as cytochrome P450 and Rieske oxygenases presumably use formal Fe(V) intermediates to oxidize inert substrates, including alkanes or arenes. Cytochrome P450 has been shown to use an oxoiron(IV) porphyrin cation radical intermediate termed compound I (cpd I) in C-H oxidation reactions 4 , whereas Rieske oxygenases may use a non-detectable oxoiron(V) intermediate in the syn-dihydroxylation of arenes and in metabolic C-H oxidations, although no direct evidence has been reported thus far [5][6][7] . Oxoiron(V) complexes are extremely challenging targets for synthetic inorganic chemistry because of their high reactivity. Accordingly, no crystal structure is available, and spectroscopically characterized examples remain exceedingly rare [8][9][10][11][12][13] .
Inspired by iron oxygenases, chemists have intensively exploited iron coordination complexes as catalysts, also thanks to the availability of this metal 14 . Complexes with tetradentate aminopyridine ligands are particularly interesting because they can use hydrogen peroxide to catalyze enzyme-like stereoretentive C-H and C=C oxidations (Fig. 1a) [15][16][17] . Extensive mechanistic studies based on product analysis, isotopic labeling and computations have indirectly shown that these complexes operate via Fe V (O)(X) (X = alkyl carboxylate or OH) reactive species [18][19][20][21] .
In addition, this catalyst shows outstanding selectivity properties; for example, olefins are highly chemoselectively syn-dihydroxylated while epoxidation is largely minimized. Moreover, electron-deficient olefins, and arenes, unreactive to Os-based reagents, are instantaneously dihydroxylated, thus indicating the involvement of extraordinarily powerful oxidizing species.
Before the present study, Fe V (O)(OH) species have been detected only by mass spectrometry (MS), and their formulation was derived from experiments using isotopically labeled reagents (H 2 18 O and H 2 18 O 2 ) 30,31 . Although their reactivity has been inferred from MS analysis of catalytic reaction mixtures, their spectroscopic characterization and direct assessment of their reactivity has not been performed yet.
Herein, we spectroscopically characterized the proposed [Fe V (O)(OH)( 5tips3 tpa)] 2+ reactive intermediate in the gas phase by helium tagging infrared photodissociation (IRPD) spectroscopy 32 . We conclusively identify the terminal Fe V =O and Fe V -OH stretching vibrations of the Fe(O)(OH) unit. Furthermore, we confirm that [Fe V (O)(OH)( 5tips3 tpa)] 2+ hydroxylates C-H bonds in a rebound mechanism and performs the syn-dihydroxylation of alkenes and arenes. These reactions, previously described in enzymes and bioinspired oxidation catalysts, have only been previously understood based on product analysis and computational methods 5,6,19,30,[33][34][35] .
Thus, the present study reports the experimental characterization of the Fe V (O)(OH) species and demonstrates its chemical competence in bioinspired reactions, particularly in reactions relevant to Rieske oxygenases.

Results
Generation and ion-spectroscopy characterization of intermediates. The reaction of 1 (0.4 mM) with H 2 O 2 (10 equiv.) in acetonitrile at −40°C, monitored by ultraviolet-visible (UV-vis) spectroscopy, produces a metastable purple species 2 (λ max = 544 nm, ε = 1300 M −1 cm −1 ) ( Fig. 2 and Supplementary Fig. 2). After 2 was formed in acetonitrile solution, the reaction mixture was analyzed by electrospray ionization mass spectrometry (ESI-MS). Two peaks at m/z = 444 and 424 stand out in the ESI-MS spectrum ( Supplementary Fig. 1 (3 2+ ), wherein the O-O bond has been broken. Using heliumtagging IRPD spectroscopy 32 we were able to measure IR spectra of the mass-selected ions with m/z 424 generated by electrospray ionization from the solution of 2 and ascertain that these indeed correspond to [Fe V (O)(OH)( 5tips3 tpa)] 2+ (3 2+ ).
