Why intermolecular nitric oxide (NO) transfer? Exploring the factors and mechanistic aspects of NO transfer reaction

Small molecule activation and their transfer reactions in biological or catalytic reactions are greatly influenced by the metal-centers and the ligand frameworks. Here, we report the metal-directed nitric oxide (NO) transfer chemistry in low-spin mononuclear {Co(NO)}8, [(12-TMC)CoIII(NO−)]2+ (1-CoNO, S = 0), and {Cr(NO)}5, ([(BPMEN)Cr(NO)(Cl)]+) (4-CrNO, S = 1/2) complexes. 1-CoNO transfers its bound NO moiety to a high-spin [(BPMEN)CrII(Cl2)] (2-Cr, S = 2) and generates 4-CrNOvia an associative pathway; however, we did not observe the reverse reaction, i.e., NO transfer from 4-CrNO to low-spin [(12-TMC)CoII]2+ (3-Co, S = 1/2). Spectral titration for NO transfer reaction between 1-CoNO and 2-Cr confirmed 1 : 1 reaction stoichiometry. The NO transfer rate was found to be independent of 2-Cr, suggesting the presence of an intermediate species, which was further supported experimentally and theoretically. The experimental and theoretical observations support the formation of μ-NO bridged intermediate species ({Cr–NO–Co}4+). Mechanistic investigations using 15N-labeled-15NO and tracking the 15N-atom established that the NO moiety in 4-CrNO is derived from 1-CoNO. Further, to investigate the factors deciding the NO transfer reactivity, we explored the NO transfer reaction between another high-spin CrII-complex, [(12-TMC)CrII(Cl)]+ (5-Cr, S = 2), and 1-CoNO, showing the generation of the low-spin [(12-TMC)Cr(NO)(Cl)]+ (6-CrNO, S = 1/2); however, again there was no opposite reaction, i.e., from Cr-center to Co-center. The above results advocate clearly that the NO transfer from Co-center generates thermally stable and low-spin and inert {Cr(NO)}5 complexes (4-CrNO & 6-CrNO) from high-spin and labile Cr-complexes (2-Cr & 5-Cr), suggesting a metal-directed NO transfer (cobalt to chromium, not chromium to cobalt). These results explicitly highlight that the NO transfer is strongly influenced by the labile/inert behavior of the metal-centers and/or thermal stability rather than the ligand architecture.


Introduction
In the biological system, the choice of metal-centers and the ligand frameworks signicantly inuence coordination chemistry, redox property, and various metalloenzymes' reactivity. 1 When it comes to small molecule activation, enzymatic and catalytic reactions, these properties become more prominent for selective biological reactions. For example, heme systems of particular proteins (NiRs) bind with nitric oxide (NO); 2 however, other proteins with similar heme groups do not show NO-activation, 3 and it is rare to have NO activation on different metal centers having the same coordination sphere. 4 Together, these parameters provide an excellent opportunity or challenge to the scientic community to study/explore the small molecules' interactions with metals in different ligand frameworks. Consequently, the transfer of coordinated ligands to another metal center has become the area of interest for the past few decades. Halide transfer reactions are one of the most explored reactions. 5 Some examples of intermolecular transfer of bound carbon monoxide (CO), 6 superoxide (O 2 c À ), 7 and peroxide (O 2 2À ) 8 were also reported. Nowadays, atom transfer reactions are an area of fundamental importance, particularly apparent in biological systems. 9 A number of metalloenzymes were reported to mediate the oxygen atom transfer (OAT) reactions, 10 specially molybdoenzymes such as nitrate reductase (NRs) 11 and C-H activation reactions. 12,13 NO activation became an area of interest for chemists and biochemists due to the vast presence of metal-nitrosyls (M-NOs) in the biological system. Being a radical species, NO is widely known as a signal transduction molecule for its involvement in major biological processes, such as neurotransmission, vascular regulation, platelet disaggregation, and immune response to bacterial infections. 