Oxygen atom transfer promoted nitrate to nitric oxide transformation: a step-wise reduction of nitrate → nitrite → nitric oxide

Abstract

Nitrate reductases (NRs) are molybdoenzymes that reduce nitrate (NO₃⁻) to nitrite (NO₂⁻) in both mammals and plants. In mammals, the salival microbes take part in the generation of the NO₂⁻ from NO₃⁻, which further produces nitric oxide (NO) either in acid-induced NO₂⁻ reduction or in the presence of nitrite reductases (NiRs). Here, we report a new approach of VCl₃ (V³⁺ ion source) induced step-wise reduction of NO₃⁻ in a Co^II-nitrato complex, [(12-TMC)Co^II(NO₃⁻)]⁺ (2,{Co^II–NO₃⁻}), to a Co^III–nitrosyl complex, [(12-TMC)Co^III(NO)]²⁺ (4,{CoNO}⁸), bearing an N-tetramethylated cyclam (TMC) ligand. The VCl₃ inspired reduction of NO₃⁻ to NO is believed to occur in two consecutive oxygen atom transfer (OAT) reactions, i.e., OAT-1 = NO₃⁻ → NO₂⁻ (r₁) and OAT-2 = NO₂⁻ → NO (r₂). In these OAT reactions, VCl₃ functions as an O-atom abstracting species, and the reaction of 2 with VCl₃ produces a Co^III-nitrosyl ({CoNO}⁸) with V^V-Oxo ({V^V=O}³⁺) species, via a proposed Co^II-nitrito (3, {Co^II–NO₂⁻}) intermediate species. Further, in a separate experiment, we explored the reaction of isolated complex 3 with VCl₃, which showed the generation of 4 with V^V-Oxo, validating our proposed reaction sequences of OAT reactions. We ensured and characterized 3 using VCl₃ as a limiting reagent, as the second-order rate constant of OAT-2 (k₂^/) is found to be ∼1420 times faster than that of the OAT-1 (k₂) reaction. Binding constant (K_b) calculations also support our proposition of NO₃⁻ to NO transformation in two successive OAT reactions, as K_{b(Co^II–NO2⁻)} is higher than K_{b(Co^II–NO3⁻)}, hence the reaction moves in the forward direction (OAT-1). However, K_{b(Co^II–NO2⁻)} is comparable to K_b{CoNO}⁸, and therefore sequenced the second OAT reaction (OAT-2). Mechanistic investigations of these reactions using ¹⁵N-labeled-¹⁵NO₃⁻ and ¹⁵NO₂⁻ revealed that the N-atom in the {CoNO}⁸ is derived from NO₃⁻ ligand. This work highlights the first-ever report of VCl₃ induced step-wise NO₃⁻ reduction (NRs activity) followed by the OAT induced NO₂⁻ reduction and then the generation of Co-nitrosyl species {CoNO}⁸.

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Introduction

The mechanism of microbial denitrification is still one of the most mysterious subjects, despite being explored in detail in both in vivo and in vitro systems. Based on the extensive research and literature available, denitrification has been well accepted to be a four-step reductive process of nitrate (NO₃⁻) to dinitrogen (N₂) conversion {NO₃⁻ → NO₂⁻ → NO → N₂O → N₂}, through a series of intermediate gaseous nitrogen oxide products.¹ In mammals and bacteria, inorganic NO₃⁻ and nitrite (NO₂⁻) serve as a fundamental storage material of NO for its bio-physiological processes.^2,3 However, in humans, an excessive amount of NO₃⁻ has been discovered to cause gastric cancer and other disorders.⁴ To maintain an optimal NO₃⁻ level in the bio-system, commensal bacteria in the human oral cavity play a vital role in converting NO₃⁻ to NO₂⁻ (eqn (1)).² In bacteria, molybdenum-based nitrate reductase (NRs) enzymes generate NO₂⁻via an OAT reaction from NO₃⁻ anion.⁵ At the bio-physiological level, NO₂⁻ serves as a pool of NO and can easily be transformed to NO, either (i) in non-enzymatic acid-catalyzed NO₂⁻ reduction in the stomach^6,7 or (ii) by Fe and Cu based nitrite reductase (NiRs) enzymes catalyzed reactions (eqn (2)).^8,9

NO₃⁻ + Mo^IV → NO₂⁻ + Mo^VI=O (1)

M–NO₂⁻ + 2H⁺ + e⁻ → M–NO + H₂O (2)

M–NO₂ + R₂S → M–NO + R₂SO (3)

M–NO₂ + RSH → M–NO + R(O)SH (4)

M–NO₂ + PPh₃ → M–NO + OPPh₃ (5)

