Self-Assembly-Assisted Kinetically Controlled Papain-Catalyzed Formation of mPEG-b-Phe(Leu)_x

Abstract

Self-assembling peptide materials are promising next-generation materials with applications that include tissue engineering scaffolds, drug delivery, bionanomedicine, and enviro-responsive materials. Despite these advances, synthetic methods to form peptides and peptide−polymer conjugates still largely rely on solid-phase peptide synthesis (SPPS) and N-carboxyanhydride ring-opening polymerization (NCA-ROP), while green methods remain largely undeveloped. This work demonstrates a protease-catalyzed peptide synthesis (PCPS) capable of directly grafting leucine ethyl ester (Leu-OEt) from the C-terminus of a methoxy poly(ethylene glycol)-phenylalanine ethyl ester macroinitiator in a one-pot, aqueous reaction. By using the natural tendency of the growing hydrophobic peptide segment to self-assemble, a large narrowing of the (Leu) x distributions for both mPEG 45 -b-Phe(Leu) x and oligo(Leu) x coproducts, relative to oligo(Leu) x synthesized in the absence of a macroinitiator (mPEG 45 -Phe-OEt), was achieved. A mechanism is described where in situ β-sheet coassembly of mPEG 45 -b-Phe(Leu) x and oligo(Leu) x coproducts during polymerization prevents peptide hydrolysis, providing a means to control the degree of polymerization (DP) and dispersity of diblock (Leu) x segments (matrix-assisted laser desorption time-of-flight (MALDI-TOF) x = 5.1, dispersity ≤ 1.02). The use of self-assembly to control the uniformity of peptides synthesized by PCPS paves the way for precise peptide block copolymer architectures with various polymer backbones and amino acid compositions synthesized by a green process.

Amphiphilic mPEG-Modified Oligo-Phenylalanine Nanoparticles Chemoenzymatically Synthesized via Papain

“…Papain is commercially available, inexpensive, and extracellularly stable, showing a high affinity for hydrophobic amino acids like l -Phe. The synthesis of polypeptides catalyzed by papain is under kinetic control. , The yield of the kinetically controlled reaction is in direct relationship with the enzyme/substrate ratio. , The effect of various concentrations of the monomer ( l -Phe-OMe·HCl) on the amount of OPhe NPs produced per 100 mg of l -Phe-OMe·HCl was investigated under the conditions of 4 U/mL papain, 15% (v/v) dimethyl sulfoxide (DMSO) in the buffer (pH 8.0, 200 mmol/L) at 40 °C for 3 h. The results are shown in Figure A. The yield reached the maximal value (36.9 ± 0.6 mg/100 mg l -Phe-OMe·HCl) at 200 mg/mL l -Phe-OMe·HCl.…”

“…The synthesis of polypeptides catalyzed by papain is under kinetic control. 6,27 Scheme 1. Preparation of mPEG-g-OPhe NPs and the Micellar Self-Aggregation of the NPs a a OPhe NPs, oligo-phenylalanine nanoparticles; mPEG-g-OPhe NPs, methoxypolyethylene glycol-modified oligo-phenylalanine NPs; and mPEG-SC, methoxypolyethylene glycol succinimidyl carbonate ester.…”

Monomer Choice Influences N-Acryloyl Amino Acid Grafter Conversion via Protease Catalysis

“…The method used to determine papain activity was adapted from a literature procedure. 34 In brief, to a 2 mL microcentrifuge tube (n = 3), 0.25 mL of a 2.5% (w/v) azocasein solution (50 mM PBS, pH 8) was added. An additional 0.25 mL of 50 mM PBS (pH 8) was added to the blank.…”

“…22−24 Homo-oligopeptides, 25 co-oligopeptides with random, 26,27 block, 28 and alternating sequences, 29,30 terminalmodified oligopeptides, 31,32 and peptide-polymer conjugates have been prepared using PCPS. 33,34 Given the published work thus far, PCPS is a promising method for scale-up in nonspecialized reactors to provide peptides at costs compatible with higher-volume specialty applications such as coating and specialty adhesives. 35 Modification of amino acids and peptide segments at the N terminus with acryloyl moieties is an efficient method to synthesize peptide-conjugates with unique properties via grafting-to or grafting-through strategies.…”

“…Kinetically controlled PCPS by cystine proteases (papain) is normally performed in aqueous buffer, using amino acid ethyl ester (AA-OEt) monomers, without protection–deprotection of the N-termini of the growing chain and amino acid (AA) functional R-groups (e.g., lysine, ε and α amine) . Generally, PCPS is conducted at a pH of about 8 and temperature 40 °C for seconds to hours with peptide yields between 30 and 80%. − Homo-oligopeptides, co-oligopeptides with random, , block, and alternating sequences, , terminal-modified oligopeptides, , and peptide-polymer conjugates have been prepared using PCPS. , Given the published work thus far, PCPS is a promising method for scale-up in non-specialized reactors to provide peptides at costs compatible with higher-volume specialty applications such as coating and specialty adhesives …”