13 The deciency of NO in the natural system hamper the processes described above and leads to several biological disorders, including atherothrombosis, 14 diabetic hypertension, 15 and chronic kidney disease. 16,17 In this regard, nitric oxide synthases (NOSs) 18 and nitrite reductases (NiRs) 2,19 accomplish the constant biosynthesis of NO. NOSs are a family of enzymes that catalyze the guanidine nitrogen oxidation in L-arginine to produce NO. 20 Whereas, in some mammalian and bacterial systems, NiRs catalyze the production of NO by NO 2 À reduction in the presence of protons, i.e., NO 2 À + e À + 2H + / NO + H 2 O. 21 In the biological system, NO acts as a double-edged sword, and overproduction can lead to the generation of several cytotoxic molecules. 22 The coordination chemistry of M-NOs has a long history of understanding the fundamental aspects of binding, electronic arrangements, and reactivity. 23 Until now, several M-NOs have been synthesized and explored for different reactivities, 4b,24 including even a few reports on intermolecular NO transfer; 25 however, the mechanistic aspects of these reactions have rarely been explored. An extensive study of the NO transfer process may enlighten the NO transport process in the biological system. Earlier, Armor reported acid-promoted metaldependent NO transfer from Co III to Cr II involving a labile aqua complex. 25e Further studies suggest the transfer of the NO ligand at high acidic conditions due to axial NO ligand protonation. 26 Caulton and co-workers, in a study of NO transfer reactivity from Co to various metal complexes of Fe, Ru, Rh, and Co complexes, proposed that the NO transfers readily to the coordinatively unsaturated metal complexes, either via a simple NO transfer or via halide exchange pathway. 25c Mu and Kadish speculated the effect of metal-center on a NO transfer reaction. 27 An intermediate species formation was also proposed before NO transfer, suggesting an association mechanism. 25a-e Cook and co-workers, for the rst time, reported a dissociative pathway in a NO transfer reaction from Co(NO)(dmgH) 2 to Hemoglobin (Hb). 28 Lippard and co-workers also testied a dissociated NO transfer pathway from Mn to Fe bearing a tropocarnado ligand. 25d A m-NO bridge intermediate was discussed in a NO transfer reaction of Ru to Fe-center of Hb and myoglobin (Mb); however, this has not been well identied or conrmed with experimental evidence. 25a Recently, Nam and co-workers reported NO transfer reactivity inuenced by ligand size and the spin-state of the acceptor/donor metal complexes. 29 These results suggest that the reaction mechanism and the intermediate involved in the intermolecular NO transfer reaction are inconsistent and depend on various factors; however, these reports lack a detailed mechanistic investigation. Here, the spectroscopic signatures for the proposed m-NO bridge intermediate species need to be characterized in detail, which will surely help the scientic community understand the mechanistic insights of NO transfer reactions.

Synthesis of Cr-nitrosyl complex [(BPMEN)Cr(NO)(Cl)]Cl (4-CrNO)
The initial Cr II -complex [(BPMEN)Cr II (Cl) 2 ] was synthesized by adding BPMEN ligand to a stirring solution of CrCl 2 (ESI, † Experimental Section (ES)). The addition of excess NO (g) (Fig. S1 in the ESI † shows a schematic diagram of the NO (g) purication and handling process) to the CH 3 CN solution of 2-Cr at RT under an Ar atmosphere resulted in the generation of 4-CrNO (l max ¼ 600, 3 ¼ 110 M À1 cm À1 , red line) within 5 min and isolated as a solid product ( Fig. 1a and S2a, ESI, † ES) (Scheme 1, reaction I). Electrospray ionization mass spectrometry (ESI-MS) of 4-CrNO showed a prominent ion peak at m/z 387.1, whose mass and isotope distribution patterns correspond to [(BPMEN) Cr(NO)(Cl)] + (calcd m/z 387.1) (Fig. 1b). When the reaction was carried out with , the mass peak corresponding to [Cr(BPMEN)( 15 NO)(Cl)] + appears at m/z 388.1 (calcd m/z 388.1) (inset, Fig. 1b and S2b; ESI †). This one unit shi in mass value upon substitution of NO by 15 NO indicates that 4-CrNO contains an NO moiety attached to the Cr-center. The FT-IR spectrum is also consistent with the exact formulation of 4-CrNO, revealing a linear NO with a typical M-NO stretching at 1690 cm À1 , which shied to 1650 cm À1 when 4-CrNO was prepared using 15 NO (inset, Fig. 1a, S2c and S2d; ESI †). 