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Determination of the solution conformation of both small organic molecules and peptides in water remains a substantial hurdle in using NMR solution conformations to guide drug design due to the lack of easy to use alignment media. Herein we report the design of a flexible compressible chemically cross-linked poly-4-acrylomorpholine gel that can be used for the alignment of both small molecules and cyclic peptides in water. To test the new gel, residual dipolar couplings (RDCs) and J-coupling constants were used in the configurational analysis of strychnine hydrochloride, a molecule that has been studied extensively in organic solvents as well as a small cyclic peptide that is known to form an α-helix in water. The conformational ensembles for each molecule with the best fit to the data are reported. Identification of minor conformers in water that cannot easily be determined by conventional NOE measurements will facilitate the use of RDC experiments in structure-based drug design.
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Reactivos de Enlaces Cruzados/química , Morfolinas/química , Péptidos/análisis , Polímeros/química , Estricnina/análisis , Agua/química , Geles/química , Espectroscopía de Resonancia Magnética , Estructura MolecularRESUMEN
We present a method to use long-range CH coupling constants to derive the correct diastereoisomer from the molecular constitution of small molecules. A set of 79 2 JCH and 3 JCH values collected from a single HSQMBC experiment on a sample of strychnine were used in the CASE-3D (computer-assisted 3D structure elucidation) protocol. In addition to the most commonly used 3 JCH coupling constants, the subset of 32 2 JCH values alone showed an excellent degree of configuration selection. The study is mainly based on comparison of DFT-calculated 2,3 JCH values with experimental ones, critical for the case of 2 JCH . But the configuration selection also works well using 3 JCH values predicted from a semi-empirical Karplus-based equation limited to H-C-C-C fragments. The robustness, shown using strychnine as a proof of concept, makes the J-based CASE-3D analysis a viable option for the application in fields such as peptide and carbohydrate research, organic synthesis, natural-product identification and analysis, as well as medicinal chemistry.
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Residual dipolar couplings and other anisotropic NMR parameters are powerful tools for molecular structure elucidation when conventional techniques do not suffice. With current liquid crystalline preparations it is necessary to prepare two samples to extract isotropic and anisotropic data from spectra and to derive the residual dipolar couplings. Here, we present the preparation, measurement, and interpretation of a novel biphasic liquid crystalline phase where a single sample can be used to generate both isotropic and anisotropic data. First, we introduce the synthesis of the chiral polymer leading to the biphasic liquid crystal. Second, we present two approaches to measure spatially selective CLIP-HSQC spectra. From these spectra, we extracted the couplings, performed an assignment of diastereotopic protons, and achieved the enantiomeric discrimination of isopinocampheol as a well-studied test molecule.
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Pulse sequences in NMR spectroscopy sometimes require the application of pulses with effective flip angles different from 90° and 180°. Previously (Magn. Reson. Chem. 2015, 53, 886-893), offset-compensated broadband excitation pulses with RF-amplitude-dependent effective flip angles (RADFA) were introduced that are applicable in such cases. However, especially RF-amplitude-restricted RADFA pulses turned out to perform not as good as desired in terms of achievable bandwidths. Here, a class of RF-amplitude-restricted RADFA pulses with linear phase slope is introduced that allows excitation over much larger bandwidths with better performance. In this theoretical work, the basic principle of the pulse class is explained, their physical limits explored, and their properties, also compared with other pulse classes, discussed in detail. Copyright © 2017 John Wiley & Sons, Ltd.
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Differentiating enantiomers using 2D bench-top NMR spectroscopy. Spectrometers working with permanent magnets at 1 T field strength allow the acquisition of 2D data sets. In conjunction with previously reported chiral alignment media, this setup allows the measurement of enantiomeric excess via residual dipolar couplings in stretched gelatine as a result of the reduced line width obtained by 2D J-resolved spectroscopy.
