Simultaneous nitrosylation and N-nitrosation of a Ni-thiolate model complex of Ni-containing SOD

Nitric oxide reacts with a NiSOD model complex to yield a thiolate-ligated/N-nitrosated {NiNO}10 species with unusually labile Ni–NO bonds.


Introduction
Nitric oxide (NO) and its derivatives (termed reactive nitrogen species or RNS) play a vital role in a variety of mammalian (and in some cases bacterial) physiological and pathological processes. [1][2][3][4] Additionally, this gaseous free radical has applications in fundamental research, especially in bioinorganic chemistry, where it is utilized as a structural/spectroscopic probe of O 2 (and other reactive oxygen species, e.g., O 2 c À and H 2 O 2 ) binding/activating metalloenzymes. [5][6][7][8][9] In general, this approach is employed because metal-nitrosyl (MNO) bonds are highly covalent, and hence more stable, than metal-dioxygen (M-O 2 ) adducts. 10 The use of NO as an O 2 analogue is based on similar electronic structures between these diatoms and their reduced derivatives. 3 For example, 3 NO À (termed the nitroxyl anion), the one electron reduced analogue of NO, is isoelectronic with O 2 with two unpaired p* electrons in the HOMO. Additionally, NO, while not isoelectronic with O 2 c À , has the same ground state electronic structure with a singly occupied p* MO. Thus, NO interactions with the active sites of O 2activating/ROS-breakdown enzymes report coordination (innersphere substrate binding) and the extent of substrate bond activation from vibrational spectroscopic measurements of the N-O and M-NO stretching frequencies.
Since 2009, our lab has designed and constructed numerous low molecular weight models of the active site of Ni-containing superoxide dismutase (NiSOD). [11][12][13][14][15][16][17][18] NiSOD is an unprecedented SOD due to Ni III/II -coordination to cysteinato-S (CysS) and peptido-N donors (Chart 1), the former of which is susceptible to oxidative modication by the substrate (O 2 c À ) and products (O 2 and H 2 O 2 ) of the SOD catalyzed reaction. 19,20 Few models employ ligands with the correct spatial disposition and electronic nature of the unique N 3 S 2 donor set found in the active site. [21][22][23] Moreover, fewer report reversible electrochemical and/or spectroscopic evidence for the Ni III oxidation state due to redox associated with the coordinated thiolates. One model from our lab, namely Et 4 N[Ni(nmp)(SPh-o-NH 2 -p-CF 3 )] (1; nmp 2À ¼ dianion of the N 2 S ligand N-(2-mercaptoethyl) picolinamide; see Chart 1) displays a reversible redox-event at À0.43 V (vs. Fc/Fc + in DMF) that, based on EPR, UV-vis, MCD, and DFT computations, represents the electrochemical conversion from Ni II in 1 to a Ni II -thiyl 4 Ni III -thiolate resonance species termed 1 ox . 16 Because substrate binding to Ni in NiSOD has not been dened, although most reports favor an outer-sphere mechanism, 15,24 we were curious to use NO as an O 2 c À probe to dene potential intermediates that may be traversed in the NiSOD mechanism. We report here, for the rst time, the reactions and product characterization of NO (and NO + ) with 1 and the well-dened analogue of NiSOD ox (1 ox ). NO/ NO + oxidize the aromatic thiolate ligand in 1 ox and 1, respectively. However, introduction of NO to 1 affords the green dimeric {NiNO} 10 complex (Et 4 N) 2 [{Ni(k 2 -SPh-o-NNO-p-CF 3 )(NO)} 2 ] (2) via NO-induced loss of nmp 2À as the disulde and N-nitrosation of the aromatic thiolate (Chart 1). While 2 bears little resemblance to NiSOD, its formation indicates how reactive NiSOD models such as 1 are in the presence of redoxactive diatoms and suggest similar paths for other biological Ni-thiolate sites. Additionally, 2 contains a labile Ni-NO bond, a new feature for the {NiNO} 10 formulation that appears to be controlled by the presence of the thiolate ligands. We describe the synthesis, spectroscopy, electronic structure, reactivity and mechanistic insight into the formation of the Ni-nitrosyl in this account.