31 The 1 H NMR of 4-CrNO does not exhibit any signal for aromatic protons, suggesting a paramagnetic Cr-center (ESI, † Fig. S3a). Further, the magnetic moment calculated by Evans' method revealed a magnetic moment of 1.68, which corresponds to a low-spin Cr-centre (d 5 , S ¼ 1/2) in complex 4-CrNO (ESI, † Fig. S3b), which can be assigned as {Cr(NO)} 5 . 32 Further, 4-CrNO was structurally characterized via single-crystal X-ray crystallography. The NO ligand is coordinated to a Cr-center in an endon fashion with a distorted octahedral geometry. The Cr(1)-N(5)-O(1) bond angle of 4-CrNO was found to be 174.0(11) suggesting sp hybridization of N-atom, as explained in other reports. 31a Hence, NO coordination to the low-spin Cr-center was conrmed, which was further consistent with our assignment of this complex as {Cr(NO)} 5 species (Fig. 2, S4, Tables S1 and S2; ESI, † ES). 33

NO transfer reaction from 1-CoNO to 2-Cr
To further understand the NO-coordination chemistry, we explored NO transfer reactions between cobalt (1-CoNO) and chromium complexes (2-Cr & 5-Cr), having different or alike ligand frameworks to determine the fundamental parameters behind NO transfer chemistry. When CH 3 CN solution of 4-CrNO reacted with an equivalent amount of 3-Co under Ar atmosphere, we did not observe any signicant change in the UV-Visible spectra except a new band at 485 nm, corresponding to 3-Co (ESI, † Fig. S5). In contrast to the NO transfer from complex 4-CrNO to 3-Co, the reverse reaction, i.e., NO transfer from 1-CoNO to 2-Cr, showed a visible color change from pink to bluish-green. The addition of one equivalent of 2-Cr to 1-  CoNO immediately showed the generation of a new band (l max ¼ 700 nm, blue line) (Fig. 3a), proposed to be an intermediate, which then slowly changed to a new absorption band (l max ¼ 600 nm, red line), which corresponds to the absorption spectrum of 4-CrNO ( Fig. 3b and S6a; ESI †) (Scheme 2, reaction I & II). The nal UV-Visible spectrum of the above NO transfer reaction was conrmed as the sum of complexes 3-Co and 4-CrNO, further supporting the NO transfer from Co to Cr-center (ESI, † Fig. S6b). The NO transfer reaction was established by the spectroscopic titration at 370 nm for the decomposition of 1-CoNO, demonstrating that the ratio-metric equivalent of complex 2-Cr to 1-CoNO was 1 : 1 (Fig. 4). We further characterized the generation of this intermediate by FT-IR spectroscopy to follow the reaction mechanism. The addition of 2-Cr to the solution of 1-CoNO showed a new peak at 1520 cm À1 , which shied to 1491 cm À1 when the reaction was performed with 1-Co 15 NO (Inset, Fig. 3a), suggesting the generation of m-NO bridged species 34 {BPMEN-Cr-NO-Co-12-TMC} 4+ ({Cr-NO-Co} 4+ ), in the NO transfer reaction from Co to Cr-center. Additionally, EPR measurements were performed to trap the intermediate. In the low-temperature EPR measurements, we observed the formation of an intermediate species (g ¼ 2.03) that shied to a signal (g ¼ 1.99) (inset, Fig. 3b) characteristic to the EPR signal of 4-CrNO. This EPR spectrum suggests that the NO moiety is more on the Cr-center than Co-center in the {Cr-NO-Co} 4+ intermediate species, as both 1-CoNO and 2-Cr are EPR silent and only 4-CrNO showed a peak in this range of gvalues. Then, we further tried to characterize the intermediate via ESI-MS. However, being a thermally unstable moiety, the intermediate species decomposes under the mass spectrometry instrumental environment even at low temperatures and gives a peak for 4-CrNO and 3-Co, and efforts to characterize the intermediate become futile.
