Rapid nitrite reduction enabled by secondary sphere hydrogen bonds within non-heme iron complexes

Abstract

A non-heme iron(II) complex bearing a ligand with secondary sphere hydrogen bond (H-bond) donors, (tris(6-phenylaminopyridylmethyl)amine, TPA^NHPh, rapidly reduces nitrite (NO₂⁻) to nitric oxide (NO) in the absence of exogenous additives, affording a Fe(III)₂(μ-O)₂ diamond core. An electronically analogous complex containing a ligand without H-bonds (tris(6-methylpyridylmethylamine), TPA^Me, also reduces NO₂⁻ to NO and forms an Fe(III)₂(μ-O)₂ core, but is four orders of magnitude slower, highlighting the impact of H-bonds to promote NO₂⁻ reduction. We compare the structural and spectroscopic differences of the two Fe(III)₂(μ-O)₂ complexes and show that H-bonding interactions weaken the Fe–O bonds, perturb the electronic structure of the Fe₂O₂ cores, and thereby engender distinct reductive stability profiles.

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Introduction

Nitrite (NO₂⁻) is a source for physiological production of nitric oxide (NO), which is an important bioregulatory signalling molecule.¹ NO has multiple functions as a vasodilator, neurotransmitter, and has important roles in mammalian immune responses.^1,2 In these biological contexts, an array of Fe- and Cu-based metalloenzymes (e.g. cytochrome cd1- or cytochrome c nitrite reductases, cd1-NiR/cc-NiR, and Cu-NiR) catalyze the reduction of NO₂⁻ to NO or to NH₃ (ccNiR).³ Mutagenesis and computational studies implicate a critical role of specific hydrogen-bonding (H-bonding) amino acids close to the active sites of these enzymes (i.e., in the secondary sphere) for enzymatic function.⁴ Removal of these H-bonding residues in cd1- or cc-NiR inhibits catalytic activity, affording up to a 99% decrease in catalytic rates, highlighting their roles to both enable and accelerate enzymatic NO₂⁻ reduction.⁵ Although the dependence on H-bonds for overall function is established, the molecular-level details of these interactions are challenging to clarify because removal induces larger structural changes, obfuscating their role(s) on individual reaction steps.^4a

Inorganic model complexes can provide insight into the effects of secondary sphere H-bonds,⁶ and by extension, the mechanisms of enzymatic NO₂⁻ reduction. Complexes that do not contain H-bond donors often require the addition of an exogenous Brønsted acid and/or (electro)chemical reducing equivalents to promote NO release.⁷ In contrast, systems containing secondary sphere H-bond donors can induce spontaneous NO₂⁻ reduction without exogenous acids.⁸ Seminal work by the Fout group reported facile NO₂⁻ reduction mediated by Fe in a tripodal azafulvine-imine ligand scaffold, producing NO and a monomeric Fe(III)–O(H), a result that was attributed to the pendent-imino groups acting as H-bond donors (Fig. 1a).^8b–d,9 Related reports from the Gilbertson group highlighted Fe-pyridinediimine complexes with tethered amines that reduced NO₂⁻ to afford an {Fe(NO)₂}^9,10 and the Hung group reported an N-confused porphyrin that similarly reduced NO₂⁻ to form an {Fe(NO)}^6/7.¹¹

Fig. 1 (a) Previously reported Fe-based systems with secondary sphere H⁺/H-bond donors that spontaneously reduce NO₂⁻,^8b–d,9,10 (b) this work.

While these prior studies demonstrated synthetic examples of H-bond promoted NO₂⁻ reduction, direct comparisons between ligands that either contain or omit H-bonds are notably absent. Modification of the secondary sphere of a complex can impact its primary coordination sphere via perturbations to ligand field environments, redox potentials, or spin states.^5a,12 These competing effects present challenges when assessing the role(s) of H-bonds during NO₂⁻ reduction. Our group has attempted to decouple these parameters by comparing reaction outcomes with electronically similar ligands that differ in their secondary sphere,¹³ and we previously showed that a scaffold containing aniline groups as H-bond donors, (tris(6-phenylaminopyridy-lmethyl)amine, TPA^NHPh), promoted capture/activation of O₂ (with Fe, Cu, and Zn)^13b,f,g in addition to tandem ClO₄⁻ reduction/C–H oxygenation (with Fe).^13a,c We also reported a Cu(I) complex with a related ligand containing appended OH groups, tris(6-hydroxypyridylmethyl)amine TPA^OHCu(I), which reduced NO₂⁻ to NO, an example of ligand-promoted H⁺/e⁻ transfer.¹⁴ In this report, we expand these efforts to Fe-mediated NO₂⁻ reduction.