M–NO₂ + Sub + hν → M–NO + (O)Prod (6)

NO is a gaseous secondary messenger in animals, plants, fungi, and bacteria. In plants, NO is involved in different physiological processes, such as plant growth & development, metabolism, aging, defense against pathogens, biotic and abiotic trauma.¹⁰ However, NO regulates various physiological processes in mammals.¹¹ For instance, NO inadequacy possibly will aggravate the pathogenic effects associated with atherosclerosis, diabetic hypertension, etc.12 Also, the immune response of NO towards the harmful pathogens is related to the oxidized NO species,¹³i.e., peroxynitrite (PN, ONOO⁻)^13a,14 or/nitrogen dioxide (˙NO₂).^15,16 Hence, balanced production of NO is required to maintain a normal homeostatic bio-physiological condition. In bio-systems, enzymes, i.e., NiRs^8,9,17 and endothelial nitric oxide synthases (eNOSs)^17,18 are available for NO generation. The NOSs enzymes catalyze the biosynthesis of NO from L-arginine.¹⁸

In the case of NO overproduction, nitric oxide dioxygenase (NODs) generates NO₃⁻ in the reaction of the iron–dioxygen adduct with NO via a proposed PN intermediate,¹⁹ as explored in other biomimetic systems.^19d,20 Also, there are various reports on NO₂⁻ formation in nitric oxide monooxygenation (NOM) reaction from metal-nitrosyls in the presence of O₂, O₂˙⁻ and OH⁻.^19a,20b,21 Oxidized species of NO (NO₃⁻ & NO₂⁻) may also generate via different oxidative processes (vide supra). However, NO₃⁻ to NO₂⁻ transformation and NO₃⁻/NO₂⁻ to NO conversion (vise versa) are critical steps of the denitrification process.¹

In an attempt to mimic the denitrification process, R. H. Holm and co-workers reported NRs activity, molybdenum, and tungsten-based catalyst for catalytic reduction of NO₃⁻ to NO₂⁻ with metal-Oxo species.²² Similarly, S. Sarkar and co-workers have reported Mo^IV mediated reduction of NO₃⁻ to NO₂⁻ to mimic NRs activity.²³ Eunsuk Kim proposed the reduction of NO₃⁻ to NO₂⁻ using the Lewis acid Sc³⁺ to activate the OAT reaction from NO₃⁻ to Mo metal center.²⁴ On the other hand, nitrite reduction chemistry is explored widely; Ford and co-workers examined the acid-induced reduction of NO₂⁻ to N₂O in a Fe–porphyrin complex.²⁵ Warren group described the NiR by using the thiol group.²⁶ Recently, we have reported acid-induced nitrite reduction to NO.²⁷ However, the conversion of NO₃⁻ to NO using a single catalyst is barely explored; Yunho Lee and co-workers testified Ni catalyzed the transformation of inorganic NO₃⁻ to N₂via NO₂⁻ intermediate using the carbon monoxide (CO) as oxophilic species.²⁸

In biological systems^3,5a and biomimetic^22,23,27,29/or catalytic reactions,²⁴ the approach towards converting NO₃⁻ to NO is usually a two-enzymes/-catalysts-induced two-step process in two different reactions (i.e., NO₃⁻ reduction followed by NO₂⁻ reduction).^5a However, only a few reports simultaneously carry out both the NO₃⁻ and NO₂⁻ reduction using a single metal center in a biomimetic system. Here, our eagerness is to mimic the NRs enzymatic reaction followed by the NO₂⁻ reduction, sponsored by the same reagent (i.e., single metal-induced two-step NO₃⁻ to NO conversion). Herein, we report the NO₃⁻ reduction chemistry of a Co^II–NO₃⁻ complex, [(12-TMC)Co^II(NO₃)]⁺ (2), bearing a 12-TMC ligand (12-TMC = 1,4,7,10-tetramethyl-1,4,7,10-tetraazacyclododecane) via the two consecutive oxygen atom transfer (OAT) reactions using VCl₃ as an oxophilic compound (Scheme 1, reaction II & III). Complex 2 reacts with VCl₃ to form corresponding Co^III-nitrosyl complex, [(12-TMC)Co^III(NO)]²⁺ (ref. 19d, 20b, 21a, 27 and 30) (4), with V^V-Oxo ({V^V=O}³⁺) species, which further decomposes to V₂O₅, via the formation of a presumed Co^II–nitrito (3,{Co^II–NO₂⁻) intermediate in MeOH or H₂O at 298 K (Scheme 1, reaction II). Interpretation of various spectral measurements, we have confirmed the generation of 3 with V^V-Oxo by the transfer of one O-atom from NO₃⁻ moiety of 2 to VCl₃ (OAT-1). Further, we observed the generation of 4 with V^V-Oxo from 3 upon reaction with VCl₃ (OAT-2), under similar reaction conditions, showing the similar OAT induced NO₂⁻ reduction reactivity as reported in the case of PPh₃ and sulphur based compounds (thiols and thioethers) (eqn (3)–(6)).^26,31 Combining these two OAT reactions, we can predict the reaction sequences, i.e., the first OAT from NO₃⁻ moiety of 2 to VCl₃ and the generation of 3, {OAT-1 = Co^II–NO₃⁻ + VCl₃ → Co^II–NO₂⁻ + VOCl₃ (r₁)}. Subsequently, the second OAT from NO₂⁻ moiety of 3 to another VCl₃ moiety and the generation of 4 {OAT-2 = Co^II–NO₂⁻ + VCl₃ → {CoNO}⁸ + VOCl₃ (r₂)}. Mechanistic investigation using ¹⁵N-labeled-¹⁵NO₃⁻ & ¹⁵NO₂⁻ confirmed clearly that the N-atom in the {CoNO}⁸ is derived from NO₃⁻ anion of 2. To the best of our knowledge, the present work reports the first example of VCl₃ induced conversion of Co^II–NO₃⁻ to {CoNO}⁸ in two successive OAT reactions, demonstrating a new mechanistic approach for one-metal induced NO₃⁻ to NO₂⁻ reduction (NRs activity) followed by NO₂⁻ to NO conversion (OAT induced NO₂⁻ reduction).

