15N NMR Research Articles

Tracking nitrite's deviation from Stokes-Einstein predictions with pulsed field gradient 15N NMR spectroscopy.

Predicting the behavior of oxyanions in radioactive waste stored at the Department of Energy legacy nuclear sites requires the development of novel analytical methods. This work demonstrates 15N pulsed field gradient nuclear magnetic resonance spectroscopy to quantify the diffusivity of nitrite. Experimental results, supported by molecular dynamics simulations, indicate that the diffusivity of free hydrated nitrite exceeds that of free hydrated sodium despite the greater hydrodynamic radius of nitrite. Investigations are underway to understand how the compositional and dynamical heterogeneities of the ion networks at high concentrations affect rheological and transport properties.

Biomimetic catalysis of nitrite reductase enzyme using copper complexes in chemical and electrochemical reduction of nitrite.

Copper nitrite reductase mimetics were synthesized using three new tridentate ligands sharing the same N,N,N motif of coordination. The ligands were based on L-proline modifications, attaching a pyridine and a triazole to the pyrrolidine ring, and differ by a pendant group (R = phenyl, n-butyl and n-propan-1-ol). All complexes coordinate nitrite, as evidenced by cyclic voltammetry, UV-Vis, FTIR and electron paramagnetic resonance (EPR) spectroscopies. The coordination mode of nitrite was assigned by FTIR and EPR as κ2O chelate mode. Upon acidification, EPR experiments indicated a shift from chelate to monodentate κO mode, and 15N NMR experiments of a Zn2+ analogue, suggested that the related Cu(II) nitrous acid complex may be reasonably stable in solution, but in equilibrium with free HONO under non catalytic conditions. Reduction of nitrite to NO was performed both chemically and electrocatalytically, observing the highest catalytic activities for the complex with n-propan-1-ol as pendant group. These results support the hypothesis that a hydrogen bond moiety in the secondary coordination sphere may aid the protonation step.

The extracellular chaperone clusterin prevents primary and secondary nucleation of an amyloidogenic variant of β2-microglobulin

Amyloid fibril formation by the extracellular protein β2-microglobulin (β2m) and its subsequent accumulation in periarticular tissues have been linked to dialysis-related amyloidosis. A natural variant of human β2m responsible for aggressive systemic amyloidosis contains an aspartate to asparagine mutation at residue 76 (i.e. D76N β2m), which readily forms amyloid fibrils in vitro under physiological conditions. In this study, we examined the role of the extracellular molecular chaperone clusterin in modulating D76N β2m fibril formation in vitro under physiological conditions. The presence of extrinsic charged amino acids modulated D76N β2m fibril formation, implying that electrostatic interactions are involved in the protein’s aggregation. Thioflavin T (ThT) and 1-anilinonaphthalene-8-sulfonate fluorescence assays indicated that clusterin interacts via hydrophobic and electrostatic forces with the monomeric, prefibrillar and fibrillar species of D76N β2m. As a result, clusterin was incorporated into D76N β2m aggregates during the latter’s fibril formation, as indicated by SDS-PAGE of depolymerised fibrils. SYPRO Orange and ThT fluorescence assays suggested that, compared to pure D76N β2m fibrils, those formed in the presence of clusterin are chemically more stable with a reduced ability to act as nucleation seeds. Detailed 15N NMR relaxation studies of mixtures of 15N-labelled β2m with clusterin confirmed that the chaperone interacts transiently and non-specifically with monomeric β2m. Clusterin inhibits both primary and secondary nucleation of D76N β2m fibril formation. In doing so, clusterin binds to D76N β2m fibrils and stabilises them to prevent possible fragmentation. In vivo, the multifaceted chaperone action of clusterin may delay, if not prevent, β2m amyloid proliferation and deposition in tissues.

Solvent- and functional-group-assisted tautomerism of 3-alkyl substituted 5-(2-pyridyl)-1,2,4-triazoles in DMSO-water.

