15N Nuclear Magnetic Resonance Spectra Research Articles

Effects of structures of lower aliphatic alcohols and concentrations on CO2 chemical absorption and desorption reactions in various alcohol solutions of 1,8-diazabicyclo[5.4.0]undec-7-ene

Alcohol solutions of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) have received considerable interest as chemical absorbers of CO2 and switchable solvents because of their reversible reaction with CO2 and the polarity change triggered by CO2 absorption/desorption. However, there have been only a few systematic studies on the CO2 absorption and desorption behaviors of DBU–alcohol solutions. In this study, we determined the solubility of CO2 in binary solutions of DBU and various lower aliphatic alcohols at different temperatures. Except for the 2-methyl-2-propanol solution, which showed very low reactivity with CO2, all the DBU–alcohol solutions exhibited almost the same solubilities of CO2 at 298.2 K, independent of the alkyl groups present on the alcohols. However, their solubilities varied with DBU concentration. Moreover, we examined the amounts of CO2 recovery from the CO2-saturated solutions at high temperatures up to 393.2 K or the boiling points. The amount of CO2 per mole of DBU desorbed by heating was markedly influenced by both the alkyl group on the alcohols and the DBU concentration, indicating that at the studied concentrations, the temperature dependence of CO2 solubilities is strongly affected by the type of alcohol. To clarify this unique desorption behavior, we measured the 1H, 13C, and 15N nuclear magnetic resonance spectra of the DBU–alcohol solutions with and without CO2 at 298.2 K. In the absence of CO2, the nitrogen atoms at the 8-position of the DBU molecules strongly interact with the hydroxyl hydrogen atoms of the alcohol molecules via hydrogen bonding. As the DBU–alcohol solutions were saturated with CO2, the formation of protonated DBU ([DBUH]+) and alcohol-CO2 adduct (alkyl carbonate, [ROCO2]–) was confirmed. [DBUH]+ is stabilized by the solvation of free alcohol molecules, and the reverse reaction of [DBUH]+ with [ROCO2]– for CO2 release is hindered. Our findings are beneficial for designing and tuning DBU–alcohol systems for CO2 separation.

Surface Acidity Characterization of Metal-Doped Amorphous Silicates via Py-FTIR and 15N NMR Simulations

Metal-doped amorphous silicates are effective acidic catalysts for numerous industrial applications, including dehydration of alcohols, hydrodeoxygenation of biomass, and metathesis of olefins. The acidity of these catalysts is derived from the metal dopant and is experimentally characterized using a number of techniques, such as ammonia temperature-programmed desorption (NH3-TPD), pyridine Fourier transform infrared (Py-FTIR), and 15N nuclear magnetic resonance (NMR) spectroscopy of either probe molecule. Although these measurements provide information regarding the amount and strength of the acid sites, they alone do not provide any detail about the local structure around the metal center, which hampers the ability to systematically design better catalysts. In this study, we present density functional theory (DFT) simulations of Py-FTIR and 15N NMR spectra to characterize the local structure and acidity strength of metal-doped silicate clusters. We perform calculations on the model structures compiled in the METal-doped Amorphous SIlicate Library (METASIL), which aims at providing realistic amorphous structures for Zr-, Nb-, and W-doped silicates. By matching the calculated normal mode frequencies with experimental IR spectra, we reproduce the differentiation between Lewis and strong Brønsted acid sites. However, our simulations show that the coordination of Py to weak Brønsted acids is indistinguishable from that to metal sites, as they elicit similar frequency shifts. On the other hand, 15N NMR can distinguish between Py interacting with metal centers, weak and strong Brønsted acid sites, and it may be able to identify the structure of metal dopants. Therefore, these results can be used to provide theoretical support for the design of new catalytic materials.