Furthermore, intermolecular NO transfer was conrmed by various spectroscopic techniques. The FT-IR spectrum of the reaction mixture obtained aer the completion of the reaction of 2-Cr with 1-CoNO, in the KBr palette, exhibited a peak at 1690 cm À1 , which shied to 1650 cm À1 when the reaction was carried out using 15 (Fig. 3c). When the above reaction was performed by using 1-Co 15 NO, the mass peak shied by one unit  and exhibited a prominent peak at m/z 388.1, which corresponds to [(BPMEN)Cr(Cl)( 15 NO)] + and conrms the transfer of Co-bound NO to Cr-center ( Fig. 3c and S7c, ESI †). The 1 H-NMR of the reaction mixture does not show any signals for the protons of the 12-TMC ring, which was initially present for 1-CoNO (d 6 , S ¼ 0), suggesting the generation of 3-Co (low spin d 7 , S ¼ 1/2) (ESI, † Fig. S8). 4b,25f Further, 1 H-NMR titration also conrmed the 1 : 1 stoichiometry of 2-Cr to 1-CoNO (ESI, †  Fig. S9). Based on the above evidence, we can conclude that complexes 3-Co and 4-CrNO were generated in the reaction of 1-CoNO with 2-Cr.

DFT calculations and experimental details for mechanistic insight into the NO transfer reaction of 1-CoNO with 2-Cr
From the spectroscopic and kinetic results, it is evident that the reaction is going through the formation of a bridged NO intermediate species. However, due to the instability of this intermediate, we were unable to characterize it structurally. Therefore, we investigated the reaction pathway of the intermediate formation by carrying out a DFT calculation. DFT calculation suggests the formation of an isonitrosyl-Co complex in the rst transition state (TS 1 ) with an energy barrier of 14.66 kcal mol À1 (Fig. 5). In the next step, NO binds to the Cr via the N-donor site, which is À2.25 kcal mol À1 lower in energy than the reactant and therefore formed a thermodynamically favored side-on bound {Cr-NO-Co} 4+ intermediate species. 35 The experimental results well justied this; the inuence of both the metal-centers leads to the lower stretching frequency from 1703 cm À1 for 1-CoNO to 1520 cm À1 for the side on bonded {Cr-NO-Co} 4+ species. In addition to IR spectroscopy, a parallel trend was observed in low-temperature EPR measurements. When we reacted 1-CoNO with 2-Cr, a new peak formation for the intermediate species was observed with g ¼ 2.03, corresponding to a low spin Cr-center having a strong interaction of the N-atom of NO moiety to the Cr-center than Cocenter (distance of N-atom of NO in intermediate, Cr-N ¼ 228 pm and Co-N ¼ 303 pm). This peak shied to g ¼ 1.99 (inset: Fig. 3b), corresponding to 4-CrNO when the NO transfer completes. Further, the coordinated NO moiety undergoes dissociation leading to the nal NO transfer product. The highest lying transition state TS 2 along the reaction prole is 42.77 kcal mol À1 , more elevated than the reactant, signicantly higher than the 14.66 kcal mol À1 barrier of TS 1, indicating that the dissociation of the intermediate to the product is the ratedetermining step. This high-energy TS 2 leads the reaction to a rst-order reaction independent of the concentration of both 1-CoNO and 2-Cr.  Cr(NO)(Cl)] + (6-CrNO) (calcd m/z 345.1), and [(12-TMC)Co(Cl)] + (calcd m/z 322.1), respectively (Fig. 6b). The mass peaks shied by one unit to m/z 346.1 corresponds to [(12-TMC)Cr( 15 NO)(Cl)] + (calcd m/z 346.1) when the reaction is performed with 1-Co 15 NO conrms the NO transfer from complex 1-CoNO to 5-Cr (ESI, †  Fig. S12). Further, stoichiometric titration for NO transfer reaction was followed at 370 nm for the decomposition of 1-CoNO, which showed that the ratio-metric equivalent of complex 5-Cr to 1-CoNO was 1 : 1 (ESI, † Fig. S13).