Results and discussion
In the anticipation of isolating a Ni-nitrosyl as an analogue of a potential Ni-superoxo/peroxo catalytic intermediate of NiSOD, we examined the reaction of 1 with NOBF 4 and in situ prepared 1 ox with NO (Scheme 1). In theory, both reactions yield the same product. For example, nitrosonium (NO + ; a strong oxidant, E ¼ +0.56 V vs. Fc/Fc + in DMF 25 ) will oxidize 1 to 1 ox and form NO in the process. The newly generated 1 ox (S ¼ 1/2) then reacts with NO to form the Ni-nitrosyl, formally a {NiNO} 8 complex, assuming binding of NO and no other coordination sphere changes, using the notation dened by Enemark and Feltham. 26 Likewise, NO will readily intercept paramagnetic 1 ox to generate the same species. Mixing a DMF solution of 1 with NOBF 4 (1 : 1) resulted in instantaneous bleaching of the solution, consistent with oxidation of the RS À ligand to disulde (RSSR), and the appearance of a dark-red precipitate that was spectroscopically identied to be the neutral S,S-bridged tetramer [Ni 4 (nmp) 4 ] (Scheme 1). 16 This outcome is typical for all [Ni(nmp)(SR)] À complexes when treated with chemical oxidants, i.e., Soxidation of the coordinated monodentate thiolate to RSSR. 15 Incidentally, the same result was obtained when introducing NO(g) into a DMF solution of in situ generated 1 ox . In this case, formation of the disulde may traverse a eeting, and yet to be characterized, RSNO intermediate that releases NO via homolytic cleavage of the RS-NO bond (Scheme 1). 27 Overall, a Ninitrosyl was not isolated. This result may not be too surprising considering that all known Ni-nitrosyls are in the {NiNO} 10 Enemark-Feltham (EF) classication, 28 although a {NiNO} 9/8 species is not entirely unrealistic in light of the strong donors present in 1 and in NiSOD, i.e., peptido-N and alkyl-thiolato-S.
As a control, we also explored the reaction of Ni II complex 1 with NO. In general, NO does not react with square-planar [Ni(nmp)(SR)] À (R ¼ simple aryl or alkyl groups) complexes due to their diamagnetic nature. However, when R contains a potentially bidentate chelate, as in 1, a different course takes place. For instance, exposing a DMF solution of 1 with NO(g) for 30 s resulted in a gradual change of the solution from dark-red to green over several minutes. Workup of this reaction indicated a Ni-nitrosyl based on the strong double-humped peak in the N-O stretching (n NO ) region of the IR spectrum (vide infra). Subsequent crystallization of the bulk material from MeCN/ Et 2 O at À20 C resulted in green crystals of a dinuclear thiolate-  Table 1). Complex 2 is analogous to the limited number of four-coordinate/S-bound Ni-nitrosyls, 30 Structures of coordinated nitroso-Nmetal complexes (vs. amine-N as in 2) also afford similar structural parameters in the RNNO. 48 In contrast, O-bound nitrosamine complexes appear to favor more of a resonance delocalized structure as the N-N (1.275-1.288Å) and N-O (1.251-1.275Å) distances in a series of [Fe III (P)(ONNR 2 ) 2 ] + (P ¼ porphyrin) complexes are nearly identical and result in a single 15 N-sensitive peak in the IR due to overlapping n NN /n NO modes. [49][50][51] Complex 2 was characterized by a variety of spectroscopic methods. The solid-state IR spectrum (KBr matrix) of 2 exhibits two closely spaced, but well-resolved, n NO at 1759 and 1743 cm À1 (1724, 1708 cm À1 for 2-15 NO; Dn NO : 35 cm À1 ; see Fig. 2). These values fall in the range of known tetrahedral, neutral, and anionic {NiNO} 10 complexes. 