Scheme 1

Results and discussion

Preparation of Co^II–nitrate complex, [(12-TMC)Co^II(NO₃⁻)]⁺ (2)

The primary Co^II–nitrate complex, [(12-TMC)Co^II(NO₃⁻)]⁺ (2), was prepared by reacting Co^II-complex, [(12-TMC)Co^II(NCCH₃)]²⁺ (1), with 1 equivalent of NaNO₃ in H₂O/CH3CN (Scheme 1, the reaction I; also see ESI† and Experimental section (ES)). Further, 2 was characterized by various spectroscopic techniques, including the single-crystal X-ray structure determination. UV-vis absorption band of 1 (λ_max = 485 nm) changed to a new band (λ_max = 480 nm, ε = 25 M⁻¹ cm⁻¹) upon addition of 1 equivalent NaNO₃ in CH₃CN at RT, suggesting the formation of 2 (Fig. 1a). FT-IR spectrum of 2 showed a characteristic peak for Co^II-bound NO₃⁻ anion at 1384 cm⁻¹ and shifted to 1358 cm⁻¹ when exchanged with ¹⁵N-labeled-NO₃⁻ (¹⁵N¹⁶O₃⁻) (Fig. 1a; ESI, Fig. S1†). A wide range ¹H-NMR spectrum of 2 showed fairly clean paramagnetic proton-signals (Figure S2a†), suggesting a magnetically active Co-center. The spin state of 2 was determined by calculating the magnetic moment of the Co^II metal-center by the Evans' method and found to be 4.46 BM, suggesting a high spin Co^II-ion in complex 2 (ES, ESI, Fig. S2b†). Electrospray ionization mass spectrum (ESI-MS) of 2 showed a prominent peak at m/z 349.1, which shifted to m/z 350.1 when prepared with ¹⁵N-labeled Na¹⁵NO₃, and their mass and isotope distribution pattern corresponds to [(12-TMC)Co^II(¹⁴NO₃)]⁺ (calc. m/z 349.1) and [(12-TMC)Co^II(¹⁵NO₃)]⁺ (calc. m/z 350.1), respectively (Fig. 1a; ESI, Fig. S3†). In addition to the above experimental characterization, 2 was structurally characterized by single-crystal X-ray crystallography. Complex 2 has a six-coordinate distorted octahedral geometry around the Co^II-center, possessing O, O^/-chelated bi-dentate NO₃⁻ anion (Fig. 2; ESI, ES, Fig. S4, Tables T1 and T2†).

Fig. 1 (a) UV-vis spectra of 1 (0.50 mM, brick red line) and 2 (0.50 mM, black line) in CH₃CN under Ar at 298 K. Inset: IR spectra of 2-¹⁴NO₃⁻ (red line) and 2-¹⁵NO₃⁻ (blue line) in KBr. (b) ESI-MS spectra of 2. The peak at 349.1 is assigned to [(12TMC)Co^II(NO₃)]⁺ (calcd m/z 349.1). Inset: isotopic distribution pattern for 2-¹⁴NO₃⁻ (red line) and 2-¹⁵NO₃⁻ (blue line).

