Sensitivity-Enhanced 13C-NMR Spectroscopy for Monitoring Multisite Phosphorylation at Physiological Temperature and pH

Abundant phosphorylation events control the activity of nuclear proteins involved in gene regulation and DNA repair. These occur mostly on disordered regions of proteins, which often contain multiple phosphosites. Comprehensive and quantitative monitoring of phosphorylation reactions is theoretically achievable at a residue-specific level using ¹H-¹⁵N NMR spectroscopy, but is often limited by low signal-to-noise at pH>7 and T>293 K. We have developed an improved ¹³Cα-¹³CO correlation NMR experiment that works equally at any pH or temperature, that is, also under conditions at which kinases are active. This allows us to obtain atomic-resolution information in physiological conditions down to 25 μm . We demonstrate the potential of this approach by monitoring phosphorylation reactions, in the presence of purified kinases or in cell extracts, on a range of previously problematic targets, namely Mdm2, BRCA2, and Oct4.     

Miscibility of Polyoxymethylene Blends as Revealed by High-resolution Solid-state 13C-NMR Spectroscopy

The miscibility of polyvinylphenol (PVPh) or terpenephenol (TPh) with polyoxymethylene (POM) was examined by high-resolution solid-state ¹³C nuclear magnetic resonance (NMR) spectroscopy. It was found that the driving force for the mixing of POM and PVPh is the hydrogen-bonding interaction between the phenolic OH group of PVPh and the ether oxygen of POM, and that the mixing is preferentially induced in the noncrystalline phase. ¹H relaxation time experiments indicated that POM/PVPh blends were homogeneous on a scale of 20–30 nm but heterogeneous on a scale of 2–3 nm. On the other hand, Fourier transform infrared and cross-polarization/magic-angle-spinning ¹³C-NMR (nuclear magnetic resonance) spectra revealed that POM and TPh are also mixed in the noncrystalline phase through the intermolecular hydrogen-bonding interaction, while some fraction of POM is still crystallizable. Moreover, the domain size of the micro-phase separation was estimated to be about 1 nm by the direct ¹H spin-diffusion measurements, suggesting almost homogeneous mixing on a molecular level in the noncrystalline phase. © 1997 John Wiley & Sons, Ltd.     

Pulse Fourier Transform 13C-NMR Spectroscopy,Principles and Applications

As a result of the development of pulse Fourier transform NMR spectroscopy ¹³C resonance has become accepted as a means of elucidating the structures of organic compounds having the natural ¹³C abundance. Assignment of the signals is facilitated by broad-band and off resonance proton decoupling and by knowledge of longitudinal relaxation times. The pulse Fourier technique, which is more sensitive and faster than conventional NMR spectroscopy because of multi-channel excitation, allows ¹³C measurements within a short time even on dilute samples of high molecular weight compounds without prior enrichment in ¹³C.     

13C-NMR. Spectroscopy of Naturally Occuring Substances. XLII. Conformational analysis of quebrachamine-like indole alkaloids and related substances

The ¹³C shifts of 16α- and 16β-substituted derivatives of quebrachamine, 14,15-dehydroquebrachamine, cleavamine, 15,20α-dihydrocleavamine and 15,20β-dihydrocleavamine are determined and correlated with possible conformations of these tetracycles. The method of analysis of the C(16) configuration of these compounds, which emanated from this study, is used for the determination of the configuration of the site of coupling of vindoline and cleavamine β-chloroindolenine.     

Conformational Analysis by Photoelectron Spectroscopy

The conformation of organic molecules may be determined by means of photoelectron (PE) spectroscopy if there are orbital interactions present which depend on a dihedral angle. Straightforward application of the method requires that all ionization bands of interest can be assigned unambiguously and that inductive effects and secondary orbital interactions do not occur or can be accounted for appropriately. In many cases, this method complements conventional methods of conformational analysis. PE conformational analysis is particularly suited to compounds containing vicinal lone pairs or π systems.     

Conformational studies of 5-substituted 3-aryl-1,2,3-oxathiazolidine 2-oxides using 13C-NMR

The title compounds were prepared and their ¹³C-nmr spectra were examined. On the basis of the data for the magnitudes of the γ shifts and the δ effects, four isomeric conformations of the compounds were discussed.     

13C-NMR. Spectroscopy of Naturally Occurring Substances. XXXV. Labdanic diterpenes

A total carbon shift analysis of several representatives of the labdane diterpene family of natural products is presented. The shift assignment is based on the prior shift designation of some synthetic trans-decalin derivatives.     

13C-NMR. Spectroscopy of naturally occurring substances. XLV. Iboga alkaloids.

The carbon shifts of the Iboga-type alkaloids catharanthine, voacangine, coronaridine, ibogaine, dihydrocatharanthine and epiibogamine were recorded and correlated with the conformation of the natural bases. A ¹³C-NMR. analysis of heyneanine determined its C(19) configuration and a similar study of epiheyneanine proved its structure.     

High-Resolution Solid-State 13C-NMR Spectroscopy of Polymers [New Analytical Methods (37)]

Until a few years ago, solid-state nuclear resonance yielded spectra containing broad lines only. Meanwhile, CP/MAS-NMR spectroscopy has provided a method which gives narrow nuclear resonance lines from a solid-state specimen as well. Using this technique, it is now possible to produce spectra of “rare” nuclei (¹³C, ²⁹Si, ¹⁵N etc.) which are resolved in terms of chemical structure. The analytical capabilities of NMR spectroscopy can be applied to the solid state: it may be that it is necessary to identify compounds in the solid state because, for example, a solvent would alter the coordination sphere, or that it is desired to monitor chemical reactions in the solid state, for example the baking of an enamel. Where a substance in the solid state is concerned, high-resolution ¹³C-NMR spectroscopy provides not only information about the chemical structure, but also about the solid state itself. To mention just a few examples, information on the conformation, crystal structure and molecular dynamics, as well as molecular miscibility is given. This opens up a broad spectrum of applications, from a statement concerning the crystal modification of an active substance in ready-to-use pharmaceutical preparations, e.g. tablets, to the question of whether two polymers are miscible with one another at a molecular level.