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DiPODS: A Reagent for Site-Specific Bioconjugation via the Irreversible Rebridging of Disulfide Linkages
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2020-11-19 , DOI: 10.1021/acs.bioconjchem.0c00590 Elaheh Khozeimeh Sarbisheh 1 , Guillaume Dewaele-Le Roi 2, 3, 4 , Whitney E Shannon 1 , Sally Tan 2 , Yujia Xu 2, 3 , Brian M Zeglis 2, 3, 4 , Eric W Price 1
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2020-11-19 , DOI: 10.1021/acs.bioconjchem.0c00590 Elaheh Khozeimeh Sarbisheh 1 , Guillaume Dewaele-Le Roi 2, 3, 4 , Whitney E Shannon 1 , Sally Tan 2 , Yujia Xu 2, 3 , Brian M Zeglis 2, 3, 4 , Eric W Price 1
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Chemoselective reactions with thiols have long held promise for the site-specific bioconjugation of antibodies and antibody fragments. Yet bifunctional probes bearing monovalent maleimides—long the “gold standard” for thiol-based ligations—are hampered by two intrinsic issues: the in vivo instability of the maleimide–thiol bond and the need to permanently disrupt disulfide linkages in order to facilitate bioconjugation. Herein, we present the synthesis, characterization, and validation of DiPODS, a novel bioconjugation reagent containing a pair of oxadiazolyl methyl sulfone moieties capable of irreversibly forming covalent bonds with two thiolate groups while simultaneously rebridging disulfide linkages. The reagent was synthesized from commercially available starting materials in 8 steps, during which rotamers were encountered and investigated both experimentally and computationally. DiPODS is designed to be modular and can thus be conjugated to any payload through a pendant terminal primary amine (DiPODS–PEG4–NH2). Subsequently, the modification of a HER2-targeting Fab with a fluorescein-conjugated variant of DiPODS (DiPODS–PEG4–FITC) reinforced the site-specificity of the reagent, illustrated its ability to rebridge disulfide linkages, and produced an immunoconjugate with in vitro properties superior to those of an analogous construct created using traditional stochastic bioconjugation techniques. Ultimately, we believe that this work has particularly important implications for the synthesis of immunoconjugates, specifically for ensuring that the attachment of cargoes to immunoglobulins is robust, irreversible, and biologically and structurally benign.
中文翻译：
DiPODS：通过二硫键不可逆转的特定位点生物缀合的试剂
长期以来，与硫醇的化学选择性反应对于抗体和抗体片段的位点特异性生物缀合具有广阔的前景。然而，带有单价马来酰亚胺的双功能探针（长期用于基于硫醇的连接的“金标准”）受到两个内在问题的阻碍：体内马来酰亚胺-硫醇键的不稳定性以及需要永久破坏二硫键以促进生物缀合。在本文中，我们介绍了DiPODS的合成，表征和验证，DiPODS是一种新型生物缀合试剂，其中含有一对恶二唑基甲基砜部分，能够与两个硫醇盐基团不可逆地形成共价键，同时使二硫键重新键合。该试剂由市售起始原料分8个步骤合成，在此过程中遇到了旋转异构体，并进行了实验和计算研究。DiPODS被设计为模块化的，因此可以通过末端伯胺侧基（DiPODS–PEG 4 –NH 2）。随后，用荧光素偶联的DiPODS（DiPODS–PEG 4 –FITC）修饰的靶向HER2的Fab增强了试剂的位点特异性，说明了该试剂重新桥接二硫键的能力，并与体外产生了免疫偶联物具有优于使用传统随机生物共轭技术创建的类似构建体的特性。最终，我们认为这项工作对免疫缀合物的合成具有特别重要的意义，特别是对于确保货物与免疫球蛋白的结合牢固，不可逆且在生物学和结构上是良性的。
更新日期：2020-12-16
中文翻译：
DiPODS：通过二硫键不可逆转的特定位点生物缀合的试剂
长期以来，与硫醇的化学选择性反应对于抗体和抗体片段的位点特异性生物缀合具有广阔的前景。然而，带有单价马来酰亚胺的双功能探针（长期用于基于硫醇的连接的“金标准”）受到两个内在问题的阻碍：体内马来酰亚胺-硫醇键的不稳定性以及需要永久破坏二硫键以促进生物缀合。在本文中，我们介绍了DiPODS的合成，表征和验证，DiPODS是一种新型生物缀合试剂，其中含有一对恶二唑基甲基砜部分，能够与两个硫醇盐基团不可逆地形成共价键，同时使二硫键重新键合。该试剂由市售起始原料分8个步骤合成，在此过程中遇到了旋转异构体，并进行了实验和计算研究。DiPODS被设计为模块化的，因此可以通过末端伯胺侧基（DiPODS–PEG 4 –NH 2）。随后，用荧光素偶联的DiPODS（DiPODS–PEG 4 –FITC）修饰的靶向HER2的Fab增强了试剂的位点特异性，说明了该试剂重新桥接二硫键的能力，并与体外产生了免疫偶联物具有优于使用传统随机生物共轭技术创建的类似构建体的特性。最终，我们认为这项工作对免疫缀合物的合成具有特别重要的意义，特别是对于确保货物与免疫球蛋白的结合牢固，不可逆且在生物学和结构上是良性的。
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