Enantiomer Differentiation by Interaction-Specific Prediction of Residual Dipolar Couplings in Spherical-like Molecules.

Residual Dipolar Couplings (RDCs) are averaged dipolar couplings between nuclear spins of atoms in a molecule that can be measured by nuclear magnetic resonance (NMR) spectroscopy upon partial alignment by a chiral alignment medium. The estimation of differences in alignment of enantiomers may, in principle, enable the determination of absolute configuration. Here, we use molecular dynamics (MD) simulations to mimic the alignment of chiral molecules (i.

Hydrogen bond formation may enhance RDC-based discrimination of enantiomers.

The distinction of enantiomers based on residual anisotropic parameters obtained by alignment in chiral poly-γ-benzyl-L-glutamate (PBLG) is among the strongest in high-resolution NMR spectroscopy. However, large variations in enantiodifferentiation among different solutes are frequently observed. One hypothesis is that the formation of hydrogen bonds between solute and PBLG is important for the distinction of enantiomers.

Band-selective universal 90° and 180° rotation pulses covering the aliphatic carbon chemical shift range for triple resonance experiments on 1.2 GHz spectrometers.

Biomolecular NMR spectroscopy requires large magnetic field strengths for high spectral resolution. Today's highest fields comprise proton Larmor frequencies of 1.2 GHz and even larger field strengths are to be expected in the future.

Pure shift amide detection in conventional and TROSY-type experiments of C,N-labeled proteins.

Large coupling networks in uniformly C,N-labeled biomolecules induce broad multiplets that even in flexible proteins are frequently not recognized as such. The reason is that given multiplets typically consist of a large number of individual resonances that result in a single broad line, in which individual components are no longer resolved. We here introduce a real-time pure shift acquisition scheme for the detection of amide protons which is based on C-BIRD.

SORDOR pulses: expansion of the Böhlen-Bodenhausen scheme for low-power broadband magnetic resonance.

A novel type of efficient broadband pulse, called second-order phase dispersion by optimised rotation (SORDOR), has recently been introduced. In contrast to adiabatic excitation, SORDOR-90 pulses provide effective transverse 90 rotations throughout their bandwidth, with a quadratic offset dependence of the phase in the plane. Together with phase-matched SORDOR-180 pulses, this enables the Böhlen-Bodenhausen broadband refocusing approach for linearly frequency-swept pulses to be extended to any type of 90/180 pulse-delay sequence.

Concurrent J-evolving refocusing pulses.

Conventional refocusing pulses are optimised for a single spin without considering any type of coupling. However, despite the fact that most couplings will result in undesired distortions, refocusing in delay-pulse-delay-type sequences with desired heteronuclear coherence transfer might be enhanced considerably by including coupling evolution into pulse design. We provide a proof of principle study for a Hydrogen-Carbon refocusing pulse sandwich with inherent J-evolution following the previously reported ICEBERG-principle with improved performance in terms of refocusing performance and/or overall effective coherence transfer time.

Selective excitation enables encoding and measurement of multiple diffusion parameters in a single experiment.

Band selectivity to address specific resonances in a spectrum enables one to encode individual settings for diffusion experiments. In a single experiment, this could include different gradient strengths (enabling coverage of a larger range of diffusion constants), different diffusion delays, or different gradient directions (enabling anisotropic diffusion measurement). In this report, a selective variant of the bipolar pulsed gradient eddy current delay (BPP-LED) experiment, enabling selective encoding of three resonances, was implemented.

Selective H NMR Methods Reveal Functionally Relevant Proline cis/trans Isomers in Intrinsically Disordered Proteins: Characterization of Minor Forms, Effects of Phosphorylation, and Occurrence in Proteome.

It is important to identify proline cis/trans isomers that appear in several regulatory mechanisms of proteins, and to characterize minor species that are present due to the conformational heterogeneity in intrinsically disordered proteins (IDPs). To obtain residue level information on these mobile systems we introduce two H -detected, proline selective, real-time homodecoupled NMR experiments and analyze the proline abundant transactivation domain of p53. The measurements are sensitive enough to identify minor conformers present in 4-15 % amounts; moreover, we show the consequences of CK2 phosphorylation on the cis/trans-proline equilibrium.

Virtual decoupling to break the simplification versus resolution trade-off in nuclear magnetic resonance of complex metabolic mixtures.

The heteronuclear single quantum correlation (HSQC) experiment developed by Bodenhausen and Ruben (1980) in the early days of modern nuclear magnetic resonance (NMR) is without a doubt one of the most widely used experiments, with applications in almost every aspect of NMR including metabolomics. Acquiring this experiment, however, always implies a trade-off: simplification versus resolution. Here, we present a method that artificially lifts this barrier and demonstrate its application towards metabolite identification in a complex mixture.

