Reversible Polymer-Protein Functionalization by Stepwise Introduction of Amine-Reactive, Reductive-Responsive Self-Immolative End Groups onto RAFT-Derived Polymers.

Many promising therapeutic protein or peptide drug candidates are rapidly excreted from an organism due to their small size or their inherent immunogenicity. One way to counteract these effects is PEGylation, in which the biopolymer is shielded by synthetic polymers exploiting their stealth properties. However, these modifications are often accompanied by a reduction in the biological function of the protein. By using responsive moieties that bridge the polymer to the protein, a reversible character is provided to this type of conjugation. In this regard, the reductive-responsive nature of disulfides can be exploited via self-immolative structures for reversible linkage to aminic lysine residues and the N-terminus on the protein surface. They enable a traceless release of the intact protein without any further modification and thus preserve the protein's bioactivity. In this study, we demonstrate how this chemistry can be made broadly accessible to RAFT-derived water-soluble polymers like poly(N,N-dimethylacrylamide) (pDMA) as a relevant PEG alternative. A terminal reactive imidazole carbamate with an adjacent self-immolative motif was generated in a gradual manner onto the trithiocarbonate chain transfer moiety of the polymer by first substituting it with a disulfide-bridged alcohol and subsequently converting it into an amine reactive imidazole carbamate. Successful synthesis and complete characterization were demonstrated by NMR, size exclusion chromatography, and mass spectrometry. Finally, two model proteins, lysozyme and a therapeutically relevant nanobody, were functionalized with the generated polymer, which was found to be fully reversible under reductive conditions in the presence of free thiols. This strategy has the potential to extend the generation of reversible reductive-responsive polymer-protein hybrids to the broad field of available functional RAFT-derived polymers.