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The design and synthesis of analogs of natural products can be a valuable source of medicinal preparations for the pharmaceutical industry. In the present study, the structural elucidation of eleven derivatives of 2,4-dihalogeno substituted synthetic analogues of the natural compound carvacrol was carried out by means of NMR experiments, and of another thirteen by DFT calculations. By selective NOE experiments and the irradiation of CH signals of the isopropyl group, individual conformers were assigned as syn and anti. By comparing GIAO/B3LYP/6-311++G(d,p)-calculated and experimentally measured vicinal 3JCH spin-spin constants, this assignment was confirmed. An unusual relationship is reported for proton-carbon vicinal couplings: 3JCH (180°) < 3JCH (0°). The conformational mobility of carvacrols was studied by 2D EXSY spectra. The application of homonuclear decoupling technique (HOBS) to these spectra simplifies the spectra, improves resolution without reducing the sensitivity, and allows a systematic examination of the rotational barrier of all compounds via their CH signals of the isopropyl group in a wider temperature interval. The rate constants of the isopropyl rotation between syn and anti conformers were determined and the corresponding energy barriers (14-17 kcal/mol) were calculated. DFT calculations of the energy barriers in carvacrol derivatives allowed the determination of the steric origin of the restricted isopropyl rotation. The barrier height depends on the size of the 2- and 4-position substituents, and is independent of the derivatization of the OH group.

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Signals undergoing chemical or conformational exchange in one-dimensional NMR spectra are often identified by deuterium exchange. In order to obtain quantitative information about the dynamic processes involved, one frequently used method is EXchange SpectroscopY (EXSY). To detect all exchange processes, the EXSY experiment requires the acquisition of time-consuming two-dimensional spectra. Here we report a faster alternative, an experiment which uses spatial encoding to extract similar information in a 1D exchange-edited experiment. Thereby, all protons are observed at once, but in different slices of the detection volume. The experiment can be carried out in a single scan to identify exchanging sites in a 1D spectrum by changes in signal intensity indicating exchange processes. If the exchanging partner, for example water is in molar excess the exchange-editing method easily identifies mobile protons by negative signals in the 1D 1H NMR spectrum.

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Cyclosporin is an 11-amino acid cyclic peptide with pharmacologically valuable properties which has a variety of actual and potential applications. Its activity relies on the cell membrane permeability which, in turn, depends on the structure of cyclosporin and its ability to change the conformation. In this work, conformational exchange processes occurring in cyclosporin C were studied using one- and two-dimensional nuclear magnetic resonance spectroscopy. The free energy barrier separating two major conformers observed in polar solution (acetonitrile) was found to be 77 ± 2 kJ/mol. Less populated conformation states are also present in the solution, which agrees with the ease of formation of multiple forms revealed by MD simulations of cyclosporin C.

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EXSY (exchange spectroscopy) NMR provides the residue-specific equilibrium constants, K, and residue-specific kinetic rate constants, k, of a polypeptide chain in a two-state exchange in the slow exchange regime. A linear free energy relationship (LFER) discovered in a log k versus log K plot is considered to be a physicochemical basis for smooth folding and conformational changes of protein molecules. For accurate determination of the thermodynamic and kinetic parameters, the measurement bias arising from state-specific differences in the R1 and R2 relaxation rates of 1H and other nuclei in HSQC and EXSY experiments must be minimized. Here, we showed that the time-zero HSQC acquisition scheme (HSQC0) is effective for this purpose, in combination with a special analytical method (Π analysis) for EXSY. As an example, we applied the HSQC0 + Π method to the two-state exchange of nukacin ISK-1 in an aqueous solution. Nukacin ISK-1 is a 27-residue lantibiotic peptide containing three mono-sulfide linkages. The resultant bias-free residue-based LFER provided valuable insights into the transition state of the topological interconversion of nukacin ISK-1. We found that two amino acid residues were exceptions in the residue-based LFER relationship. We inferred that the two residues could adopt special conformations in the transition state, to allow the threading of some side chains through a ring structure formed by one of the mono-sulfide linkages. In this context, the two residues are a useful target for the manipulation of the physicochemical properties and biological activities of nukacin ISK-1.