28 Because 2 is of C 2 symmetry (cis NO, syn bridging thiolates), two IR-active N-O vibrational modes are expected. The other feasible isomer of 2 would be of C i symmetry (trans NO, anti bridging thiolates) and would display one IR-active N-O stretch. Indeed, the IR spectrum of 2 in DMSO exhibits one n NO at 1784 cm À1 suggesting possible cis/ trans-NO conversion in solution (or an averaged n NO value due to rapid tumbling) or thiolate-bridge splitting to yield a four-   (Fig. 3). The bond lengths (Ni-S: 2.2072Å, Ni-N: 1.896Å) and angles (Table  S3 †) are similar to other planar Ni II -N 2 S 2 complexes that contain k 2 -N,S-o-aminobenzenethiolate ligands. [59][60][61] The Ni-N distance in 3 is shorter than the typical Ni-N amine bond and reects the enhanced donor strength of the deprotonated nitrosamino-N, which is comparable to, although weaker than, a Ni-N carboxamido ($1.86Å). 15,58 No evidence for a coordinated ligand radical is evident from the X-ray structure (i.e., short C-S, C-N distances of the coordinated o-aminobenzenethiolate 62  To conrm that NO(g) is released from 2 (forming 3 among other products), solutions of 2 were mixed with the NO(g) trap [Co(T(-OMe)PP)] (T(-OMe)PP ¼ 5,10,15,20-tetrakis(4-methoxyphenyl)-21H,23H-porphine). 63 For example, mixing 2 and the Co II -P (1 : 2) in CH 2 Cl 2 at RT for 24 h resulted in the {CoNO} 8 complex [Co(T(-OMe)PP)(NO)] in $70% avg. yield as quantied by 1 H NMR (CD 2 Cl 2 ) and further veried by IR spectroscopy using 2-15 NO (Fig. S21-S23 †). Notably, the reaction mixture becomes red over the course of the reaction. Workup of this solution aer separating the Co-P compounds (MeOHinsoluble) reveals the presence of 3 (MeOH-soluble) via 1 H NMR to conrm the fate of the {NiNO} 10 complex 2. To eliminate bimolecular NO-transfer via a putative Co/NO/Ni intermediate, NO(g) release was further veried by vial-to-vial trapping reactions wherein a CH 2 Cl 2 solution of the Co II -P was separated from an MeCN solution of 2 (Co II -P in excess, see the ESI †). Carrying out this reaction conrmed that NO(g) is indeed released from 2 (or 2-15 NO) to generate the {CoNO} 8 porphyrin complex (80% avg. yield) as shown by 1 H NMR and IR measurements (Fig. S24 †). In contrast, no reaction takes place between THF solutions of 2 with [Fe(TPP)Cl] (1 : 2; TPP ¼ 5,10,15,20-tetraphenylporphyrin), a common HNO (or NO À ) trap. 64 Although {NiNO} 10 has not been characterized as a particularly labile EF notation, we note that the majority of these complexes are cationic or neutral without coordinated thiolate ligands. 28 Indeed, the thiolate-ligated {NiNO} 10 complex III photochemically releases NO to [Co(TPP)] in MeCN suggesting some lability in the Ni-NO bond. Furthermore, the RN-NO bond is quite stable (as noted by formation of 3) and the energetically stabilized MOs that contribute to the electronic structure of 2 and 3 where HOMOÀ3 represents a bonding MO with primary contributions from s-NR and s-NO orbitals (Fig. S25 †).
