Synthesis of fully active biotinylated analogues 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 alpha-Boc-Lys(N epsilon-Fmoc) at a selected position in the sequence, followed by selective side-chain deprotection and biotinylation of the epsilon-amino group, permitted modification of the specific lysine only. Biotinylated analogues of [Nle8,18,Tyr34]bPTH(1-34)NH2 (analogue 1a) were prepared by modification of Lys13 with a biotinyl group (analogue 1) or a biotinyl-epsilon-aminohexanoyl group (analogue 2) or at Lys26 with a biotinyl-epsilon-aminohexanoyl group (analogue 3). A biotinylated PTHrP antagonist [Leu11,D-Trp12,Lys13(N epsilon-(biotinyl-beta-Ala))]PTHrP(7-34)NH2 (analogue 5), was also prepared. In a different synthetic approach, selective modification of the thiol group of [Cys35]PTHrP(1-35)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 (Kb = 1.5 nM, Km = 0.35 nM) and in porcine renal membranes (Kb = 0.70 nM) identical or similar to those of its parent peptide, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Roubini
E
E
Merck, Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Duong
L T
LT
Gibbons
S W
SW
Leu
C T
CT
Caulfield
M P
MP
Chorev
M
M
Rosenblatt
M
M
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin; D002462:Cell Membrane / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007672:Kidney Cortex / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D012516:Osteosarcoma / Q000378:metabolism; D010281:Parathyroid Hormone / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018016:Receptors, Parathyroid Hormone; D013552:Swine; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D007457:Iodine Radioisotopes; C470121:PTHLH protein, human; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; D001710:Biotin; D000242:Cyclic AMP; D008239:Lysine