Pegylated peptides III. Solid-phase synthesis with pegylating reagents of varying molecular weight: synthesis of multiply pegylated peptides

Abstract

N α-Terminal solid-phase pegylation studies were carried out by coupling PEG nCH 2COOH [PEG = monomethoxypoly(ethyleneglycol); n = 750, 2000, 5000, 10000] to either N-terminally hindered (Ile) or unhindered (Gly) model peptide-resin to study the effect of the molecular weight of the pegylating reagent on the efficiency of coupling. The coupling proceeded to completion (quantitative ninhydrin determination) with hindered peptide-resin within 8 h using PEG 750CH 2COOH and proceeded almost as rapidly using PEG 2000CH 2COOH. However, acylations with PEG 5000CH 2COOH and PEG 10000CH 2COOH were much slower and did not proceed to completion even after 72 h. N α-Pegylation of unhindered peptide-resin proceeded to completion more rapidly (within 4 h) and was successfully carried out with higher-molecular-weight functionalized poly(ethylene glycol), PEG 750CH 2COOH, PEG 2000CH 2COOH and PEG 5000CH 2COOH. However, pegylation of unhindered peptideresin did not proceed to completion with PEG 10000CH 2COOH even after 72 h. The feasibility of multiply pegylating peptides by the 9-fluorenylmethyloxycarbonyl/ tert.-butyl (Fmoc/tBu) solid-phase procedure was also examined. Dipegylation, in which PEG 2000 was inserted at the N-terminal and C-terminal positions, or at the side-chain and C-terminal positions, were successfully achieved by this method. Two model dipegylated peptides, PEG 2000CH 2CONleGlyIleAsnAsnTyrLysAsnProLys LeuOrn(PEG 2000CH 2CO)NH 2 and HIleLeuAsnGlyIleAsnAsnTyrLys(PEG 2000CH 2CONle) AsnProLysLeuOrn(PEG 2000CH 2CO)NH 2, were synthesized by the Fmoc/tBu solid-phase procedure. The model peptides, fragments of interleukin-2 [IL-2(47–56)-NH 2 and IL-2(44–56)-NH 2], were chosen since they possess several trifunctional amino acids and offer various sites for multiple pegylation. The synthesis of the dipegylated peptides was achieved through the initial attachment of FmocOrn(PEGCH 2CO)OH to the solid support, followed by Fmoc/tBu solid-phase peptide synthesis. N-Terminal pegylation was carried out by coupling PEGCH 2CONleOH to the pegylated undecapeptide-resin. The side-chain pegylation of Lys-52 was achieved by coupling FmocLys(PEGCH 2CONle)OH to the COOH-pegylated pentapeptide-resin, followed by Fmoc/tBu solid-phase assemblage of the dipegylated 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 acid analysis, analytical HPLC, 1H nuclear magnetic resonance (NMR) and laser desorption ionization mass spectrometry (MS). Attempts to synthesize the corresponding dipegylated peptides using functionalized PEG 5000 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 sterically hindered ( e.g. Ile) failed to couple using functionalized PEG 2000. These studies demonstrate that solid-phase pegylation proceeds more efficiently with functionalized poly(ethylene glycol) of lower molecular weight and that coupling is less efficient to sterically hindered residues.