Heteronuclear cross-relaxation effect modulated by the dynamics of N-functional groups in the solid state under 15N DP-MAS DNP

In a typical magic-angle spinning (MAS) dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) experiment, several mechanisms are simultaneously involved when transferring much larger polarization of electron spins to NMR active nuclei of interest. Recently, specific cross-relaxation enhancement by active motions under DNP (SCREAM-DNP) [Daube et al. JACS 2016] has been reported as one of these mechanisms. Thereby 13C enhancement with inverted sign was observed in a direct polarization (DP) MAS DNP experiment, caused by reorientation dynamics of methyl that was not frozen out at 100 K. Here, we report on the spontaneous polarization transfer from hyperpolarized 1H to both primary amine and ammonium nitrogens, resulting in an additional positive signal enhancement in the 15N NMR spectra during 15N DP-MAS DNP. The cross-relaxation induced signal enhancement (CRE) for 15N is of opposite sign compared to that observed for 13C due to the negative sign of the gyromagnetic ratio of 15N. The influence on CRE efficiency caused by variation of the radical solution composition and by temperature was also investigated.

Structure of melanins from the fungi Ochroconis lascauxensis and Ochroconis anomala contaminating rock art in the Lascaux Cave

Two novel species of the fungal genus Ochroconis, O. lascauxensis and O. anomala have been isolated from the walls of the Lascaux Cave, France. The interest in these fungi and their melanins lies in the formation of black stains on the walls and rock art which threatens the integrity of the paintings. Here we report solid-state cross polarization magic-angle spinning 13C and 15N nuclear magnetic resonance (NMR) spectroscopy and surface-enhanced Raman spectroscopy (SERS) of the melanins extracted from the mycelia of O. lascauxensis and O. anomala in order to known their chemical structure. The melanins from these two species were compared with those from other fungi. The melanins from the Ochroconis species have similar SERS and 13C and 15N NMR spectra. Their chemical structures as suggested by the data are not related to 3,4-dihydroxyphenylalanine, 5,6-dihydroxyindole or 1,8-dihydroxynaphthalene precursors and likely the building blocks from the melanins have to be based on other phenols that react with the N-terminal amino acid of proteins. The analytical pyrolysis of the acid hydrolysed melanin from O. lascauxensis supports this assumption.

Enhanced NMR Discrimination of Pharmaceutically Relevant Molecular Crystal Forms through Fragment-Based Ab Initio Chemical Shift Predictions.

Chemical shift prediction plays an important role in the determination or validation of crystal structures with solid-state nuclear magnetic resonance (NMR) spectroscopy. One of the fundamental theoretical challenges lies in discriminating variations in chemical shifts resulting from different crystallographic environments. Fragment-based electronic structure methods provide an alternative to the widely used plane wave gauge-including projector augmented wave (GIPAW) density functional technique for chemical shift prediction. Fragment methods allow hybrid density functionals to be employed routinely in chemical shift prediction, and we have recently demonstrated appreciable improvements in the accuracy of the predicted shifts when using the hybrid PBE0 functional instead of generalized gradient approximation (GGA) functionals like PBE. Here, we investigate the solid-state 13C and 15N NMR spectra for multiple crystal forms of acetaminophen, phenobarbital, and testosterone. We demonstrate that the use of the hybrid density functional instead of a GGA provides both higher accuracy in the chemical shifts and increased discrimination among the different crystallographic environments. Finally, these results also provide compelling evidence for the transferability of the linear regression parameters mapping predicted chemical shieldings to chemical shifts that were derived in an earlier study.

Probing the Carbonyl Functionality of a Petroleum Resin and Asphaltene through Oximation and Schiff Base Formation in Conjunction with N-15 NMR.

