Nuclear Overhauser Effect Measurements Research Articles

Combining Desferriferrioxamine B and 1-Hydroxy-2-Piperidone ((PIPO)H) to Chelate Zirconium. Solution Structure of a Model Complex of the [89Zr]Zr-DFOcyclo*-mAb Radioimmunoconjugate.

89Zr-immunoPET is a hot topic as 89Zr cumulates the advantages of 64Cu and 124I without their drawbacks. We report the synthesis of a model ligand of a chiral bioconjugable tetrahydroxamic chelator combining the desferriferrioxamine B siderophore and 1-hydroxy-2-piperidone ((PIPO)H), a chiral cyclic hydroxamic acid derivative, and the study by NMR spectroscopy of its zirconium complex. Nuclear Overhauser effect measurements (ROESY) indicated that the complex exists in the form of two diastereomers, in 77 : 23 ratio, resulting from the combination of the central chiralities at the 3-C of the (PIPO)H component and at the Zr4+ cation. The 44 lowest energy structures out of more than 1000 configurations/conformations returned by calculations based on density functional theory were examined. Comparison of the ROESY data and the calculated interatomic H⋅⋅⋅H distances allowed us to select the most probable configuration and conformations of the major complex.

The chemokines CCL5 and CXCL12 exhibit high-affinity binding to N-terminal peptides of the non-cognate receptors CXCR4 and CCR5, respectively.

CC and CXC chemokines are distinct chemokine subfamilies. CC chemokines usually do not bind CXC-chemokine receptors and vice versa. CCR5 and CXCR4 receptors are activated by CCL5 and CXCL12 chemokines, respectively, and are also used as HIV-1 coreceptors. CCL5 contains one conserved binding site for a sulfated tyrosine residue, whereas CXCL12 is unique in having two additional sites for sulfated/nonsulfated tyrosine residues. In this study, N-terminal (Nt) CXCR4 peptides were found to bind CCL5 with somewhat higher affinities in comparison to those of short Nt-CCR5(8-20) peptides with the same number of sulfated tyrosine residues. Similarly, a long Nt-CCR5(1-27)(s Y3,s Y10,s Y14) peptide cross reacts with CXCL12 and with lower KD in comparison to its binding to CCL5. Intermolecular nuclear overhauser effect (NOE) measurements were used to decipher the mechanism of the chemokine/Nt-receptor peptide binding. The Nt-CXCR4 peptides interact with the conserved CCL5 tyrosine sulfate-binding site by an allovalency mechanism like that observed for CCL5 binding of Nt-CCR5 peptides. Nt-CCR5 peptides bind CXCL12 in multiple modes analogous to their binding to HIV-1 gp120 and interact with all three tyrosine/sulfated tyrosine-binding pockets of CXCL12. We suggest that the chemokine-receptors Nt-segments bind promiscuously to cognate and non-cognate chemokines and in a mechanism that is dependent on the number of binding pockets for tyrosine residues found on the chemokine. In conclusion, common features shared among the chemokine-receptors' Nt-segments such as multiple tyrosine residues that are potentially sulfated, and a large number of negatively charged residues are the reason of the cross binding observed in this study.

Hyaluronan-arginine enhanced and dynamic interaction emerges from distinctive molecular signature due to electrostatics and side-chain specificity

Hyaluronan is a natural carbohydrate polymer with a negative charge that fosters gel-like conditions crucial for its cellular functions and industrial applications. As a recognized ligand for proteins, understanding their mutual interactions provides solid ground to tune hyaluronan's gel properties using biocompatible peptides. This work employs NMR and molecular dynamics simulations to identify molecular motifs relevant to hyaluronan–peptide interactions using arginine, lysine, and glycine oligopeptides. Arginine-rich peptides exhibit the strongest binding to hyaluronan according to chemical shift perturbation measurements, followed distantly by the similarly charged lysine. This difference highlights the significance of electrostatics and the peculiarities of the guanidinium side chain in arginine, capable of non-polar interactions that further stabilize the binding. Additional nuclear Overhauser effect measurements do not show stable interaction partners, precluding strong and well-defined complexes. Finally, molecular simulations support our findings and show an extended but significant interaction region, especially for arginine, responsible for the observed enhanced binding, which can also promote cross-linking of hyaluronan polymers. Our findings pave the way for optimizing biocompatible peptides to alter hyaluronan gels' properties efficiently and also explain why hyaluronan–protein interaction typically involves positively charged arginine-rich regions also capable of forming hydrogen bonds and non-polar interactions.