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Comparisons of G protein-coupled receptor (GPCR) complexes with agonists and antagonists based on X-ray crystallography and cryo-electron microscopy structure determinations show differences in the width of the orthosteric ligand binding groove over the range from 0.3 to 2.9 Å. Here, we show that there are transient structure fluctuations with amplitudes up to at least 6 Å. The experiments were performed with the neurokinin 1 receptor (NK1R), a GPCR of class A that is involved in inflammation, pain, and cancer. We used 19F-NMR observation of aprepitant, which is an approved drug that targets NK1R for the treatment of chemotherapy-induced nausea and vomiting. Aprepitant includes a bis-trifluoromethyl-phenyl ring attached with a single bond to the core of the molecule; 19F-NMR revealed 180° flipping motions of this ring about this bond. In the picture emerging from the 19F-NMR data, the GPCR transmembrane helices undergo large-scale floating motions in the lipid bilayer. The functional implication is of extensive promiscuity of initial ligand binding, primarily determined by size and shape of the ligand, with subsequent selection by unique interactions between atom groups of the ligand and the GPCR within the binding groove. This second step ensures the wide range of different efficacies documented for GPCR-targeting drugs. The NK1R data also provide a rationale for the observation that diffracting GPCR crystals are obtained for complexes with only very few of the ligands from libraries of approved drugs and lead compounds that bind to the receptors.

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Recent NMR studies of the exchangeable protons of GAGs in aqueous solution, including those of the amide, sulfamate, and hydroxyl moieties, have demonstrated potential for the detection of intramolecular hydrogen bonds providing insights into secondary structure preferences. GAG amide protons are observable by NMR over wide pH and temperature ranges; however, specific solution conditions are required to reduce the exchange rate of the sulfamate and hydroxyl protons and allow their detection by NMR. Building on the vast body of knowledge on detection of hydrogen bonds in peptides and proteins, a variety of methods can be used to identify hydrogen bonds in GAGs including temperature coefficient measurements, evaluation of chemical shift differences between oligo- and monosaccharides, and relative exchange rates measured through line shape analysis and EXSY spectra. Emerging strategies to allow direct detection of hydrogen bonds through heteronuclear couplings offer promise for the future. Molecular dynamic simulations are important in this effort both to predict and confirm hydrogen bond donors and acceptors.

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The conformational equilibria and guest exchange process of a resorcin[4]arene derived self-folding cavitand receptor have been characterized in detail by molecular dynamics simulations (MD) and 1 H EXSY NMR experiments. A multi-timescale strategy for exploring the fluxional behaviour of this system has been constructed, exploiting conventional MD and accelerated MD (aMD) techniques. The use of aMD allows the reconstruction of the folding/unfolding process of the receptor by sampling high-energy barrier processes unattainable by conventional MD simulations. We obtained MD trajectories sampling events occurring at different timescales from ns to s: 1) rearrangement of the directional hydrogen bond seam stabilizing the receptor, 2) folding/unfolding of the structure transiting partially open intermediates, and 3) guest departure from different folding stages. Most remarkably, reweighing of the biased aMD simulations provided kinetic barriers that are in very good agreement with those determined experimentally by 1 H NMR. These results constitute the first comprehensive characterization of the complex dynamic features of cavitand receptors. Our approach emerges as a valuable rational design tool for synthetic host-guest systems.

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Dynamics in complexes of porphyrin cage compounds and viologen-derived guest molecules are investigated by selective exchange NMR spectroscopy (1D EXSY). Exchange rates were found to be independent of excess guest concentration, revealing a dissociative exchange mechanism, which is accompanied by negative activation entropies, indicating significant reorganization of the host-guest complex during dissociation. Nonsymmetric viologen guests with bulky head groups had more unidirectional binding and slower exchange rates than guests with less-bulky head groups. Thermodynamic and kinetic studies revealed that the exchange process is primarily driven by the thermodynamics of binding and that guest binding can be influenced by introducing steric and electronic groups on the host . Exchange studies with guests bearing a polymer chain revealed that both slippage and full dissociation takes place and the rate constants for both processes were determined. The slippage rate constant revealed that for smaller guests exchange takes place nearly exclusively under thermodynamic control.

