Photoaffinity, biotinyl, and iodo analogues as probes for vasotocin receptors

Vasotocin (AVT) analogues with tyrosine or phenylalanine in position 9, i.e., [9-tyrosine]AVT, [2-phenylalanine,-9-tyrosine]AVT, and 1-desamino[9-(p-aminophenylalanine)]AVT, were synthesized by the solid-phase method. These compounds showed a high biological activity in the hydroosmotic toad bladder assay. Using the chemically reactive functional groups on tyrosine and p-aminophenylalanine in position 9, we prepared iodinated, photoreactive, and affinity ligands, i.e., [2-phenylalanine,9-(iodotyrosine)]AVT, 1-desamino[9-(p-azidophenylalanine)]AVT, and 1-desamino[9-(biotinylphenylalanine)]AVT, respectively. Half-maximal hydroosmotic responses (ED-50 values) were obtained with 2.5 X 10(-9) M for the iodinated analogue, with 0.9 X 10(-10) M for the photoaffinity analogue, and with 1.2 X 10(-8) M for the biotinyl analogue. The hydroosmotic activity of the biotinyl analogue was reversed following addition of avidin, whereas the photoaffinity analogue induced a persistent response following UV irradiation that was not reversed upon repeated and prolonged periods of washout. These analogues of vasotocin are the most potent that have been synthesized to date, and they should serve as useful probes in the isolation and characterization of vasotocin receptors in toad bladders and tissues from other species that use vasotocin as an antidiuretic/pressor principle. The photoaffinity and biotinyl analogues had a rat antidiuretic activity of 66 and 40 units/mg, respectively. These compounds are, therefore, also suitable for the isolation of V-2 vasopressin receptors from mammalian tissues.