Density functional theory (DFT) computations have provided a deeper understanding of the electronic structure of a variety of metal nitrosyls, 65,66 and we have employed them here for 2 and 3 at the OLYP/def2-TZVPP level of theory. Pure functionals such as BP86 and OLYP were used for geometry optimization and single point energy calculations, respectively, as these functionals have been established to deliver better matches with experimental geometries in MNO systems. 67-69 Geometry optimization of 2 was performed with coordinates from the crystal structure to yield DFT-optimized complex 2* (Fig. 4, Tables 1, S5 and S7 in the ESI †). Structurally, 2* replicates the metrics of 2 well, suggesting the computational model is reasonable. While the distances in 2* are within AE0.025Å of experimental values, the bond angles (especially S-Ni-S: À6.6 , and Ni-N-O: +3.6 from 2) are slightly beyond the allowable tolerances for satisfactory DFT performance in small molecules (i.e., distances AE0.03Å; angles AE1 ). 70 However, these rules may be broken to some degree because of the enhanced complexity arising from the covalent MNO unit in 2. The computations also reasonably match the two closely spaced N-O stretching frequencies for the symmetric and asymmetric n NO in the IR at 1730 and 1708 cm À1 , respectively. The $30 cm À1 downshi from 2 is likely due to a slight overestimation of Ni-NO bond covalency arising from Ni-dp backbonding. Previous calculations on three-71 and fourcoordinate 43,72 {NiNO} 10 complexes support a Ni II -3 NO À (S tot ¼ 0, antiferromagnetically coupled) oxidation state assignment. This is comparable to high-spin nonheme {FeNO} 7 systems that are classied as Fe III -3 NO À (S tot ¼ 3/2). 6,66,73 In the Fe case, 3 NO À serves as a strong p-donor to afford a highly covalent Fe-NO bond. 74 The strength of this interaction originates from the effective nuclear charge on the metal, which is controlled by the basicity of the supporting ligands. 75 Thus, electron rich supporting ligands attenuate the p-basicity of 3 NO À to result in diminished M-NO bond covalency. This property has been established in the {FeNO} 7 case, but not yet for {NiNO} 10 . Indeed, examination of the frontier MOs of 2* show that, much like other {NiNO} 10 systems with Tp ligands 43,72 (Tp ¼ tris(pyrazolyl)borate), the LUMO is a p* MO primarily comprised of antibonding interactions between Ni-dp and NO-p* orbitals (Fig. S25 †). On the other hand, the HOMO (Fig. 4) and HOMOÀ1 have little contribution from NO, but large contributions from Ni-ds (38.0%) and S-ps (19.3%) orbitals of the Ni(m-SR) 2 Ni core. The HOMO is antibonding in nature and suggests a thermally unstable structure. As expected from analogous {FeNO} 7 systems, based on the increased donor strength of the anionic nitrosamine-N/thiolate-S supporting ligands in 2*, the covalency in the Ni-N-O unit is less than in TpNi-NO complexes and rationalizes the observed lability of the Ni-NO bond and the Ni(m-SR) 2 Ni core in 2.
DFT computations on 3* were performed in the same fashion as for 2*. Geometry optimized 3* is square-planar (s 4 ¼ 0.09) with metric parameters on-par with the X-ray structure of 3 and within the error of the DFT method (Table S9 †). Unlike 2*, the p* HOMO of 3* is comprised primarily of Ni(dp)/S(pp) contributions (Fig. S26 †), typical of planar Ni II -N 2 S 2 complexes with strong-eld ligands and suggests a highly covalent Ni-SR bond. 15 The formation of 2 likely follows a mechanistic path analogous to those observed in the reductive nitrosylation of Cuamine systems, where one-equiv. of NO reacts with Cu II -NR 2 complexes to yield R 2 N-NO and deligated Cu I . 76,77 The difference here is that the nitrosated ligand remains coordinated and the resulting paramagnetic Ni binds NO radical. Our working model is depicted in Scheme 2. Complex 1 is likely in resonance with a distorted tetrahedral species which places the anilido-N in the coordination sphere. This proposal is supported by the presence of low intensity peaks in the 1 H NMR spectrum of 1 and may explain the difficulty in crystallizing this complex. 