Cleavable Polyethylene Glycol: 3,4-Epoxy-1-butene as a Comonomer to Establish Degradability at Physiologically Relevant pH

Polyethylene glycol (PEG) has been used for decades to improve the pharmacokinetic properties of protein drugs, and several PEG-protein conjugates are approved by the FDA. However, the nondegradability of PEG restricts its use to a limiting molecular weight to permit renal excretion. In this work, we introduce a simple strategy to overcome the nondegradability of PEG by incorporating multiple pH-sensitive vinyl ether moieties into the polyether backbone. Copolymerization of 3,4-epoxy-1-butene (EPB) with ethylene oxide via anionic ring-opening polymerization (AROP) provides access to allyl moieties that can be isomerized to pH-cleavable propenyl units (<i>iso</i>EPB). Well-defined P­(EPB-<i>co</i>-EG) copolymers (<i>Đ</i> = 1.05–1.11) with EPB contents of ∼4 mol% were synthesized in a molecular weight range of 3000 to 10000 g mol^–1. ¹H NMR kinetic studies served to investigate acidic hydrolysis in a pH range of 4.4 to 5.4 and even allowed to distinguish between the hydrolysis rates of (<i>E</i>)- and (<i>Z</i>)-<i>iso</i>EPB units, demonstrating faster hydrolysis of the (<i>Z</i>)-isomer. SEC analysis of degradation products revealed moderate dispersities <i>Đ</i> of 1.6 to 1.8 and consistent average molecular weights <i>M</i>_n of ∼1000 g mol^–1. The presence of a defined hydroxyl end group permits attachment to other functional molecules. The novel pH-degradable PEGs combine various desirable properties such as excellent long-term storage stability and cleavage in a physiologically relevant pH-range that render them promising candidates for biomedical application

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

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

<i><i>N,N</i></i>-Diallylglycidylamine: A Key Monomer for Amino-Functional Poly(ethylene glycol) Architectures

The first application of <i><i>N,N</i></i>-diallylglycidylamine (DAGA) as a monomer for anionic ring-opening polymerization is presented. The monomer is obtained in a one-step procedure using epichlorohydrin and <i><i>N,N</i></i>-diallylamine. Both random and block copolymers consisting of poly­(ethylene glycol) and poly­(<i><i>N,N</i></i>-diallylglycidylamine) with adjusted DAGA ratios from 2.5 to 24% have been prepared, yielding well-defined materials with low polydispersities (<i>M</i>_w/<i>M</i>_n) in the range 1.04–1.19. Molecular weights ranged between 2600 and 10 300 g mol^–1. Isomerization of allylamine to enamine structures during polymerization depending on time, temperature, and counterion has been realized. The kinetics of the formation of the copolymer structure obtained by random copolymerization was investigated, using time-resolved ¹H NMR measurements and ¹³C NMR triad sequence analysis. A tapered character of the monomer incorporation was revealed in the course of the concurrent copolymerization of EO and DAGA. The thermal behavior of the copolymers in both bulk and aqueous solution has been studied, revealing LCSTs in the range 29–94 °C. Quantitative removal of protective groups via double-bond isomerization mediated by Wilkinson’s catalyst and subsequent acidic hydrolysis yielded multiamino-functional PEG copolymers with tapered or block structure. Accessibility of liberated primary amines for further transformation was demonstrated in a model reaction by derivatization with acetic anhydride. In contrast to previous approaches, the DAGA monomer permits the synthesis of block copolymers with PEG block combined with multiamino-functional polyether block

Universal Concept for the Implementation of a Single Cleavable Unit at Tunable Position in Functional Poly(ethylene glycol)s

Poly­(ethylene glycol) (PEG) with acid-sensitive moieties gained attention particularly for various biomedical applications, such as the covalent attachment of PEG (PEGylation) to protein therapeutics, the synthesis of stealth liposomes, and polymeric carriers for low-molecular-weight drugs. Cleavable PEGs are favored over their inert analogues because of superior pharmacodynamic and/or pharmacokinetic properties of their formulations. However, synthetic routes to acetal-containing PEGs published up to date either require enormous efforts or result in ill-defined materials with a lack of control over the molecular weight. Herein, we describe a novel methodology to implement a single acetaldehyde acetal in well-defined (hetero)­functional poly­(ethylene glycol)­s with total control over its position. To underline its general applicability, a diverse set of initiators for the anionic polymerization of ethylene oxide (cholesterol, dibenzylamino ethanol, and poly­(ethylene glycol) monomethyl ether (mPEG)) was modified and used to synthesize the analogous labile PEGs. The polyether bearing the cleavable lipid had a degree of polymerization of 46, was amphiphilic and exhibited a critical micelle concentration of 4.20 mg·L^–1. From dibenzylamino ethanol, three heterofunctional PEGs with different molecular weights and labile amino termini were generated. The transformation of the amino functionality into the corresponding squaric acid ester amide demonstrated the accessibility of the cleavable functional group and activated the PEG for protein PEGylation, which was exemplarily shown by the attachment to bovine serum albumin (BSA). Furthermore, turning mPEG into a macroinitiator with a cleavable hydroxyl group granted access to a well-defined poly­(ethylene glycol) derivative bearing a single cleavable moiety within its backbone. All the acetal-containing PEGs and PEG/protein conjugates were proven to degrade upon acidic treatment

Ferrocenyl Glycidyl Ether: A Versatile Ferrocene Monomer for Copolymerization with Ethylene Oxide to Water-Soluble, Thermoresponsive Copolymers

The first ferrocene-containing epoxide monomer, ferrocenyl glycidyl ether (fcGE), is introduced. The monomer has been copolymerized with ethylene oxide (EO). This leads to electroactive, water-soluble, and thermoresponsive poly­(ethylene glycol) (PEG) derived copolyethers. Anionic homo- and copolymerization of fcGE with EO was possible. Molecular weights could be varied from 2000 to 10 000 g mol^–1, resulting in polymers with narrow molecular weight distribution (<i>M</i>_w/<i>M</i>_n = 1.07–1.20). The ferrocene (fc) content was varied from 3 to 30 mol %, obtaining water-soluble materials up to 10 mol % incorporation of the apolar ferrocenyl comonomer. Despite the steric bulk of fcGE, random copolymers were obtained, as confirmed via detailed ¹H NMR kinetic measurements as well as ¹³C NMR studies of the polymer microstructure, including detailed triad characterization. In addition, the poly­(fcGE) homopolymer has been prepared. All water-soluble copolyethers with fc side chains exhibited a lower critical solution temperature (LCST) in the range 7.2–82.2 °C in aqueous solution, depending on the amount of fcGE incorporated. The LCST is further tunable by oxidation/reduction of ferrocene, as demonstrated by cyclic voltammetry. Investigation of the electrochemical properties by cyclovoltammetry revealed that the iron centers can be oxidized reversibly. Further, to evaluate the potential for biomedical application, cell viability tests of the fc-containing PEG copolymers were performed on a human cervical cancer cell line (HeLa), revealing good biocompatibility only in the case of low amounts of fcGE incorporated (below 5%). Significant cytotoxic behavior was observed with fcGE content exceeding 5%. The ferrocene-substituted copolyethers are promising for novel redox sensors and create new options for the field of organometallic (co)­polymers in general