Fig. 2 Displacement ellipsoid plot (20% probability) of 2 at 100 K. Disorder C-atoms of TMC ring, anion and H-atoms have been removed for clarity.

OAT reaction of Co^II–nitrate complex (2)

So as to understand the NO₃⁻ reduction chemistry of 2, we explored its reaction with VCl₃ to mimic the OAT based NRs enzymatic reaction. We observed a visible color change from pink to wine-red in the reaction of 2 with VCl₃ and a new absorption band (at 370 nm) formed, which is corresponding to the characteristic absorption band of a Co^III-nitrosyl ({CoNO}⁸, 4) (Fig. 3a and 6a).^{19d,20b,21a,27,30,32} Astonishingly, 2 upon reaction with VCl₃ generated corresponding Co^III-nitrosyl complex 4, ({CoNO}⁸),^{19d,20b,21a,27,30} with V^V-Oxo species in both aqueous/or methanol medium at 298 K (Scheme 1; reaction II). It is important to note that 2 did not show any spectral changes in the absence of VCl₃, suggesting that 2 is highly stable in H₂O/or MeOH and at 298 K (ESI, ES, and Fig. S5†). Finally, the product of NO₃⁻ reduction, formed in the reaction of 2 and VCl₃, was established to be {CoNO}⁸ (4) based on various spectroscopic (UV-vis, FT-IR, ESI-MS, NMR) and structural characterization (vide infra).^{19d,20b,21a,27,30,32-33} The FT-IR spectrum of 2 showed a peak at 1384 cm⁻¹, characteristic to the Co^II bound NO₃⁻ stretching frequency which shifted to 1703 cm⁻¹ when 2 was reacted with VCl₃, which is characteristic of NO stretching frequency of {CoNO}⁸ (4).^{19d,20b,21a,27,30} The peak at 1703 cm⁻¹ shifted to 1673 cm⁻¹ when 4 was prepared by reacting ¹⁵N-labeled-NO₃⁻ (Co^II–¹⁵NO₃⁻) with VCl₃, evidently suggesting the formation of {Co¹⁵NO}⁸ (inset: Fig. 3a; ESI and Fig. S6†). The shifting of NO stretching frequency (Δ = 30 cm⁻¹) indicates that N-atom in NO ligand is derived from Co^II–NO₃⁻. The ESI-MS spectrum of 4 showed a prominent peak at m/z 404.2, [(12-TMC)Co^III(NO)(BF₄)]⁺ (calcd m/z 404.2), and shifted to 405.2, [(12-TMC)Co^III(¹⁵NO)(BF₄)]⁺ (calcd m/z 405.2) when the reaction was performed with Co^II–¹⁵NO₃⁻ (Fig. 3b; ESI, Fig. S7†); suggests clearly that NO moiety in 4 is derived from NO₃⁻ moiety. The ¹H-NMR spectrum of 4 showed the peaks for the protons of 12-TMC ligand frameworks, confirming a low spin diamagnetic Co^III center (d⁶, S = 0) in complex 4 (ESI, Fig. S8†).^32,33 Further, we have calculated the yield of 4 from NMR spectra using benzene as an internal standard and found to be 90 ± 3% (ESI, Fig. S9†). As a final point, the exact conformation of 4 was provided by its single-crystal X-ray crystallographic analysis (ESI, ES, Fig. S10, Tables T1 and T2†) and comparable with previously reported Co^III–NO⁻/M–NO⁻ having sp² hybridized N-atom.^{20b,21a,27,30,34} The lone pair present on N-atom is responsible for the significant bending of the Co^III–NO⁻ moiety, with Co(1)–N(5)–O(1) bond angles of 128.52 (18)° for 4 and, therefore, further consistent with the assignment of 4 as {CoNO}⁸ species.

Fig. 3 (a) UV-vis spectral changes of 2 (0.50 mM, black line) upon addition of VCl₃ (2.2 equiv.) in H2O at 298 K. Black line (2) changed to a red line (4) upon addition of VCl₃. Inset: IR spectra 4-14NO (blue line) and 4-15NO (red line) in KBr. (b) ESI-MS spectra of 4. The peak at 404.2 is assigned to [(12TMC)Co^III(NO)(BF4)]⁺ (calcd m/z 404.2). Inset: isotopic distribution pattern for 4-¹⁴NO (red line) and 4-¹⁵NO (blue line).

