Abstract：
Achieving efficient and site-specific conjugation of therapeutic protein to polymer is crucial to augment their applicability in the realms of biomedicine by improving their stability and enzymatic activity. In this study, we exploited tetrazine bioorthogonal chemistry to achieve the site-specific conjugation of bottlebrush polymers to urate oxidase (UOX), a therapeutic protein for gout treatment. An azido-functionalized zwitterionic bottlebrush polymer (N3-ZBP) using a \"grafting-from\" strategy involving RAFT and ATRP methods was synthesized, and a trans-cyclooctene (TCO) moiety was introduced at the polymer end through the strain-promoted azide-alkyne click (SPAAC) reaction. The subsequent coupling between TCO-incorporated bottlebrush polymer and tetrazine-labeled UOX using a fast and safe bioorthogonal reaction, inverse electron demand Diels-Alder (IEDDA), led to the formation of UOX-ZBP conjugates with a 52% yield. Importantly, the enzymatic activity of UOX remained unaffected following polymer conjugation, suggesting a minimal change in the folded structure of UOX. Moreover, UOX-ZBP conjugates exhibited enhanced proteolytic resistance and reduced antibody binding, compared to UOX-wild type. Overall, the present findings reveal an efficient and straightforward route for synthesizing protein-bottlebrush polymer conjugates without compromising the enzymatic activity while substantially reducing proteolytic degradation and antibody binding.
摘要：
实现治疗性蛋白质与聚合物的有效和位点特异性缀合对于通过改善其稳定性和酶活性来增强其在生物医学领域中的适用性至关重要。在这项研究中,我们利用四嗪生物正交化学来实现瓶刷聚合物与尿酸氧化酶（UOX）的位点特异性缀合，痛风治疗的治疗性蛋白质。使用涉及RAFT和ATRP方法的“接枝自”策略合成了叠氮基官能化的两性离子瓶刷聚合物（N3-ZBP），并且通过应变促进的叠氮化物-炔点击(SPAAC)反应在聚合物末端引入反式-环辛烯(TCO)部分。随后使用快速安全的生物正交反应，将TCO掺入的瓶刷聚合物与四嗪标记的UOX偶联，反电子需求狄尔斯-阿尔德(IEDDA)，导致UOX-ZBP缀合物的形成,产率为52%。重要的是,UOX的酶活性在聚合物缀合后保持不受影响，表明UOX的折叠结构变化最小。此外，UOX-ZBP缀合物表现出增强的蛋白水解抗性和降低的抗体结合,与UOX野生型相比。总的来说,本研究发现揭示了一种有效和直接的途径，用于合成蛋白质-瓶刷聚合物缀合物，而不损害酶活性，同时大大减少蛋白水解降解和抗体结合。