Results and discussion

Introduction of excess (ca. 10 equiv.) [Bu₄N][NO₂] to an MeCN solution of TPA^NHPhFe(II) bis(bis-trifluoromethylsulfonylazanide), (TPA^NHPhFe(NTf₂)₂, I-NTf₂, Fig. 2a), under ambient conditions afforded a two-step reaction sequence: a rapid color change from colorless to yellow (seconds), followed by a gradual transition to reddish-brown (hours). Removal of MeCN in vacuo followed by precipitation from CH₂Cl₂/Et₂O provided a single product, as assessed by ¹H-NMR spectroscopy. A solution-phase IR spectrum of the product contained no Fe–NO stretches between 1600–1850 cm⁻¹, but did exhibit broadened and bathochromically shifted N–H stretches (ν_NH = 3232 and 3188 cm⁻¹) relative to I-NTf₂ (ν_NH = 3361 and 3278 cm⁻¹), consistent with strengthened H-bond interactions.¹⁵ To examine whether NO was released during this reaction, we allowed I-NTf₂ to react with excess [Bu₄N][NO₂] in the presence of CoTPP (TPP = tetraphenyl porphyrin), which provided quantitative yield of CoTPP(NO) after 5 h.¹⁶ These data are consistent with spontaneous NO₂⁻ reduction to NO by I-NTf₂, potentially with concomitant formation of an Fe–O unit, as we did not observe Fe–NO bond formation.

Fig. 2 (a) Reaction of TPA^NHPhFe(X)₂ (X = OTf⁻ or NTf₂⁻; I-OTf or I-NTf₂) with NO₂⁻ to form NO(g) and TPA^NHPh₂Fe(III)₂(μ-O)₂²⁺ (II). (b) Reaction of TPA^MeFe(NTf₂)₂ (III-NTf₁) with NO₂⁻ to form TPA^Me₂Fe(III)₂(μ-O)₂²⁺(IV) and NO_(g), (c) molecular structures of II and IV (NO₂⁻-derived); 50% probability ellipsoids, short H-bond contacts highlighted in blue, phenyl groups are wireframed, H-atoms not involved in H-bonds, and outer sphere anions omitted for clarity (d). Bond metrics of II, IV^a and IV^b (a: this work, b: ref. 19g, *denotes an average of 3 bond distances).

To determine the molecular composition of the product, we performed a single-crystal X-ray diffraction (XRD) experiment on crystals grown from CH₂Cl₂/Et₂O. Rather than a monomeric Fe–O, the refined structure revealed a [Fe(III)₂(μ-O)₂]²⁺ diamond core (II, Fig. 2c) enveloped by moderate strength H-bonding interactions (average O₁–N₃ distance of 2.9 ± 0.2 Å).¹⁷ This Fe₂O₂ structural motif is reminiscent of higher valent intermediates within non-heme di-iron enzymes (e.g. soluble methane monooxygenase (sMMO-Q) in the Fe(IV)₂ state and ribonucleotide reductase (RNR-X) in the Fe(III)Fe(IV) state)¹⁸ but is comparatively rare in synthetic systems.¹⁹ Que and coworkers reported the first isolated Fe(III)₂(μ-O)₂ core, supported by two tris(6-methylpyridylmethyl)amine) ligands, (TPA^Me₂Fe(III)₂(μ-O)₂²⁺IV), which was prepared via more standard oxygenation reagents (tBuOOH and NEt₃).^19g Another related example, reported by Masuda, is a TPA^NH₂ analogue that formed an H-bonded Fe(III)₂(μ-O)₂ species from O₂.^19c We found that IV could also be prepared directly from [Bu₄N][NO₂], albeit over a longer time frame (1 week, Fig. 2b). Importantly, TPA^Me provides an analogous primary sphere environment as TPA^NHPh, but does not contain pendent H-bond donors.^13b Thus, we propose that differences in the chemical properties of II and IV can be attributed to the effects of secondary sphere H-bonds.

