SYNTHESIS, RECEPTOR-BINDING, AND CROSS-LINKING OF PHOTOACTIVE ANALOGSOF NEUROPEPTIDE-Y

Five photoactive analogues of porcine neuropeptide Y (NPY), a 36 amino acid hormone of the pancreatic polypeptide family, have been synthesi zed by solid phase peptide synthesis method, Fmoc/tBu strategy and car efully characterized. The analogues contain the photoactivatable amino acid -trifluoromethyl)-3H-diazirine-3-yl-phenyl-alanine ((Tmd)Phe) in dividually at different positions (1, 20, 21, 27 or 36) instead of tyr osine in the wildtype sequence. Affinity to membranes prepared from SM S-KAN cells, which stably express the Y-2 receptor has been investigat ed by measuring the displacement of I-125-Bolton Hunter-NPY. After inc ubation of the membranes with different concentrations of the crosslin ker and subsequent photolysis, the specific binding of I-125-Bolton Hu nter-NPY at those membranes was tested in order to quantify the crossl inking efficiency. Whereas [(Tmd)Phe(20)] NPY, [(Tmd)Phe(21)] NPY and [(Tmd)Phe(27)] NPY revealed highest affinity to the Y-2 receptor, cros slinking was most efficient when Tyr(36) was replaced by (Tmd)Phe. Thi s is in good agreement with the previously suggested C-terminal-bindin g site of neuropeptide Y.