Despite recent advances in spectroscopic techniques, there is uncertainty regarding the nature of the carbonyl groups in the asphaltene and resin fractions of crude oil, information necessary for an understanding of the physical properties and environmental fate of these materials. Carbonyl and hydroxyl group functionalities are not observed in natural abundance 13C nuclear magnetic resonance (NMR) spectra of asphaltenes and resins and therefore require spin labeling techniques for detection. In this study, the carbonyl functionalities of the resin and asphaltene fractions from a light aliphatic crude oil that is the source of groundwater contamination at the long term USGS study site near Bemidji, Minnesota, have been examined through reaction with 15N-labeled hydroxylamine and aniline in conjunction with analysis by solid and liquid state 15N NMR. Ketone groups were revealed through 15N NMR detection of their oxime and Schiff base derivatives, and esters through their hydroxamic acid derivatives. Anilinohydroquinone adducts provided evidence for quinones. Some possible configurations of the ketone groups in the resin and asphaltene fractions can be inferred from a consideration of the likely reactions that lead to heterocyclic condensation products with aniline and to the Beckmann reaction products from the initially formed oximes. These include aromatic ketones and ketones adjacent to quaternary carbon centers, β-hydroxyketones, β-diketones, and β-ketoesters. In a solid state cross polarization/magic angle spinning (CP/MAS) 15N NMR spectrum recorded on the underivatized asphaltene as a control, carbazole and pyrrole-like nitrogens were the major naturally abundant nitrogens detected.

Identification and Physicochemical Characteristics of Temozolomide Process-Related Impurities

In this article the crystal structures of the starting material TZ-5 and the key intermediate TZ-6 of temozolomide (TZ-7), an anticancer therapeutic agent, are presented, together with their spectroscopic and thermal characteristics. Both compounds crystallize in the triclinic P-1 space group. X-ray crystallography studies proved that the compound TZ-6 exists as a monohydrate. A complete structural assignment was obtained for the signals in the 1H-, 13C- and 15N-nuclear magnetic resonance spectra and the structures were confirmed by Fourier-Transform infrared and Raman spectroscopy. The article describes the importance of the high purity of TZ-6 during the small-scale plant production of TZ-7 in a desired polymorphic form III with the purity higher than 99.50%, according to an HPLC method.

<b>Experimental and theoretical NMR study of 4-(1-pyrrolidinyl)piperidine</b>

The possible stable forms and molecular structure of 4-(1-pyrrolidinyl)piperidine (4-pypp) have been studied experimentally and theoretically using nuclear magnetic resonance (NMR) spectroscopy. 1H, 13C, 15N, DEPT, COSY and HETCOR NMR spectra of 4-pypp (C9H18N2) have been reported. Solvent effects on nuclear magnetic shielding tensors have been investigated using chloroform-d, methanol-d, acetone-d, dimethylsulfoxide-d and water-d. The magnitude of nJ(C,H) (n = 1, 2, 3) coupling constants of 4-pypp have been determined with selective 1H decoupled 13C NMR techniques. 1H, 13C, 15N NMR chemical shifts and 1-3J(C,H) coupling constants have also been calculated for the most stable two conformers, equatorial-equatorial (e-e) and axial-equatorial (a-e) forms of 4-pypp using DFT/6-311++G(d,p)//6-31G(d) level of theory. Results from experimental and theoretical data have showed that the molecular geometry and the mole fractions of stable conformers of 4-pypp are solvent dependent.KEY WORDS: 4-(1-Pyrrolidinyl)piperidine, NMR, DFT, Axial, Equatorial Bull. Chem. Soc. Ethiop. 2011, 25(3), 437-442.

Bicelle-Bound Solid-State NMR Structural Studies and Membrane-Permeabilizing Activities of Piscidin 1 and Piscidin 3: Implications for Mode of Antimicrobial Action

Bicelles represent a novel preparation of hydrated lipid bilayers, which can be used to study membrane-associated proteins under physiologically-relevant conditions. Large bicelles can be oriented within a magnetic field, enabling the determination of high-resolution peptide structures and angles of insertion within a lipid membrane via solid-state Nuclear Magnetic Resonance (NMR). Piscidin, an amphipathic, antimicrobial peptide found in hybrid striped bass, plays a major role in host defense. It is effective against a wide range of pathogens, including methicillin-resistant Staphylococcus aureus and HIV-1. The peptide is known to have an alpha-helical conformation when bound to anionic lipid membranes that mimic the surface of bacterial membranes. We have investigated the use of bicelles in the study of piscidin. 15N NMR spectra show that piscidin has been successfully aligned in magnetically oriented bicelles. 31P NMR studies, which show that piscidin disrupts bicelle-forming lipids, have helped us better understand its mode of action. To complement these backbone solid-state NMR studies of piscidin, we have used fluorescent-dye leakage experiments with various phospholipids and have performed solution NMR to determine the charge state of the histidine side chains in the presence of micelles and investigate their possible role in mediating important peptide-lipid interactions. The long term goal of this project is to improve our understanding of structure function relationships in an interesting family of antimicrobial peptides. This knowledge could be used to design potent antimicrobial pharmaceuticals that minimize bacterial resistance.

