Homodimeric Protein–Polymer Conjugates via the Tetrazine–trans-Cyclooctene Ligation

Tetrazine end-functionalized telechelic polymers were synthesized by controlled radical polymerization (CRP) and employed to generate T4 lysozyme homodimers. Mutant T4 lysozyme (V131C), containing a single surface-exposed cysteine, was modified with a protein-reactive trans -cyclooctene (T4L-TCO). Reversible addition–fragmentation chain transfer (RAFT) polymerization yielded poly( N -isopropylacrylamide) (pNIPAAm) with a number-average molecular weight ( M n by 1 H NMR) of 2.0 kDa and a dispersity ( Đ by GPC) of 1.05. pNIPAAm was then modified at both ends by postpolymerization with 6-methyltetrazine. For comparison, 2.0 kDa bis-tetrazine poly(ethylene glycol) (PEG) and 2.0 kDa bis-maleimide pNIPAAm were synthesized. Ligation of T4L-TCO to bis-tetrazine pNIPAAm or bis-tetrazine PEG resulted in protein homodimer in 38% yield and 37% yield, respectively, after only 1 h, whereas bis-maleimide pNIPAAm resulted in only 5% yield of dimer after 24 h. This work illustrates the advantage of employing tetrazine ligation over maleimide thiol–ene chemistry for the synthesis of protein homodimer conjugates.