The structural metrics in II are similar to those in IV, with a few notable exceptions (Fig. 2d).²⁰ The Fe₂O₂ diamond core in II displays two distinct Fe–O bonds (Fe₁–O₁: 1.886(3) Å, and Fe₁–O₂: 1.925(3) Å), an Fe₁–Fe₂ separation of 2.729(1) Å, and an Fe₁–O₁–Fe₂ angle of 91.5(2)°. In comparison, IV has a 0.038(4) Å shorter Fe₁–O₁ bond (1.848(2) Å) and a 0.034(2) Å shorter Fe–Fe distance (2.695(1) Å), but the other Fe₂O₂ core metrical parameters are similar (Fe₁–O₂: 1.925(2) Å, and ∠Fe₁–O₁–Fe₂: 91.1(1)°). The elongated Fe₁–O₁ bond likely exhibits a weaker trans-influence, which is manifest by the contracted Fe₁–N₁ distance of II (2.178(3) Å) compared to IV (2.215(2) Å). We attribute these structural differences to the trifurcated H-bonds surrounding each μ-O²⁻ ligand in II, which are proposed to reduce the basicity of oxo ligands, thereby weakening the Fe–O bonds.^19c,21,22

To clarify the electronic differences that are imparted by H-bonding interactions, we examined the electronic absorption spectra of II and IV. Their respective spectra are distinct: II exhibits a prominent shoulder band at 433 nm (ε = 2.6 mM⁻¹ cm⁻¹) while IV displays a similarly intense shoulder band at 375 nm (ε = 2.0 mM⁻¹ cm⁻¹), both assigned to LMCT transitions.^19b These spectral shifts suggest that H-bonding interactions alter the electronic structure of the Fe₂(μ-O)₂ cores, which we interrogated further by Mössbauer spectroscopy. The zero-field Mössbauer spectrum (Fig. 3, top) of II as a solid (at 298 K) displays a symmetric doublet with an isomer shift (δ) of 0.33 mm s⁻¹ and quadrupole splitting (ΔE_q) of 1.422 mm s⁻¹, consistent with two identical high spin Fe(III) centers.^19a,23 These data are distinct from the reported Mössbauer parameters of IV: (symmetric, δ = 0.50 mm s⁻¹; ΔE_q = 1.93 mm s⁻¹). We attribute the differences in δ (lower δ for II) to more oxidized/Lewis acidic Fe(III) centers,²² and the differences in ΔE_q values to H-bond induced charge redistribution and/or orbital rehybridization of the μ-O ligands.²⁴ These effects may also be partially responsible for the decrease in antiferromagnetic coupling in II (J = −28 cm⁻¹) relative to IV (J = − 54 cm⁻¹).^19g,25

Fig. 3 Top: 298 K Mössbauer spectrum of II and associated spectral data for both II and IV.^26,27 Bottom: cyclic voltammograms of II and IV (6 mM [Fe₂(μ-O)₂, 0.1 M [Bu₄N][NTf₂] in MeCN, 100 mV s⁻¹).

The differences in electronic structures of the Fe(III)₂(μ-O)₂ cores within II and IV allude to distinct redox behaviors, which we investigated by cyclic voltammetry (CV, Fig. 3, bottom). The CV of II exhibited three principal features: a reversible 1e⁻ reduction at −0.78 V (vs. Fc^0/+, assigned as a Fe(III)₂/Fe(III)Fe(II) couple), and two irreversible events at −1.2 V and +0.92 V, see SI. The well-behaved reduction event of II is in stark contrast to IV, where the CV was not well-defined, and instead, displayed broad features with an E_onset = −0.80 V (Fig. 3, see SI).^28,29