From the final spectrum (black line in Fig. 3a), we have calculated the amount of 4 (90 ± 2%) by comparing its ε (M⁻¹ cm⁻¹) value at 370 nm, since V^V-Oxo and VCl₃ species does not show any absorption at 370 nm. This value is also in good agreement with the yield calculated from NMR spectroscopy (ESI, Fig. S9†). Further, we had also determined the isolated yield of the formation of 4 and found it to be 90 (±2)%, depicting clearly VCl₃ induced NO₃⁻ to NO transformation. The reduction of NO₃⁻ was observed to be slow; however, it enhanced with an increase in VCl₃ amount, suggesting that the NO₃⁻ to NO transformation follows the second-order reaction. Upon adding 10 equivalents of VCl₃ to the solution of 2 (0.5 mM), the UV-visible band at 370 nm starts forming with a pseudo-first-order rate constant, k_obs = 1.2 × 10⁻¹ s⁻¹, and showed the isosbestic points at 418 and 497 nm (Fig. 3a). Upon increasing the concentration of VCl₃, the pseudo-first-order rate constants increased proportionally, allowing us to determine a second-order rate constant (k₂) of 2.4 × 10⁻² M⁻¹ s⁻¹ (Fig. 4a) for the reaction of 2 with the various equivalents of VCl₃ (5, 10, 15, 20, 25).

Fig. 4 Plot of k_obsversus the concentration of VCl₃ to determine the second order rate constant in the OAT reaction of (a) 2 (b) 3 in H₂O at 298 K.

Confirming V^V-Oxo generation in NO₃⁻ reduction reaction via OAT

In order to authenticate our proposition of OAT promoted NO₃⁻ to NO reduction, we should observe the generation of V^V-Oxo species during this transformation. In this regard, we have confirmed the conversion of VCl₃ to V^V-Oxo species in the NO₃⁻ reduction reaction by ⁵¹V-NMR. We observed the characteristic peaks of V^V-Oxo species in the ⁵¹V-NMR spectrum for the reaction mixture obtained after the completion of the reaction of 2 (4 mM) with VCl₃ (8 mM) in CD₃OD, at −365, −525, and −598 assignable to VOCl₃, VOCl(OMe₂)₂ and VO(OMe₂)₃, respectively, as reported previously (ESI and Fig. S11†).³⁵ The observation of V^V-Oxo species in the VCl₃ promoted NO₃⁻ reduction reaction indisputably illustrates that the VCl₃ sponsored NO₃⁻ to NO conversion should proceed via the two consecutive OAT reactions, where OAT-1 mimics the NRs enzymatic reaction,^5b,36 while OAT-2 mimics the phosphorus or sulphur induced OAT transfer reactions (Schemes 1 and 2).^26,31

Scheme 2

Mechanistic investigation of NO₃⁻ reduction

In the biological system, the conversion of NO₃⁻ to NO proceeds via a common NO₂⁻ intermediate in two consecutive steps (vide supra). In this report, it is dreadfully clear that the formation of NO from NO₃⁻ could only be accomplished via the VCl₃ induced two consecutive OAT reactions (vide supra), i.e., OAT-1 & OAT-2. Hence, the conversion of NO₃⁻ to NO is likely to proceed via a Co^II–NO₂⁻ intermediate (3). Although we were unable to isolate the intermediate 3; however, we were able to show its generation by using VCl₃ as a limiting reagent (ES). In the reaction of 2 with 1.0-fold of VCl₃, we observed the generation of 4 with Co^II–NO₃⁻ and Co^II–NO₂⁻ and confirmed with various spectroscopic measurements. The FT-IR spectrum of the above reaction mixture showed the characteristic peaks for Co^II–NO₃⁻ (at 1385 cm⁻¹), Co^II–NO₂⁻ (at 1272 cm⁻¹) and {Co¹⁴NO}⁸ (at 1703 cm⁻¹), those shifted to 1358 cm⁻¹, 1245 cm⁻¹ and 1673 cm⁻¹ when ¹⁵N-labeled-nitrate complex (3-¹⁵NO₃⁻) reacted with VCl₃, respectively (ESI, Fig. S12a and b†). Also, we have recorded the ESI-MS spectrum of the reaction mixture, which showed the prominent peaks at m/z 404.2, [(12-TMC)Co^III(NO)(BF₄)]⁺ (calcd m/z 404.2), 333.1, [(12-TMC)Co^II(NO₂⁻)]⁺ (calcd m/z 333.1) and [(12-TMC)Co^II(NO₃⁻)]⁺ (calcd m/z 349.1), those shifted to 405.2, [(12-TMC)Co^III(¹⁵NO)(BF₄)]⁺ (calcd m/z 405.2), 334.1, [(12-TMC)Co^II(¹⁵NO₂⁻)]⁺ (calcd m/z 334.1) and [(12-TMC)Co^II(¹⁵NO₃⁻)]⁺ (calcd m/z 350.1), when the reaction was performed with Co^II–¹⁵NO₃⁻ and VCl₃, respectively (ESI, Fig. S12c and d†). Further, when we reacted 2 with 1.5-fold of VCl₃, we observed the generation of 4 with Co^II–NO₂⁻ and confirmed by FT-IR and ESI-MS measurements. The FT-IR spectrum showed the characteristic peaks for Co^II–NO₂⁻ (at 1272 cm⁻¹) and {Co(¹⁴NO)⁸ (at 1703 cm⁻¹), and shifted to 1245 cm⁻¹ (Co^II–¹⁵NO₂⁻) and 1673 cm⁻¹ ({Co¹⁵NO}⁸) when using ¹⁵N-labeled-nitrate complex (3-¹⁵NO₃⁻) (SI, Fig. S13a and b†). The ESI-MS spectrum, for the above reaction mixture, showed the prominent peaks at m/z 404.2, [(12-TMC)Co^III(NO)(BF₄)]⁺ (calcd m/z 404.2), and 333.1, [(12-TMC)Co^II(NO₂⁻)]⁺ (calcd m/z 333.2), and shifted to 405.2, [(12-TMC)Co^III(¹⁵NO)(BF₄)]⁺ (calcd m/z 405.2) and 333.1, [(12-TMC)Co^II(¹⁵NO₂⁻)]⁺ (calcd m/z 334.1), when using Co^II–¹⁵NO₃⁻ as starting reacting material in OAT reaction, correspondingly (ESI, Fig. S13d†). Together, the FT-IR and ESI-MS spectra confirmed that VCl₃ induced reduction of NO₃⁻ to NO is going through a Co^II–NO₂⁻ (3) intermediate. Furthermore, as described above, when 2 reacted with 2.2-fold of VCl₃, we had observed the generation of only complex 4 with nearly 90 ± 2% yield (vide supra).

