Conformational relevant NMR parameters such as NOE values and coupling constants depend in a nonlinear way on distances and dihedral angles. This may be misleading in the determination of molecular conformations when a conformational equilibrium exists with rates that are fast on the NMR time scale: short nuclear distances are overemphasized when distances obtained by NOEs are used as a tool for modelling the conformation in solution. Antamanide, a cyclic decapeptide, is shown to be such a case. However, molecular dynamics calculations with NOE constraints can be used to identify crucial NOE values and prove the evidence of a conformational equilibrium. In addition, homonuclear and heteronuclear coupling constants provide additional support for the existence of "the" conformation or the conformational equilibrium, respectively.
The conformation of the cyclic decapeptide antamanide (1) in CDCl3 solution was determined by means of NMR‐spectroscopic methods. Distances obtained from 40 negative NOE values at 500 MHz at low temperature were used as constraints in calculations with the GROMOS molecular dynamics program package to elucidate the most stable backbone conformation in solution. It turned out that there is a fast conformational equilibrium between up to four conformations which differ in a flip about φ and ψ around the two amide bonds Ala4–Phe5 and Phe9–Phe10. Side chain conformations were determined using homo‐ and heteronuclear coupling constants. Carbon relaxation times provide information about the internal flexibility of 1. Three new antamanide derivatives in which Pro3, Pro7, or Pro8 have been substituted by γ‐thiaproline (Thz) were synthesized and crystallized from acetone/water. The X‐ray analyses of these Thz derivatives demonstrate that in the presence of water antamanide adopts a preferred structure which includes four water molecules for building up a compact hydrogen‐bonded structure, which differs from the conformation in chloroform sulution.
A procedure is proposed for obtaining coupling constants from phase-sensitive H,H-COSY spectra. For this purpose, firstly pseudo-one-dimensional spectra are created by co-addition of individual traces of cross-peaks, and by a similar addition of diagonal peaks in a spectrum which is phase corrected to pure-absorption line shapes. The sum and difference of the pseudo-one-dimensional spectra of a cross-peak and its diagonal peak give two spectra, with a pattern in which the active coupling ceases to exist ('spin decoupling') but is shifted by half the value of the same. If diagonal peaks are not available owing to overlapping signals, sums and differences of the pseudo-one-dimensional spectra of two cross-peaks can be applied to simpliy spectral patterns for the evaluation of J values. The procedure is demonstrated with the example of the AMXY spin system of a tyrosine, and the complex pattern of the Pro8 spin system of the cyclic decapeptide antamanide. The conformation of the Pro8 ring, derived from the coupling constants thus obtained, is of the ,E type. Recently, techniques have been reported to achieve
Constitution and conformation determine the biological activity of a peptide, as demonstrated by comparative investigations of the inhibition of cholate uptake in liver cells by somatostatin, antamanide, cyclolinopeptide A and 1 and 2. The crystal structure of 1 is the same as that of 2 over a wide range and both react strongly cytoprotectively.
Homo‐ and heteronuclear one‐ and two‐dimensional techniques have been used to assign the 1H, 13C, and 15N NMR spectra of the cyclic decapeptide antamanide (1) in CDCl3. The assignment includes the aromatic resonances of phenylalanine and the non‐protonated nitrogen atoms of proline by means of heteronuclear two‐dimensional NMR spectroscopy. Sequence analysis of the four phenylalanine and four proline amino acid residues was performed by heteronuclear proton – carbon long‐range couplings using the COLOC sequence. Three new thiazolidine analogues of antamanide confirm the sequence analysis of prolines and open the possibility for the extraction of extended spectral parameters. The interpretation of NOE data, obtained from one‐ and two‐dimensional spectra at high field and low temperatures, demonstrate possible misinterpretations of this effect respecting sequence assignment, if only NOEs between NH protons and α‐protons are considered.
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