Squaric Acid Mediated Synthesis and Biological Activity of a Library of Linear and Hyperbranched Poly(Glycerol)–Protein Conjugates

Abstract

Polymer-protein conjugates generated from side chain functional synthetic polymers are attractive because they can be easily further modified with, for example, labeling groups or targeting ligands. The residue specific modification of proteins with side chain functional synthetic polymers using the traditional coupling strategies may be compromised due to the nonorthogonality of the side-chain and chain-end functional groups of the synthetic polymer, which may lead to side reactions. This study explores the feasibility of the squaric acid diethyl ester mediated coupling as an amine selective, hydroxyl tolerant, and hydrolysis insensitive route for the preparation of side-chain functional, hydroxyl-containing, polymer-protein conjugates. The hydroxyl side chain functional polymers selected for this study are a library of amine end-functional, linear, midfunctional, hyperbranched, and linear-block-hyperbranched polyglycerol (PG) copolymers. These synthetic polymers have been used to prepare a diverse library of BSA and lysozyme polymer conjugates. In addition to exploring the scope and limitations of the squaric acid diethylester-mediated coupling strategy, the use of the library of polyglycerol copolymers also allows to systematically study the influence of molecular weight and architecture of the synthetic polymer on the biological activity of the protein. Comparison of the activity of PG-lysozyme conjugates generated from relatively low molecular weight PG copolymers did not reveal any obvious structure-activity relationships. Evaluation of the activity of conjugates composed of PG copolymers with molecular weights of 10000 or 20000 g/mol, however, indicated significantly higher activities of conjugates prepared from midfunctional synthetic polymers as compared to linear polymers of similar molecular weight.

Protein–polymer conjugation — moving beyond PEGylation

“…A unique benefit of PG is that it carries multiple side-chain hydroxyls, which readily enables further functionalization or architectural variation [25]. A variety of architectures have been demonstrated with PG, including linear, hyperbranched [24], midfunctional, linear-block-hyperbranched [26] and side-chain functional [27]. Klok and coworkers conjugated PG with various architectures to lysozyme through squaric acid diethyl ester mediated coupling to reactive amine groups in the lysine side chains of the protein.…”

“…Klok and coworkers conjugated PG with various architectures to lysozyme through squaric acid diethyl ester mediated coupling to reactive amine groups in the lysine side chains of the protein. A conjugate with midfunctional PG demonstrated significantly higher activity than its linear counterpart at a similar MW [26]. Harth and coworkers were the first to demonstrate in situ grafting of branched PG from Bovine Serum Albumin (BSA) using Ring Opening Polymerization (ROP) with the ability to control the degree of branching [28].…”

Molecular Sieving on the Surface of a Protein Provides Protection Without Loss of Activity

“…[ 15 , 18 ] Unfortunately, little systematic modulation of the structure of these polymers has been examined. In a recent study, Wurm et al [ 18 ] have observed only a small effect of polymer architecture on bioactivity within a library of hyperbranched poly(glycerol)-protein conjugates. In their study however, grafting density is quite low and their fi ndings corroborate those herein at low grafting densities.…”

Protein–polymer therapeutics: a macromolecular perspective

“…55 Another interesting class of PEG-like polymers, which, however, so far has been barely explored for the synthesis of protein-polymer conjugates, are polyglycerols (PGs). 56 PGs can be prepared with linear or hyperbranched structures thus providing access to libraries of chemically identical but structurally different polymers for optimizing the pharmacokinetic properties and improving therapeutic effects. 56 In the pursuit for PEG alternatives, oligosaccharides, inspired by the glycosylation of proteins present in nature, have been also considered as PEG analogues.…”

“…56 PGs can be prepared with linear or hyperbranched structures thus providing access to libraries of chemically identical but structurally different polymers for optimizing the pharmacokinetic properties and improving therapeutic effects. 56 In the pursuit for PEG alternatives, oligosaccharides, inspired by the glycosylation of proteins present in nature, have been also considered as PEG analogues. 57,58 Likewise, oligosaccharides provide a large hydration shell capable of protecting proteins from proteolysis and introducing prolonged circulation times.…”