The mechanistic insights of the NO transfer reaction
To elaborate on the chemistry involved in the NO transfer reaction of 1-CoNO with 2-Cr, why is it happening? We then explored the NO transfer reaction's mechanistic insight. There are two possible mechanistic approaches for the NO transfer reactions in M-NOs, i.e., associative or dissociative pathways (vide infra). 25a,25d,25f As shown in Fig. 3, the reaction of 1-CoNO with 2-Cr is proceeding via the formation of an intermediate species (Fig. 3a, blue line) before generating the nal product (Fig. 3b, red line), suggesting an associative mechanistic pathway. The UV-Visible spectrum of this intermediate species is different from the sum of the spectra of complexes 1-CoNO + 2-Cr, indicating that it is a new species rather than the mixture of 1-CoNO & 2-Cr (ESI, † Fig. S10). Suppose NO transfer is going through an associative pathway, then the dissociation of the intermediate species should be the rate-determining step. In that case, the reaction rate will be independent of 2-Cr. 25f Kinetics study revealed that the reaction rate was independent of the concentration of 2-Cr, suggesting an associative intermediate species before NO transfer. In addition, the DFT calculation suggests an energy barrier of 42.77 kcal mol À1 in TS 2 , a 28 kcal mole À1 higher-lying transition state than TS 1 . Due to this higher energy barrier, the formation of the product from intermediate via TS 2 becomes slow and makes the reaction rst order. Further, the formation of a peak at 1520 cm À1 in IR spectroscopy also supports the generation of a m-NO bridged 34 transient species (Scheme 2) in the above reaction. 25b,25d,36 The NO transfer reaction between 1-CoNO and 2-Cr was found to be following the rst-order kinetic with a rst-order rate constant (k 1 ) of 9 Â 10 À3 s À1 . Hence, the dissociation of the proposed {Cr-NO-Co} 4+ intermediate is supposed to be a rate-determining step, which is $10 3 times slower than the earlier reported second-order formation rate-constant of 1-CoNO (k 2 ¼ 12(1) M À1 s À1 ). 4b To nd the driving forces of the NO transfer reaction, the second-order rate constant (k 2 / ) and equilibrium constant (K eq / ) of the formation of 4-CrNO were determined and found to be 51.7(5) M À1 s À1 and 2.27 Â 10 3 M À1 (ESI, † Fig. S11), respectively, which are $4 times and $2.5 times larger than the k 2 (12(1) M À1 s À1 ) and K eq (8.90 Â 10 2 M À1 ) of 1-CoNO, respectively. This comparison of the rate-constants and equilibrium constants suggests clearly that the dissociation of the {Cr-NO-Co} 4+ intermediate should generate thermodynamically more stable molecules 4-CrNO & 3-Co, as proposed by the second-order rate constant and equilibrium constant for 1- Further, various thermodynamic properties were also determined for the generation of 1-CoNO & 4-CrNO (Table 1), which suggests that the formation of 4-CrNO (DG ‡ ¼ 15.71 kcal mol À1 , DG 0 ¼ À4.57 kcal mol À1 ) is thermodynamically more favorable than the formation of 1-CoNO (DG ‡ ¼ 16.43 kcal mol À1 , DG 0 ¼ À4.02 kcal mol À1 ). The thermodynamic stability of these molecules can also be explained based on the labile and inert behavior of the metal-centers. 37 The generation of an inert species 4-CrNO (low spin d 5 , S ¼ 1/2) from a highly labile 2-Cr (high spin d 4 , S ¼ 2) is one of the major driving forces of NO transfer, as reported in many other reports. 25f,37 In addition to labile and inert parameters and second-order formation rate constants for 1-CoNO & 4-CrNO, thermodynamic parameters (Table 1) further support our hypothesis as the generation of 3-Co & 4-CrNO from 1-CoNO & 2-Cr is due to the lower free energy for 4-CrNO than 1-CoNO, making the NO transfer reaction more spontaneous. Thus, apart from the various parameters (vide supra), other factors may affect the NO transfer reactivity. Logically thinking about the NO transfer chemistry claries that this reaction depends not only on various metal-center parameters, 25d but also on the ligand frameworks, which may inuence the transfer of NO from Co to  With an aim to determine the effect of the ligand frameworks, the NO transfer reaction was performed between Co and Cr bearing the same 12-TMC ligand framework. Complex 1-CoNO also showed NO transfer to 5-Cr and generated 6-CrNO. While further exploring the reaction mechanism, we have investigated the reaction of 5-Cr with 1-CoNO in various ratios. The characteristic UV-Visible peaks of 1-CoNO overlap with 5-Cr made it difficult to perform the kinetic comparison in the presence of the higher amounts of 5-Cr. Complex 1-CoNO is thermodynamically stable and does not show any natural decay (ESI, † Fig. S14); hence, the NO transfer's plausible mechanism from 1-CoNO to 5-Cr suggests that the reaction may go through a transient intermediate species. We did not observe any intermediate species formation in the UV-Visible or FT-IR spectral measurements (Fig. 6a), which suggests that the strain between the 12-TMC ligand frameworks of 1-CoNO & 5-Cr causes a fast transfer of NO from Co to Cr-center. Since the NO transfer is rst-order in nature, the formation of 6-CrNO is believed to be the rate-determining step, which is equal to the overall reaction rate with a rate constant (k 1 / ) of 3.1 Â 10 À2 s À1 .
