Synthesis, purification and biological activity of (Ser10-phosphatidyl)-urodilatin (phosphourodilatin).
Based on the global phosphorylation approach, a selective synthesis of (Ser10-phosphatidyl)-urodilatin (phosphourodilatin), which contains 32 amino acid residues and a disulfide loop is described. The peptide was assembled stepwise on a polyethyleneglycol-polystyrene support using Fmoc-chemistry. The phosphorylation was performed on-resin by phosphitylation with a large excess of di-tert-butyl-N,N-diethylphosphoramidite within 1 hour, followed by oxidation with tert-butylhydroperoxide to the protected phosphopeptide. After cleavage and deprotection the disulfide bridge was introduced without side reactions by iodine titration of the mono-acetamidomethyl protected crude peptide. During the synthetic pathway, the acylation with side chain-unprotected Fmoc-serine and the phosphitylation satisfactorily yielded the expected intermediates. In some phosphorylation experiments a by-product having a reduced mass corresponding to the H-phosphonate was observed. Illustrated with the synthesis of phosphourodilatin, this type of by-product, which could not be separated by HPLC, and the difficult amino acid sequence make the synthesis of a large phosphopeptide a more delicate task than the synthesis of short phosphopeptides, which do not contain oxidation-sensitive amino acids, difficult sequences or additional structural elements such as disulfide loops. The biological activity of phosphourodilatin was compared with non-phosphorylated urodilatin in two assay systems. Both peptides revealed a vasorelaxant effect on aortic smooth muscle strips and induced a cGMP-generation in RFL-6 cells with increasing dose dependency.
Mostafavi
H
H
Lower Saxony Institute for Peptide Research, Hannover, Germany.
Adermann
K
K
Austermann
S
S
Raida
M
M
Meyer
M
M
Forssmann
W G
WG
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000187:drug effects; D009320:Atrial Natriuretic Factor / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006152:Cyclic GMP / Q000096:biosynthesis; D004220:Disulfides / Q000737:chemistry; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000494:pharmacology; D010766:Phosphorylation; D011817:Rabbits; D012694:Serine / Q000737:chemistry; D014664:Vasodilation / Q000187:drug effects
D004220:Disulfides; D010446:Peptide Fragments; C103122:phospho-urodilatin; D012694:Serine; C056452:Ularitide; D009320:Atrial Natriuretic Factor; D006152:Cyclic GMP