PEGA supports for combinatorial peptide synthesis and solid-phase enzymatic library assays

Abstract

Permeable resins cross-linked with long PEG chains were synthesized for 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 synthesized by partial or di-acryloylation of bis-amino-PEG derivatives. Bis/mono-acrylamido-PEG were copolymerized along with acrylamide by inverse suspension copolymerization to yield a less cross-linked resin (Type I, compounds 6-9). Furthermore, acryloyl-sarcosin ethyl ester was co-polymerized along with bis-acrylamido PEG to obtain more crosslinked capacity resin (Type II, compounds 13-19). N,N-Dimethylacrylamide was 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 and dry conditions. The supports showed good mechanical properties and were characterized by studying the swelling properties, size distribution of beads, and by estimating the amino group capacity. Depending on the PEG chain length, the monomer composition and the degree of cross-linking the PEGA supports showed a high degree of swelling in a broad range of solvents, including water, dichloromethane, DMF, acetonitril, THF and toluene: no swelling was observed in diethyl ether. The PEGA 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 were synthesized on the new polymer using a multiple column library synthesizer 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 for MMP-9. After treatment with ethylene diamine, high-loaded resins (Type II) have been employed in continuous flow peptide synthesis to yield peptides in excellent yield and purity.

Making PEG-based Microparticles for Applications in Biology and Medicine

“…This is in line with the loadings observed in commercially available PEGA macrobeads [12]. The optical micrographs appear very similar to that of environmental scanning electron (ESEM), the only difference being that the diameters of the particles are much larger, between the range of about 5-25 µm.…”

Improved Fmoc‐based solid‐phase synthesis of homologous peptide fragments of human and mouse prion proteins

“…Although this hydrophilic copolymer resin was originally described by Meldal et al as a convenient support for post‐synthetic screening of peptides in aqueous environments—including for assays entailing large biomolecules (e.g. enzymes or other proteins)32–34—, we found that it also performs very well for synthesising peptide sequences prone to self‐associate on‐resin.…”

“…However, Cardona et al use polystyrene Wang resin combined with Dmb‐peptides and single 30‐min coupling reactions. In our case, we chose the PEGA resin, composed of a hydrophilic copolymer because it has better swelling properties—including in aqueous solvents34—compared to the Wang resin, which is polystyrene based. In addition, it also avoids protected peptide–resin interaction because of its polar nature12, 38; features a low to medium level of functionalisation; is also amenable to automated peptide synthesis; is highly flexible, owing to its internal structure32, 33; and, lastly, is compatible with biological assays32, 39, 40.…”

Synthesis of Substituted γ‐ and δ‐Lactams through Mannich‐Type Reactions of Solid‐Supported N‐Acyliminium Ions

“…With masked aldehyde building blocks 2a and 2b in hand, cyclization precursors 4a and 4b were assembled on the 4-(hydroxymethyl)benzoic acid (HMBA) modified PEGA 800 (polyethylene glycol dimethyl acrylamide) resin [27] using standard Fmoc (fluorenylmethoxycarbonyl) based solid-phase protocols and couplings mediated by TBTU {N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium tetrafluoroborate N-oxide} amide (Scheme 3). The attachment of the first glycine residue was carried out by esterification mediated by MSNT [1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole].…”