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

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Abstract
The synthesis, purification, and structural analysis of the major compounds resulting from photoderivatization of [Tyr36]-parathyroid hormone related peptide (1-36)amide[[Tyr36]PTHrP(1-36)amide] are described. The reaction of the synthetic peptide with 4-fluoro-3-nitrophenyl azide under nonaqueous conditions yields three major products (peaks D-1, D2, 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-1 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-1 and G were derivatized on Lys13 and Lys11, respectively. Compound D-2 was N-blocked, indicating that this compound is derivatized on the .alpha.-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 .times. 10-9 M and 0.6 .times. 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(1-36)amide. Compound G had the same adenylate cyclase stimulating potency as the underivatized, synthetic [Tyr36]PTHrP(1-36)amide, whereas compound D-2 was only a partial agonist, having about 25% of the maximal cAMP production. Compound D-1, 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,N.epsilon.-(4-azido-2-nitrophenyl)Lys13,Nle18,Tyr34]bPTH(1-34)amide (NAP-NlePTH) and D-1 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 80,000-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(1-36)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.

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