Butterfly Effect in Cytarabine: Combined NMR-NQR Experiment, Solid-State Computational Modeling, Quantitative Structure-Property Relationships and Molecular Docking Study.

Cytarabine (Ara-C) is a synthetic isomer of cytidine that differs from cytidine and deoxycytidine only in the sugar. The use of arabinose instead of deoxyribose hinders the formation of phosphodiester linkages between pentoses, preventing the DNA chain from elongation and interrupting the DNA synthesis. The minor structural alteration (the inversion of hydroxyl at the 2' positions of the sugar) leads to change of the biological activity from anti-depressant and DNA/RNA block builder to powerful anti-cancer. Our study aimed to determine the molecular nature of this phenomenon. Three 1H-14N NMR-NQR experimental techniques, followed by solid-state computational modelling (Quantum Theory of Atoms in Molecules, Reduced Density Gradient and 3D Hirshfeld surfaces), Quantitative Structure-Property Relationships, Spackman's Hirshfeld surfaces and Molecular Docking were used. Multifaceted analysis-combining experiments, computational modeling and molecular docking-provides deep insight into three-dimensional packing at the atomic and molecular levels, but is challenging. A spectrum with nine lines indicating the existence of three chemically inequivalent nitrogen sites in the Ara-C molecule was recorded, and the lines were assigned to them. The influence of the structural alteration on the NQR parameters was modeled in the solid (GGA/RPBE). For the comprehensive description of the nature of these interactions several factors were considered, including relative reactivity and the involvement of heavy atoms in various non-covalent interactions. The binding modes in the solid state and complex with dCK were investigated using the novel approaches: radial plots, heatmaps and root-mean-square deviation of the binding mode. We identified the intramolecular OH···O hydrogen bond as the key factor responsible for forcing the glycone conformation and strengthening NH···O bonds with Gln97, Asp133 and Ara128, and stacking with Phe137. The titular butterfly effect is associated with both the inversion and the presence of this intramolecular hydrogen bond. Our study elucidates the differences in the binding modes of Ara-C and cytidine, which should guide the design of more potent anti-cancer and anti-viral analogues.

Exploring mitochondrial biomarkers for Friedreich's ataxia: a multifaceted approach.

This study presents an in-depth analysis of mitochondrial enzyme activities in Friedreich's ataxia (FA) patients, focusing on the Electron Transport Chain complexes I, II, and IV, the Krebs Cycle enzyme Citrate Synthase, and Coenzyme Q10 levels. It examines a cohort of 34 FA patients, comparing their mitochondrial enzyme activities and clinical parameters, including disease duration and cardiac markers, with those of 17 healthy controls. The findings reveal marked reductions in complexes II and, specifically, IV, highlighting mitochondrial impairment in FA. Additionally, elevated Neurofilament Light Chain levels and cardiomarkers were observed in FA patients. This research enhances our understanding of FA pathophysiology and suggests potential biomarkers for monitoring disease progression. The study underscores the need for further clinical trials to validate these findings, emphasizing the critical role of mitochondrial dysfunction in FA assessment and treatment.

Identification of complex III, NQR, and SDH as primary bioenergetic enzymes during the stationary phase of Pseudomonas aeruginosa cultured in urine-like conditions.

Pseudomonas aeruginosa is a common cause of urinary tract infections by strains that are often multidrug resistant, representing a major challenge to the world's health care system. This microorganism has a highly adaptable metabolism that allows it to colonize many environments, including the urinary tract. In this work, we have characterized the metabolic strategies used by stationary phase P. aeruginosa cells cultivated in urine-like media to understand the adaptations used by this microorganism to survive and produce disease. Our proteomics results show that cells rely on the Entner-Duodoroff pathway, pentose phosphate pathway, the Krebs cycle/ glyoxylate shunt and the aerobic oxidative phosphorylation to survive in urine-like media and other conditions. A deep characterization of the oxidative phosphorylation showed that the respiratory rate of stationary phase cells is increased 3-4 times compared to cells in the logarithmic phase of growth, indicating that the aerobic metabolism plays critical roles in the stationary phase of cells grown in urine like media. Moreover, the data show that respiratory complex III, succinate dehydrogenase and the NADH dehydrogenase NQR have important functions and could be used as targets to develop new antibiotics against this bacterium.

C- and N-Phosphorylated Enamines-An Avenue to Heterocycles: NMR Spectroscopy.

