PEGA SUPPORTS FOR COMBINATORIAL PEPTIDE-SYNTHESIS AND SOLID-PHASE ENZYMATIC LIBRARY ASSAYS

Permeable resins cross-linked with long PEG chains were synthesized fo r use in solid-phase enzyme library assays. High molecular weight bis- amino-polyethylene glycol (PEG) 4000, 6000, 8000 were synthesized by a three-step reaction starting from PEG-bis-OH. Macromonomers were synt hesized by partial or di-acryloylation of bis-amino-PEG derivatives. B is/mono-acrylamido-PEG were copolymerized along with acrylamide by inv erse suspension copolymerization to yield a less cross-linked resin (T ype I, compounds 6-9). Furthermore, acryloyl-sarcosin ethyl ester was co-polymerized along with bis-acrylamido PEG to obtain more crosslinke d capacity resin (Type II, compounds 13-19). N,N-Dimethylacrylamide wa s used as a co-monomer in some cases. The polymer was usually obtained in a well-defined beaded form and was easy to handle under both wet a nd dry conditions. The supports shelved good mechanical properties and were characterized by studying the swelling properties, size distribu tion of beads, and by estimating the amino group capacity. Depending o n the PEG chain length, the monomer composition and the degree of cros s-linking the PEGA supports showed a high degree of swelling in a broa d range of solvents, including water, dichloromethane, DMF, acetonitri l, THF and toluene; no swelling was observed in diethyl ether. The PEG A resins (Type I) with an amino acid group capacity between 0.07 and 1 .0 mmol/g could be obtained by variation of the monomer composition in the polymerization mixture. Fluorescent quenched peptide libraries we re synthesized on the new polymer using a multiple column library synt hesizer and incubated with the matrix metalloproteinase MMP-9 after it had been activated by 4-aminophenyl mercuric acetate resulting in 67/ 83 kDa active enzyme. The bright beads were separated manually under a fluorescence microscope and sequenced to obtain peptide substrates fo r MMP-9. After treatment with ethylene diamine, high-loaded resins (Ty pe II) have been employed in continuous flow peptide synthesis to yiel d peptides in excellent yield and purity. (C) 1998 European Peptide So ciety and John Wiley & Sons, Ltd.