Feasibility of transient nuclear Overhauser effect imaging in brain at 7 T.

The nuclear Overhauser effect (NOE) quantification from the steady-state NOE imaging suffers from multiple confounding non-NOE-specific sources, including direct saturation, magnetization transfer, and relevant chemical exchange species, and is affected by B0 and B1 + inhomogeneities. The B0 -dependent and B1 + -dependent data needed for deconvolving these confounding effects would increase the scan time substantially, leading to other issues such as patient tolerability. Here, we demonstrate the feasibility of brain lipid mapping using an easily implementable transient NOE (tNOE) approach. This 7T study used a frequency-selective inversion pulse at a range of frequency offsets between 1.0 and 5.0 parts per million (ppm) and -5.0 and -1.0 ppm relative to bulk water peak. This was followed by a fixed/variable mixing time and then a single-shot 2D turbo FLASH readout. The feasibility of tNOE measurements is demonstrated on bovine serum albumin phantoms and healthy human brains. The tNOE measurements from bovine serum albumin phantoms were found to be independent of physiological pH variations. Both bovine serum albumin phantoms and human brains showed broad tNOE contributions centered at approximately -3.5 ppm relative to water peak, with presumably aliphatic moieties in lipids and proteins being the dominant contributors. Less prominent tNOE contributions of approximately +2.5 ppm relative to water, presumably from aromatic moieties, were also detected. These aromatic signals were free from any CEST signals. In this study, we have demonstrated the feasibility of tNOE in human brain at 7 T. This method is more scan-time efficient than steady-state NOE and provides NOE measurement with minimal confounders.

A C 54 B 2 Polycyclic π-System with Bilayer Assembly and Multi-Redox Activity

A C ₅₄ B ₂ Polycyclic π-System with Bilayer Assembly and Multi-Redox Activity

Molecular Capture and Conformational Change of Diketopiperazines Containing Proline Residues by Epigallocatechin-3-O-gallate in Water.

The addition of an aqueous solution of diketopiperazine cyclo(Pro-Xxx) (Xxx: amino acid residue) to an aqueous solution of (-)-epigallocatechin-3-O-gallate (EGCg) led to precipitation of the complex of EGCg and cyclo(Pro-Xxx). The molecular capture abilities of cyclo(Pro-Xxx) using EGCg were evaluated by the ratio of the amount of cyclo(Pro-Xxx) included in the precipitates of the complex with EGCg to that of the total cyclo(Pro-Xxx) used. Stronger hydrophobicity of the side chain of the amino acid residue of cyclo(Pro-Xxx) led to a higher molecular capture ability. Furthermore, the molecular capture ability decreased when the side chain of the amino acid residue had a hydrophilic hydroxyl group. When diketopiperazine cyclo(Pro-Xxx), excluding cyclo(D-Pro-L-Ala), was taken into the hydrophobic space formed by the three aromatic A, B, and B' rings of EGCg, and formed a complex, their conformation was maintained in the hydrophobic space. Based on nuclear Overhauser effect (NOE) measurement, the 3-position methyl group of cyclo(D-Pro-L-Ala) in D2O was axial, whereas that of cyclo(L-Pro-L-Ala) was equatorial. When cyclo(D-Pro-L-Ala) was taken into the hydrophobic space of EGCg and formed a 2 : 2 complex, its 3-position methyl group changed from the axial position to the equatorial position due to steric hindrance by EGCg.