With the intention of further validate our concept of Co^II–NO₂⁻ species formation in the NO₃⁻ reduction reaction, in a control experiment, we explored the VCl₃ induced transformation of Co^II–NO₂⁻ to one-electron oxidized {CoNO}⁸ species;³⁷ possibly by the release of one electron which usually gets solvated as observed in other cases,³⁸ and trailed the fate of OAT reaction (Fig. 5). For this reaction, the initial Co^II–NO₂⁻ complex was prepared by following the reported literature (Scheme 1, reaction VIa).^21a,27 Upon addition of one fold VCl₃ to a solution of 3 in MeOH/H₂O at RT, the color of the reaction solution immediately changed from light pink to wine red, suggesting the generation of {CoNO}⁸ (4),³⁷ and its characteristic absorption band (at 370 nm) appeared in ∼1 minute as shown in Fig. 5 and 6b (Scheme 1, reaction VIb). Also, we have calculated the yield of 4 by comparing its ε (M⁻¹ cm⁻¹) value at 370 nm and found it to be (>95 ± 2%). Further, spectral titration data confirmed that the stoichiometric ratio of 3 with VCl₃ was 1 : 1 (ESI, Fig. S14†). Furthermore, the final product (4) was confirmed by the FT-IR and ¹H-NMR spectroscopy (ESI, Fig. S15 and S16†).^{19d,20b,21a,27,30} Also, the generation of V^V-Oxo species was confirmed by the ⁵¹V-NMR (ESI, Fig. S17†). Speedy conversion of 3 to 4 in the presence of VCl₃ justifies our inability to isolate intermediate 3 in the VCl₃ induced conversion of 2 to 4 and supports our supposition of two consecutive OAT reactions in reducing NO₃⁻ to NO. We have also determined the second-order rate constant for the reduction of NO₂⁻ to NO to understand the reaction mechanism and NO formation fate. The second-order rate constant (k₂^/) for NO₂⁻ reduction was determined by plotting the pseudo-first-order rate constant against various equivalents of VCl₃ (5, 10, 15, 20, and 25) and found to be 34.2 M⁻¹ s⁻¹ (Fig. 4b). Our efforts to isolate the Co^II–NO₂⁻ intermediate in the NO₃⁻ reduction reaction is unsuccessful due to the high reactivity of Co^II–NO₂⁻ with VCl₃ (OAT-2), which is ∼1420 times faster than the second-order rate constant of VCl₃ induced NO₃⁻ to NO reduction (k₂ = 2.4 × 10⁻² M⁻¹ s⁻¹).

Fig. 5 UV-vis spectral changes of 3 (0.50 mM, blue line) upon addition of VCl₃ (1 equiv.) in H₂O at 298 K. Blue line (3) changed to red line (4) upon addition of VCl₃. Inset: IR spectra 4-¹⁴NO (blue line) and 4-¹⁵NO (red line) in KBr.