The tautomerism of a series of 5-alkyl substituted 3-(2-pyridyl)-1,2,4-triazoles in DMSO-d6-containing water has been investigated by 1H, 13C and 15N NMR spectroscopy. The populations of the three possible regioisomers in the tautomeric equilibrium (A [3-alkyl-5-(2-pyridyl)-1H], B [5-alkyl-3-(2-pyridyl)-1H] and C [5-alkyl-3-(2-pyridyl)-4H]) were determined. Isomers A (17-40%) and B (54-79%) are the major components and their ratio is insensitive to the substitution pattern, except for the unsubstituted and the methoxymethyl substituted derivatives. The isomer C (3-5%) has been fully characterised for the first time by NMR spectroscopy. Activation energies of tautomerisation (14.74-16.78 kcal mol-1) were determined by EXSY experiments, which also supported the involvement of water in the tautomerisation. Substituent effects on the 15N chemical shifts are relatively small. The DFT study of the tautomerism in DMSO-water showed that both A/B and B/C interconversions are assisted by the pyridine substituent and catalysed by solvent molecules. The NH-A/NH-B tautomerisation takes place via a relayed quadruple proton transfer mediated by three water molecules in the hydrogen-bonded cyclic substructure of a triazole·4H2O complex. The equilibrium B ⇄ C involves three steps: NH-B transfer to the pyridyl nitrogen mediated by a water molecule in a 1 : 1 cyclic complex, rotamerisation to bring the pyridinium NH close to N4 of the triazole catalysed by complexation to a DMSO molecule and transfer of the NH from the pyridinium donor to the N4 acceptor via a 1 : 1 complex with a bridging water molecule. This mechanism of 1,3-prototropic shift in triazoles is unprecedented in the literature.

Processing on crystal growth, structure, thermal property, and nuclear magnetic resonance of organic-inorganic hybrid perovskite type [NH3(CH2)6NH3]ZnCl4 crystal.

Organic-inorganic hybrid compounds have recently gained significant attention in recent years due to their diverse applications. Herein, [NH3(CH2)6NH3]ZnCl4 crystals were grown, and their triclinic structure, phase transition temperature (TC = 408 K), and high thermal stability (Td = 584 K) was determined using X-ray diffraction (XRD), differential scanning calorimetry, and thermogravimetry measurements. By analyzing the chemical in response to temperature changes, we observed that the coordination geometry around 1H and 13C were highly symmetric below TC, whereas their symmetry was lowered above TC. The change of N-H⋯Cl hydrogen bond from XRD results and the change of 14N NMR chemical shifts was due to the changes to the coordination geometry of Cl- around Zn2+ in the ZnCl4 anion. The activation energy of 1H was three times greater than that of 13C, and this result indicates that the energy transfer of 13C was easier than those of 1H. We compared the results for [NH3(CH2)nNH3]ZnCl4 (n = 6) studied here with those for n = 2, 3, 4, and 5 obtained from previous studies. The characteristics of the length of CH2 in the methylene chain are expected to be used for potential applications in the near future.

Cosolvent Dimethyl Sulfoxide Influences Protein–Ligand Binding Kinetics via Solvent Viscosity Effects: Revealing the Success Rate of Complex Formation Following Diffusive Protein–Ligand Encounter

Protein-ligand-exchange kinetics determines the duration of biochemical signals and consequently plays an important role in drug design. Binding studies commonly require solubilization of designed ligands in solvents such as dimethyl sulfoxide (DMSO), resulting in residual amounts of DMSO following titration of solubilized ligands into aqueous protein samples. Therefore, it is critical to establish whether DMSO influences protein-ligand binding. Here, we address the general and indirect effect of DMSO on protein-ligand binding caused by solvent viscosity, which is strongly dependent on the relative concentrations of DMSO and water. As a model system, we studied the binding of a drug-like ligand to the carbohydrate recognition domain of galectin-3 in the presence of variable amounts of DMSO. We used isothermal titration calorimetry to characterize binding thermodynamics and 15N NMR relaxation to monitor kinetics. The binding enthalpy is not affected, but we observe a subtle trend of increasingly unfavorable entropy of binding, and consequently decreased affinity, with increasing DMSO concentration. The increasing concentration of DMSO results in a reduced association rate of binding, while the dissociation rate is less affected. The observed association rate is inversely proportional to the viscosity of the DMSO-water mixture, as expected from theory, but significantly reduced from the diffusion-controlled limit. By comparing the viscosity dependence of the observed association rate with that of the theoretical diffusion-controlled association rate, we estimate the success rate of productive complex formation following an initial encounter of proteins and ligands, showing that only one out of several hundred binding "attempts" are successful.

1H, 13C and 15N assignment of the human mitochondrial paramagnetic iron-sulfur protein CISD3.