To probe differences in reductive stability, we evaluated chemical reductions of II and IV. The reaction of II with 1 equiv. Na⁰ (as 5% Na/NaCl) in frozen THF afforded a mixture of products (as assessed by ¹H-NMR spectroscopy) but did not induce demetalation. An analogous reduction of IV underwent immediate demetalation, generating free ligand as the sole TPA^Me containing product. In contrast, introduction of milder reagents capable of delivering H⁺/e⁻ equivalents (i.e. H-atom donors) provided tractable reactivity with both II and IV. Addition of 1.5 equiv. 1,2-diphenylhydrazine (DPH, N–H_BDFE = 68 kcal mol⁻¹)³⁰ to II (23 °C, MeCN) provided quantitative formation of TPA^NHPhFe(II)(OH)⁺ (I-OH, Fig. 4) and azobenzene after 24 h, a net 2H⁺/2e⁻ reduction of II.³¹ In comparison, addition of 1.5 equiv. DPH to IV immediately (ca. 15 min, 23 °C) produced a new species (assigned as TPA^MeFe(II)(H₂O)_x(MeCN)_y²⁺, III-H₂O),³² with a low conversion to azobenzene (∼20%). The slower reduction of II by DPH relative to IV is consistent with distinct reductive stability profiles of these Fe(III)₂(μ-O)₂ cores as a result of secondary sphere H-bonding interactions.

Fig. 4 Reductions of II and IV with 1,2-diphenylhydrazine (DPH).

The H-bond dependent differences in reaction times for reduction of II and IV noted above are opposite from observations in Fig. 2 (NO₂⁻ reactivity). To investigate the role of H-bonds during the initial NO₂⁻ reduction step, we examined reactions between NO₂⁻ and the control compound TPA^MeFe(NTf₂)₂ (III-NTf₂), which maintains a similar primary sphere environment to I-NTf₂ but does not contain secondary sphere H-bonds (Fig. 1b).^13b Introduction of excess (ca. 10 equiv.) [Bu₄N][NO₂] to a colorless solution of III-NTf₂ in MeCN immediately produced a yellow solution that exhibited five broad ¹H-NMR resonances (assigned as NO₂⁻ binding). After 7 days, the solution turned orange and developed a characteristic UV-vis shoulder feature at 470 nm, corresponding to IV in 93% yield (see SI), and a separate NO trapping experiment provided CoTPP(NO) in 80% yield.

Formation of NO from NO₂⁻ can occur through multiple distinct pathways (inner- or outer-sphere 2H⁺/1e⁻ reduction or through H⁺-mediated disproportionation);³³ thus, we executed additional control experiments to provide further clarity into the mechanism for NO₂⁻ reduction in this system. To probe a disproportionation process, we introduced an exogenous acid, 1-methyl-2-(phenylamino)pyridinium³⁴ ([HA], Fig. 5), which has a similar structure and charge, and therefore acidity/H-bond donor strength as the appended NHPh groups in I-NTf₂, to excess [Bu₄N][NO₂] in MeCN. NO did not form in appreciable amounts after 5 h (3% yield of NO (via CoTPP(NO)). To examine outer-sphere reduction, we introduced ferrocene³⁵ to a mixture of 10 equiv. [Bu₄N][NO₂], TPA^NHPh, and Zn(OTf)₂, and again did not observe NO formation after 5 h. Collectively, these control experiments suggest that neither disproportionation nor outer-sphere reduction pathways proceed at rates comparable to those occurring during NO₂⁻ reduction with I-NTf₂ or III-NTf₂. Because the O-atoms from NO₂⁻ reduction are incorporated into the terminal Fe-containing products (II and IV), we propose that an inner-sphere reduction pathway is most likely.

Fig. 5 Assessment of alternative pathways for NO production from NO₂⁻.

Since both I-NTf₂ and III-NTf₂ enabled spontaneous reduction of NO₂⁻ to NO, we quantified the effects of H-bonds on their kinetic profiles (Fig. 6). Evolution of NO (monitored via CoTPP trapping) from a reaction between I-NTf₂ and 10 equiv. [Bu₄N][NO₂] fit well to a first-order exponential model with k_obs = (2.3 ± 0.2) × 10⁻⁴ s⁻¹. In contrast, with III-NTf₂, we observed a slow reaction (linear fit) with k_obs ≈ (3) × 10⁻⁸ s⁻¹, four orders of magnitude slower than I-NTf₂. To clarify the extent to which the N–O bond cleavage step is promoted by weak Brønsted acids (including the appended NHPh H-bond donors), we performed a control experiment with [HA] and III-NTf₂. Introduction of 3 equiv. [HA] to a mixture of III-NTf₂ and 10 equiv. [Bu₄N][NO₂] marginally enhanced NO₂⁻ reduction, affording k_obs = (7.0 ± 0.9) × 10⁻⁸ s⁻¹ and a concomitant increase in NO yield to 21% (5 h). These results contrast with the rapid reaction rate and quantitative NO yield (5 h) afforded by I-NTf₂, suggesting a proximity requirement for the weakly acidic-HNPh groups to provide large rate accelerations for NO₂⁻ reduction activity as an H-bond donor.