Flexibility and Cell Permeability of Cyclic Ras-Inhibitor Peptides Revealed by the Coupled Nosé-Hoover Equation.

Quantifying the cell permeability of cyclic peptides is crucial for their rational drug design. However, the reasons remain unclear why a minor chemical modification, such as the difference between Ras inhibitors cyclorasin 9A5 and 9A54, can substantially change a peptide's permeability. To address this question, we performed enhanced sampling simulations of these two 11-mer peptides using the coupled Nosé-Hoover equation (cNH) we recently developed. The present cNH simulations realized temperature fluctuations over a wide range (240-600 K) in a dynamic manner, allowing structural samplings that were well validated by nuclear Overhauser effect measurements. The derived structural ensembles were comprehensively analyzed by all-atom structural clustering, mapping the derived clusters onto principal components (PCs) that characterize the cyclic structure, and calculating cluster-dependent geometric and chemical properties. The planar-open conformation was dominant in aqueous solvent, owing to inclusion of the Trp side chain in the main-chain ring, while the compact-closed conformation, which favors cell permeation due to its compactness and high polarity, was also accessible. Conformation-dependent cell permeability was observed in one of the derived PCs, demonstrating that decreased cell permeability in 9A54 is due to the high free energy barrier separating the two conformations. The origin of the change in free energy surface was determined to be loss of flexibility in the modified residues 2-3, resulting from the increased bulkiness of their side chains. The derived molecular mechanism of cell permeability highlights the significance of complete structural dynamics surveys for accelerating drug development with cyclic peptides.

REDCRAFT: A computational platform using residual dipolar coupling NMR data for determining structures of perdeuterated proteins in solution.

Nuclear Magnetic Resonance (NMR) spectroscopy is one of the three primary experimental means of characterizing macromolecular structures, including protein structures. Structure determination by solution NMR spectroscopy has traditionally relied heavily on distance restraints derived from nuclear Overhauser effect (NOE) measurements. While structure determination of proteins from NOE-based restraints is well understood and broadly used, structure determination from Residual Dipolar Couplings (RDCs) is relatively less well developed. Here, we describe the new features of the protein structure modeling program REDCRAFT and focus on the new Adaptive Decimation (AD) feature. The AD plays a critical role in improving the robustness of REDCRAFT to missing or noisy data, while allowing structure determination of larger proteins from less data. In this report we demonstrate the successful application of REDCRAFT in structure determination of proteins ranging in size from 50 to 145 residues using experimentally collected data, and of larger proteins (145 to 573 residues) using simulated RDC data. In both cases, REDCRAFT uses only RDC data that can be collected from perdeuterated proteins. Finally, we compare the accuracy of structure determination from RDCs alone with traditional NOE-based methods for the structurally novel PF.2048.1 protein. The RDC-based structure of PF.2048.1 exhibited 1.0 Å BB-RMSD with respect to a high-quality NOE-based structure. Although optimal strategies would include using RDC data together with chemical shift, NOE, and other NMR data, these studies provide proof-of-principle for robust structure determination of largely-perdeuterated proteins from RDC data alone using REDCRAFT.

Allovalency observed by transferred NOE: interactions of sulfated tyrosine residues in the N‐terminal segment of CCR5 with the CCL5 chemokine