The review presents extensive data (from the works of the author and literature) on the structure of C- and N-chlorophosphorylated enamines and the related heterocycles obtained by multipulse multinuclear 1H, 13C, and 31P NMR spectroscopy. The use of phosphorus pentachloride as a phosphorylating agent for functional enamines enables the synthesis of various C- and N-phosphorylated products that are heterocyclized to form various promising nitrogen- and phosphorus-containing heterocyclic systems. 31P NMR spectroscopy is the most convenient, reliable and unambiguous method for the study and identification of organophosphorus compounds with different coordination numbers of the phosphorus atom, as well as for the determination of their Z- and E-isomeric forms. An alteration of the coordination number of the phosphorus atom in the phosphorylated compounds from 3 to 6 leads to a drastic screening of the 31P nucleus from about +200 to -300 ppm. The unique structural features of nitrogen-phosphorus-containing heterocyclic compounds are discussed.

3D to 0D cesium lead bromide: A 79/81Br NMR, NQR and theoretical investigation.

Inorganic metal halides offer unprecedented tunability through elemental variation of simple three-element compositions, but can exhibit complicated phase behaviour, degradation, and microscopic phenomena (disorder/dynamics) that play an integral role for the bulk-level chemical and physical properties of these materials. Understanding the halogen chemical environment in such materials is crucial to addressing many of the concerns regarding implementing these materials in commercial applications. In this study, a combined solid-state nuclear magnetic resonance, nuclear quadrupole resonance and quantum chemical computation approach is used to interrogate the Br chemical environment in a series of related inorganic lead bromide materials: CsPbBr3, CsPb2Br5, and Cs4PbBr6. The quadrupole coupling constants (CQ) were determined to range from 61 to 114 MHz for 81Br, with CsPbBr3 exhibiting the largest measured CQ and Cs4PbBr6 the smallest. GIPAW DFT was shown to be an excellent pre-screening tool for estimating the EFG of Br materials and can increase experimental efficiency by providing good starting estimates for acquisition. Finally, the combination of theory and experiment to inform the best methods for expanding further to the other quadrupolar halogens is discussed.

A sulfur-33 nuclear quadrupole resonance study of 33 S2 -labeled L-cystine.

The sulfur electric-field-gradient tensor for a disulfide bond in 33 S2 -labeled L-cystine has been investigated by 33 S nuclear quadrupole resonance (NQR). 33 S2 -labeled L-cystine is synthesized by introduction of disulfide ions prepared from elemental 33 S-sulfur into an amino acid derivative, the side chain of which is iodinated. In its NQR spectrum, sharp single peaks are observed at between 24.63 and 24.90 MHz in the temperature range from 80 to 298 K. The two-dimensional nutation echo 33 S NQR experiment is carried out at 160 K, and the quadrupole coupling constant, CQ , and the asymmetric parameter, ηQ , are obtained to be 46.9(9) MHz and 0.6(1), respectively. The calculated 33 S electric-field-gradient tensor components with respect to the molecular frame is briefly discussed.

Detecting and Characterizing Interactions of Metabolites with Proteins by Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR) Spectroscopy.

Saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy is an established technique for detecting and characterizing the binding of small molecules, such as metabolites, to biological macromolecules like proteins and nucleic acids. STD NMR allows detection of binding in complex mixtures of potential ligands, which is often used for library screening in the pharmaceutical industry but may also be beneficial for binding studies with metabolite mixtures. The nature of the ligand is normally restricted to small molecules in terms of NMR spectroscopy, and the size of the macromolecule on the other side should be larger than 10-15 kDa. This technique is especially applicable to detecting binders of intermediate to low affinity with the dissociation constant (KD) above 1 µM. In this chapter, we focus on recent developments and the applications of STD NMR to studying interactions of natural products and metabolites, in particular. The reader is also referred to excellent reviews of the field and the literature cited therein. This chapter also provides a detailed experimental protocol for performing the STD NMR measurement based on the example of the subunit A of the Na+-transporting NADH/ubiquinone oxidoreductase (Na+-NQR) from V. cholerae interacting with its natural quinone substrate and inhibitors.

Identification of the riboflavin cofactor-binding site in the Vibrio cholerae ion-pumping NQR complex: A novel structural motif in redox enzymes.

