Honcharenko Dmytro, Druceikaite Kristina, Honcharenko Malgorzata, Bollmark Martin, Tedebark Ulf, Strömberg Roger
Department of Biosciences and Nutrition, Karolinska Institutet, 14183 Huddinge, Sweden.
RISE Chemical Process and Pharmaceutical Development, Forskargatan 20J, 15136 Södertälje, Sweden.
ACS Omega. 2020 Dec 18;6(1):579-593. doi: 10.1021/acsomega.0c05075. eCollection 2021 Jan 12.
Oligonucleotide (ON) conjugates are increasingly important tools for various molecular diagnostics, nanotechnological applications, and for the development of nucleic acid-based therapies. Multiple labeling of ONs can further equip ON-conjugates and provide improved or additional tailored properties. Typically, the preparation of ON multiconjugates involves additional synthetic steps and/or manipulations in post-ON assembly. This report describes the simplified methodology allowing for multiple labeling of ONs on a solid support and is compatible with phosphodiester as well as phosphorothioate (PS) ONs. The current approach utilizes two novel alkyne- and amino-functionalized linker phosphoramidites that can be readily synthesized from a common aminodiol intermediate in three steps. The combination of new linkers provides orthogonal functionalities, which allow for multiple attachments of similar or varied moieties. The linkers are incorporated into ONs during automated solid-phase ON synthesis, and the conjugation with functional entities is achieved by either amide bond formation or by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The versatility of the approach is demonstrated by the synthesis of 5'-site ON multiconjugates with small molecules, peptides, and fatty acids as well as in the preparation of an internal peptide-ON conjugate.
寡核苷酸（ON）缀合物对于各种分子诊断、纳米技术应用以及基于核酸的疗法的开发而言，正日益成为重要工具。对寡核苷酸进行多重标记可进一步增强寡核苷酸缀合物的功能，并赋予其改良的或额外的定制特性。通常，制备寡核苷酸多缀合物需要在寡核苷酸组装后进行额外的合成步骤和/或操作。本报告描述了一种简化方法，该方法可在固相载体上对寡核苷酸进行多重标记，并且与磷酸二酯以及硫代磷酸酯（PS）寡核苷酸兼容。当前方法利用了两种新型的炔基和氨基功能化的连接亚磷酰胺，它们可以通过三步从常见的氨基二醇中间体轻松合成。新连接子的组合提供了正交功能，这使得类似或不同部分能够进行多重连接。在自动化固相寡核苷酸合成过程中，将连接子掺入寡核苷酸中，与功能实体的缀合通过酰胺键形成或铜（I）催化的叠氮化物-炔烃环加成反应（CuAAC）实现。通过合成具有小分子、肽和脂肪酸的5'-位点寡核苷酸多缀合物以及制备内部肽-寡核苷酸缀合物，证明了该方法的通用性。