Fig. 6 (a) Time course of the formation of 4 (blue circles) monitored at 370 nm upon addition VCl₃ (2.2 equiv.) to a solution of 2 (0.5 mM) in H₂O at 298 K. (b) Time course of the formation of 4 (Red circles) monitored at 370 nm upon addition VCl₃ (1 equiv.) to a solution of 3 (0.5 mM) in H₂O at 298 K.

Spectroscopic and kinetic measurements undeniably confirmed that the reaction of 3 with VCl₃ generates 4 ({CoNO}⁸) plus V^V-Oxo species with a second-order rate constant (k₂^/ = 34.2 M⁻¹ s⁻¹), suggesting a rapid conversion (Fig. 6b). However, the transformation of 2 to 4 was observed to be a prolonged reaction, based on the spectral measurements (vide infra), in two sequential OAT reactions with a second-order rate-constant (k₂ = 2.4 × 10⁻² M⁻¹ s⁻¹) via a Co^II–NO₂⁻ intermediate. This comparison of rate constants (k₂ ⋘ k₂^/) suggests that the formation of NO₂⁻ from NO₃⁻ is a rate-determining step in the NO₃⁻ to NO reduction chemistry. Kinetic measurements (vide supra) confirmed clearly that the first step of the reaction (Scheme 1, pathway II) is the slowest step of the reaction; hence, a rate-determining step. Therefore, the overall second-order rate constant (k₂ = 2.4 × 10⁻² M⁻¹ s⁻¹) is equal to the rate constant of the conversion of 2 to 3 (Fig. 4a and 6a). Additionally, the binding constants (K_{b(Co^II–NO3⁻)}, K_{b(Co^II–NO2⁻)}, & K_b{(CoNO)⁸) for the generation of different species, Co^II–NO₃⁻, Co^II–NO₂⁻ & {CoNO}⁸ in the reaction of [Co^II(CH₃CN)(12TMC)]²⁺ with NO₃⁻, NO₂⁻ & NO, were determined by using Benesi–Hildebrand equation³⁹ and found to be 2.3 × 10² M⁻¹, 2.5 × 10³ M⁻¹ & 2.4 × 10³ M⁻¹ (ESI, ES, and Fig. S18†), respectively (Scheme 2). Structural parameters and ambiphilic nature of O-atom in coordinated NO₃⁻ & NO₂⁻species, in their respective complexes,²⁸ and the binding constants calculations further support our proposal of two OAT reactions with different reaction rates (r₂ ⋙ r₁). The K_{b(Co^II–NO2⁻)} is higher than that of K_{b(Co^II–NO3⁻)}; hence the reaction moves in the forward direction once the NO₂⁻ generates from NO₃⁻ in OAT-1. However, the abstraction of the first non-coordinated O-atom from NO₃⁻ using VCl₃ is somewhat hard due to its less electrophilic nature and more bond strength (bond length_(N–_O) = 1.215 Å) compare to other more electrophilic Co²⁺-coordinated O-atoms (bond length_(N–_O) = 1.267 Å & 1.269 Å); therefore showed a slower rate of OAT-1 (r₁) than OAT-2 (r₂) reaction, as proposed theoretically in CO induced Ni–NO₃⁻ reduction chemistry, suggesting the slow rate of NO₃⁻ to NO₂⁻ than NO₂⁻ to NO.²⁸ Kim and co-workers reported that the alteration of O-atom's electrophilic behavior in NO₃⁻ species, induced by the Sc³⁺ metal (Lewis acid) binding, showed the NO₃⁻ to NO₂⁻ reduction, which was not observed in the absence of Sc³⁺ ion, suggesting the O-atom activation upon Sc³⁺ binding. Discussion on the OAT chemistry²⁸ and metal-induced activation of O-atom²⁴ (vide supra) undoubtedly support our supposition of a faster OAT-2 than the OAT-1, as non-co- ordinated O-atom of NO₃⁻ is difficult to abstract by VCl₃ than Co²⁺-co-ordinated O-atoms of nitrite moiety.^24,28 Recent reports on Lewis acid induced OAT reactions showed an increase in the oxidizing power of M–oxygen adducts and their OAT reaction rate,⁴⁰ which coincides with the activation of N₂ by Lewis acid.^28,41 Further, the difference in the rates of the NO₃⁻ & NO₂⁻ reduction were supported by the inert and labile behavior of Co-complexes. High spin Co^II-complexes, 2 and 3, are labile (d⁷, S = 3/2);⁴² hence the conversion of 2 to 3 was found to be slow as there is not much change in the CFSE; however, the conversion of labile 3 to an inert 4 (d⁶, S = 0) found to be very fast as there is a huge change in the CFSE, in order to achieve more stable inert electronic configuration.^19d,43 These results verify our theory of step-wise conversion of NO₃⁻ to NO₂⁻, which further reduces to NO in the presence of VCl₃ in two consecutive OAT reactions.

