Abstract
Five photoactive analogues of porcine neuropeptide Y (NPY), a 36 amino acid hormone of the pancreatic polypeptide family, have been synthesized by solid phase peptide synthesis method, Fmoc/tBu strategy and carefully characterized. The analogues contain the photoactivatable amino acid 4-(3-trifluoromethyl)-3H-diazirine-3-yl-phenyl-alanine ((Tmd)Phe) individually at different positions (1, 20, 21, 27 or 36) instead of tyrosine in the wildtype sequence. Affinity to membranes prepared from SMS-KAN cells, which stably express the Y2 receptor has been investigated by measuring the displacement of 125I-Bolton Hunter-NPY. After incubation of the membranes with different concentrations of the crosslinker and subsequent photolysis, the specific binding of 125I-Bolton Hunter-NPY at those membranes was tested in order to quantify the crosslinking efficiency. Whereas [(Tmd)Phe20] NPY, [(Tmd)Phe21] NPY and [(Tmd)Phe27] NPY revealed highest affinity to the Y2 receptor, crosslinking was most efficient when Tyr36 was replaced by (Tmd)Phe. This is in good agreement with the previously suggested C-terminal binding site of neuropeptide Y.