We measured the IRPD spectrum of the ions (corresponding to the iron(V) intermediate 3 2+ ) generated from the reaction mixture of 1 and H 2 Fig. 4e). We also considered the doublet state, but the calculations predict that this state is 12.3 kcal mol −1 higher in energy than the quartet state. Its predicted IR spectrum is quite similar to that of the quartet state complex, except for a higher frequency of the Fe=O stretching vibration (Supplementary Fig. 4b).
The The isotopic composition of 3 2+ shows that both oxygen atoms originate from a single H 2 O 2 molecule, thus indicating that its formation is not assisted by a water molecule (Fig. 1b). In contrast, isotopic analyzes of diol products formed in catalytic  is formed in solution with the assistance of a water molecule, that is, 3 2+ contains one oxygen atom from H 2 O 2 and another from water 26 . Therefore, 3 2+ must be formed by different mechanisms in solution and in gas phase. DFT calculations suggest that 3 2+ is more than 6 kcal mol −1 lower in energy than 2a 2+ in the gas phase (see Supplementary Table 1). Therefore, elimination of acetonitrile from 2 2+ in the gas phase may likely lead directly to the rearranged product 3 2+ . The electronic spectrum of 3 2+ could be also determined by photodissociation spectroscopy (Supplementary Fig. 5). The spectrum is characterized by two absorption bands at 440 nm and 530 nm, corresponding to a charge transfer transition, which is also well reproduced by TD-DFT calculations of 4 3 2+ .
The complex 4 3 2+ is one of the few spectroscopically characterized Fe V =O complexes thus far (Table 1)  Fe=O bond is along z-axis). Interestingly, the related iron(V) complex with oxo and acyloxo ligands had the doublet ground state (Table 1) 13 . According to our calculations, replacing of OH by CH 3  Reactivity studies. After establishing the structure of the iron(V) intermediate, we probed its reactivity with a series of substrates in collisional experiments in the gas phase 43,44 . We studied reactions of mass-selected ions where each ion interacted with only one molecule of a given reactant R. The detected ionic products are thus formed from a well-defined reactant complex [ 4 3 2+ •R] without involvement of any additional molecules such as water or another reactant molecule. The reactions of 4 3 2+ proceed efficiently, attesting its high reactivity. Remarkably, when the ion corresponding to [Fe III (OOH)(CH 3 CN)( 5tips3 tpa)] 2+ (2 2+ , m/z = 444.3) was tested in similar experiments, no reactivity was observed ( Supplementary Fig. 7). This lack of reactivity of the hydroperoxide species against organic molecules reproduces well the rather sluggish oxidant character of these species in solution 25,45 .
The reaction of 4 3 2+ with 1,3-cyclohexadiene also dominantly leads to the adduct resulting from the dihydroxylation of an olefinic site alongside with the oxygen transfer reaction (Supplementary Table 2). The addition reaction is accompanied in approximately 10% by subsequent water elimination probably driven by restoring the conjugated double bond system.
We rationalized the reaction pathways based on experiments with deuterated complex ([Fe V (O)(OD)( 5tips3 tpa)] 2+ ( 4 3 2+ ( 2 H), Fig. 4b). The deuterium atom allows us to follow the subsequent fragmentation pathways. The initially formed adduct complex (the dihydroxylation product) is long-lived and therefore allows for hydrogen scrambling (complex is isolated in the gas phase and does not interact with any other molecules/ions) 46,47 . Note that this complex is isolated in the gas phase, therefore contains the   Fig. 4 Ion-molecule reactivity of 3 2+ and 3 2+ ( 2 H) in the gas phase. a 0.1 mTorr of cyclohexene, b 0.1 mTorr of 1,3-cyclohexadiene, c < 0.1 mTorr of naphthalene, and d 0.2 mTorr benzene (asterisks indicate impurities from previous measurements). All reactions were measured at nominally zerocollision energy determined from the retarding potential analysis energy released by the exothermic interaction between the reactants and cannot dissipate this energy by interaction with other molecules. The subsequent dehydration of the adduct thus features as elimination of HDO or H 2 O in the 3:2 ratio.