On the choice of optimal protocol for calculation of 13C and 15N NMR isotropic chemical shifts in nitramine systems

Calculated nuclear magnetic resonance (NMR) chemical shifts ( 13C and 15N) are reported for the RDX conformers and additional nine cyclic and acyclic nitramines. In order to establish a convenient and consistent protocol to be employed for confirming the experimental 13C and 15N NMR spectra of nitramine compounds, different combinations of models and basis sets were considered. The most reliable results were obtained at B3LYP/6-311+G(2d,p) level and CSGT model and can be used to calculate 13C and 15N NMR chemical shifts with a very high accuracy for nitramine compounds. These results show that the agreement between theoretical and experimental 15N NMR chemical shieldings of nitramines could be used for the evaluation of the intrinsic relationship between structure and explosive properties.

Solid-state NMR investigations of Si-29 and N-15 enriched silicon nitride

We compare 29Si magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectra from the two modifications of silicon nitride, α-Si 3N 4 and β-Si 3N 4, with that of a fully ( 29Si, 15N)-enriched sample 29Si 3 15N 4, as well as 15N NMR spectra of Si 3 15N 4 (having 29Si at natural abundance) and 29Si 3 15N 4. We show that the 15N NMR peak-widths from the latter are dominated by J( 29Si– 15N) through-bond interactions, leading to significantly broader NMR signals compared to those of Si 3 15N 4. By fitting calculated 29Si NMR spectra to experimental ones, we obtained an estimated coupling constant J( 29Si– 15N) of 20 Hz. We provide 29Si spin-lattice ( T 1) relaxation data for the 29Si 3 15N 4 sample and chemical shift anisotropy results for the 29Si site of β-Si 3N 4. Various factors potentially contributing to the 29Si and 15N NMR peak-widths of the various silicon nitride specimens are discussed. We also provide powder X-ray diffraction (XRD) and mass spectrometry data of the samples.

Characterization of a nitrogen-rich fulvic acid and its precursor algae from solid state NMR

A nitrogen-rich fulvic acid (FA) from Pony Lake, a coastal pond in Antarctica, was investigated using advanced solid state nuclear magnetic resonance (NMR) techniques and compared with the precursor algal material. A significant fraction of non-protonated alkyl carbons, some bonded to nitrogen, was detected; most must be humification products, since they were undetectable in the lake algae. The corresponding branched alkyl structures account for 25–50% of carbon in the FA. Spectral editing detected methylenes (20% of all C) and ethyl groups attached to a branch point. COOH groups are also enriched (∼10% of all C); 15N NMR and spectral editing of CH and CN groups indicates peptides (∼1/4 of all C, ∼2/5 of all N), but the 15N NMR spectrum also exhibits bands up- and downfield from the peptide signal; these must be associated with a 157 ppm sp 2-hybridized carbon bonded to three heteroatoms, of which at least two are N, according to 13C{ 14N} SPIDER (saturation pulse induced dipolar exchange with recoupling) NMR. This component, possibly a degradation product of penguin guano, accounts for nearly half the N. OCH, O–CH–O and OCH 2 moieties, typically found in sugar rings, were identified using spectral editing (∼12% of all C). The quantitative 13C spectrum shows an aromaticity of 8%; many of the aromatic carbons are bonded to N. 1H– 13C NMR with 1H spin diffusion shows that most components are within 1 nm from non-polar alkyl segments, which excludes polysaccharide or aromatic domains. Overall, the structural differences between the algal precursor and the FA are striking.

