Site-specific labeling and conjugation of antibodies are highly desirable for fundamental research and for developing more efficient diagnostic and therapeutic methods. We report here a general and robust chemoenzymatic method that permits a one-pot site-specific functionalization of antibodies. A series of selectively modified disaccharide oxazoline derivatives were designed, synthesized, and evaluated as donor substrates of different endoglycosidases for antibody Fc glycan remodeling. We found that among several endoglycosidases tested, wild-type endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) exhibited remarkable activity in transferring the functionalized disaccharides carrying site-selectively modified azide, biotin, or fluorescent tags to antibodies without hydrolyzing the resulting transglycosylation products. This discovery, together with the excellent Fc deglycosylation activity of Endo-S2 on recombinant antibodies, allowed direct labeling and functionalization of antibodies in a one-pot manner without the need of intermediate and enzyme separation. The site-specific introduction of varied numbers of azide groups enabled a highly efficient synthesis of homogeneous antibody–drug conjugates (ADCs) with a precise control of the drug-to-antibody ratio (DAR) ranging from 2 to 12 via a copper-free strain-promoted click reaction. Cell viability assays showed that ADCs with higher DARs were more potent in killing antigen-overexpressed cells than the ADCs with lower DARs. This new method is expected to find applications not only for antibody–drug conjugation but also for cell labeling, imaging, and diagnosis.

中文翻译：

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用于聚糖介导的抗体位点特异性标记和偶联的通用且稳健的化学酶法：均质抗体-药物偶联物的简便合成

抗体的位点特异性标记和偶联对于基础研究和开发更有效的诊断和治疗方法是非常需要的。我们在这里报告了一种通用且稳健的化学酶法，该方法允许对抗体进行一锅式位点特异性功能化。设计、合成和评估了一系列选择性修饰的二糖恶唑啉衍生物，作为抗体 Fc 聚糖重塑的不同内切糖苷酶的供体底物。我们发现，在测试的几种内切糖苷酶中，来自化脓性链球菌的野生型内切糖苷酶血清型 M49 (Endo-S2) 在将带有位点选择性修饰的叠氮化物、生物素或荧光标签的功能化二糖转移到抗体而不水解产生的转糖基产物方面表现出显着的活性。这一发现连同 Endo-S2 对重组抗体的出色 Fc 去糖基化活性，允许以一锅法直接标记和功能化抗体，无需中间体和酶分离。位点特异性引入不同数量的叠氮基团使得能够高效合成均相抗体-药物偶联物 (ADC)，并通过无铜方法精确控制药物抗体比 (DAR) 在 2 到 12 之间应变促进的点击反应。细胞活力测定表明，与具有较低 DAR 的 ADC 相比，具有较高 DAR 的 ADC 在杀死抗原过表达细胞方面更有效。预计这种新方法不仅可以用于抗体-药物偶联，还可以用于细胞标记、成像和诊断。