The N-terminal segment of the chemokine receptor Human CC chemokine receptor 5 (CCR5), Nt-CCR5, contains four tyrosine residues, Y3, Y10, Y14, and Y15. Sulfation of at least two of these tyrosine residues was found to be essential for high-affinity binding of CCR5 to its chemokine ligands. Here, we show that among the monosulfated Nt-CCR5(8-20) peptide surrogates (sNt-CCR5) those sulfated at Y15 and Y14 have the highest affinity for the CC chemokine ligand 5 (CCL5) chemokine in comparison with monosulfation at position Y10. Sulfation at Y3 was not investigated. A peptide sulfated at both Y14 and Y15 has the highest affinity for CCL5 by up to a factor of 3, in comparison with the other disulfated (sNt-CCR5) peptides. Chemical shift perturbation analysis and transferred nuclear Overhauser effect measurements indicate that the sulfated tyrosine residues interact with the same CCL5-binding pocket and that each of the sulfated tyrosines at positions 10, 14, and 15 can occupy individually the binding site on CCL5 in a similar manner, although with somewhat different affinity, suggesting the possibility of allovalency in sulfated Nt-CCR5 peptides. The affinity of the disulfated peptides to CCL5 could be increased by this allovalency and by stronger electrostatic interactions.

Potentially Mistaking Enantiomers for Different Compounds Due to the Self-Induced Diastereomeric Anisochronism (SIDA) Phenomenon

The NMR phenomenon of self-induced diastereomeric anisochronism (SIDA) was observed with an alcohol and an ester. The alcohol exhibited large concentration-dependent chemical shifts (δ’s), which initially led us to erroneously consider whether two enantiomers were in fact atropisomers. This highlights a potential complication for the analysis of chiral compounds due to SIDA, namely the misidentification of enantiomers. A heterochiral association preference for the alcohol in CDCl3 was determined by the intermolecular nuclear Overhauser effect (NOE) and diffusion measurements, the same preference as found in the solid state. The ester revealed more subtle effects, but concentration-dependent δ’s, observation of intermolecular NOE’s, as well as distinct signals for the two enantiomers in a scalemic sample all indicated the formation of associates. Intermolecular NOE and diffusion measurements indicated that homochiral association is slightly preferred over heterochiral association in CDCl3, thus masking association for enantiopure and racemic samples of equal concentration. As observed with the alcohol, heterochiral association was preferred for the ester in the solid state. The potential problems that SIDA can cause are highlighted and constitute a warning: Due care should be taken with respect to conditions, particularly the concentration, when measuring NMR spectra of chiral compounds. Scalemic samples of both the alcohol and the ester were found to exhibit the self-disproportionation of enantiomers (SDE) phenomenon by preparative TLC, the first report of SDE by preparative TLC.

Anion Binding Affinity: Acidity versus Conformational Effects.

High-level quantum chemical calculations were used to elucidate the gas- and solution-phase conformational equilibria for a series of symmetrically substituted (thio)ureas, (thio)squaramides, and croconamides. Gas-phase calculations predict that the thermodynamic conformer of many of these anion receptors is not the dual-hydrogen-bond-facilitating anti-anti conformer as is commonly assumed. For N,N'-diaryl thiosquaramides and croconamides, the syn-syn conformer is typically the predominant conformer. Solution-phase calculations show that the anti-anti conformer is increasingly stabilized as the polarity of the solvent increases. However, the syn-syn conformer remains the lowest energy conformation for croconamides. These predictions are used to explain the acidity versus chloride binding affinity correlations recently reported for some of these compounds. The chloride binding constants for thioureas and croconamides are significantly lower than expected on the basis of their pKa values, and this may be due in part to the need for these receptors to reorganize into the anti-anti conformer. Experimental NMR nuclear Overhauser effect (NOE) measurements of an asymmetrically substituted squaramide and its thio analogue are consistent with the syn-syn conformation being predominant at 298 K. The conformational equilibria should therefore be an important consideration for the design and development of future anion receptors and organocatalysts.

Ascorbigen A-NMR identification.