CISD3 is a mitochondrial protein that contains two [2Fe-2S] clusters. This protein is overexpressed in some types of cancer, so it has emerged as a potential drug target. A detailed characterization of this protein is crucial to understand how CISD3 is involved in these physiopathologies. In this study, isotopically labeled human CISD3 was expressed in Escherichia coli. A set of double and triple resonance experiments performed with standard parameters/datasets provided the assignment of 40% of the HN resonances, 47% of Cα, and 46% of C' resonances. Tailored paramagnetic HSQC, CON and CACO experiments extended up to 59% for HN, 70% for Cα and 69% for C'. The 1H, 13C and 15N NMR chemical shift assignment of human CISD3 is reported here.

Nitrogen‐Rich Oxetanes Based on the Combination of Azides and Tetrazoles

AbstractLiterature known energetic oxetane derivatives have a nitrogen content of up to 49.98 %. Through the introduction of azide and tetrazole functionalities attached to an oxetane ring, energetic oxetanes with higher nitrogen contents than previously reported in the literature were obtained. The newly synthesized oxetane derivatives were extensively characterized via 1H NMR, 13C{1H} NMR, 14N NMR, 15N NMR, 1H‐15N HMBC, FT‐IR spectroscopy and/or DTA. Their crystal structures were elucidated using X‐ray diffraction, their sensitivities towards impact, friction and electrostatic discharge were determined and their energetic properties were calculated using the EXPLO5 code.

15N NMR study of azo-hydrazo tautomerism: Past and present

15N NMR data (1J(15N, 1H) coupling constants and 15N chemical shifts) seem to be the most suitable ones to characterize azo-hydrazo tautomerism quantitatively. The reason is that nitrogen atoms of –NN−/−NH-N = fragments are directly involved in azo-hydrazo equilibrium.First data was measured and published in 1970 and 15N selectively enriched compounds had to be prepared since electro magnets were used in NMR spectrometers at that time providing very low resonance frequencies (from 60 to 100 MHz for protons) and have had, consequently, very low sensitivity. Moreover, a choice of 15N enriched 15N anilines was very limited and these compounds were relatively very expensive.At present, the situation is completely different. There has been a huge increase of NMR spectrometer frequencies used for measurement of NMR spectra and even routinely used NMR spectrometers having 400–600 MHz proton frequencies are sufficiently sensitive to provide 1J(15N, 1H) coupling constants and 15N chemical shifts at the natural abundance level of15N. Inverse detection of resonances is now routinely used. The lower magnetogyric ratio of appropriate nucleus the higher signal enhancement is achieved: this is the very case of the 15N isotope. Theoretical enhancement factor obtained for inverse detection of 15N compared with direct one is ca 300! Application of cryoprobes where electronics is chilled either by liquid nitrogen or even liquid helium reducing electric noise allows to reduce time of accumulation from 8 to 16 times!Some representative examples are presented both from past to present demonstrating the fact that thorough analyses of NMR spectra allow determining the proper constitutions of compounds studies and excluding the wrong ones.

Synthesis, characterization and computational investigation of the phosphatase activity of a dinuclear Zinc(II) complex containing a new heptadentate asymmetric ligand

We report the synthesis of a new asymmetric heptadentate ligand based on the 1,3-diaminopropan-2-ol backbone. The ligand 3-[[3-(bis-pyridin-2-ylmethyl-amino)-2-hydroxy-propyl]-(2-carbamoyl-ethyl)-amino]-propionamide (HL1) contains two amide and two pyridine groups attached to the 1,3-diaminopropan-2-ol core. Reaction between HL1 and Zn(ClO4)2.6H2O resulted in the formation of the dinuclear [Zn2(L1)(μ-OAc)](ClO4)2 complex, characterized by single crystal X-ray diffraction, 1H, 13C and 15N NMR, ESI-(+)-MS, CHN elemental analysis as well as infrared spectroscopy. The phosphatase activity of the complex was studied in the pH range 6–11 employing pyridinium bis(2,4-dinitrophenyl)phosphate (py(BDNPP)) as substrate. The complex exhibited activity dependent on the pH, presenting an asymmetric bell shape profile with the highest activity at pH 9; at high pH ligand exchange is rate-limiting. The hydrolysis of BDNPP− at pH 9 displayed behavior characteristic of Michaelis-Menten kinetics, with kcat = 5.06 × 10−3 min−1 and Km = 5.7 ± 1.0 mM. DFT calculations map out plausible reaction pathways and identify a terminal, Zn(II)-bound hydroxide as likely nucleophile.

