PEGYLATED PEPTIDES .3. SOLID-PHASE SYNTHESIS WITH PEGYLATING REAGENTSOF VARYING MOLECULAR-WEIGHT - SYNTHESIS OF MULTIPLY PEGYLATED PEPTIDES

N(alpha)-Terminal solid-phase pegylation studies were carried out by c oupling PEG(n)-CH2-COOH [PEG = monomethoxypoly(ethylene glycol); n = 7 50, 2000, 5000, 100001 to either N-terminally hindered (Ile) or unhind ered (Gly) model peptide-resin to study the effect of the molecular we ight of the pegylating reagent on the efficiency of coupling. The coup ling proceeded to completion (quantitative ninhydrin determination) wi th hindered peptide-resin within 8 h using PEG750-CH2-COOH and proceed ed almost as rapidly using PEG2000-CH2-COOH. However, acylations with PEG5000-CH2-COOH and PEG10000-CH2-COOH were much slower and did not pr oceed to completion even after 72 h. N(alpha)-Pegylation of unhindered peptide-resin proceeded to completion more rapidly (within 4 h) and w as successfully carried out with higher-molecular-weight functionalize d poly(ethylene glycol), PEG750-CH2-COOH, PEG2000-CH2-COOH and PEG5000 -CH2-COOH. However, pegylation of unhindered peptide-resin did not pro ceed to completion with PEG10000-CH2-COOH even after 72 h. The feasibi lity of multiply pegylating peptides by the 9-fluorenylmethyloxycarbon yl/tert.-butyl (Fmoc/tBu) solid-phase procedure was also examined. Dip egylation, in which PEG2000 was inserted at the N-terminal and C-termi nal positions, or at the side-chain and C-terminal positions, were suc cessfully achieved by this method. Two model dipegylated peptides, Asn -Tyr-Lys-Asn-Pro-Lys-Leu-Orn(PEG2000-CH2CO)-NH2 and CH2CO-Nle)-Asn-Pro -Lys-Leu-Orn(PEG2000-CH2CO)-NH2, were synthesized by the Fmoc/tBu soli d-phase procedure. The model peptides, fragments of interleukin-2 [IL- 2(47-56)-NH2 and IL-2(44-56)-NH2], were chosen since they possess seve ral trifunctional amino acids and offer various sites for multiple peg ylation. The synthesis of the dipegylated peptides was achieved throug h the initial attachment of Fmoc-Orn(PEG-CH2CO)-OH to the solid suppor t, followed by Fmoc/tBu solid-phase peptide synthesis. N-Terminal pegy lation was carried out by coupling PEG-CH2CO-Nle-OH to the pegylated u ndecapeptide-resin. The side-chain pegylation of Lys-52 was achieved b y coupling Fmoc-Lys(PEG-CH2CO-Nle)-OH to the COOH-pegylated pentapepti de-resin, followed by Fmoc/tBu solid-phase assemblage of the dipegylat ed peptide-resin. Following cleavage by trifluoroacetic acid (TFA) and purification by reversed-phase high-performance liquid chromatography (HPLC), the dipegylated peptides were fully characterized by amino ac id analysis, analytical HPLC, H-1 nuclear magnetic resonance (NMR) and laser desorption ionization mass spectrometry (MS). Attempts to synth esize the corresponding dipegylated peptides using functionalized PEG5 000 were unsuccessful due to a combination of steric hindrance and the high-molecular-weight PEG that was employed. In addition, attempts to carry out a second pegylation in which the N-terminus residue is ster ically hindered (e.g. Ile) failed to couple using functionalized PEG20 00. These studies demonstrate that solid-phase pegylation proceeds mor e efficiently with functionalized polyethylene glycol) of lower molecu lar weight and that coupling is less efficient to sterically hindered residues.