The ion-pumping NQR complex is an essential respiratory enzyme in the physiology of many pathogenic bacteria. This enzyme transfers electrons from NADH to ubiquinone through several cofactors, including riboflavin (vitamin B2). NQR is the only enzyme reported that is able to use riboflavin as a cofactor. Moreover, the riboflavin molecule is found as a stable neutral semiquinone radical. The otherwise highly reactive unpaired electron is stabilized via an unknown mechanism. Crystallographic data suggested that riboflavin might be found in a superficially located site in the interface of NQR subunits B and E. However, this location is highly problematic, as the site does not have the expected physiochemical properties. In this work, we have located the riboflavin-binding site in an amphipathic pocket in subunit B, previously proposed to be the entry site of sodium. Here, we show that this site contains absolutely conserved residues, including N200, N203, and D346. Mutations of these residues decrease enzymatic activity and specifically block the ability of NQR to bind riboflavin. Docking analysis and molecular dynamics simulations indicate that these residues participate directly in riboflavin binding, establishing hydrogen bonds that stabilize the cofactor in the site. We conclude that riboflavin is likely bound in the proposed pocket, which is consistent with enzymatic characterizations, thermodynamic studies, and distance between cofactors.

The side chain of ubiquinone plays a critical role in Na+ translocation by the NADH-ubiquinone oxidoreductase (Na+-NQR) from Vibrio cholerae.

The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is an essential bacterial respiratory enzyme that generates a Na+ gradient across the cell membrane. However, the mechanism that couples the redox reactions to Na+ translocation remains unknown. To address this, we examined the relation between reduction of UQ and Na+ translocation using a series of synthetic UQs with Vibrio cholerae Na+-NQR reconstituted into liposomes. UQ0 that has no side chain and UQCH3 and UQC2H5, which have methyl and ethyl side chains, respectively, were catalytically reduced by Na+-NQR, but their reduction generated no membrane potential, indicating that the overall electron transfer and Na+ translocation are not coupled. While these UQs were partly reduced by electron leak from the cofactor(s) located upstream of riboflavin, this complete loss of Na+ translocation cannot be explained by the electron leak. Lengthening the UQ side chain to n-propyl (C3H7) or longer significantly restored Na+ translocation. It has been considered that Na+ translocation is completed when riboflavin, a terminal redox cofactor residing within the membrane, is reduced. In this view, the role of UQ is simply to accept electrons from the reduced riboflavin to regenerate the stable neutral riboflavin radical and reset the catalytic cycle. However, the present study revealed that the final UQ reduction via reduced riboflavin makes an important contribution to Na+ translocation through a critical role of its side chain. Based on the results, we discuss the critical role of the UQ side chain in Na+ translocation.

Molecular dynamics modeling of the Vibrio cholera Na+-translocating NADH:quinone oxidoreductase NqrB-NqrD subunit interface.

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is the major Na+ pump in aerobic pathogens such as Vibrio cholerae. The interface between two of the NQR subunits, NqrB and NqrD, has been proposed to harbor a binding site for inhibitors of Na+-NQR. While the mechanisms underlying Na+-NQR function and inhibition remain underinvestigated, their clarification would facilitate the design of compounds suitable for clinical use against pathogens containing Na+-NQR. An in silico model of the NqrB-D interface suitable for use in molecular dynamics simulations was successfully constructed. A combination of algorithmic and manual methods was used to reconstruct portions of the two subunits unresolved in the published crystal structure and validate the resulting structure. Hardware and software optimizations that improved the efficiency of the simulation were considered and tested. The geometry of the reconstructed complex compared favorably to the published V. cholerae Na+-NQR crystal structure. Results from one 1 µs, three 150 ns and two 50 ns molecular dynamics simulations illustrated the stability of the system and defined the limitations of this model. When placed in a lipid bilayer under periodic boundary conditions, the reconstructed complex was completely stable for at least 1 µs. However, the NqrB-D interface underwent a non-physiological transition after 350 ns.

Electrostatics and water occlusion regulate covalently-bound flavin mononucleotide cofactors of Vibrio cholerae respiratory complex NQR.