Most interestingly, in the formal oxygen transfer reaction, 4 3 2+ ( 2 H) yielded not only the expected product ([Fe III (OD) ( 5tips3 tpa)] 2+ , m/z = 416.3), but also a product in which the OD group was replaced by OH (i.e. [Fe III (OH)( 5tips3 tpa)] 2+ , m/z = 415.8). This observation can be explained by a two-step rebound mechanism. In the first step, hydrogen atom abstraction generates [Fe IV (OD)(OH)( 5tips3 tpa)] 2+ and a short-lived carboncentered radical. The radical can be then rebound with either OH or OD from the Fe(OH)(OD) unit, to finally form the corresponding alcohol. The observation of rebound mechanism contrasts with the previously-reported reactivity of iron(IV)-oxo complexes in the gas phase, where the observed oxygen transfer is exclusively due to the epoxidation of C=C double bonds 36 . The opening of the C-H activation pathway in the reaction with 1,3-cyclohexadiene increases the overall abundance of the formal oxygen atom transfer channel over cyclohexene (see Supplementary Table 2). This path occurs in the reaction with 1,3-cyclohexadiene but not with cyclohexene because the latter has a stronger C-H bond (BDE C-H = 74.3 vs 87.0 kcal mol −1 ) 48 . Exactly the same product pattern, identified in all previous reaction channels, was also observed in the reaction of 4 3 2+ ( 2 H) with 1,4-cyclohexadiene ( Supplementary Fig. 6).
Lastly, we investigated reactions of 4 3 2+ with aromatic compounds. Reactions with benzene and naphthalene yield addition products followed by water elimination. Furthermore, only in the case of naphthalene, we also observed a single electron transfer reaction, yielding the naphthalene radical cation and a product of single-electron reduced 4 3 2+ 49,50 . Because gas phase reactions only occur when they are exothermic, the electron affinity of 4 3 2+ must be higher than ionization energy of naphthalene (8.14 eV.) 51 . In turn, this value is higher than the electron affinities of oxoiron(IV) porphyrin cation radicals (cpdI models), which are always lower than 7.5 eV, thus indicating that 4 3 2+ is a stronger one-electron oxidant than oxoiron(IV) porphyrin cation radicals 50,52 . The addition/water elimination reaction is similar to reactions with cyclohexadiene reactants, but the reaction fully shifts towards final water elimination. The final product regains aromaticity, thereby likely driving the dehydration step kinetically and thermodynamically. This is particularly relevant in the gas phase because the initially formed syn-dihydroxylated product cannot be stabilized by interaction with solvent molecules. On the contrary, we observed the catalytic syn-dihydroxylation of naphthalene by complex 1 and H 2 O 2 in solution (see supporting information) as also previously observed in reactions with the [Fe(CH 3 CN) 2 (tpa)] 2+ complex 53 . We also probed the reaction of 3 2+ with D 6 -benzene, and we observed addition followed by HDO elimination with almost 100% selectivity (Fig. 4d). This reaction is highly interesting because these substrates are inert against high-valent Ru and Os oxides and, therefore, show the uniquely powerful oxidation ability of 4 3 2+ .

