Synthesis of Fully Active Biotinylated Analogs of Parathyroid Hormone and Parathyroid Hormone-Related Protein as Tools for the Characterization of Parathyroid Hormone Receptors

The synthesis, purification, and characterization of biotinylated analogues of parathyroid hormone (PTH) and PTH-related protein (PTHrP) are described. A novel methodology was developed which allowed the selective biotinylation during solid-phase synthesis of either the Lys¹³ or Lys²⁶ residue in PTH/PTHrP sequences. Incorporation of orthogonally protected N^α-Boc-Lys(N^e-Fmoc) at a selected position in the sequence, followed by selective side-chain deprotection and biotinylation of the €-amino group, permitted modification of the specific lysine only. Biotinylated analogues of [Nle^{8, 18}, Tyr³⁴]bPTH(l-34)NH₂ (analogue la) were prepared by modification of Lys¹³ with a biotinyl group (analogue 1) or a biotinyl-€-aminohexanoyl group (analogue 2) or at Lys²⁶ with a biotinyl-€-aminohexanoyl group (analogue 3). A biotinylated PTHrP antagonist [Leu¹¹, D-Trp¹², Lys¹³(N^€-(biotinyl-β-Ala))]PTHrP(7-34)NH₂ (analogue 5), was also prepared. In a different synthetic approach, selective modification of the thiol group of [Cys³⁵]PTHrP(l-35)NH₂, in solution, with N-biotinyl-N'-(6-maleimidohexanoyl)hydrazide, resulted in analogue 4. The high affinities of the biotinylated analogues for PTH receptors present in human osteosarcoma B-10 cells or in porcine renal cortical membranes (PRCM), were comparable to those of the underivatized parent peptides. The analogues were also highly potent in stimulation of cAMP formation (analogues 1-4) or inhibition of PTH-stimulated adenylyl cyclase (analogue 5) in B-10 cells. The most potent analogue (analogue 1) had potencies in B-10 cells (K_b = 1.5 nM, K_m = 0.35 nM) and in porcine renal membranes (K_b = 0.70 nM) identical or similar to those of its parent peptide, respectively. Furthermore, these high binding affinities were retained in the presence of either avidin, streptavidin, or anti-biotin (1 μM). Radioiodination of PTH analogues (analogues 1-2) generated highly potent radioligands which bind to a single class (B_max= 390-450 fmol/mg of protein) of high affinity (K_d = 0.24-0.61 nM) PTH receptors on renal membranes and which also bind to immobilized streptavidin or anti-biotin. Cross-linking of biotinylated radioligands (analogues 1-3) and analysis by SDS-PAGE and autoradiography showed specific labeling of renal PTH receptors (M_r = 75000). The biotinylated analogues prepared in this study, and their radiolabeled derivatives, serve as useful tools for the identification and isolation of PTH receptors.