Fig. 6 Kinetic profiles of NO evolution and lines of best fit for NO₂⁻ reduction with I-NTf₂ (red circles, 1st order exponential fit), III-NTf₂ (orange triangles, linear fit) or III-NTf₂ + 3 equiv. [HA] and 10 equiv. [NO₂⁻] (green squares, linear fit). Error bars represent the range of yields from reactions executed in triplicate.

Conclusions

In conclusion, we have reported the first examples of spontaneous NO₂⁻ reduction to afford Fe(III)₂(μ-O)₂²⁺ cores, both in the presence and absence of appended H-bonds (II and IV respectively). The appended H-bonds within I-NTf₂ provide a four-order of magnitude rate acceleration for NO₂⁻ reduction, relative to III-NTf₂, which does not contain H-bonds. Control reactions using exogenous reagents of similar H⁺/e⁻ strengths as I-NTf₂ illustrate the requirement of preorganization of H-bonding units to facilitate rapid nitrite reduction.

The H-bond interactions surrounding the resulting Fe₂(μ-O)₂ core of II imparts distinct electronic and chemical properties relative to IV, and as a consequence, II is more challenging to reduce with H-atom donors. These observations illustrate the interplay between Fe–O stabilization and subsequent H⁺/e⁻ transfer necessary to promote a net reductive transformation, of particular relevance to nitrite reductases and reaction intermediates containing Fe₂(μ-O)₂ cores of dioxygenases, as those found in sMMO-Q or RNR-X. Ongoing work in our lab is focusing on the effects of H-bonds on the electronic structure and reactivity of Fe(III)₂(μ-O)₂ cores, as well as their application toward catalytic nitrogen-oxyanion reduction.

Author contributions

The manuscript was written through the contributions of all authors. Project conceptualization: J. D. G. and N. K. S. collection of experimental data: A. R. L., J. E. G., W. S., and J. D. G. Research supervision: N. K. S.

Conflicts of interest

There are no conflicts of interest to declare.

Data availability

CCDC 2448814 and 2448815 contain the supplementary crystallographic data for this paper.^36a,b

The data supporting this article have been included as part of the SI. Supplementary information: Experimental procedures and characterization of all species and reaction products. See DOI: https://doi.org/10.1039/d5sc04153h.

Acknowledgements

This work was supported by the NIGMS of the NIH under awards R35GM136360 (N. K. S.), and 2R15GM123380-02 (J. D. G). A. R. L. is an NSF-GRFP Fellow (DGE-2241144). Prof. Takele Seda is acknowledged for assistance in obtaining the Mössbauer spectrum of II, Dr Reza Loloee at Michigan State University for collection of SQUID magnetometry data, and Prof. Nicolai Lehnert and Claire Patterson for discussions relating to analysis of magnetometry data.

