Lorenzo Maltish M, Decker Caitlin G, Kahveci Muhammet U, Paluck Samantha J, Maynard Heather D
Department of Chemistry and Biochemistry and California NanoSystems Institute, University of California, Los Angeles, 607 Charles E. Young Drive South, Los Angeles, California 90095-1569, United States.
Macromolecules. 2016 Jan 12;49(1):30-37. doi: 10.1021/acs.macromol.5b02323. Epub 2015 Dec 23.
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 -cyclooctene (T4L-TCO). Reversible addition-fragmentation chain transfer (RAFT) polymerization yielded poly(N-isopropylacrylamide) (pNIPAAm) with a number average molecular weight ( by H-NMR) of 2.0 kDa and a dispersity (Đ by GPC) of 1.05. pNIPAAm was then modified at both ends by post-polymerization with 6-methyl tetrazine. 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 hour, 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.
通过可控自由基聚合（CRP）合成了四嗪端基功能化的遥爪聚合物，并将其用于生成T4溶菌酶同二聚体。含有单个表面暴露半胱氨酸的突变型T4溶菌酶（V131C）用蛋白质反应性的环辛烯（T4L-TCO）进行修饰。可逆加成-断裂链转移（RAFT）聚合得到数均分子量（通过H-NMR）为2.0 kDa且分散度（通过GPC测得的Đ）为1.05的聚（N-异丙基丙烯酰胺）（pNIPAAm）。然后通过与6-甲基四嗪进行后聚合反应，在pNIPAAm的两端进行修饰。为作比较，合成了2.0 kDa的双四嗪聚乙二醇（PEG）和2.0 kDa的双马来酰亚胺pNIPAAm。仅1小时后，T4L-TCO与双四嗪pNIPAAm或双四嗪PEG连接分别产生了产率为38%和37%的蛋白质同二聚体，而双马来酰亚胺pNIPAAm在24小时后仅产生了5%产率的二聚体。这项工作说明了在合成蛋白质同二聚体缀合物方面，采用四嗪连接法相对于马来酰亚胺硫醇-烯化学法的优势。
