Conjugation of biologics with polymers modulates their pharmacokinetics, with polyethylene glycol (PEG) as the gold standard. We compared alternative polymers and two types of cyclooctyne linkers (BCN/DBCO) for bioconjugation of interferon-α2a (IFN-α2a) using 10 kDa polymers including linear mPEG, poly­(2-ethyl-2-oxazoline) (PEtOx), and linear polyglycerol (LPG). IFN-α2a was azide functionalized via amber codon expansion and bioorthogonally conjugated to all cyclooctyne linked polymers. Polymer conjugation did not impact IFN-α2a’s secondary structure and only marginally reduced IFN-α2a’s bioactivity. In comparison to PEtOx, the LPG polymer attached via the less rigid cyclooctyne linker BCN was found to stabilize IFN-α2a against thermal stress. These findings were further detailed by molecular modeling studies which showed a modulation of protein flexibility upon PEtOx conjugation and a reduced amount of protein native contacts as compared to PEG and LPG originated bioconjugates. Polymer interactions with IFN-α2a were further assessed via a limited proteolysis (LIP) assay, which resulted in comparable proteolytic cleavage patterns suggesting weak interactions with the protein′s surface. In conclusion, both PEtOx and LPG bioconjugates resulted in a similar biological outcome and may become promising PEG alternatives for bioconjugation.

In a two-step synthesis, five different alkyl-substituted morpholine-2,5-dione monomers were synthesized from the natural amino acids glycine, alanine, valine, leucine, and isoleucine. The heterocyclic compounds crystallize in a boat-like conformation and are polymerized via 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD)-catalyzed ring-opening polymerization (ROP) in tetrahydrofuran. Well-defined polymers could be obtained from the monomers based on valine, leucine, and isoleucine at a feed ratio of M/I/TBD = 100/1/0.5. Kinetic studies of the ROP reveal that the molar masses and dispersities (Đ < 1.2) could be well controlled, as confirmed by size exclusion chromatography and H NMR spectroscopy. At conversions above 50%, the polymerization rate decreases and the dispersity slightly increases, presumably due to transesterification. Matrix-assisted laser desorption time-of-flight mass spectrometry indicates the presence of polymer chains with α-end groups derived from the initiator.

Poly­(N-isopropylacrylamide) (PNiPAm) and poly­(2-ethyl-2-oxazoline) (PEtOx) represent two polymer types that are well-known for their lower critical solution temperature (LCST) behavior in aqueous media. To synthesize triblock copolymers containing both polymers, a crossover of two different polymerization methods was applied using a bifunctional initiator for the living cationic ring-opening polymerization (CROP) of EtOx. Quantitative end-functionalization with a trithiocarbonate resulted in a bifunctional PEtOx macro chain transfer agent (CTA). A series of well-defined PNiPAm-b-PEtOx-b-PNiPAm triblock copolymers were obtained by subsequent reversible addition–fragmentation chain transfer (RAFT) polymerization of NiPAm. The influence of the PNiPAm to PEtOx ratio on the thermoresponsive properties was intensively investigated via turbidimetry, dynamic light scattering, cryo transmission electron microscopy, and 1H NMR studies, revealing hydrogen bonds between both copolymer segments that strongly lower the phase separation temperature of aqueous solutions.

No abstract

Interleukin-4 (IL-4) is a potentially interesting anti-inflammatory therapeutic, which is rapidly excreted. Therefore, serum half-life extension by polymer conjugation is desirable, which may be done by PEGylation. Here, we use PEtOx as an alternative to PEG for bioconjugate engineering. We genetically extended murine IL-4 (mIL-4) with the d-domain of insulin-like growth factor I (IGF-I), a previously identified substrate of transglutaminase (TG) Factor XIIIa (FXIIIa). Thereby, engineered mIL-4 (mIL-4-TG) became an educt for TG catalyzed C-terminal, site-directed conjugation. This was deployed to enzymatically couple an azide group containing peptide sequence to mIL-4, allowing C-terminal bioconjugation of polyethylene glycol or poly­(2-ethyl-2-oxazoline). Both bioconjugates had wild-type potency and alternatively polarized macrophages.