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The COSY experiment is an essential homonuclear 2D NMR experiment for the assignment of resonances. Its multiplet line shape, however, is often overly complicated, potentially leads to signal intensity losses, and is responsible for long minimum overall acquisition times. Herein, we present CLIP-COSY, a COSY-type experiment yielding clean in-phase peaks. It can be recorded within a few minutes and benefits from enhanced signal intensities for most cross-peaks. In combination with non-uniform sampling, the experiment times can be further reduced, and the in-phase multiplets enable the application of modern homonuclear decoupling techniques in both dimensions. As antiphase cancelations are avoided, CLIP-COSY can also be applied to macromolecules and other samples with broadened lines.
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Pulse sequences in NMR spectroscopy sometimes require the adjustment of effective flip angles with respect to experiment-specific or sample-specific parameters. Here, we present a quality factor for efficient optimization of offset-compensated broadband excitation pulses with RF amplitude-dependent effective flip angles (RADFA). After proof of principle, physical limits of RF amplitude-restricted and RF power-restricted broadband RADFA pulses are explored and corresponding pulse shapes and performances characterized in detail.
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Espectroscopía de Resonancia Magnética/métodos , Simulación por ComputadorRESUMEN
Deuterium NMR imaging was used to evaluate the spatial distribution of the degree of alignment in different types of alignment media by monitoring the deuterium quadrupolar splitting using spatially resolved NMR techniques in conventional liquid state NMR instruments. These images allow the unambiguous distinction of magnetic field and alignment inhomogeneities present in partially aligned samples, revealing the underlying reasons for linebroadening within an alignment medium that cannot be explained by the sole analysis of 1D (2)H NMR spectra. For example, alignment inhomogeneities due to broken gels or the presence of concentration gradients in liquid crystalline solutions are clearly detected by the imaging methods proposed in this work.
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Deuterio/química , Espectroscopía de Resonancia Magnética , Geles/química , Cristales Líquidos/química , Imagen por Resonancia MagnéticaRESUMEN
The melanocortin-4 receptor (MC4R) is a centrally expressed, class A GPCR that plays a key role in the regulation of appetite and food intake. Deficiencies in MC4R signaling result in hyperphagia and increased body mass in humans. Antagonism of MC4R signaling has the potential to mitigate decreased appetite and body weight loss in the setting of anorexia or cachexia due to underlying disease. Herein, we report on the identification of a series of orally bioavailable, small-molecule MC4R antagonists using a focused hit identification effort and the optimization of these antagonists to provide clinical candidate 23. Introduction of a spirocyclic conformational constraint allowed for simultaneous optimization of MC4R potency and ADME attributes while avoiding the production of hERG active metabolites observed in early series leads. Compound 23 is a potent and selective MC4R antagonist with robust efficacy in an aged rat model of cachexia and has progressed into clinical trials.
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Apetito , Receptor de Melanocortina Tipo 4 , Ratas , Humanos , Animales , Caquexia/tratamiento farmacológico , Anorexia/tratamiento farmacológico , Conformación MolecularRESUMEN
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. Pulses are optimised using optimal control theory with a newly derived quality factor and z-controls as an efficient tool to speed up calculations. Pulses are characterised in theory and experiment and compared to conventional concurrent refocusing pulses, clearly showing an improvement for the J-evolving pulse sandwich. As a side-product, also efficient J-compensated resfocusing pulse sandwiches - termed BUBU pulses following the nomenclature of previous J-compensated BUBI and BEBEtr pulse sandwiches - have been optimised.
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Nuclear Overhauser Effect (NOE) methods in NMR are an important tool for 3D structural analysis of small molecules. Quantitative NOE methods conventionally rely on reference distances, known distances that have to be spectrally separated and are not always available. Here we present a new method for evaluation and 3D structure selection that does not require a reference distance, instead utilizing structures optimized by molecular mechanics, enabling NOE evaluation even on molecules without suitable reference groups.