Treacherous Tetrahedron

The relative strength of the triple bond in N2 renders compounds with three or more catenated nitrogen atoms unstable, often explosively so. Banert et al. have succeeded in the careful preparation and isolation of the nitrogen-rich, dangerously explosive tetraazidomethane, C(N3)4, as a colorless liquid at room temperature. The stable, readily available trichloroacetonitrile molecule proved the most convenient precursor, affording the product after an 18-hour reaction with sodium azide in acetonitrile solvent. Cycloadducts with three and four equivalents of cyclooctyne could be isolated in ∼5% yield and were characterized crystallographically. Reaction with norbornene, however, yielded unusual tetrazole derivatives in place of expected 1,3-dipolar cycloaddition adducts. Despite the compound's instability, the authors acquired clean 13C and 15N nuclear magnetic resonance spectra, as well as vibrational and mass spectral data, and an estimated boiling point of 165°C. Both Brønsted and Lewis acids accelerated exchange with free azide. — JSY

Thermal Transformations of Nitrogen and Sulfur Forms in Peat Related to Coalification

The chemical pathways for nitrogen and sulfur transformations during coalification are elucidated by comparing the chemical forms of unaltered peats, lignites, and coals and pyrolyzed peats. Nitrogen forms are characterized by a combination of X-ray photoelectron spectroscopy (XPS) and 15N nuclear magnetic resonance (NMR). In unaltered peats, the 15N NMR and XPS nitrogen (1s) spectra are consistent with the presence of amide nitrogen. When peat is pyrolyzed, the main peak in the 15N NMR spectrum broadens and shifts from −260 ppm to −245 ppm, which is consistent with the loss of some amide nitrogen and the appearance of pyrrolic nitrogen forms. The pyrolyzed peat shows a new XPS peak that appears at 398.6 eV, which is characteristic of pyridinic nitrogen. These results indicate that a thermal transformation of amide nitrogen into pyrrolic and pyridinic forms occurs after thermal stress that is roughly equivalent to lignitification. High total nitrogen levels are found in pyrolyzed peats relative to lignite...

Organic N forms of a subtropical Acrisol under no-till cropping systems as assessed by acid hydrolysis and solid-state NMR spectroscopy

This study was conducted to investigate the influence of land-use systems (grassland and cropland) and of long-term no-till cropping systems [bare soil, oat/maize (O/M), pigeon pea+maize (P+M)] on the composition of organic N forms in a subtropical Acrisol. Soil samples collected from the 0- to 2.5-cm layer in the study area (Eldorado do Sul RS, Brazil) were submitted to acid hydrolysis and cross-polarization magic angle spinning (CPMAS) 15N and 13C nuclear magnetic resonance (NMR) spectroscopies. The legume-based cropping system P+M contained the highest contents of non-hydrolysable C and N, hydrolysable C and N, amino acid N and hydrolysed unknown N. The relative proportion of non-hydrolysable N was higher in bare soil (30.0%) and decreased incrementally in other treatments based on the total C and N contents. The amino acid N corresponded to an average of 37.2% of total N, and was not affected by land use and no-till cropping systems. The non-hydrolysable residue contained lower O-alkyl and higher aromatic C concentrations, as revealed by CPMAS 13C NMR spectroscopy, and higher C:N ratio than the bulk soil. No differences in the bulk soil organic matter composition could be detected among treatments, according to CPMAS 13C and 15N NMR spectra. In the non-hydrolysable fraction, grassland showed a lower concentration of aromatic and a higher concentration of alkyl C than other treatments. From CPMAS 15N NMR spectra, it could be concluded that amide N from peptide structures are the main organic N constituent. Amide structures are possibly protected through encapsulation into hydrophobic sites of organic matter and through organomineral interaction.

