PREPARATION AND CHARACTERIZATION OF [N-ALPHA-(4-AZIDO-2-NITROPHENYL)ALA1,TYR36]-PARATHYROID HORMONE RELATED PEPTIDE (1-36)AMIDE - A HIGH-AFFINITY, PARTIAL AGONIST HAVING HIGH CROSS-LINKING EFFICIENCY WITH ITS RECEPTOR ON ROS 17/2.8 CELLS

The synthesis, purification, and structural analysis of the major compounds resulting from photoderivatization of [Tyr36]-parathyroid hormone related peptide (l-36)amide [[Tyr36]PTHrP(l-36)amide] are described. The reaction of the synthetic peptide with 4-fluoro-3-nitrophenyl azide under nonaqueous conditions yields three major products (peaks D-l, D-2, and G), which were purified to homogeneity by reverse-phase high-performance liquid chromatography. Subsequent amino acid analysis showed that the peptides of peaks D-l and G each lack one lysine residue, while the peptide in peak D-2 lacks one alanine residue, suggesting that these residues are chemically modified by photoderivatization. Sequence analysis of the photoderivatized peptides revealed that compounds D-l and G were derivatized on Lys13 and Lys11, respectively. Compound D-2 was N-blocked, indicating that this compound is derivatized on the α-amino function of Ala1. Both Lys residues of D-2 were quantitatively recovered upon sequencing after digestion with endoproteinase Glu-C. Compounds D-2 and G had apparent Kds of 1 × 10−9 M and 0.6 × 10−9 M, respectively, for their receptors on ROS 17/2.8 cells, which are identical with or similar to that of the underivatized [Tyr36]PTHrP(l-36)amide. Compound G had the same adenylate cyclase stimulating potency as the underivatized, synthetic [Tyr36]PTHrP(l-36)amide, whereas compound D-2 was only a partial agonist, having about 25% of the maximal cAMP production. Compound D-l, which is modified on Lys13, retained only 2-4% of its receptor binding affinity and biological activity relative to that of its parent compound. After radioiodination and incubation with ROS 17/2.8 cells in the dark, D-2 and G bound equivalently to the receptor protein (specific binding 18.0 ± 3.6% and 16.2 ± 1.3%, respectively), whereas [Nle8,Nϵ-(4-azido-2-nitrophenyl)Lys13,Nle18,Tyr34]bPTH(l-34)amide (NAP-NlePTH) and D-l bound less well (specific binding 5.6 ± 2.9% and 3.7 ± 2.4%, respectively), as was predicted from their dissociation constants. After photolysis and analysis by SDS-PAGE and autoradiography, labeling of the 80000-dalton receptor protein was approximately 5-10 times more efficient with D-2 than with G. This improved cross-linking efficiency of Ala1-photoderivatized [Tyr36]PTHrP(l-36)amide presumably reflects a steric advantage for this derivative, such as a closer proximity of the azide function at that position to the receptor protein, and suggests that this compound potentially is useful for receptor identification and isolation. © 1990, American Chemical Society. All rights reserved.