The connectivities of all atoms in ascorbigen A, an important metabolite, were determined unambiguously for the first time. The connectivity between carbon atoms was established by 2D INADEQUATE, and one-bond 13 C-13 C coupling constants were determined for all pairs of directly connected carbon atoms except for two strongly coupled carbon pairs. The 13 C-13 C coupling in one of the pairs was proved by a modification of standard INADEQUATE; however, the signals from the other pair were too weak to be observed. The connectivity within the two strongly coupled C-C pairs was confirmed by a combination of COSY and gHSQC; the latter experiment also identified all C-H bonds. The proton nuclear magnetic resonance (1 H NMR) spectra in dry dimethyl sulfoxide allowed identification and assignment of the signals due to NH and OH protons. The derived structure, 3-((1H-indol-3-yl)methyl)-3,3a,6-trihydroxytetrahydrofuro[3,2-b]furan-2(5H)-one, agrees with the structure suggested for ascorbigen A in 1966. The density functional theory (DFT) calculations showed that among 16 possible stereoisomers, only two complied with the almost zero value of the measured 3 J(H6-H6a). Of the two stereoisomers, 3S,3aS,6S,6aR and 3R,3aR,6R,6aS, the latter was excluded on synthetic grounds. The nuclear Overhauser effect measurements unveiled close proximity between H2' proton of the indole and the H6a proton of the tetrahydrofuro[3,2-b]furan part. Detailed structural interpretation of the measured NMR parameters by means of DFT NMR was hampered by rotational flexibility of the indole and tetrahydrofuro[3,2-b]furan parts and inadequacy of Polarizable Continuum Model (PCM) solvent model.

Folding Simulations of a Nuclear Receptor Box-Containing Peptide Demonstrate the Structural Persistence of the LxxLL Motif Even in the Absence of Its Cognate Receptor.

Regulation of nuclear receptors by their coactivators involves the recognition and binding of a specific sequence motif contained in the coactivator sequence. This motif is known as the nuclear receptor (NR) box and contains a conserved LxxLL subsequence, where L is leucine and x is any amino acid residue. Crystallographic studies have shown that the LxxLL motifs adopt an α-helical conformation when bound to their cognate nuclear receptors. Here we use an extensive set of folding molecular dynamics simulations to examine whether the α-helical conformation demonstrated by the LxxLL motifs in the bound state may represent a persistent structural preference of these peptides even in the absence of their cognate receptors. To this end, we have performed a grand total of 35 μs of adaptive tempering folding simulations of an NR-box-containing peptide derived from Drosophila's fushi tarazu segmentation gene product. Our simulations-performed using full electrostatics and an explicit representation of two different solvents (water and a TFE/water mixture)-clearly indicate the presence of a persistent helical preference of the LxxLL motif with a concomitant native-like structure and contacts between the motif's leucine residues. To lend further support to our findings, we compare the simulation-derived peptide dynamics with experimental NMR-derived nuclear Overhauser effect (NOE) measurements that had been previously obtained for the same peptide in the same two solvents. The comparison demonstrates a quantitative agreement between simulation and experiment with average upper bound NOE violations of less than 0.084 Å, thus independently validating our main conclusion concerning the intrinsic preference of NR-box motifs to form helical structures even in the absence of their cognate receptors.

Effect of the Flexible Regions of the Oncoprotein Mouse Double Minute X on Inhibitor Binding Affinity.

The oncoprotein MdmX (mouse double minute X) is highly homologous to Mdm2 (mouse double minute 2) in terms of their amino acid sequences and three-dimensional conformations, but Mdm2 inhibitors exhibit very weak affinity for MdmX, providing an excellent model for exploring how protein conformation distinguishes and alters inhibitor binding. The intrinsic conformation flexibility of proteins plays pivotal roles in determining and predicting the binding properties and the design of inhibitors. Although the molecular dynamics simulation approach enables us to understand protein-ligand interactions, the mechanism underlying how a flexible binding pocket adapts an inhibitor has been less explored experimentally. In this work, we have investigated how the intrinsic flexible regions of the N-terminal domain of MdmX (N-MdmX) affect the affinity of the Mdm2 inhibitor nutlin-3a using protein engineering. Guided by heteronuclear nuclear Overhauser effect measurements, we identified the flexible regions that affect inhibitor binding affinity around the ligand-binding pocket on N-MdmX. A disulfide engineering mutant, N-MdmXC25-C110/C76-C88, which incorporated two staples to rigidify the ligand-binding pocket, allowed an affinity for nutlin-3a higher than that of wild-type N-MdmX (Kd ∼ 0.48 vs Kd ∼ 20.3 μM). Therefore, this mutant provides not only an effective protein model for screening and designing of MdmX inhibitors but also a valuable clue for enhancing the intermolecular interactions of the pharmacophores of a ligand with pronounced flexible regions. In addition, our results revealed an allosteric ligand-binding mechanism of N-MdmX in which the ligand initially interacts with a compact core, followed by augmenting intermolecular interactions with intrinsic flexible regions. This strategy should also be applicable to many other protein targets to accelerate drug discovery.

