DESIGN AND SYNTHESIS OF SIDE-CHAIN CONFORMATIONALLY RESTRICTED PHENYLALANINES AND THEIR USE FOR STRUCTURE-ACTIVITY STUDIES ON TACHYKININ NK-1 RECEPTOR

Constrained analogues of phenylalanine have been conceptually designed for analyzing the binding pockets of Phe(7) (S-7) and Phe(8) (S-8), t wo aromatic residues important for the pharmacological properties of S P, i.e., L-tetrahydroisoquinoleic acid, L-diphenylalanine, L-9-fluoren ylglycine (Flg), 2-indanylglycine, the diastereomers of L-1-indanylgly cine (Ing) and L-1-benz[f]indanylglycine (Bfi), and the Z and E isomer s of dehydrophenylalanine (Delta(z)Phe, Delta(E)Phe). Binding studies were performed with appropriate ligands and tissue preparations allowi ng the discrimination of the three tachykinin binding sites, NK-1, NK- 2, and NK-3. The potencies of these agonists were evaluated in the gui nea pig ileum bioassay. According to the binding data, we can conclude that the S-7 subsite is small, only the gauche (-) probe [(2S,3S)-Ing (7)]SP presents a high affinity for specific NK-1 binding sites. Surpr isingly, the [Delta(E)Phe(7)]SP analogue, which projects the aromatic ring toward the trans orientation, is over 40-fold more potent than th e Z isomer, [Delta(Z)Phe(7)]SP. A plausible explanation of these confl ictual results Is that either the binding protein quenches the minor t rans rotamer of [(2S,3S)-Ing(7)]SP in solution or this constrained ami no acid side chain rotates when inserted in the protein. In position 8 , the high binding affinities of [Flg(8)]SP and [(2S,3S)-Bfi(8)]SP sug gest that the S-8 subsite is large enough to accept two aromatic rings in the gauche (-) and one aromatic ring in the trans direction. Pepti des bearing two conformational probes in positions 7, 8, or 9 led to p ostulate that S-7, S-8, and S-9 subsites are independent from each oth er. The volumes available for side chains 7 and 8 can be estimated to be close to 110 and 240 Angstrom(3), respectively. The large volume of the S-8 subsite raises question on the localization of the SP-binding site in the NK-1 receptor. If SP were to bind in the transmembrane do mains, the cleft defined by the seven transmembrane segments must rear range during the binding process in order to bind a peptide in an ac-h elical structure and at least one large binding subsite in position 8. Thus, indirect topographical analysis with constrained amino acids mi ght contribute to the analysis of the receptor/ligand dynamics. Finall y, this study demonstrates that a good knowledge of the peptidic backb one structure and a combination of constrained amino acids are prerequ isites to confidently attribute the preferred orientation(s) of an ami no acid side chain.