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ß-N-methyl-amino-L-alanine (BMAA) in the presence of bicarbonate (HCO3-) undergoes structural modifications generating two carbamate species, α-carbamate and ß-carbamate forms of BMAA. The chemical structure of BMAA and BMAA-carbamate adducts strongly suggest they may interact with divalent metal ions. The ability of BMAA to cross the blood-brain barrier and possibly interact with divalent metal ions may augment the neurotoxicity of these molecules. To understand the effects of divalent metal ions (Mg2+, Zn2+, and Cu2+) on the overall dynamic equilibrium between BMAA and its carbamate adducts, a systematic study using nuclear magnetic resonance (NMR) is presented. The chemical equilibria between BMAA, its carbamate adducts, and each of the divalent ions were studied using two-dimensional chemical exchange spectroscopy (EXSY). The NMR results demonstrate that BMAA preferentially interacts with Zn2+ and Cu2+, causing an overall reduction in the production of carbamate species by altering the dynamic equilibria. The NMR-based spectral changes due to the BMAA interaction with Cu2+ is more drastic than with the Zn2+, under the same stoichiometric ratios of BMAA and the individual divalent ions. However, the presence of Mg2+ does not significantly alter the dynamic equilibria between BMAA and its carbamate adducts. The NMR-based results are further validated using circular dichroism (CD) spectroscopy, observing the n ➔ π interaction in the complex formation of BMAA and the divalent metal ions, with additional verification of the interaction with Cu2+ using UV-Vis spectroscopy. Our results demonstrate that BMAA differentially interacts with divalent metal ions (Mg2+ < Zn2+ < Cu2+), and thus alters the rate of formation of carbamate products. The equilibria between BMAA, the bicarbonate ions, and the divalent metal ions may alter the total population of a specific form of BMAA-ion complex at physiological conditions and, therefore, add a level of complexity of the mechanisms by which BMAA acts as a neurotoxin.

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For the first time, hyperpolarized (HP) 129Xe NMR measurements are utilized to explore porous structures of porous starch (PS) successfully. Some micropores resided inside the mesopore walls of PS were detected by variable temperature (VT) HP 129Xe NMR, and the pore sizes of micropores were also estimated using the empirical relationship. Furthermore, the interconnectivity of pores was investigated in detail by two-dimensional (2D) exchange spectroscopy (EXSY). The exchange process of xenon from microporosity within pore walls to the free gas space was occurred at the mixing time of ≥12 ms at 173 K, which indicated the well interconnectivity between micropores and mesopores. This study not only exhibits a new approach for investigation of pores and hollows of PS, but also provides a better understanding of porous structures for rational design in adsorbing functional compounds.

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We carried out a detailed investigation of the local ordering and dynamics of the lithium intercalation in paramagnetic LixVOPO4.2H2O (with 0 < x ≤ 1) materials. This question was addressed using a combination of X-ray diffraction, 31P and 7Li MAS NMR experiments. We first studied the structure of the fully ordered end-member of the series, Li1VOPO4.2H2O, revisiting the X-ray single crystal diffraction data on the basis of the information provided by 31P MAS NMR. We then carried out 7Li MAS and exchange NMR experiments and 31P MAS experiments on the polycrystalline powders obtained after partial lithium insertion in VOPO4.2H2O phases. These experiments evidenced an unexpected ageing of the material related with lithium dynamics between the VOPO4 layers and a V4+/V5+ charge ordering mechanism within the layers.

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We report the acquisition of 2D NMR EXSY spectra with ultrahigh resolution, which allows for probing the slow conformational exchange process in a pharmaceutical compound. The resolution enhancement is achieved by implementing interferogram based PSYCHE homonuclear decoupling to generate a pure shift proton spectrum along the direct domain of the resulting data. The performance of this pure shift EXSY pulse sequence is compared to the standard experiment recorded under identical conditions. It is found that although being less sensitive and requiring a longer acquisition time, the quality of pure shift spectra allows for extracting exchange rates values that are coherent with the ones determined by standard approach, on a temperature range that demonstrates the robustness of the chosen homonuclear decoupling method. The resolution enhancement provided by the simplification of proton line shape allows for probing a higher number of proton sites whose analysis would have been biased using a standard method. These results open the way to a thorough and accurate study of chemical exchange processes based on a multi-site analysis of 2D pure shift EXSY spectra.