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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. It was originally derived in the ASAP-HMQC and recently received renewed attention in the ASAP-HSQC. Sharing the polarization of active protons with the surrounding reservoir can result in seemingly instant polarization recovery and therefore enormous gains in sensitivity, but can also lead to a slight reduction of polarization and spectral intensity, depending on sample and setup. A second type of setup has been introduced with the so-called Alternate SOFAST (ALSOFAST-) HMQC and ALSOFAST-HSQC for natural abundance 1H,13C-correlation experiments and in the SOFAST-HMQC for 1H,15N-correlations. In these cases, the reservoir spins are only maintained through the pulse sequence without Hartmann-Hahn-type mixing. A model for the estimation of the available polarization in the fast repetition schemes could be a valuable tool for experimentalists and pulse sequence developers. Starting from the well-known Ernst angle model, we derive in this article several mathematical models that describe the polarization over the course of ALSOFAST and ASAP type experiments. The models can be used to visualize the initial scans of an experiment and even more importantly, show the polarization and achievable signal intensity in the steady state of an experiment. In this way the two extreme applications of ASAP- and ALSOFAST-type acquisition schemes are covered: (i) acquisition using progressive excitation for experiments with few increments and shortest possible overall acquisition times and (ii) steady-state-type experiments with ultrahigh resolution and correspondingly large number of increments. The two resulting excitation strategies are applied to maximize SNR in different situations. To test the models, experimental data was obtained by special pulse sequences and examples are shown for different spin environments. The results show good agreement between theory and experiment.
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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 30â¯s. Alternatively, it allows the acquisition of highly carbon-resolved spectra (several Hz digital resolution) on the order of minutes. An ASAP-HSQC-TOCSY-IPAP variant, finally, allows the sign-sensitive extraction of heteronuclear long-range coupling constants from a pair of highly resolved spectra in less than an hour. Pulse sequences, several example spectra, and a discussion of results are given.
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Novel CLIP-COSY based homo- and heteronuclear correlation experiments are reported for the rapid, semi-automated NMR assignment of small to medium-sized molecules. The homonuclear CLIP-COSY and corresponding relayed experiments employ the perfect-echo based mixing sequence for in-phase coherence transfer between directly and/or indirectly coupled proton spins. The combined analysis of the resulting CLIP-COSY and relayed spectra made it possible to easily track down, layer by layer, the proton-proton connectivity network. In larger molecules the narrow chemical shift range of protons may, however, compromise the efficacy of the homonuclear correlation based assignment strategy. To overcome this limitation, an HSQC variant of the CLIP-COSY experiment has now been devised. Combined treatment of HSQC-CLIP-COSY (relayed) and standard HSQC spectra facilitates simultaneous and semi-automatic assignment of 1 H and 13 C resonances of medium-sized molecules, such as pentasaccharides. The recently introduced PSYCHE broadband homonuclear decoupling scheme has been also implemented into the devised homo- and heteronuclear CLIP-COSY based experiments, resulting in fully decoupled high-resolution pure-shift correlation spectra.
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A variety of new racemic alcohol and ketone cyclooctadiene derivatives was prepared for their complexation with platinum to generate a new class of platinum(ii) complexes.
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Previously we introduced two novel NMR experiments for small molecules, the so-called ASAP-HSQC and ALSOFAST-HSQC (Schulze-Sünninghausen et al., 2014), which allow the detection of heteronuclear one-bond correlations in less than 30s at natural abundance. We propose an improved symmetrized pulse scheme of the basic experiment to minimize artifact intensities and the combination with non-uniform sampling to enable the acquisition of conventional HSQC spectra in as short as a couple of seconds and extremely 13C-resolved spectra in less than ten minutes. Based on steady state investigations, a first estimate to relative achievable signal intensities with respect to conventional, ASAP-, and ALSOFAST-HSQC experiments is given. In addition, we describe several extensions to the basic pulse schemes, like a multiplicity-edited version, a revised symmetrized CLIP-ASAP-HSQC, an ASAP-/ALSOFAST-HSQC sequence with broadband BIRD-based 1H,1H decoupling, and a symmetrized sequence optimized for water suppression. Finally, RF-power considerations with respect to the high duty cycle of the experiments are given.