Squaric ester amides as hydrolysis-resistant functional groups for protein-conjugation of RAFT-derived polymers

We report on the synthesis of amine-reactive polymers, for the purpose of protein conjugation. Poly(N,N-dimethylacrylamide) (polyDMA) was prepared by RAFT polymerization using either a squaric ester amide functionalized chain transfer agent (CTA) or a phthalimide functionalized CTA which served as a precursor for post-polymerization introduction of a squaric ester amide moiety. Polymers were characterized for their end group fidelity by 1H-NMR and MALDI-TOF MS, which showed the use of a squaric ester amide functionalized CTA to be superior. The performance of these polymers for lysine-targeted protein modification was assessed using lysozyme as model protein and compared to N-hydroxysuccinimide (NHS)-functionalized polymers as the golden standard in bio-conjugation. We found that squaric ester amides were much less prone to hydrolysis, which yielded higher protein-conjugation efficiency in aqueous medium.