Preparation of a photoreactive analog of parathyroid hormone [Nle8,Lys(N-ε-4-azido-2-nitrophenyl)13,Nle18,Tyr34]bovine parathyroid hormone-(1-34)NH2, a selective, high-affinity ligand for characterization of parathyroid hormone receptors

Photoaffinity radiolabeling techniques have been widely used to characterize the properties of peptide hormone receptors. However, the identity of authentic receptors is often uncertain because many macromolecules are labeled. These ambiguities are due, in part, to the use of a heterogeneous mixture of photoreactive photoligands, many of which have no or low affinity for the relevant hormone receptor. In this report, we describe the synthesis, purification, and structural analysis of the photoreactive parathyroid hormone analog, [Nle8,Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]-bovine parathyroid hormone-(1-34)NH2. The sulfur-free, oxidation-resistant, synthetic analog of bovine parathyroid hormone (PTH), [Nle8,Nle18,Tyr34]bovine PTH-(1-34)NH2 (NlePTH), was reacted with 4-fluoro-3-nitrophenylazide under nonaqueous conditions to yield several derivatives which were separated by reverse-phase high-performance liquid chromatography and analyzed by amino acid compositional analysis, thin-layer chromatography, and ultraviolet and visible absorption spectroscopy. Among the NlePTH derivatives generated, one of the least hydrophobic was shown to retain the highest potency as assessed in the canine renal cortical membrane radioreceptor assay. Sequence analysis of this peptide, after it had been derivatized with 4-fluoro-3-nitro-[2,6-3H]phenylazide and purified to homogeneity, permitted us to determine that the structure of this analog is [Nle8,Lys(N-epsilon-4-azide-2-nitrophenyl)13,Nle18,Tyr34]bovine PTH-(1-34)NH2. We emphasize the importance of using photoreactive ligands which are purified and subjected to detailed chemical and biological analyses for characterizing the properties of parathyroid hormone receptors and receptors for other peptide hormones.