Four-Component Relativistic Calculations of NMR Shielding Constants of the Transition Metal Complexes—Part 2: Nitrogen-Coordinated Complexes of Cobalt

Both four-component relativistic and nonrelativistic computations within the GIAO-DFT(PBE0) formalism have been carried out for 15N and 59Co NMR shielding constants and chemical shifts of a number of the nitrogen-coordinated complexes of cobalt. It was found that the total values of the calculated nitrogen chemical shifts of considered cobalt complexes span over a range of more than 580 ppm, varying from -452 to +136 ppm. At that, the relativistic corrections to nitrogen shielding constants and chemical shifts were demonstrated to be substantial, changing accordingly from ca. -19 to +74 ppm and from -68 to +25 ppm. Solvent effects on 15N shielding constants and chemical shifts were shown to have contributions no less important than the relativistic effects, namely from -35 to +63 ppm and from -74 to +23 ppm, respectively. Cobalt shielding constants and chemical shifts were found to vary in the ranges of, accordingly, -20,157 to -11,373 ppm and from +3781 to +13,811. The relativistic effects are of major importance in the cobalt shielding constants, resulting in about 4% for the shielding-type contributions, while solvent corrections to cobalt shielding constants appeared to be of less significance, providing corrections of about 1.4% to the gas phase values.

Connecting Energetic Nitropyrazole and Nitrobenzene Moieties with C−C Bonds using Suzuki Cross‐Coupling Reaction: A Novel Route to Thermally Stable Energetic Materials

AbstractThis work describes a new route to thermally stable energetic materials by connecting different types of energetic moieties (nitropyrazole and polynitrobenzene) with C−C bonds, where the first step involves a Suzuki cross‐coupling reaction between 4‐bromo‐3,5‐dinitro‐1H‐pyrazole and 4‐chlorophenylboronic acid. Further nitration, amination, oxidation, and salt formation reactions on the resulting C−C coupled framework resulted in various thermally stable energetic materials. All the compounds were fully characterized using IR, NMR [1H, 13C{1H}], differential scanning calorimetry (DSC), elemental analysis, and HRMS studies. Compounds 7 and 9 were further characterized by 15N NMR, and compounds 3 and 6 were characterized by single‐crystal X‐ray diffraction studies. Theoretical heats of formation and energetic performance for all the energetic compounds were calculated using Gaussian 09 and EXPLO5 v6.06 programs, respectively. This research introduces a novel method for synthesizing C−C coupled energetic materials that are not accessible by any other routes.

Evaluation of the Stability of Diamine-Appended Mg2(dobpdc) Frameworks to Sulfur Dioxide.

Diamine-appended Mg2(dobpdc) (dobpdc4- = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate) metal-organic frameworks are a promising class of CO2 adsorbents, although their stability to SO2─a trace component of industrially relevant exhaust streams─remains largely untested. Here, we investigate the impact of SO2 on the stability and CO2 capture performance of dmpn-Mg2(dobpdc) (dmpn = 2,2-dimethyl-1,3-propanediamine), a candidate material for carbon capture from coal flue gas. Using SO2 breakthrough experiments and CO2 isobar measurements, we find that the material retains 91% of its CO2 capacity after saturation with a wet simulated flue gas containing representative levels of CO2 and SO2, highlighting the robustness of this framework to SO2 under realistic CO2 capture conditions. Initial SO2 cycling experiments suggest dmpn-Mg2(dobpdc) may achieve a stable operating capacity in the presence of SO2 after initial passivation. Evaluation of several other diamine-Mg2(dobpdc) variants reveals that those with primary,primary (1°,1°) diamines, including dmpn-Mg2(dobpdc), are more robust to humid SO2 than those featuring primary,secondary (1°,2°) or primary,tertiary (1°,3°) diamines. Based on the solid-state 15N NMR spectra and density functional theory calculations, we find that under humid conditions, SO2 reacts with the metal-bound primary amine in 1°,2° and 1°,3° diamine-appended Mg2(dobpdc) to form a metal-bound bisulfite species that is charge balanced by a primary ammonium cation, thereby facilitating material degradation. In contrast, humid SO2 reacts with the free end of 1°,1° diamines to form ammonium bisulfite, leaving the metal-diamine bond intact. This structure-property relationship can be used to guide further optimization of these materials for CO2 capture applications.

From Peptide Nucleic Acids to Supramolecular Structures of Nucleic Acid Derivatives.