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In this report we present site-specific measurements of amide hydrogen-deuterium exchange rates in a protein in the solid state phase by MAS NMR. Employing perdeuteration, proton detection and a high external magnetic field we could adopt the highly efficient Relax-EXSY protocol previously developed for liquid state NMR. According to this method, we measured the contribution of hydrogen exchange on apparent 15N longitudinal relaxation rates in samples with differing D2O buffer content. Differences in the apparent T1 times allowed us to derive exchange rates for multiple residues in the type III secretion system needle protein.

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Hydroxamic acids (HAs) perform tasks in medicine and industry that require bidentate metal binding. The two favored conformations of HAs are related by rotation around the C(=O)-N bond. The conformations are unequal in stability. Recently, we reported that the most stable conformation of a small secondary HA in water places the oxygen atoms anti to one another. The barrier to C-N bond rotation may therefore modulate metal binding by secondary HAs in aqueous media. We have now determined the activation barrier to C-N rotation from major to minor conformation of a small secondary HA in D2O to be 67.3 kJ/mol. The HA rotational barrier scales with solvent polarity, as is observed in amides, although the HA barrier is less than that of a comparable tertiary amide in aqueous solution. Successful design of new secondary HAs to perform specific tasks requires solid understanding of rules governing HA structural behavior. Results from this work provide a more complete foundation for HA design efforts.

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(133)Cs nuclear magnetic resonance (NMR) spectroscopy was conducted on (133)Cs(+) in gelatin hydrogels that were either relaxed or stretched. Stretching generated a septet from this spin-7/2 nucleus, and its nuclear magnetic relaxation was studied via z-spectra, and two-dimensional nuclear Overhauser (NOESY) spectroscopy. Various spectral features were well simulated by using Mathematica and the software package SpinDynamica. Spectra of CsCl in suspensions of human erythrocytes embedded in gelatin gel showed separation of the resonances from the cation inside and outside the cells. Upon stretching the sample, the extracellular (133)Cs(+) signal split into a septet, while the intracellular peak was unchanged, revealing different alignment/ordering properties of the environment inside and around the cells. Differential interference contrast light microscopy confirmed that the cells were stretched when the overall sample was elongated. Analysis of the various spectral features of (133)Cs(+) reported here opens up applications of this K(+) congener for studies of cation-handling by metabolically-active cells and tissues in aligned states.

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The thermodynamics and kinetics of the cis/trans isomerism of N-methylacetohydroxamic acid (NMAH) and its conjugated base (NMA(-)) have been reinvestigated in aqueous media by (1)H NMR spectroscopy. Hindered rotation around the central C-N bond due to electronic delocalization becomes slow enough on the NMR time scale to observe both rotamers in equilibrium in D2O at room temperature. By properly assigning the methyl group resonances, evidence for the prevalence of the E over the Z form is unambiguously provided [K300=[E]/[Z]=2.86(2) and 9.63(5) for NMAH and NMA(-), respectively], closing thereby a long-lasting dispute about the most stable conformer. To that end, calculations of the chemical shifts by density functional theory (DFT), which accurately reproduced the experimental data, turned out to be a much more reliable method than the direct computation of the relative energy for each conformer. The Z ⇌ E interconversion dynamics was probed at 300 K in D2O by 2D exchange-correlated spectroscopy (EXSY), affording the associated rate constants [kZE=9.0(2) s(-1) and kEZ=3.14(5) s(-1) for NMAH, kZE=0.96(3) s(-1) and kEZ=0.10(2) s(-1) for NMA(-)] and activation barriers at 300 K [ΔG(≠)ZE=68.0 kJ mol(-1) and ΔG(≠)EZ=70.6 kJ mol(-1) for NMAH, ΔG(≠)ZE=73.6 kJ mol(-1) and ΔG(≠)EZ=79.2 kJ mol(-1) for NMA(-)]. For the first time, mono- and bis-chelated uranium(VI) complexes of NMA(-) have been isolated. Crystals of [UO2(NMA)(NO3)(H2O)2] and [UO2(NMA)2(H2O)] have been characterized by X-ray diffractometry, infrared and Raman spectroscopies.