Selective catalytic reduction of NO x with ammonia on gallium-exchanged ferrierites

Selective catalytic reduction of NO x on Ga 3+ exchanged ferrierite zeolites (FER) using ammonia as reductant shows high rates of NO x conversion, although neither the catalytic reaction mechanism nor the role of the gallium is understood. We investigate the features of the structure of the catalyst and the mechanism of the catalytic reduction of NO x to N 2 using nuclear magnetic resonance (NMR) spectroscopy, complemented by temperature programmed desorption (TPD) and catalytic conversion measurements. The NMR studies are enabled by the diamagnetic nature of the gallium-exchanged zeolites in contrast to many of the transition metal exchanged zeolites which are often paramagnetic. Two different ferrierite samples were chosen for comparison, one containing significant quantities of K + and Na +, the other being largely alkali-free. The gallium-exchanged zeolites and their parent materials were characterized using 29Si, 27Al, 71Ga, and 129Xe NMR. Reactions run under batch-mode conditions using ammonia as the reducing agent clearly show the formation of nitrogen in the 15N NMR spectra. Our results show that the acidity of the zeolite is more important for catalytic reduction than the presence of gallium when NO x is supplied to the catalyst as a 1:1 mixture of NO and NO 2.

Fire-induced transformation of C- and N- forms in different organic soil fractions from a Dystric Cambisol under a Mediterranean pine forest ( Pinus pinaster)

High intensity forest fires in Mediterranean ecosystems probably have long-term effects on the organic matter (OM) of the forest soils. Therefore, we analyzed the quality and quantity of humic materials extracted from the A horizons (0–15 cm) of a fire-affected (FA) and a control fire-unaffected (FU) Dystric Cambisol from the Sierra de Aznalcóllar (Spain). 13C and 15N solid-state nuclear magnetic resonance (NMR) spectra of the samples from the FA and FU sites confirmed that aromatic and newly formed heterocyclic N-forms are important fire-induced products. Wildfire resulted in a doubling of organic C and N concentration in the A horizon (0–15 cm) of FA. Solid-state 13C NMR spectroscopy revealed that all C compound classes were enriched, including O- and N-alkyl C, with the highest for aromatic C (enrichment factor: 2.9). This suggests that the inputs from charred biomass and black carbon particles incorporated into the A horizon contained considerable amounts of unburned or partly charred remains. This conclusion is supported by the identification of diagnostic lignin-derived pyrolysis products in the hydrofluoric acid (HF) treated soils by analytical pyrolysis gas chromatography mass spectrometry (Py-GC/MS). Most of the partially charred material in addition to slightly altered plant necromass accumulated in the NaOH insoluble fraction (RES). Although the yield of alkali soluble OM from site FA was higher than that from site FU, the qualitative fire-induced alterations in C composition were slight. This may indicate that within this fraction, the OM was derived mainly from slightly charred or unburned materials. Fire also changed the chemical composition of the N-fraction of all humic extracts. These accumulated as pyrrole-type N compounds, although amide N was found to be the dominant form. This suggests that not all peptide-structures of the necromass were transformed by the fire. It is also possible that those amides are part of the melanoidized compounds, known to be formed during thermal treatment of mixtures containing sugars and amino acids. The fact that pyrrole-type N forms were not detected in the solid-state 15N NMR spectra of FU supports their pyrogenic origin. Apart from the importance of the alkali-insoluble fractions of wildfire-affected soils as a source of molecular descriptors of the thermal effect, pyrrole identified in soil organic matter (SOM) by means of solid-state 15N NMR spectroscopy could be a molecular marker for the presence of pyromorphic humic material in the soil.

Analysis of carbon and nitrogen forms in soil fractions after the addition of 15N-compost by 13C and 15N nuclear magnetic resonance.

