Monitoring fast chemical processes by reaction-interrupted excitation transfer (ExTra) NMR spectroscopy.

NMR spectroscopy is generally used to investigate molecules under equilibrium conditions. Despite recent technological and methodogical developments to study on-going reactions, tracing the fate of individual atoms during an irreversible chemical reaction is still a challenging and elaborate task. Reaction-interrupted excitation transfer (ExTra) NMR provides a selective tracking of resonances from atoms, which undergo chemical conversion. We show that reactions triggered either by rapid mixing or by photo-excitation can be conveniently followed at a sub-second time scale using standard NMR equipment. In ExTra NMR we use the selectively inverted magnetization of a selected atom to follow its conversion in the course of a fast chemical reaction. The chemical reaction has to be started within the relaxation period of an initial inverting 180° pulse. The presented protocol provides a generally applicable NMR method for reaction monitoring.

Determination of unresolved heteronuclear scalar coupling constants by J(up)-HSQMBC.

Long-range heteronuclear scalar coupling constants provide important structural information, which is necessary for obtaining stereospecific assignment or dihedral angle information. The measurement of small proton-carbon splittings is particularly difficult due to the low natural abundance of carbon-13 and the presence of homonuclear couplings of similar size. Here we present a real-time J-upscaled HSQMBC, which allows the measurement of heteronuclear coupling constants even if they are hidden in the signal linewidth of a regular spectrum.

Faster and cleaner real-time pure shift NMR experiments.

Real-time pure shift experiments provide highly resolved proton NMR spectra which do not require any special processing. Although being more sensitive than their pseudo 2D counterparts, their signal intensities per unit time are still far below regular NMR spectra. In addition, scalar coupling evolution during the individual data chunks produces decoupling sidebands. Here we show that faster and cleaner real-time pure shift spectra can be obtained through the implementation of two parameter alterations. Variation of the FID chunk lengths between individual transients significantly suppresses decoupling sidebands for any kind of real-time pure shift spectra and thus allows for example the analysis of minor components in compound mixtures. Shifting the excitation frequency between individual scans of real-time slice-selective pure shift spectra increases their sensitivity obtainable in unit time by allowing faster repetitions of acquisitions.

Uniform Reduction of Scalar Coupling by Real-Time Homonuclear J-Downscaled NMR.

Scalar coupling in proton NMR spectra provides important information for the structural analysis. However, the low resolution due to the resulting signal splitting, together with the rather narrow spectral range of hydrogen, often prevents the extraction of J-coupling information. Here we present a method to achieve real-time homonuclear J-downscaling. Thereby, all J-values are uniformly reduced by an arbitrary scaling factor. In the resulting one-dimensional spectra, signal overlap is reduced, while scalar coupling information is still available.

Visualizing unresolved scalar couplings by real-time J-upscaled NMR.

Scalar coupling patterns contain a wealth of structural information. The determination, especially of small scalar coupling constants, is often prevented by merging the splittings with the signal line width. Here we show that real-time J-upscaling enables the visualization of unresolved coupling constants in the acquisition dimension of one-dimensional (1D) or multidimensional NMR spectra. This technique, which works by introducing additional scalar coupling evolution delays within the recording of the FID (free induction decay), not only stretches the recorded coupling patterns but also actually enhances the resolution of multiplets, by reducing signal broadening by magnetic field inhomogeneities during the interrupted data acquisition. Enlarging scalar couplings also enables their determination in situations where the spectral resolution is limited, such as in the acquisition dimension of heteronuclear broadband decoupled HSQC (heteronuclear single quantum correlation) spectra.

Structural and functional implications of the interaction between macrolide antibiotics and bile acids.

Macrolide antibiotics, such as azithromycin and erythromycin, are in widespread use for the treatment of bacterial infections. Macrolides are taken up and excreted mainly by bile. Additionally, they have been implicated in biliary system diseases and to modify the excretion of other drugs through bile. Despite mounting evidence for the interplay between macrolide antibiotics and bile acids, the molecular details of this interaction remain unknown. Herein, we show by NMR measurements that macrolides directly bind to bile acid micelles. The topology of this interaction has been determined by solvent paramagnetic relaxation enhancements (solvent PREs). The macrolides were found to be bound close to the surface of the micelle. Increasing hydrophobicity of both the macrolide and the bile acid strengthen this interaction. Both bile acid and macrolide molecules show similar solvent PREs across their whole structures, indicating that there are no preferred orientations of them in the bile micelle aggregates. The binding to bile aggregates does not impede macrolide antibiotics from targeting bacteria. In fact, the toxicity of azithromycin towards enterotoxic E. coli (ETEC) is even slightly increased in the presence of bile, as was shown by effective concentration (EC50 ) values.

Directly decoupled diffusion-ordered NMR spectroscopy for the analysis of compound mixtures.

For the analysis of compound mixtures by NMR spectroscopy, it is important to assign the different peaks to the individual constituents. Diffusion-ordered spectroscopy (DOSY) is often used for the separation of signals based on their self-diffusion coefficient. However, this method often fails in the case of signal overlap, which is a particular problem for (1)H-detected DOSY spectra. Herein, an approach that allows the acquisition of homonuclear broadband-decoupled DOSY spectra without the introduction of an additional decoupling dimension, by instant decoupling during acquisition, is presented. It was demonstrated on a mixture of six alcohols, and the investigation of the binding of a dodecapeptide to membrane mimetics.

A general method for diagonal peak suppression in homonuclear correlated NMR spectra by spatially and frequency selective pulses.

Homonuclear two- and multidimensional NMR spectra are standard experiments for the structure determination of small to medium-sized molecules. In the large majority of homonuclear correlated spectra the diagonal contains the most intense peaks. Cross-peaks near the diagonal could overlap with huge tails of diagonal peaks and can therefore be easily overlooked. Here we present a general method for the suppression of peaks along the diagonal in homonuclear correlated spectra. It is based on a spatially selective excitation followed by the suppression of magnetization which has not changed the frequency during the mixing process. In addition to the auto correlation removal, these experiments are also less affected by magnetic field inhomogeneities due to the slice selective excitation, which on the other side leads to a reduced intensity compared to regular homonuclear correlated spectra.