Structure determination and by-product profile of the NK(2) receptor antagonist nepadutant, a bicyclic glycopeptide.

We have synthesized and fully characterized the NK(2) receptor antagonist nepadutant and its by-products using nuclear magnetic resonance (NMR) and restrained molecular dynamics. The agent consists of an active bicyclic hexapeptide combined with a sugar residue. Analysis of the high-performance liquid chromatogram and the mass spectroscopy spectra yields traces of three by-products with the same molecular weight as the main product. The conformation of the molecules in the bicyclic hexapeptide segment, the active region, is well defined, whereas the sugar moiety is disordered. For the peptide region of nepadutant and all of its by-products, the NMR observables can be described by a single backbone conformation, more specifically a betaI, betaII-turn arrangement. The active dipeptide unit Trp-Phe occupies the i+1 and i+2 position of a betaI-turn. The by-product profile is characterized by different forms of sugars which are caused mainly by isomerization in the process of ring opening.

Mimicry of beta II'-turns of proteins in cyclic pentapeptides with one and without D-amino acids.

The solution structure of eight cyclic pentapeptides has been determined by two-dimensional 1H-NMR spectroscopy combined with spectra simulations and restrained molecular dynamic simulations. Six of the cyclic pentapeptides were derived from the C-terminal cholecystokinin fragment CCK-4 enlarged with Asp1 resulting in the sequence (Asp-Trp-Met-Asp-Phe), one L-amino acid after the other was substituted by its D-analog. In addition, two peptides, including an all-L-amino-acid-containing cyclic pentapeptide, cyclo(Asp-Phe-Lys-Ala-Thr) and cyclo(Asp-Phe-Lys-Ala-D-Thr) were investigated. All D-amino-acid-containing peptides show beta II'-turn conformations with the D-amino acid in the i + 1 position, excepting the D-aspartic-acid-containing peptides. These two peptides are characterized by the lack of beta-turns at pH values less than 4, suggesting that D-aspartic acid in the full-protonized state avoids the formation of beta-turns in these compounds. At pH values greater than 5, a conformational change into the beta II'-turn conformation was also observed for these peptides. Conformations without beta-turns are expected for cyclic all-L pentapeptides, but both cyclo(Asp-Phe-Lys-Ala-Thr) and the D-Thr analog cyclo(Asp-Phe-Lys-Ala-D-Thr) exhibit beta II'-turn conformations around Thr-Asp and D-Thr-Asp. Thus cyclic all-L pentapeptides and those with one D-amino acid are able to form similar structures preferably with a beta II'-turn. The beta-turn formation in cyclic pentapeptides containing a D-aspartic acid is dependent on the ionization state. The relevance of the work to the design of beta'-turn mimetics is discussed.

Interaction between paramagnetic metal complexes and intracellular water of single cells.

Oocytes from Xenopus laevis were used as well-established model to investigate spatially resolved changes in relaxation time (T1) within a cell during exposure to copper complexes by means of 1H-NMR-microscopy. T1 relaxation of intracellular water was shortened in dependence on the complex concentration, the water content, and the water mobility in various cell compartments. A relatively constant T1 decrease was observed in the cytoplasm of the vegetal pole, irrespective of the type and concentration of the permeable complexes used. Since the lowest content of free mobile water molecules was detected at the vegetal pole, we concluded that this quantity was as important for the relaxation time decrease as that of the chelated paramagnetic ions. Experiments using 14C-labeled inulin demonstrated that the paramagnetic metal complexes entered the oocytes without gross injury to their plasma membranes.

Estimation of water content and water mobility in the nucleus and cytoplasm of Xenopus laevis oocytes by NMR microscopy.

NMR microscopy is a noninvasive approach for studying cell structure and properties. Spatially resolved measurements of the relaxation times T1 and T2 provided information on the water proton spin density and water mobility in different parts of Xenopus laevis oocytes. The spin-lattice relaxation time T1 was determined using a saturation-recovery sequence and the common spin-echo sequence with increasing repetition times, while the transverse relaxation time T2 was measured by means of the spin-echo sequence with varying echo times. From the relaxation times, the mole fractions of possible reorientational correlation times tau c for different types of intracellular water were calculated according to a simple two-phase model. The values for T1, T2, and proton spin density (i.e., water content) are: nucleus >> animal cytoplasm > vegetal cytoplasm. Based on the estimation of tau c, nearly 90% of the nuclear water and 74.4% of the water of the animal pole was considered as free mobile water, whereas 55.5% of the water of the vegetal pole appeared as bound water.

Study of the membrane permeability of a paramagnetic metal complex on single cells by NMR microscopy.

A new procedure has been developed for investigating the ability of paramagnetic metal complexes to penetrate the plasma membrane of eukaryotic cells without decomposition. Defolliculated Xenopus laevis oocytes formed the biological system to test N,N-ethylenebis-(1,5,5-trimethyltetramic-acid-3-acetiminato) copper (II). An increase of the signal intensities in spin-echo (SE) images of oocytes treated with the tested substance indicated that the complex was able to penetrate biological membranes due to the arrangement of hydrophobic and hydrophilic groups within the ligand. In contrast, the treatment with the commonly used contrast agent gadolinium-DTPA/dimeglumine did not enhance the signal intensity in NMR images of oocytes after time periods of exposure comparable to those used for the copper complex. After microinjection into Xenopus oocytes the copper complex was released into the extracellular medium without degradation, as shown by HPLC measurements.

[Identification of an antimicrobially active constituent isolated from propolis (author's transl)]. / Identifizierung eines aus Propolis isolierten, antimikrobiell wirksamen Inhaltsstoffes.

On isolating and identifying antimicrobially active propolis constituents, the authors detected a substance that must be regarded as a mixture of caffeic acid esters. The caffeic acid moiety was unequivocally identified by various chemical and spectroscopic methods. The results obtained from the gas chromatographic study of the alcohol fraction after hydrolysis of the isolated compound identify benzyl alcohol, phenyl ethyl alcohol and cinnamic alcohol as ester components. Furthermore, the mass spectroscopic findings are indicative of the presence of a caffeic acid pentenyl ester in the mixture. Attempts to separate this ester mixture by recrystallization and thin-layer chromatography failed.