Expedited Nuclear Magnetic Resonance Assignment of Small- to Medium-Sized Molecules with Improved HSQC-CLIP-COSY Experiments.

Resonance assignment is a pivotal step for any nuclear magnetic resonance (NMR) analysis, such as structure elucidation or the investigation of protein-ligand interactions. Both H-C heteronuclear single quantum correlation (HSQC) and H-H correlation spectroscopy (COSY) two-dimensional (2D) experiments are invaluable for H NMR assignment, by extending the high signal dispersion of C chemical shifts onto H resonances and by providing a high amount of through-bond H-H connectivity information, respectively. The recently introduced HSQC-CLIP(Clean In-Phase)-COSY method combines these two experiments, providing COSY correlations along the high-resolution C dimension with clean in-phase multiplets.

Power of Pure Shift HαCα Correlations: A Way to Characterize Biomolecules under Physiological Conditions.

Intrinsically disordered proteins (IDPs) constitute an important class of biomolecules with high flexibility. Atomic-resolution studies for these molecules are essentially limited to NMR spectroscopy, which should be performed under physiological pH and temperature to populate relevant conformational ensembles. In this context, however, fundamental problems arise with established triple resonance NMR experiments: high solvent accessibility of IDPs promotes water exchange, which disfavors classical amide H-detection, while C-detection suffers from significantly reduced sensitivity.

Determination of Configuration and Conformation of a Reserpine Derivative with Seven Stereogenic Centers Using Molecular Dynamics with RDC-Derived Tensorial Constraints*.

NMR-based determination of the configuration of complex molecules containing many stereocenters is often not possible using traditional NOE data and coupling patterns. Making use of residual dipolar couplings (RDCs), we were able to determine the relative configuration of a natural product containing seven stereocenters, including a chiral amine lacking direct RDC data. To identify the correct relative configuration out of 32 possible ones, experimental RDCs were used in three different approaches for data interpretation: by fitting experimental data based singular value decomposition (SVD) using a single alignment tensor and either (i) a single conformer or (ii) multiple conformers, or alternatively (iii) using molecular dynamics simulations with tensorial orientational constraints (MDOC).

Comprehensive and High-Throughput Exploration of Chemical Space Using Broadband F NMR-Based Screening.

Fragment-based lead discovery has become a fundamental approach to identify ligands that efficiently interact with disease-relevant targets. Among the numerous screening techniques, fluorine-detected NMR has gained popularity owing to its high sensitivity, robustness, and ease of use. To effectively explore chemical space, a universal NMR experiment, a rationally designed fragment library, and a sample composition optimized for a maximal number of compounds and minimal measurement time are required.

Urinary NMR Profiling in Pediatric Acute Kidney Injury-A Pilot Study.

Acute kidney injury (AKI) in critically ill children and adults is associated with significant short- and long-term morbidity and mortality. As serum creatinine- and urine output-based definitions of AKI have relevant limitations, there is a persistent need for better diagnostics of AKI. Nuclear magnetic resonance (NMR) spectroscopy allows for analysis of metabolic profiles without extensive sample manipulations.

Probing Long-Range Anisotropic Interactions: a General and Sign-Sensitive Strategy to Measure H- H Residual Dipolar Couplings as a Key Advance for Organic Structure Determination.

Residual dipolar couplings (RDCs) are amongst the most powerful NMR parameters for organic structure elucidation. In order to maximize their effectiveness in increasingly complex cases such as flexible compounds, a maximum of RDCs between nuclei sampling a large distribution of orientations is needed, including sign information. For this, the easily accessible one-bond H- C RDCs alone often fall short.

Configuration determination by residual dipolar couplings: accessing the full conformational space by molecular dynamics with tensorial constraints.

Residual dipolar couplings (RDCs) and other residual anisotropic NMR parameters provide valuable structural information of high quality and quantity, bringing detailed structural models of flexible molecules in solution in reach. The corresponding data interpretation so far is directly or indirectly based on the concept of a molecular alignment tensor, which, however, is ill-defined for flexible molecules. The concept is typically applied to a single or a small set of lowest energy structures, ignoring the effect of vibrational averaging.

Molecular Dynamics with Orientational Tensorial Constraints: A New Approach to Probe the Torsional Angle Distributions of Small Rotationally Flexible Molecules.