Nucleic acids play a pivotal role in life processes. The endeavours to shed light on the essential properties of these intriguing building blocks led us to the synthesis of different analogues and the investigation of their properties. First various peptide nucleic acid monomers and oligomers have been synthesized, using an Fmoc/acyl protecting group strategy, and their properties studied. The serendipitous discovery of a side reaction of coupling agents led us to the elaboration of a peptide sequencing method. The capricious behaviour of guanine derivatives spurred the determination of their substitution pattern using 13 C, 15 N NMR, and mass spectrometric methods. The properties of guanines initiated the logical transition to the study of supramolecular systems composed of purine analogues. Thus, xanthine and uracil derivatives have been obtained and their supramolecular self-assembly properties scrutinized in gas, solid, and liquid states and at solid-liquid interfaces.

1-(Nitratomethyl)-5H-tetrazole: A Highly Sensitive Ligand with Improved Oxygen Balance for Laser-Ignitable Coordination Compounds.

For the first time, the highly sensitive 1-(nitratomethyl)-5H-tetrazole (1-NAMT) was synthesized, representing the shortest possible 1-(nitratoalkyl)-5H-tetrazole with a combined nitrogen and oxygen content of 81.4%. Compared to its related ethyl derivative, 1-(nitratoethyl)-5H-tetrazole, it exhibits improved oxygen balance, resulting in higher detonation parameters. 1-NAMT was thoroughly analyzed by single-crystal diffraction experiments accompanied by elemental analysis, IR spectroscopy, and multinuclear (1H, 13C, and 14N) NMR measurements. The thermal behavior of 1-NAMT was analyzed by differential thermal analysis supported by thermogravimetric analysis. Furthermore, energetic coordination compounds (ECCs) of Cu with different inorganic (e.g., nitrate, chlorate, and perchlorate) and nitroaromatic anions (e.g., picrate and styphnate) were synthesized and thoroughly analyzed. It is shown that the formation of ECCs with nitroaromatic anions (Tdec ∼ 180 °C) is a suitable strategy to improve the rather low thermal stability of 1-NAMT (125 °C).

Erratum for the Research Article: "Full-Nitro-Nitroamino Cooperative Action: Climbing the Energy Peak of Benzenes with Enhanced Chemical Stability" by Sun et al.

In the article “Full-Nitro-Nitroamino Cooperative Action: Climbing the Energy Peak of Benzenes with Enhanced Chemical Stability,” the authors mistakenly reported that the “chemical shifts in the 15N NMR spectra were reported relative to CH3NO2 as the external standard,” when the 15N NMR spectra were in fact reported relative to NH3. This has now been corrected in the “General methods” section of the “Materials and Methods.”

Novel “naked eye” chromofluorogenic azomethine imine chemosensors for the detection of F−, CN−, AcO− and H2PO4− anions

Novel “naked eye” chromofluorogenic chemosensors for biologically important anions F−, CN−, AcO− and H2PO4− based on 5-chloro-2-hydroxy- (1), 3,5-dichloro-2-hydroxy- (2) and 3-bromo-2-hydroxy-5-methoxybenzylidene (3) azomethine imines were synthesized. Their structure and sensing properties were studied using 1Н, COSY, 13С, HSQC, HMBC and 15N NMR, UV-Vis/fluorescence spectroscopy, X-ray diffraction analysis and DFT calculations. Compounds 1-3 exhibit low-intensity emission at 537–574 nm with an anomalous Stokes shift of 7830–8420 cm−1. In the presence of F−, CN−, AcO− and H2PO4− ions, this emission is completely quenched and a new intense fluorescence appears at 540–590 nm with values of the relative enhancement in fluorescence intensity (I/I0) 15-79 (F−) and 10-137 (CN−). Simultaneously “naked-eye” chromogenic effect is observed with a color change from pale yellow to intense yellow or orange. These spectral responses were attributed to the OH…An− bonding up to the deprotonation. The association constants found for fluoride anion are 5.5 × 108 M−2 (1), 1.3 × 103 M−1 (2) and 5.3 × 108 M−2 (3), and to cyanide anion - 2.8 × 103 M−1 (1), 2.0 × 103 M−1 (2) and 2.8 × 103 M−1 (3). UV-Vis limits of detection for the F− ion are 2.0–6.0 μM (1), and for the CN− ion – 1.0-2.0 μM.

