PEGYLATED PEPTIDES .2. SOLID-PHASE SYNTHESIS OF AMINO-CHAIN, CARBOXY-CHAIN AND SIDE-CHAIN PEGYLATED PEPTIDES

General procedures are presented for the site-specific pegylation of p eptides at the NH2-terminus, side-chain positions (Lys or Asp/Glu) or COOH-terminus using solid-phase Fmoc/tBu methodologies. A model tridec apeptide fragment of interleukin-2, IL-2(44-56)-NH2, was chosen for th is study since it possesses several trifunctional amino acids which se rve as potential sites for pegylation. The pegylation reagents were de signed to contain either Nle or Orn, which served as diagnostic amino acids for confirming the presence of 1 PEG unit per mole of peptide. N H2-Terminal pegylation was carried out by coupling PEG-CH2CO-Nle-OH to the free NH2-terminus of the peptide-resin. Side-chain pegylation of Lys or Asp was achieved by one of two pathways. Direct side-chain pegy lation was accomplished by coupling with Fmoc-Lys(PEG-CH2CO-Nle)-OH or Fmoc-Asp(Nle-NH-CH2CH2-PEG)-OH, followed by sold-phase assemblage of the pegylated peptide-resin and TFA cleavage. Alternatively, allylic p rotective groups were introduced via Fmoc-Lys(Alloc)-OH or Fmoc-Asp(O- Allyl)-OH, and selectively removed by palladium-catalyzed deprotection after assemblage of the peptide-resin. Solid-phase pegylation of the side-chain of Lys or Asp was then carried out in the final stage with PEG-CH2CO-Nle-OH or H-Nle-NH-(CH2)(2)-PEG, respectively. COOH-Terminal pegylation was achieved through the initial attachment of Fmoc-Om(PEG -CH2CO)-OH to the solid support, followed by solid-phase peptide synth esis using the Fmoc/tBu strategy. The pegylated peptides were purified by dialysis and preparative HPLC and were fully characterized by anal ytical HPLC, amino acid analysis,H-1-NMR spectroscopy and laser desorp tion mass spectrometry. (C) Munksgaard 1994.