16 On the other hand, X-ray absorption spectroscopic (XAS) characterization of 1, not reported previously, suggests a four-coordinate planar Ni II center (XANES analysis, see Fig. S3 †) with two O/N-and S-ligands at 1.90Å and 2.17Å (EXAFS, Fig. S3, Table S4 †), respectively. Thus, 1 is structurally analogous to other [Ni(nmp)(SR)] À complexes at least in the solid-state. Introduction of NO(g) can then result in either: (i) reduction of Ni II to Ni I and formation of NO + that nitrosates the coordinated amine, or (ii) nitrosylation of Ni to yield {NiNO} 10 with the electron originating from the coordinated thiolate of nmp 2À to result in the disulde. Our results do not differentiate either of these transformations, but the disulde of nmp 2À (i.e., nmpS 2 1 H NMR and IR of the reaction mixture, see Fig. S19 and S20 †) is spectroscopically observed in the reaction mixture and checked against independently synthesized nmpS 2 . Thus, the fate of one proton and one electron is reasonably conrmed. At this point these intermediates can react with another equiv. of NO to yield the three-coordinate precursor to 2. Compound 3 forms through either disproportionation (shown in Scheme 2) to yield a Ni 0 species or ligand rearrangement via the loss of a Ni I -NO fragment (not shown). In ligand rearrangement, the products would be a Ni I -N 2 S 2 precursor to 3 (3-PC), an L-Ni I -NO species (L ¼ solvent), and free NO. Ultimately this Ni I intermediate oxidizes 3-PC to generate Ni II complex 3 and an L-Ni 0 -NO complex that would presumably release NO(g) as evidenced by the NO(g) trap experiments (vide supra). While the reaction mechanism for the conversion of 2-to-3 is likely more complex, similar chemistry has been proposed for N-heterocyclic carbene (NHC) Ni-nitrosyls. 28,78 The details of this mechanism are still under investigation.

Conclusions
In conclusion, NiSOD model complex 1 reacts with NO(g) in the Ni II state to form the metastable {NiNO} 10 dimeric complex 2 via loss of the nmp 2À ligand as the disulde and N-nitrosation of the o-aminobenzenethiolate ligand. Reaction of NO with 1 ox , or NO + with 1, only yields the S,S-bridged tetrameric compound [Ni 4 (nmp) 4 ] through oxidation of the aromatic thiolate ligand. While any reaction with NO (S ¼ 1/2) is generally unexpected for square-planar (S ¼ 0) Ni II complexes, this Ni-nitrosyl likely forms due to an equilibrium mixture of 1 and a tetrahedral (S ¼ 1) or ve-coordinate derivative (Scheme 2). Even if NO were to result in an nmp-bound Ni-NO complex, the resulting {NiNO} 9 (reaction of 1 with NO) or {NiNO} 8 (reaction of 1 ox with NO) oxidation levels have yet to be dened and support an outersphere superoxide interaction in NiSOD. Although these EF notations have yet to be accessed, one would propose that NiSOD mimetics, especially with strong-eld carboxamido-N and alkyl-thiolato-S donors, would surely stabilize such an electron poor species. Furthermore, the properties of complexes such as 2 extend to biology, where analogous S-bridged mononitrosyl species, i.e., Fe-S clusters and tetrahedral (RS) 3 Fe-NO complexes are proposed as intermediates in the repair of NO-damaged clusters. [79][80][81] Complex 2 is stable in the solid-state but breaks down slowly in solution causing rupture of the Ni(m-SR) 2 Ni core and release of NO that was trapped in near quantitative yield with a Co II -porphyrin receptor. The resulting Ni II -N 2 S 2 complex 3 (coordination of two o-aminobenzenethiolate in trans conguration) was isolated and structurally/spectroscopically characterized as the ultimate Ni breakdown product with the nitrosamine unit still intact. This release may take place through a disproportionation mechanism (or through ligand rearrangement), as has been proposed in other Ni-nitrosyls, to a yet ill-dened Ni 0 complex (see Scheme 2). 28

Conflicts of interest
There are no conicts to declare.