Liquid-Phase Exfoliation of MoS2 Nanosheets: The Critical Role of Trace Water

Sonication-assisted liquid-phase exfoliation of layered materials in suitable organic solvents offers a simple scalable route for the production of 2D nanomaterials. N-methyl-2-pyrrolidone (NMP) is one of the most efficient solvents for liquid-phase exfoliation of a variety of layered solids, including MoS2. We show here that trace water present in NMP is crucial for the stability of MoS2 nanosheets in NMP dispersions. In the absence of water, the sheets are fragmented and chemically unstable. Using solution NMR techniques, 2D nuclear Overhauser effect and spin-lattice relaxation measurements, supported by classical molecular dynamics simulations, we are able to establish the role of water molecules in stabilizing the dispersion. We show that water molecules are localized at the Mo-terminated edges of the MoS2 sheets, thereby inhibiting chemical erosion of the sheets, and they also exhibit enhanced interactions with the solvent NMP molecules, leading to the stability of the dispersion.

Allostery through Protein Motion at Different Length and Time Scales

The conformational dynamics of proteins are often critical for mediating their functions. They are particularly important for ubiquitin, which must adapt its structure to interact with a plethora of cellular interaction partners. Through an in-depth relaxation dispersion (RD) analysis, we determined how backbone and side chain motions mutually affect one another through a process we term “population shuffling”. Intriguingly, these motions form an allosteric network that operates using concerted motion throughout the protein. This network is a key part of enabling multispecificity and suggests that ubiquitin regulation may be more complex than previously thought. In addition to concerted motions, simulations and crystal structures have suggested that population shuffling also operates between distant parts of the backbone, and may serve as another mechanism behind allosteric communication. In an effort to further elucidate the details of these motions, we have collected a large dataset of time-dependent Nuclear Overhauser Effect (NOE) measurements on both the backbone and side chains. Through explicit modeling of the effect that fast and slow motions have on the NOE, we gain even more insight into energy landscape of ubiquitin. By computationally combining residual dipolar coupling (RDC), RD, and NOE data our structural ensembles incorporate protein dynamics from timescales covering nine orders of magnitude.

Effect of Urea on Phase Transition of Poly(N-isopropylacrylamide) and Poly(N,N-diethylacrylamide) Hydrogels: A Clue for Urea-Induced Denaturation

1H MAS NMR spectroscopy has been applied to study the urea effect on phase transition of two similar thermosensitive polymer hydrogels: poly(N-isopropylacrylamide) (PNIPAM) and poly(N,N-diethylacrylamide) (PDEA). It is found that urea influences the phase transition of the hydrogels in opposite ways: lowering the lower critical solution temperature (LCST) of PNIPAM and hence stabilizing its globular structure, whereas raising the LCST of PDEA and destabilizing the globular structure. The self-diffusion coefficient and urea–polymer nuclear Overhauser effect (NOE) measurement reveal that urea has a stronger interaction with PNIPAM than with PDEA. Moreover, the enhanced positive water–PNIPAM NOE suggests that urea not only interacts directly with PNIPAM via hydrogen bond but also intensifies the hydrogen bonding interaction between water and PNIPAM. We suggest that different urea–polymer hydrogen bonding interaction due to the presence or absence of amide hydrogen is correlated with the distinct LCST variation of PNIPAM and PDEA.