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In this work, we present a new ultrafast method for acquiring dynamic 2D EXchange SpectroscopY (EXSY) within a single acquisition. This technique reconstructs two-dimensional EXSY spectra from one-dimensional spectra based on the phase accrual during echo times. The Ultrafast-EXSY acquisition overcomes long acquisition times typically needed to acquire 2D NMR data by utilizing sparsity and phase dependence to dramatically undersample in the indirect time dimension. This allows for the acquisition of the 2D spectrum within a single shot. We have validated this method in simulations and hyperpolarized enzyme assay experiments separating the dehydration of pyruvate and lactate-to-pyruvate conversion. In a renal cell carcinoma cell (RCC) line, bidirectional exchange was observed. This new technique revealed decreased conversion of lactate-to-pyruvate with high expression of monocarboxylate transporter 4 (MCT4), known to correlate with aggressive cancer phenotypes. We also showed feasibility of this technique in vivo in a RCC model where bidirectional exchange was observed for pyruvate-lactate, pyruvate-alanine, and pyruvate-hydrate and were resolved in time. Broadly, the technique is well suited to investigate the dynamics of multiple exchange pathways and applicable to hyperpolarized substrates where chemical exchange has shown great promise across a range of disciplines.

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The title triptycene, 6, has been isolated as the product of 9,10-cycloaddition of benzyne to 9,10-diferrocenyl-2,6-di-tert-butylanthracene, 5, whose X-ray crystal structure is reported. Each ferrocenyl unit in 6 has access to the same three non-equivalent molecular environments, and their rotations relative to the molecular paddlewheel give rise to six slowly interconverting atropisomers. Their dynamic behaviour in solution is a challenging NMR puzzle that can be successfully solved by taking advantage of the recently described very large diamagnetic anisotropy of the ferrocenyl moiety, together with the C2 symmetry of particular atropisomers. Application of one- and two-dimensional NMR techniques over a range of temperatures together, with a detailed analysis of the homo- and heteronuclear correlations in 6, resulted in unequivocal mapping of the 99 (1)H and 162 (13)C positions in the six interconverting systems. Variable-temperature 2D-EXSY measurements revealed that, while the stability of the atropisomers is almost identical, they are separated by energy barriers which the ferrocenyls must overcome in the course of their interconversions. The heights of two different rotational barriers have been identified and these experimental findings are in good agreement with DFT calculations.

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Functional RNA molecules are often very plastic and often undergo changes in base-pairing patterns to achieve alternative secondary and tertiary conformations associated with their roles in multiple events in gene expression. Solution NMR techniques are an excellent tool for the analysis of conformational heterogeneity and dynamic exchange. In this work, we measure the rates associated with spontaneous interconversion between major conformers in folded RNA sequences by use of a (19)F-(19)F EXSY NMR experiment, taking advantage of RNA samples carrying a single 5-(19)F-pyrimidine label. We first utilize this approach to determine kinetic exchange rates between conformers in a model RNA stem loop capable of adopting two conformations. We then probe the dynamics of conformational rearrangements in a larger RNA construct, the U2-U6 snRNA complex of the human spliceosome. In the case of the U2-U6 snRNA complex, such a rearrangement in the context of the intact spliceosome may have critical implications in splicing activity.

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Many noncoding RNA molecules adopt alternative secondary and tertiary conformations that are critical for their roles in gene expression. Although many of these rearrangements are mediated by other biomolecular components, it is important to evaluate the equilibrium relationship of the conformers. To measure the spontaneous interconversion in a bi-stable RNA stem loop sequence into which a single (19)F-uridine label was incorporated, a (19)F-(19)F EXSY experiment was employed. The kinetic exchange rate measured from EXSY experiments for this system was 37.3±2.8s(-1). The advantage of this approach is that exchange kinetics can be monitored in any RNA sequence into which a single (19)F nucleotide is incorporated by commercial synthesis. This method is therefore suitable for application to biologically significant systems in which dynamic conformational rearrangement is important for function and may therefore facilitate studies of RNA structure-function relationships.

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