Energetic coordination compounds of late 3d metals with 1N-(Nitromethyl)-5H-tetrazole, a ligand with astonishing properties from the rare class of N-nitromethyl azoles

The hardly accessible N-nitromethyl moiety is introduced onto the 1,5H-tetrazole scaffold, representing the fourth and fifth ever synthesized N-nitromethyl azoles. Both compounds 1-(Nitromethyl)-5H-tetrazole (2a) and 2-(Nitromethyl)-5H-tetrazole (2b) are thoroughly analyzed by low-temperature single-crystal X-ray diffraction experiments, complemented by elemental analysis, multinuclear (1H, 13C, 14N, 15N) NMR as well as IR spectroscopy. Sensitivity measurements towards external stimuli revealed two highly insensitive compounds. Additionally, 2a was applied as ligand for energetic coordination compounds of 3d transition metals (e.g. Cu, Fe, Ni, and Zn) in combination with oxidizing nitrate, chlorate and perchlorate anions. The synthesized ECCs were analyzed by low-temperature single-crystal X-ray diffraction experiments, elemental analysis and IR spectroscopy. The thermal behavior of all compounds was investigated by differential thermal analysis and the sensitivities towards impact and friction was measured. Due to the lack of information about the properties of N-nitromethyl azoles, the complete insensitivity towards external stimuli of 2a and 2b supports first hints of a sensitivity decreasing influence of the N-nitromethyl moiety, which is highly desired when designing new high explosives. Additionally, the functional group is able to introduce meltability into molecular scaffolds.

Convergent views on disordered protein dynamics from NMR and computational approaches

Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) is a class of biologically important proteins exhibiting specific biophysical characteristics. They lack a hydrophobic core, and their conformational behavior is strongly influenced by electrostatic interactions. IDPs and IDRs are highly dynamic, and a characterization of the motions of IDPs and IDRs is essential for their physically correct description. NMR together with molecular dynamics simulations are the methods best suited to such a task because they provide information about dynamics of proteins with atomistic resolution. Here, we present a study of motions of a disordered C-terminal domain of the delta subunit of RNA polymerase from Bacillus subtilis. Positively and negatively charged residues in the studied domain form transient electrostatic contacts critical for the biological function. Our study is focused on investigation of ps-ns dynamics of backbone of the delta subunit based on analysis of amide 15N NMR relaxation data and molecular dynamics simulations. In order to extend an informational content of NMR data to lower frequencies, which are more sensitive to slower motions, we combined standard (high-field) NMR relaxation experiments with high-resolution relaxometry. Altogether, we collected data reporting the relaxation at 12 different magnetic fields, resulting in an unprecedented data set. Our results document that the analysis of such data provides a consistent description of dynamics and confirms the validity of so far used protocols of the analysis of dynamics of IDPs also for a partially folded protein. In addition, the potential to access detailed description of motions at the timescale of tens of ns with the help of relaxometry data is discussed. Interestingly, in our case, it appears to be mostly relevant for a region involved in the formation of temporary contacts within the disordered region, which was previously proven to be biologically important.

Hydrogen Atom Abstraction from an OsII(NH3)2 Complex Generates an OsIV(NH2)2 Complex: Experimental and Computational Analysis of the N-H Bond Dissociation Free Energies and Reactivity.

Double hydrogen atom abstraction from (TMP)OsII(NH3)2 (TMP = tetramesitylporphyrin) with phenoxyl or nitroxyl radicals leads to (TMP)OsIV(NH2)2. This unusual bis(amide) complex is diamagnetic and displays an N-H resonance at 12.0 ppm in its 1H NMR spectrum. 1H-15N correlation experiments identified a 15N NMR spectroscopic resonance signal at -267 ppm. Experimental reactivity studies and density functional theory calculations support relatively weak N-H bonds of 73.3 kcal/mol for (TMP)OsII(NH3)2 and 74.2 kcal/mol for (TMP)OsIII(NH3)(NH2). Cyclic voltammetry experiments provide an estimate of the pKa of [(TMP)OsIII(NH3)2]+. In the presence of Barton's base, a current enhancement is observed at the Os(III/II) couple, consistent with an ECE event. Spectroscopic experiments confirmed (TMP)OsIV(NH2)2 as the product of bulk electrolysis. Double hydrogen atom abstraction is influenced by π donation from the amides of (TMP)OsIV(NH2)2 into the d orbitals of the Os center, favoring the formation of (TMP)OsIV(NH2)2 over N-N coupling. This π donation leads to a Jahn-Teller distortion that splits the energy levels of the dxz and dyz orbitals of Os, results in a low-spin electron configuration, and leads to minimal aminyl character on the N atoms, rendering (TMP)OsIV(NH2)2 unreactive toward amide-amide coupling.