Morphology study of DMPC/DHPC mixtures by solution-state 1H, 31P NMR, and NOE measurements

The temperature and mixing-ratio dependencies are studied by solution-state NMR for the morphology of aggregates composed of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC). The 1H and 31P NMR and nuclear Overhauser effect (NOE) measurements were performed at 25–60°C for molar ratio of q=1–5 (q=[DMPC]/[DHPC]). For the q=1 and 2 systems, the turbidity and viscosity of the sample are unchanged and the 1H and 31P NMR spectra are sharp over the whole temperature range examined. Combined with the NOE measurement and compared to the results for small unilamellar vesicle system, the q=1 and 2 aggregates are found to form the spherical mixed micelle and/or small bicelle over 25–60°C. When q is larger and is 2.5 or 3, the lipid-mixture system exhibits two morphological changes with temperature variation. At low temperature, the DMPC/DHPC system is of spherical mixed micelle and/or small bicelle, as evidenced from the visual observation, the 1H and 31P NMR spectra, and the NOE measurement. In the intermediate temperature range, the aggregate is the magnetically oriented bicelle since the solution is observed to be opaque and viscous with the 1H signal broadening and the 31P signal splitting into two peaks. When the temperature is high and above ~50°C, the sample becomes milky with sharp 1H and singlet 31P signals. The NOE measurement shows that the lipid dynamics of the aggregate is faster than of the large unilamellar vesicle with low curvature. These show the formation of cylindrical mixed micelle.

Determination of Intermolecular Interactions Using Polarization Compensated Heteronuclear Overhauser Effect of Hyperpolarized Spins.

The nuclear Overhauser effect (NOE) has long been used as a selective indicator for intermolecular interactions. Due to relatively small changes of signal intensity, often on the order of several percent, quantitative NOE measurements can be challenging. Hyperpolarization of nuclear spins can dramatically increase the NOE intensity by increasing population differences, but poses its own challenge in quantifying the original polarization level. Here, we demonstrate a method for the accurate measurement of intermolecular heteronuclear cross-relaxation rates by simultaneous acquisition of signals from both nuclei. Using this method, we measure cross-relaxation rates between water protons and (19)F of trifluoroacetic acid at concentrations ranging from 23 to 72 mM. A concentration-independent value of 2.46 × 10(-4) ± 1.02 × 10(-5) s(-1) M(-1) is obtained at a temperature of 301 K and validated using a nonhyperpolarized measurement. In a broader context, accurate measurement of heteronuclear cross-relaxation rates may enable the study of intermolecular interactions including those involving macromolecules where (19)F atoms can be introduced as site-selective labels.

Conformational analysis of some type XII bisbenzyltetrahydroisoquinoline alkaloids from Thalictrum alpinum assisted by quantitative noe, j coupling constants analysis and anisotropic NMR parameters

Northalrugosidine (1), a type XII bisbenzyltetrahydroisoquinoline alkaloid, exhibits in vivo efficacy in a murine model of visceral leishmaniasis (J. Nat. Prod., 2015, 78, 552 – 556). The conformational space of the macrocyclic moiety in compounds within the same subgroups of this class should provide more clues on their structure-activity relationships, which will be very beneficial for the potential future medicinal chemistry optimization of 1. The solution-state conformation of this molecule was investigated by conducting NMR spectroscopic studies on two naturally occurring analogues in the same subclass, thalrugosidine (2) and thalidasine (3). Residual dipolar couplings (RDCs), collected in compressed PMMA gel, were employed for the determination of relative spatial orientation of 1H-13C bonds, whereas 1H-1H inter-atomic distances were determined by measurement of quantitative nuclear Overhauser effects (qNOEs). The conformation of the studied molecules in solution was determined by simultaneous fitting of these NMR data and 3 J H,H coupling constants with computed molecular models.