Selective Protein Conjugation of Poly(glycerol monomethacrylate) and Poly(polyethylene glycol methacrylate) with Tunable Topology via Reductive Amination with Multifunctional ATRP Initiators for Activity Preservation

Abstract

In this study, we compare poly­(glycerol monomethacrylate) (PGMA) of different chain lengths and architectures (linear and two-arm) with poly­(poly­(ethylene glycol) methyl ether methacrylate) (PPEGMA) as an alternative polymer platform for the synthesis of a new generation of protein–polymer conjugates. Mono- and two-arm functional atom-transfer radical polymerization (ATRP) initiators were designed and selectively attached to lysozyme at the N-terminus via reductive amination. Site-specific, grafting from activator regenerated by electron transfer (ARGET) ATRP was carried out in phosphate buffer, and the reaction parameters were optimized to obtain polymer conjugates with predetermined molar mass and topology. The activity preservation under proteolytic and high-temperature conditions showed a clear dependence on the structure of the repeating unit and on the macromolecular architecture. These results highlighted the potential of PGMA as a poly­(ethylene glycol) (PEG) alternative for the half-life extension of biotherapeutics. Moreover, this synthetic approach may inspire the design of a new class of protein–polymer conjugates through an optimal combination of macromolecular composition and topology.

Enzyme‐Polymer Conjugates for Tuning, Enhancing, and Expanding Biocatalytic Activity

“…It was demonstrated that the growth of two polyglycerol methacrylate chains from the same protein anchoring‐site derives to a more compact macromolecular domain that provides less steric hinderance to the protein substrate entrance channel. These systems guaranteed protection against protease degradation, high stability in serum, and higher thermostability at 90 °C compared to native lysozyme [46] . Moreover, polymers with ramified structures, that is, dendronized, branched or comb‐shaped polymers, interact with the exposed residues of the proteins giving rise to a discontinuous layer that partially covers the surface of the protein.…”

“…These systems guaranteed protection against protease degradation, high stability in serum, and higher thermostability at 90 °C compared to native lysozyme. [46] Moreover, polymers with ramified structures, that is, dendronized, branched or comb-shaped polymers, interact with the exposed residues of the proteins giving rise to a discontinuous layer that partially covers the surface of the protein. Therefore, the size of these gaps between polymer chains can be adjusted by engineering the size and the chemistry of the polymers.…”

Tailored Branched Polymer–Protein Bioconjugates for Tunable Sieving Performance

“…To enhance the biological activity of poly­(glycerol)–protein conjugates, an optimal combination of composition, molecular weight, and polymer architecture was crucial. , Densely grafted polymers have been studied, including the implantation of proteins in molecular bottlebrushes, the use of click chemistry to conjugate globular dendrimers, − and grafting PEG macromonomers via ATRP to mask proteins . Hyperbranched polymers possess unique properties such as weak entanglement, low viscosity, and a globular conformation, making them excellent candidates for grafting from the surface of proteins .…”

Controlled Polymer Synthesis Toward Green Chemistry: Deep Insights into Atom Transfer Radical Polymerization in Biobased Substitutes for Polar Aprotic Solvents

“…Water stands as the most benign solvent in the ATRP of hydrophilic monomers. − Although polymerization in an aqueous environment is environmentally friendly, it has limitations due to certain side reactions. These include disproportionation of Cu I activator complexes, the dissociation of the halide from higher oxidation state complexes leading to the formation of inactive species that cannot undergo deactivation, and the hydrolysis of the initiator, causing the creation of dead chains. − These occurrences suggest a loss of control over the molecular weight of the final polymers.…”