Proteins like NADH:ubiquinone oxidoreductase (NQR), an essential enzyme and ion pump in the physiology of several pathogenic bacteria, tightly regulate the redox properties of their cofactors. Although flavin mononucleotide (FMN) is fully reduced in aqueous solution, FMN in subunits B and C of NQR exclusively undergo one-electron transitions during its catalytic cycle. Here, we perform ab initio calculations and molecular dynamics simulations to elucidate the mechanisms that regulate the redox state of FMN in NQR. QM/MM calculations show that binding site electrostatics disfavor anionic forms of FMNH2 , but permit a neutral form of the fully reduced flavin. The potential energy surface is unaffected by covalent bonding between FMN and threonine. Molecular dynamics simulations show that the FMN binding sites are inaccessible by water, suggesting that further reductions of the cofactors are limited or prohibited by the availability of water and other proton donors. These findings provide a deeper understanding of the mechanisms used by NQR to regulate electron transfer through the cofactors and perform its physiologic role. They also provide the first, to our knowledge, evidence of the simple concept that proteins regulate flavin redox states via water occlusion.

Polymorphism and Conformational Equilibrium of Nitro-Acetophenone in Solid State and under Matrix Conditions.

Conformational and polymorphic states in the nitro-derivative of o-hydroxy acetophenone have been studied by experimental and theoretical methods. The potential energy curves for the rotation of the nitro group and isomerization of the hydroxyl group have been calculated by density functional theory (DFT) to estimate the barriers of the conformational changes. Two polymorphic forms of the studied compound were obtained by the slow and fast evaporation of polar and non-polar solutions, respectively. Both of the polymorphs were investigated by Infrared-Red (IR) and Raman spectroscopy, Incoherent Inelastic Neutron Scattering (IINS), X-ray diffraction, nuclear quadrupole resonance spectroscopy (NQR), differential scanning calorimetry (DSC) and density functional theory (DFT) methods. In one of the polymorphs, the existence of a phase transition was shown. The position of the nitro group and its impact on the crystal cell of the studied compound were analyzed. The conformational equilibrium determined by the reorientation of the hydroxyl group was observed under argon matrix isolation. An analysis of vibrational spectra was achieved for the interpretation of conformational equilibrium. The infrared spectra were measured in a wide temperature range to reveal the spectral bands that were the most sensitive to the phase transition and conformational equilibrium. The results showed the interrelations between intramolecular processes and macroscopic phenomena in the studied compound.

Specific chemical modification explores dynamic structure of the NqrB subunit in Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae.

The Na+-pumping NADH-ubiquinone oxidoreductase (Na+-NQR) is a main ion transporter in many pathogenic bacteria. We previously proposed that N-terminal stretch of the NqrB subunit plays an important role in regulating the ubiquinone reaction at the adjacent NqrA subunit in Vibrio cholerae Na+-NQR. However, since approximately three quarters of the stretch (NqrB-Met1-Pro37) was not modeled in an earlier crystallographic study, its structure and function remain unknown. If we can develop a method that enables pinpoint modification of this stretch by functional chemicals (such as spin probes), it could lead to new ways to investigate the unsettled issues. As the first step to this end, we undertook to specifically attach an alkyne group to a lysine located in the stretch via protein-ligand affinity-driven substitution using synthetic ligands NAS-K1 and NAS-K2. The alkyne, once attached, can serve as an "anchor" for connecting functional chemicals via convenient click chemistry. After a short incubation of isolated Na+-NQR with these ligands, alkyne was predominantly incorporated into NqrB. Proteomic analyses in combination with mutagenesis of predicted target lysines revealed that alkyne attaches to NqrB-Lys22 located at the nonmodeled region of the stretch. This study not only achieved the specific modification initially aimed for but also provided valuable information about positioning of the nonmodeled region. For example, the fact that hydrophobic NAS-Ks come into contact with NqrB-Lys22 suggests that the nonmodeled region may orient toward the membrane phase rather than protruding into cytoplasmic medium. This conformation may be essential for regulating the ubiquinone reaction in the adjacent NqrA.

Optimization of NQR excitation sequences using black-box techniques.

Nuclear quadrupole resonance is a spectroscopic technique that is dependent on the measurement conditions and one of its challenges is to find the optimal excitation sequence parameters that ensure the highest signal-to-noise ratio. This is typically performed empirically and this paper proposes a different approach by using black-box optimization techniques. This work explores several optimization algorithms in a black-box environment, where no model is required for the spectrometer and measurement conditions. The techniques can be applied to any excitation sequence, they can adapt to the measurement conditions and enable the automatic and simultaneous optimization of multiple parameters. Single and multi-parameter optimization experiments are performed on sodium nitrite and the algorithms are shown to converge to similar optimal values with small standard deviations. The use of an optimized excitation sequence is shown to improve the signal-to-noise ratio by approx. 8 dB compared to the initial sequence. Further research directions are discussed and include the study of different acquisition functions and the use of reinforcement learning algorithms.

