Nuclear magnetic resonance spectra: For all nuclear magnetic resonance (NMR) spectra, common Fourier-transformed free induction decay NMR spectra were used. All NMR were registered in both D2O and DMSO in order to allow detection of OH moieties. Tetramethylsilan was used as an internal standard. First, 13C-NMR spectra were registered and the peaks were associated with the particular carbon atoms in the molecule. Next, 1H-NMR spectra were registered and the peaks identifying particular hydrogen atoms were defined. Because resonance, and thereby the chemical shift of OH, NH and SH protons, may be influenced by intramolecular hydrogen bonds and because the degree of hydrogen bonding is reduced with increasing temperature, the measurements were repeated at different temperatures (25°C and 40°C) to allow calculation of the temperature dependence of a chemical shift. This made identification of intramolecular hydrogen bonds possible. After identification of each peak in the NMR spectra, two-dimensional NMR, so-called correlational spectroscopy (COSY) spectra, were performed to detect spin-spin coupling, thus identifying neighbouring atoms. For that purpose 13C1H-COSY spectra and 1H1H-COSY spectra were registered in D2O and DMSO. The principle is that two NMR spectra coregistered with slightly different pulse protocols are plotted against each other. In these plots the normal NMR spectrum can be seen at the diagonal of the plot, and the cross-peaks identify coupling nuclei. (NMR methods are detailed in 23-26.)
Statistical analysis: All values are given as means ± SEM of six experiments. Statistical significance was analyzed using rank tests for paired and unpaired observations (Wilcoxon’s test or Mann-Whitney U test, respectively) at a level of 0.05. For multiple comparisons additional analysis of variance was determined. In the following, all alterations mentioned were significant compared with control if not stated otherwise. Chemicals: All chemicals used were of analytical grade. Chemicals for peptide synthesis were obtained from Bachem (Heidelberg, Germany). All other chemicals were obtained from Sigma. You will always come across Cheap Diskus Advair click here offered by a reliable pharmacy.