Discussion
The current study describes the vibrational and electronic spectroscopic characterization of Fe V (O)(OH) species with a key role in biomimetic oxidations. These Fe V (O)(OH) species have long been proposed to be ultimately responsible for a wide array of oxidations, including enzymatic reactions. However, the inability to accumulate them in solution has thus far prevented their spectroscopic and chemical characterization. Herein, we used gas phase ion spectroscopy methods to address this problem. The electronic and vibrational spectra of these species were finally determined, providing experimental data to unambiguously determine its atomic and electronic structure. Gas-phase reactivity analysis of these well-defined species showed their competence in C-H hydroxylation and syn-dihydroxylation of olefins and arenes. Overall, the data highlights that the particular architecture of the Fe V (O)(OH) species, featuring two reactive ligands in cis-relative positions, translates into singular reactivity properties, unattainable with hemes. For example, high-valent heme iron-oxo complexes consistently epoxidize olefins 4 . However, the current study shows that Fe V (O)(OH) species readily engage in syn-dihydroxylation rather than in epoxidation reactions and, most remarkably, react with arenes. Furthermore, gas phase studies on the hydroxylation of C-H bonds provide direct experimental evidence of a stepwise rebound mechanism, wherein rebound can occur with two different ligands at the Fe center. This behavior differs from that observed in reactions with previously described synthetic Fe IV =O complexes, which engage in HAT followed by diffusion of the carbon-centered radical 36,54,55 . Furthermore, this study provides experimental evidence of the rebound of the carbon-centered radical with the two cis-labile ligands at the iron center, which is not possible for hemes because HAT and rebound can only occur at the same oxygen atom 4 . Conversely, in non-heme iron-dependent enzymes and model complexes 56 , the presence of labile sites adjacent to the Fe=O moiety enables the transfer of the incipient hydroxyl ligand, or of ligands adjacent to the ferryl. For example, halides, azides and nitrates are transferred in non-heme halogenases 57,58 .
Finally, the report shows that gas-phase ion characterization can address questions relevant to catalysis and enzymology, related to highly reactive intermediates, currently unanswerable by other methods. Gas phase reactivity. Mass-spectrometric measurements were performed in a TSQ 7000 quadrupole-octopole-quadrupole spectrometer 43,44 . The ions were transferred to the gas phase using an ESI ion source. Ionization conditions were typically: 6 kV spray voltage, 0 V capillary voltage, 90 V tube lens voltage, 150°C capillary temperature, 30 psi sheath gas pressure, 300 l h −1 auxiliary gas flow. The spray voltage was connected directly to the solution in the vial with a stainless steel wire. The solution was kept at −33°C and was pumped into the ESI source through a 30-cm-long fused-silica capillary with a 100-µm internal diameter bỹ 1 psi overpressure of nitrogen gas in the vial with the solution. The ions of interest were mass-selected by the first quadrupole and transferred to the octopole equipped with a collision cell; collision gas pressure was determined using a baratron. The collision energy was set to nominally zero, as determined by retarding potential analysis. The products were extracted from the octopole to the second quadrupole, mass-analyzed and detected with a Daly-type detector.

Methods
Helium-tagging infrared/visible photodissociation (IRPD/visPD) spectroscopy. IRPD/visPD spectra were measured with the ISORI instrument based on the TSQ 7000 platform 32,59,60 . The ions were generated and mass selected exactly as above. The mass-selected ions were transferred via a quadrupole bender and octopole to a cryogenic ion trap operating at 3 K. The ions were trapped with 250 µs helium pulse and formed weakly bound complexes with helium. The trapped ions were irradiated by IR light from an OPO/OPA system or by visible light from a continuum laser wavelength-filtered by acusto-optic tunable filter. After irradiation, all ions were ejected from the trap, mass-analyzed in a second quadrupole and counted by a Daly-type detector. IRPD spectra are plotted as wavenumber-dependent attenuation of the number of helium complexes (1−N i (ν)/N i0 ). The total number of the helium complexes (N i0 ) was obtained in alternative cycles with blocked photon beam. The visPD spectra were corrected by dividing the attenuation by the laser power.
DFT calculations. DFT calculations were performed with Gaussian 09 61 at B3LYP-D3/def2TZVP level. All structures were fully optimized and characterized by frequency calculations. The frequencies in IR spectra were scaled by 0.99. Reported energies include zero-point vibrational energy corrections; the molecular coordinates are provided in the Supplementary Note 1 and the relative energies in Supplementary Table 3.