Na+-NQR Confers Aminoglycoside Resistance via the Regulation of l-Alanine Metabolism.

Sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) functions as a unique redox-driven sodium pump, generating membrane potential, which is related to aminoglycoside antibiotic resistance. However, whether it modulates other metabolisms to confer antibiotic resistance is unknown. The present study showed that loss of nqrA or nqrF led to differential metabolomes with elevated resistance to aminoglycoside antibiotics. Decreased alanine, aspartate, and glutamate metabolism and depressed abundance of alanine were characterized as the most impacted pathway and crucial biomarker, respectively. Further data showed that higher viability was detected in ΔnqrA and ΔnqrF mutant strains than their parent strain ATCC 33787 in the presence of gentamicin but recovered by exogenous l-alanine. It proceeds by the following events. The loss of nqrA or nqrF led to the decrease of membrane potential, ATPase activity, and then ATP and cyclic AMP (cAMP), which reduced the cAMP/CRP (cAMP receptor protein) complex. The reduced cAMP/CRP complex promoted l-alanine catabolism and inhibited l-alanine anabolism, causing reduced levels of alanine. Reduced alanine affected the expression of antiporter families Atp and Mnh genes. Our results suggest a novel mechanism by which the Na+-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner.IMPORTANCE The Na+-NQR complex functions as a unique redox-driven sodium pump, generating membrane potential directly. However, whether it mediates generation of membrane potential indirectly is unknown. The present study shows that the Na+-NQR complex impacts membrane potential through other antiporter families Atp and Mnh. It proceeds by ATP and then cAMP/CRP regulon, which inhibits l-alanine catabolism and promotes l-alanine anabolism. When the Na+-NQR complex is reduced as in antibiotic-resistant bacteria, l-alanine is depressed, which is related to the antibiotic resistance phenotypes. However, exogenous l-alanine reverts the phenotype and promotes antibiotic-mediated killing. These findings suggest a novel mechanism by which the Na+-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner.

The 1H T1 dispersion curve of fentanyl citrate to identify NQR parameters.

We report the 1H T1 dispersion curve between 0 and 5 â€‹MHz for the synthetic opioid fentanyl citrate (C28H36N2O8). The structures in the curve can be used to estimate the 14N nuclear quadrupole resonance (NQR) frequencies of the material. Density functional theory predictions of the NQR parameters of several fentanyl citrate compounds are also reported. The predictions for the aniline nitrogen are consistent with structures in the observed T1 data. To help interpret the fentanyl citrate results the T1 dispersion curve for the explosive ammonium nitrate is also presented.

Inhibitors of a Na+-pumping NADH-ubiquinone oxidoreductase play multiple roles to block enzyme function.

The Na+-pumping NADH-ubiquinone (UQ) oxidoreductase (Na+-NQR) is present in the respiratory chain of many pathogenic bacteria and is thought to be a promising antibiotic target. Whereas many details of Na+-NQR structure and function are known, the mechanisms of action of potent inhibitors is not well-understood; elucidating the mechanisms would not only advance drug design strategies but might also provide insights on a terminal electron transfer from riboflavin to UQ. To this end, we performed photoaffinity labeling experiments using photoreactive derivatives of two known inhibitors, aurachin and korormicin, on isolated Vibrio cholerae Na+-NQR. The inhibitors labeled the cytoplasmic surface domain of the NqrB subunit including a protruding N-terminal stretch, which may be critical to regulate the UQ reaction in the adjacent NqrA subunit. The labeling was blocked by short-chain UQs such as ubiquinone-2. The photolabile group (2-aryl-5-carboxytetrazole (ACT)) of these inhibitors reacts with nucleophilic amino acids, so we tested mutations of nucleophilic residues in the labeled region of NqrB, such as Asp49 and Asp52 (to Ala), and observed moderate decreases in labeling yields, suggesting that these residues are involved in the interaction with ACT. We conclude that the inhibitors interfere with the UQ reaction in two ways: the first is blocking structural rearrangements at the cytoplasmic interface between NqrA and NqrB, and the second is the direct obstruction of UQ binding at this interfacial area. Unusual competitive behavior between the photoreactive inhibitors and various competitors corroborates our previous proposition that there may be two inhibitor binding sites in Na+-NQR.

