SYNTHESIS OF PHOSPHO-URODILATIN BY COMBINATION OF GLOBAL PHOSPHORYLATION WITH THE SEGMENT COUPLING APPROACH

The chemical synthesis of biologically active phosphorylated urodilati n (CDD/ANP-95-126) was achieved by using a strategy of coupling protec ted peptide segments in solution. Three protected peptide segments cor responding to urodilatin (1-14) with side chain-unprotected Ser(10), ( 15-24) and (25-32) were prepared manually using Fmoc chemistry on an a minopropyl polystyrene resin with the super acid-labile HMPB linker. F or the coupling of segments, the carboxy group of the C-terminal segme nt (25-32) was converted into the tert-butyl ester by treatment with T BTA. The protected peptide segments were coupled in the presence of ED C/HOOBt or TBTU/HOBt to yield fully protected urodilatin with a free h ydroxy function at Ser(10). Introduction of the phosphate was performe d with Et(2)NP(OtBu)(2) and tetrazole followed by oxidation of the pho sphite. Alternatively, a prephosphorylated protected segment (1-14) wa s used in the segment condensation. Our investigations indicate that b oth pathways, phosphorylation of protected urodilatin. in solution and use of a prephosphorylated building block, are suitable methods to ob tain a large phosphopeptide of high purity without formation of H-phos phonates or other by-products. (C) Munksgaard 1996.