UCLA

Described in this thesis is the use of controlled radical polymerization techniques to synthesize tetrazine end-functionalized telechelic polymers, which were employed to generate T4 Lysozyme homodimers. A mutant of T4 Lysozyme (V131C), containing a single surface-exposed cysteine residue, was modified via Michael addition with a protein-reactive trans-cyclooctene. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to obtain telechelic poly(N-isopropylacrylamide) (pNIPAAm) polymers with Mn (1H-NMR) of 2.0 kDa and molecular weight dispersity, Đ, (GPC) of 1.05. pNIPAAm was modified at both ends with (4-(6- methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanol. As a control, a 2.0 kDa bis-carboxylic acid poly(ethylene glycol) was modified at both ends with (4-(6-methyl-1,2,4,5-tetrazin-3- yl)phenyl)methanol and a 2.0 kDa bis-maleimide pNIPAAm was also synthesized. Tetrazine- functionalized polymers were then ligated to a trans-cyclooctene modified Lysozyme (T4L-TCO). Ligation of T4L-TCO to bis-tetrazine pNIPAAm resulted in 27.27% yield of the homodimeric conjugate, whereas bis-tetrazine PEG resulted in 16.75% and bis-maleimide pNIPAAm resulted in <1% yield after 1 hour with no significant increase in yield during extended incubation times up to 24 hours. This work shows that tetrazine chemistry results in a higher yield of dimeric protein conjugation for both PEG and CRP polymers.