Hydrolytically Stable Maleimide-End-Functionalized Polymers for Site-Specific Protein Conjugation

Abstract

Site-specific conjugation to cysteines of proteins often uses ester groups to link maleimide or alkene groups to polymers. However, the ester group is susceptible to hydrolysis, potentially losing the benefits gained through bioconjugation. Here, we present a simple conjugation strategy that utilizes the amide bond stability of traditional 1-ethyl-3-(3-dimethylaminopropyl)­carbodiimide/N-hydroxysuccinimide coupling while introducing site specificity. Hydrolytically stable maleimide-end-functionalized polymers for site-specific conjugation to free cysteines of proteins were synthesized using reversible addition–fragmentation chain-transfer (RAFT) polymerization. The alpha terminus of the polymers was amidated with a furan-protected aminoethyl maleimide using carbodiimide-based chemistry. Finally, the maleimide was exposed by a retro Diels–Alder reaction to yield the maleimide group, allowing for thiol-maleimide click chemistry for bioconjugation. A thermophilic cellulase from Fervidobacterium nodosum (FnCel5a) was conjugated using various strategies, including random 1-ethyl-3-(3-dimethylaminopropyl)­carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling, site-specific hydroxyethyl maleimide (HEMI) end-functionalized coupling, hydroxyethyl acrylate (HEA) end-functionalized coupling, and amidoethyl maleimide (AEMI) end-functionalized coupling. Only the polymers conjugated by EDC and AEMI remained conjugated a week after attachment. This indicates that hydrolytically stable amide-based maleimides are an important bioconjugation strategy for conjugates that require long-term stability, while esters are better suited for systems that require debonding of polymers over time.

Investigating the Impact of Polymer Length, Attachment Site, and Charge on Enzymatic Activity and Stability of Cellulase

“…The impact of polymer attachment site on cellulase activity and stability was investigated through site-specific thiol-Michael click chemistry of a library of polymers to wild-type and mutated Fn Cel5a. As shown in a previous study, wild-type Fn Cel5a has one reactive cysteine residue on the surface of the enzyme at position 319 . In addition to this, four lysine residues at various positions around the enzyme were individually mutated to cysteine residues to create K94C, K159C, K190C, and K300C.…”

“…38,39 Various approaches also exist for attaching the polymer to the protein, with the formation of amides by attachment to lysine residues or thiol-Michael adducts by attachment to cysteine residues in the protein being arguably the most common method. 40,41 In a previous study, we found that the conjugation of well-defined functional polymers to the lysine groups of FnCel5a results in enhanced activity without sacrificing stability due to electrostatic noncovalent interactions between the attached polymer and substrate. 42,43 Here, we investigate the impact of polymer length, charge, location site, and conjugation strategy on FnCel5a enzymatic activity and stability.…”

“…As shown in a previous study, wild-type FnCel5a has one reactive cysteine residue on the surface of the enzyme at position 319. 40 In addition to this, four lysine residues at various positions around the enzyme were individually mutated to cysteine residues to create K94C, K159C, K190C, and K300C. However, the mutation at K159C, relatively close to the enzymatic active site, resulted in instability of the protein and inability for this mutant to be further used in this study.…”

“…These include grafting-to, where a polymer is synthetized and attached to the protein after polymerization, or grafting-from, where the protein is modified with an initiating site or chain-transfer agent, with polymerization occurring off the protein surface. , Historically, the lack of polymerization methods compatible with aqueous media made grafting-to the main approach. However, developments in reversible addition-fragmentation chain-transfer polymerization (RAFT) and atom-transfer radical polymerization have made grafting-from far more accessible. , Various approaches also exist for attaching the polymer to the protein, with the formation of amides by attachment to lysine residues or thiol-Michael adducts by attachment to cysteine residues in the protein being arguably the most common method. , In a previous study, we found that the conjugation of well-defined functional polymers to the lysine groups of Fn Cel5a results in enhanced activity without sacrificing stability due to electrostatic noncovalent interactions between the attached polymer and substrate. , Here, we investigate the impact of polymer length, charge, location site, and conjugation strategy on Fn Cel5a enzymatic activity and stability. Fn Cel5a is a thermophilic 36 kDa cellulase from Fervidobacterium nodosum which exhibits activity against carboxymethyl cellulose (CMC), galactomannan, regenerated amorphous cellulose, and barley β- D -glucan. , The aim of this study is to understand the impact of polymer length, attachment site, and charge on enzymatic activity and stability, ultimately helping future researchers to more rationally design protein–polymer bioconjugates with retention of enzymatic activity and improved functional stability.…”

Reproducible and controlled peptide functionalization of polymeric nanoparticles

“…In a typical carbodiimide reaction, the coupling reagent (i.e., EDC, DCC, DIC, etc.) is added in excess to ensure high conjugation efficiency (Pan et al, 2012;Biju, 2014;Wang et al, 2014;Gandhi et al, 2016;Zhang et al, 2016;Kuo et al, 2017;Palmieri et al, 2018;Shipunova et al, 2018;Wang et al, 2018;Elzahhar et al, 2019;Lim et al, 2019;Shiu et al, 2021;Wright et al, 2021), which leads to a highly activated carboxylate environment in the reaction medium. All primary amines within the peptide can participate in nucleophilic addition-elimination regardless of relative reactivities in such environments.…”