The potential of residual dipolar couplings (RDCs) in conformational studies of small molecules is now widely recognized, but current theoretical approaches for their interpretation have several limitations and there is still the need for a general method to probe the torsional angle distributions applicable to any rotationally flexible molecule. Molecular dynamics simulations with RDC-based orientational tensorial constraints (MDOC), implemented in the software COSMOS, are presented here as a conceptually new strategy. For the cases of the fluorinated anti-inflammatory drug diflunisal and the disaccharide cellobiose, we demonstrate that MDOC simulations with one-bond RDCs as tensorial constraints unveil torsion distributions and allow the determination of relative configuration in the presence of rotational flexibility.

Modulating Hinge Flexibility in the APP Transmembrane Domain Alters γ-Secretase Cleavage.

Intramembrane cleavage of the β-amyloid precursor protein C99 substrate by γ-secretase is implicated in Alzheimer's disease pathogenesis. Biophysical data have suggested that the N-terminal part of the C99 transmembrane domain (TMD) is separated from the C-terminal cleavage domain by a di-glycine hinge. Because the flexibility of this hinge might be critical for γ-secretase cleavage, we mutated one of the glycine residues, G38, to a helix-stabilizing leucine and to a helix-distorting proline.

Real-time pure shift measurements for uniformly isotope-labeled molecules using X-selective BIRD homonuclear decoupling.

We introduce a novel selective inversion element for chunked homonuclear decoupling that combines isotope selection via BIRD-filtering with band-selective inversion on the X-heteronucleus and allows efficient real-time decoupling of homonuclear and heteronuclear couplings. It is especially suitable for uniformly isotope-labeled compounds. We discuss in detail the inversion element based on band-selective refocusing on the X-nuclei (BASEREX), highlighting in particular the role of appropriate band-selective shaped refocusing pulses and the application of broadband X-pulses for an effective BIRD element during homodecoupling.

Increased H-Bond Stability Relates to Altered ε-Cleavage Efficiency and Aβ Levels in the I45T Familial Alzheimer's Disease Mutant of APP.

Cleavage of the amyloid precursor protein's (APP) transmembrane domain (TMD) by γ-secretase is a crucial step in the aetiology of Alzheimer's Disease (AD). Mutations in the APP TMD alter cleavage and lead to familial forms of AD (FAD). The majority of FAD mutations shift the preference of initial cleavage from ε49 to ε48, thus raising the AD-related Aβ42/Aβ40 ratio.

Efficient Extraction from Mice Feces for NMR Metabolomics Measurements with Special Emphasis on SCFAs.

Nuclear magnetic resonance (NMR) spectroscopy is one of the most promising methods for use in metabolomics studies as it is able to perform non targeted measurement of metabolites in a quantitative and non-destructive way. Sample preparation of liquid samples like urine or blood serum is comparatively easy in NMR metabolomics, because mainly buffer and chemical shift reference substance are added. For solid samples like feces suitable extraction protocols need to be defined as initial step, where the exact protocol depends on sample type and features.

ASAP-HSQC-TOCSY for fast spin system identification and extraction of long-range couplings.

Based on Ernst-angle-type excitation and Acceleration by Sharing Adjacent Polarization (ASAP), a fast HSQC-TOCSY experiment is introduced. In the approach, the DIPSI-2 isotropic mixing period of the ASAP-HSQC is simply shifted, which provides a TOCSY period without additional application of rf-energy. The ASAP-HSQC-TOCSY allows the acquisition of a conventional 2D in about 30 s.

Polarization recovery during ASAP and SOFAST/ALSOFAST-type experiments.

Experiments with fast repetition schemes significantly enhance the capabilities of modern NMR spectroscopy. Two schemes for heteronuclear correlation experiments that have been presented are the ASAP and the ALSOFAST method. The first method is Acceleration by Sharing Adjacent Polarization (ASAP) for samples at natural abundance isotope level.

Bulk Dynamic Spray Freeze-Drying Part 2: Model-Based Parametric Study for Spray-Freezing Process Characterization.

Spray freeze-drying is an evolving technology that combines the benefits of spray-drying and conventional lyophilization techniques to produce drug substance and drug product as free-flowing powders. The high surface-to-volume ratio associated to the submillimeter spray-frozen particles contributes to shorter drying and reconstitution times. The formation of frozen particles is the most critical part of this dehydration technique because it defines the properties of final product.

Bulk Dynamic Spray Freeze-Drying Part 1: Modeling of Droplet Cooling and Phase Change.

In spray freeze-drying (SFD), the solution is typically dispersed into a gaseous cold environment producing frozen microparticles that are subsequently dried via sublimation. This technology can potentially manufacture bulk lyophilized drugs at higher rates compared with conventional freeze-drying in trays and vials because small frozen particles provide larger surface area available for sublimation. Although drying in SFD still has to meet the material collapse temperature requirements, the final characteristics of the respective products are mainly controlled by the spray-freezing dynamics.