The second-order rate constant (k 2 // ) and equilibrium constant (K eq // ) of the formation of 6-CrNO were also determined and found to be 29.3(2) M À1 s À1 and 1.48 Â 10 3 M À1 , respectively, which are $2.5 times and $1.6 times larger than the secondorder rate constant and equilibrium constant of 1-CoNO, 25f hence explaining why NO transfers from Co to Cr-center. In addition, other thermodynamic parameters for 1-CoNO (DG ‡ ¼ 16.43 kcal mol À1 , DG 0 ¼ À4.02 kcal mol À1 ) and 6-CrNO (DG ‡ ¼ 16.17 kcal mol À1 , DG 0 ¼ À4.32 kcal mol À1 ) further supports why NO transfer reaction between 1-CoNO & 5-Cr generates 6-CrNO (Table 1). In addition to the above experimental results and their interpretations, labile and inert nature of more stable 6-CrNO (low spin d 5 , S ¼ 1/2, inert) (Fig. S15a †) and less stable 5-Cr (high spin d 4 , S ¼ 2, labile) (Fig. S15b †) suggests a huge change in the spin state, which promotes the reaction to move in the forward direction. 37,38 The above discussion suggests that the ligand frameworks may inuence the intermediate's stability and the rate of NO transfer; however, when it comes to the net transfer of coordinated NO moiety from one metal to another, it largely depends upon the stability of the metalcenter and various other physical parameters.

Conclusion
In  (k), Gibbs free energy of activation (DG), and labile-inert nature of metal-centers. The second-order rate constant (k 2 / ) and equilibrium constant (K eq / ) of the formation of 4-CrNO was $4 times and $2.5 times that of 1-CoNO; subsequently, it pushes the NO transfer from Co to Crcenter to generate 4-CrNO. The free energy of activation of 4-CrNO is lower than that of 1-CoNO; hence, driving the NO transfer reaction towards the forward direction to generate thermodynamically more inert/stable 4-CrNO. Also, the formation of an inert 4-CrNO (low spin d 5 , S ¼ 1/2) from a labile 2-Cr (high spin d 4 , S ¼ 2) is another major driving force of NO transfer to achieve more stability. 25f,37 Additionally, the structure of the intermediate was predicted from a DFT calculation. The results from DFT calculations suggest a m-NO bridged intermediate that binds in a side-on fashion to both Co and Crcenter, having a strong interaction of NO with Cr-center than Co-center, and is in qualitative agreement with other experimental results. NO transfer reaction was also analyzed by taking [(12-TMC)Cr II (Cl)] + (5-Cr) to evaluate the effect of the ligand framework. Complex 1-CoNO showed the transfer of its NO moiety to 5-Cr without observing any intermediate species generating thermodynamically stable 6-CrNO (vide supra), which may be due to the high strain caused by the methyl groups in the 12-TMC ligand and its cyclic nature. 25f The   BPMEN is an open-chain ligand with only two methyl groups, probably making it adapt/change its geometry to form the proposed intermediate. This is quite interesting because two Cr II -complexes show NO transfer reactivity due to the higher stability of the nal {CrNO} 5 adduct; however, the ligand frameworks signicantly inuence the stability of the intermediate and hence control the reaction's fate. The present results describe original ndings, where the generation of m-NO bridged intermediate was observed before NO transfer and characterized spectroscopically and theoretically.

Data availability
All the required data already provided in the ESI † and manuscript.

Author contributions
PK & SD discovered/conceptualized the initial project. SD, Kulbir, & SG carried out the different experiments and gathered the data. SR & PM carried out the DFT calculation, and PM analyzed the data. PK & TD interpreted the experimental results. PK & SD wrote the rst dra of the article. PK, TD & PM corrected the manuscript, nalized the nal dra, and guided during the revision. PK followed and guided the whole project work.

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