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.

Microfluidic Generation of Amino-Functionalized Hydrogel Microbeads Capable of On-Bead Bioassay

“…We found that the non-functionalized PEG microbeads were more compatible with various solvents due to the increase in amphiphilicity [22]. Compared with commercially available PEG-based microbeads, such as PEGA resin [23,24], TentaGel XV resin [25,26], and ChemMatrix resin [27,28], PEG-amine hydrogel microbeads did not swell relatively with various solvents because of the high degree of crosslink [4,5]. However, our PEG-amine microbeads did not need a high swelling ratio because most of the active functional groups were exposed at the shell region [22,29].…”

Correlated matrix‐assisted laser desorption/ionization mass spectrometry and fluorescent imaging of photocleavable peptide‐coded random bead‐arrays

“…Here we demonstrated the feasibility of using glass, agarose and TentaGel® beads for Bead-GPS, although other bead types that are commonly used for libraries may also be compatible (e.g. PEGA 30 acrylamide-PEG co-polymer beads). While further study is needed comparing these different bead types, several factors appear to affect the ability to obtain strong MALDI-MSI signals, well-defined spot morphology and to measure functional interaction of prey biomolecules (e.g.…”

Total Chemical Synthesis, Folding, and Assay of a Small Protein on a Water‐Compatible Solid Support

“…The highly permeable nature of the SPOCC resin allows enzymes to diffuse into the polymer matrix and perform chemical reactions 10. 18 It was possible to inhibit the enzymatic activity of trypsin by using EETI‐II that had been synthesized and folded on the SPOCC resin, thus demonstrating the ability of trypsin to diffuse into the resin. The enzyme and inhibitor can also be recovered from the resin after TFA cleavage.…”