Conclusion

Investigation of insights into the mechanistic aspects of the NO₃⁻ & NO₂⁻ reduction process became a most significant research area in modern-day chemistry as it deals with the biological and environmental aspects.¹ Reduction of NO₃⁻ to NO via NO₂⁻ intermediate species are key steps in biological NO generation (salival NRs followed by NiRs in mammalian system)^5,44 and also for the denitrification process (biogeochemical systems).^1,45 Reduction of NO₃⁻ to NO using a single metal complex is still a challenge to the scientific community as two different enzymes play the catalytic role in each step in the biological system.^2,3 In this report, for the very first time, we have shown the direct reduction of NO₃⁻ in a Co^II–nitrato complex, [(12TMC)Co^II(NO₃⁻)]⁺ (2), to a Co–nitrosyl complex {CoNO}⁸ (4), in the presence of an oxophilic reagent (VCl₃). Mechanistic investigation suggests that the reaction proceeds via a Co^II–NO₂⁻ (3) species, as observed in the case of biological NRs enzymatic chemistry,⁵ in two consecutive OAT reactions. Kinetic measurements suggest that the VCl₃ induced reduction of NO₃⁻ to NO₂⁻ is a rate-determining step (k₂ = 2.4 × 10⁻², OAT-1), mimicking the salival molybdate NRs enzymatic reaction.⁵ In the second step (OAT-2), a speedy reduction process (k₂^/ = 34.2), NO₂⁻ further reduces to NO in the presence of one-fold VCl₃. Isolation of 3 was difficult due to the fast conversion of 3 to 4; however, we could characterize it with various spectroscopic techniques. The results observed in VCl₃ induced NO₃⁻ reduction to NO in two consecutive OAT reactions are found to be in good agreement with our proposed concept. The results are explained in the light of the bond strength and the electrophilic behavior of O-atoms of NO₃⁻/NO₂⁻ ligands and based on the inert & labile nature of Co-complexes. Due to high bond strength and less electrophilic character of metal-unbound O-atom of NO₃⁻, it showed a slower OAT reaction (r1); in contrast, O-atoms of NO₂⁻ moiety is activated due to their binding with Co^II-center, and the OAT from NO₂⁻ to VCl₃ found to be very fast (r2), as observed in Ni–NO₃⁻ reduction chemistry.²⁸ Also, the conversion of high spin 2 to 3 (d⁷, S = 3/2) is slow due to a very less change in the CFSE compare to the transformation of high spin 3 to a low spin 4 (d⁶, S = 0) with much change in the CFSE, additionally support our chemistry. Furthermore, direct generation of 4 from 3 supports our proposition that Co^II–NO₂⁻ involved as an intermediate species in NO₃⁻ to NO transformation. Tracking the reactions using ¹⁵N-labeled-¹⁵NO₃⁻ and ¹⁵NO₂⁻ evidently suggests that the N-atom in the {CoNO}⁸ species is derived from NO₃⁻ moiety. N–O bond activation^{20b,21a,27,30,34a,34c} of coordinated NO₃⁻ in 2 generates 4,^19d hence implying that the OAT reaction of 2 in the presence of VCl₃ generates the Co–nitrosyl species. This work highlights the first-ever report of VCl₃ encouraged the reduction of NO₃⁻ to NO₂⁻ (NRs activity, OAT-1) followed by another OAT induced NO₂⁻ to NO transformation (OAT-2). In nature, both the reduction process needs two different enzymes for converting NO₃⁻ to NO; hence, the proposed OAT reagent (VCl₃), which is capable of doing the same in a one-shot, has border significance with respect to biological as well as environmental systems.

Author contributions

PKK & Kulbir discovered/conceptualized the initial project. Kulbir, SD, MG, PB, & MY carried out the different experiments and gathered the data. PKK, SG & TD helped in interpreting the experimental results. Kulbir and SD write the first draft of the article. PKK & TD have corrected the manuscript, finalized the final draft, and guided during the revision. PKK followed and guided the whole project work.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by Grants-in-Aid (Grant No. EEQ/2016/000466) from SERB-DST. KK, SD, MY, PB, and SG thank IISER Tirupati for financial assistance and for providing the research facility. SCS thanks DST-FIST for the single crystal facility at PU. Special thanks to Prof. K. Vijayamohanan Pillai (IISER Tirupati) for fruitful discussion and support.

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Footnote

† Electronic supplementary information (ESI) available: For the Experimental details. CCDC 2058406 and 2058407. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/d1sc00803j

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