Homogeneous fields: Double expansion method, 3D printing/CNC realization, and verification by atomic magnetometry.

In low-field magnetic resonance applications there is often an interest in creating homogeneous magnetic fields over unusual geometries, particularly when quantum magnetometers are involved. In this paper a design method is proposed, where both the surface current and magnetic field are expanded to find current coefficients that cancel out higher order field terms. Two coils are designed using this double expansion methodology: (1) a tuning field for a half-meter-long atomic magnetometer array and (2) a null field for a magnetometer to operate adjacent to an excitation solenoid. The field verification of the former shows the accuracy of CNC milling and the method proposed; a close analysis of the field signature in the latter revealed the limitations of 3D printing for precise scientific applications. Both coils are designed to be fifth-order error systems or better.

Genetic and Biochemical Analysis of Anaerobic Respiration in Bacteroides fragilis and Its Importance In Vivo.

In bacteria, the respiratory pathways that drive molecular transport and ATP synthesis include a variety of enzyme complexes that utilize different electron donors and acceptors. This property allows them to vary the efficiency of energy conservation and to generate different types of electrochemical gradients (H+ or Na+). We know little about the respiratory pathways in Bacteroides species, which are abundant in the human gut, and whether they have a simple or a branched pathway. Here, we combined genetics, enzyme activity measurements, and mammalian gut colonization assays to better understand the first committed step in respiration, the transfer of electrons from NADH to quinone. We found that a model gut Bacteroides species, Bacteroides fragilis, has all three types of putative NADH dehydrogenases that typically transfer electrons from the highly reducing molecule NADH to quinone. Analyses of NADH oxidation and quinone reduction in wild-type and deletion mutants showed that two of these enzymes, Na+-pumping NADH:quinone oxidoreductase (NQR) and NADH dehydrogenase II (NDH2), have NADH dehydrogenase activity, whereas H+-pumping NADH:ubiquinone oxidoreductase (NUO) does not. Under anaerobic conditions, NQR contributes more than 65% of the NADH:quinone oxidoreductase activity. When grown in rich medium, none of the single deletion mutants had a significant growth defect; however, the double Δnqr Δndh2 mutant, which lacked almost all NADH:quinone oxidoreductase activity, had a significantly increased doubling time. Despite unaltered in vitro growth, the single nqr deletion mutant was unable to competitively colonize the gnotobiotic mouse gut, confirming the importance of NQR to respiration in B. fragilis and the overall importance of respiration to this abundant gut symbiont.IMPORTANCEBacteroides species are abundant in the human intestine and provide numerous beneficial properties to their hosts. The ability of Bacteroides species to convert host and dietary glycans and polysaccharides to energy is paramount to their success in the human gut. We know a great deal about the molecules that these bacteria extract from the human gut but much less about how they convert those molecules into energy. Here, we show that B. fragilis has a complex respiratory pathway with two different enzymes that transfer electrons from NADH to quinone and a third enzyme complex that may use an electron donor other than NADH. Although fermentation has generally been believed to be the main mechanism of energy generation in Bacteroides, we found that a mutant lacking one of the NADH:quinone oxidoreductases was unable to compete with the wild type in the mammalian gut, revealing the importance of respiration to these abundant gut symbionts.

NMR and NQR study of polymorphism in carbamazepine.

Four polymorphic forms of carbamazepine have been simultaneously investigated by 1H NMR and 14N NQR. The results show that the proton spin-lattice relaxation time and the 14N NQR spectra can be used to differentiate between various polymorphic forms. Spontaneous transformations from Form II to Form III and from Form IV to Form III have been investigated through their influence on the 14N NQR spectrum and the proton NMR signal and spin-lattice relaxation. The 14N NQR spectra prove that in the observed polymorphic forms of carbamazepine the hydrogen bonded dimers of carbamazepine molecules are the basic elements of the crystal structure. The dimers are centrosymmetric in Forms III and IV and in metastable polymorphic form occurring during the transformation of Form IV to Form III. Two non-equivalent molecular positions are observed in Form II with the occupation ratio 1:1 and in Form I with the occupation ratio either 2:1 or 3:1. The 14N NQR data are related to the published crystal structures. Possible reasons for the mismatch of the X-ray and NQR data for Forms I and II are discussed.