A quantitative laboratory assessment of the different C and N forms in soil humus fractions was carried out by incubation of a mineral substrate after the addition of (15)N-labeled compost. The experimental design included (i) preparation of the (15)N-labeled organic matter (city refuse compost, 640 g kg(-1) wheat straw and K(15)NO(3) composted for 80 days), (ii) a further 80 day incubation of a mixture of the labeled compost with a mineral soil (32 g kg(-1)), (iii) measurement of stable isotope ratios, and (iv) isolation and structural comparison by (13)C and (15)N cross-polarization, magic-angle spinning nuclear magnetic resonance (NMR) of different organic fractions, i.e., soluble, colloidal (humic and fulvic type), and particulate (free organic matter and humin), from both the compost and the compost-treated soil. The results showed that the amide forms dominated in all of the newly formed N compounds, but an increased amount of alkali insoluble organic fractions was observed after incubation of the soil. The analysis of the insoluble, particulate fractions shows that nonextractable amides constitute the major pool of newly formed N compounds. The particulate soil fraction isolated by flotation in CHBr(3)-MeOH contained 16.8% of the total soil N and 26% of the (15)N. The (13)C NMR spectra showed that the fulvic acid-like fraction (7.6% of the soil N, 8.8% of (15)N) consisted almost completely of a C=O-containing carbohydrate material, whereas the humic acid-like fraction (20.3% of the total soil N, 8.6% of (15)N) resembled an oxidized lignoproteic fraction containing the most significant aromatic domain. The water soluble fraction was, in both soil and compost, the one with the highest isotopic abundance of (15)N (96%), but the (15)N NMR spectrum revealed minor amounts of soluble mineral N in this fraction and the remainder consisting of amide compounds.

Exploring symbiotic nitrogen fixation and assimilation in pea root nodules by in vivo 15N nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry.

Nitrogen (N) fixation and assimilation in pea (Pisum sativum) root nodules were studied by in vivo (15)N nuclear magnetic resonance (NMR) by exposing detached nodules to (15)N(2) via a perfusion medium, while recording a time course of spectra. In vivo (31)P NMR spectroscopy was used to monitor the physiological state of the metabolically active nodules. The nodules were extracted after the NMR studies and analyzed for total soluble amino acid pools and (15)N labeling of individual amino acids by liquid chromatography-mass spectrometry. A substantial pool of free ammonium was observed by (15)N NMR to be present in metabolically active, intact nodules. The ammonium ions were located in an intracellular environment that caused a remarkable change in the in vivo (15)N chemical shift. Alkalinity of the ammonium-containing compartment may explain the unusual chemical shift; thus, the observations could indicate that ammonium is located in the bacteroids. The observed (15)N-labeled amino acids, glutamine/glutamate and asparagine (Asn), apparently reside in a different compartment, presumably the plant cytoplasm, because no changes in the expected in vivo (15)N chemical shifts were observed. Extensive (15)N labeling of Asn was observed by liquid chromatography-mass spectrometry, which is consistent with the generally accepted role of Asn as the end product of primary N assimilation in pea nodules. However, the Asn (15)N amino signal was absent in in vivo (15)N NMR spectra, which could be because of an unfavorable nuclear Overhauser effect. gamma-Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized (15)N gamma-aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible.

Formation of Heteroaromatic Nitrogen after Prolonged Humification of Vascular Plant Remains as Revealed by Nuclear Magnetic Resonance Spectroscopy

In the search for the mechanisms involved in the immobilization of organic nitrogen in humified remains of vascular plants, the efforts of the present investigation were directed toward the examination of the transformation of nitrogenous compounds during the peat and coal stage by means of solid-state nuclear magnetic resonance (NMR) spectroscopy. While accumulation of heteroaromatic-N is not detected in most of the studied peat layers, a clear shoulder in the chemical shift region of pyrrole- or indole-N is observed in the solid-state 15N NMR spectrum of material from the deepest (and thus oldest) peat layer underlying the sapropel from Mangrove Lake, Bermuda (10000 years). This points to the assumption that transformation of nitrogen occurs between an advanced stage of peatification and an early stage of coalification. The observed sudden alteration in nitrogen functionality indicates that continuous accumulation of newly synthesized or selectively preserved biogenic structures is not responsible for the presence of heteroaromatic-N in these fossilized deposits. It seems rather likely that abiotic conditions, occurring during advanced sediment maturation, have an effect on the observed N transformation. With increasing coalification, pyrrole-type-N becomes the dominant form in the macromolecular coal network. Pyridine-type-N was only detected in a coal of anthracite rank.