Notes and references

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16. Relative to I-NTf₂, the reaction evolved 1 equivalent of NO gas.
17. We propose that the dimeric structure of II stays intact in solution because variable temperature ¹H-NMR spectroscopy of II in MeCN did not display significant changes between +80 to −35 °C.
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20. The structure of IV reported in this work is used for comparing structural metrics because it is of higher quality than the previous polymorph (R₁ = 3.95% versus R₁ = 6.5% in ref. 19g).
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22. V. F. Oswald, J. L. Lee, S. Biswas, A. C. Weitz, K. Mittra, R. Fan, J. Li, J. Zhao, M. Y. Hu and E. E. Alp, et al., Effects of Noncovalent Interactions on High-Spin Fe(IV)–Oxido Complexes, J. Am. Chem. Soc., 2020, 142(27), 11804–11817.
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24. E. Zars, L. Gravogl, M. R. Gau, P. J. Carroll, K. Meyer and D. J. Mindiola, Isostructural bridging diferrous chalcogenide cores [FeII(μ-E)FeII] (E = O, S, Se, Te) with decreasing antiferromagnetic coupling down the chalcogenide series, Chem. Sci., 2023, 14(24), 6770–6779,  10.1039/D3SC01094E.
25. (a) The temperature-dependent magnetic susceptibility of II (SQUID) was modelled with the exchange Hamiltonian Ĥ_ex = −J × S₁S₂ using Easyspin’s curry function.; (b) S. Stoll and A. Schweiger, EasySpin, a comprehensive software package for spectral simulation and analysis in EPR, J. Magn. Reson., 2006, 178(1), 42–55.
26. The Mössbauer spectrum of II (as a polycrystalline solid) was collected at 298 K using a constant-acceleration spectrometer (Wissel GMBH, Germany) in a horizontal transmission mode using a 50 mCi ₅₇Co source was utilized for Mössbauer spectra collection. All Mössbauer spectra were collected at room temperature at 4 mm s⁻¹ velocity with approximately 80 mg of sample loaded into an acrylic sample holder with minimal Paratone-N oil to prevent oxidation. Lorentzian line shape with the NORMOS (Wissel GMBH) least-squares fitting program was used to fit spectra, and isomer shifts were normalized to metallic iron. We attribute the slight asymmetry in the two peaks to the random orientation of the polycrystalline sample. See ref. 27.
27. E. Kreber and U. Gonser, The Problem of Texture in Mössbauer Spectroscopy, Texture, Stress, Microstruct., 1974, 1(4), 869318.
28. At −40 °C, the CV of IV has been reported to feature a reversible Fe(III)₂ to Fe(III)Fe(IV) wave (E^1/2 = 0.41 V) that we did not observe at ambient temperature. See ref. 29.
29. H. Zheng, S. J. Yoo, E. Münck and L. Que, The Flexible Fe₂(μ-O)₂ Diamond Core: A Terminal Iron(IV)−Oxo Species Generated from the Oxidation of a Bis(μ-oxo)diiron(III) Complex, J. Am. Chem. Soc., 2000, 122(15), 3789–3790.
30. J. J. Warren, T. A. Tronic and J. M. Mayer, Thermochemistry of Proton-Coupled Electron Transfer Reagents and its Implications, Chem. Rev., 2010, 110(12), 6961–7001.
31. Treatment of II with substrates containing weak C–H bonds (i.e. 9,10-dihydroanthracene or Ph₃CH) as reductants did not afford reduction of II nor C–H oxidation (to form anthracene, anthraquinone, or Ph₃COH). See SI for further details.
32. S. V. Kryatov, E. V. Rybak-Akimova, V. L. MacMurdo and L. Que, A Mechanistic Study of the Reaction between a Diiron(II) Complex [FeII₂(μ-OH)₂(6-Me₃-TPA)₂]²⁺ and O₂ to Form a Diiron(III) Peroxo Complex, Inorg. Chem., 2001, 40(10), 2220–2228.
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34. 1-Methyl-2-(phenylamino)pyridinium [HA] features an ν(NH) band at 3272 cm⁻¹, while I-NTf₂'s ν(NH) is at 3278 cm⁻¹ (see SI). This further supports our assertion that [HA] and I-NTf₂ have similar acidity/H-bond donor strengths..
35. We selected ferrocene as a reductant in this control reaction because it approximates the reducing potential of I-NTf₂ and III-NTf₂ in the presence of 10 equiv. [Bu₄N][NO₂] (0.05 V vs. Fc^0/+) see SI..
36. (a) A. R. LaDuca, J. E. Gonder, W. Sarkar, J. D. Gilbertson and N. Szymczak, CCDC 2448814: Experimental Crystal Structure Determination, 2025,  DOI:10.5517/ccdc.csd.cc2n6603; (b) A. R. LaDuca, J. E. Gonder, W. Sarkar, J. D. Gilbertson and N. Szymczak, CCDC 2448815: Experimental Crystal Structure Determination, 2025,  DOI:10.5517/ccdc.csd.cc2n6614.

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