Farzan Valentina M, Ulashchik Egor A, Martynenko-Makaev Yury V, Kvach Maksim V, Aparin Ilya O, Brylev Vladimir A, Prikazchikova Tatiana A, Maklakova Svetlana Yu, Majouga Alexander G, Ustinov Alexey V, Shipulin German A, Shmanai Vadim V, Korshun Vladimir A, Zatsepin Timofei S
Center of Translational Biomedicine, Skolkovo Institute of Science and Technology , Skolkovo, Moscow 143026, Russia.
Institute of Physical Organic Chemistry, National Academy of Sciences of Belarus , Surganova 13, Minsk 220072, Belarus.
Bioconjug Chem. 2017 Oct 18;28(10):2599-2607. doi: 10.1021/acs.bioconjchem.7b00462. Epub 2017 Oct 4.
We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI·P(OEt) as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-GalNAc alkyne gives oligonucleotide-GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high-throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2'-modified siRNA.
我们开发了一种新技术，利用固相上的铜催化炔烃-叠氮环加成反应以及CuI·P(OEt)作为催化剂，实现寡核苷酸与各种烷基叠氮化物（如荧光染料、生物素、胆固醇、N-乙酰半乳糖胺（GalNAc）等）的高效共轭。共轭反应在寡核苷酸合成仪中以全自动模式进行，并与寡核苷酸合成和柱上脱保护相结合。我们还提出了一套用于构建多种共轭物的试剂。使用季戊四醇衍生的四叠氮化物进行顺序双击程序，然后加入GalNAc或Tris-GalNAc炔烃，可高产率地得到寡核苷酸-GalNAc树枝状聚合物共轭物，且复杂炔烃试剂的过量最少。该方法适用于从荧光DNA探针到2'-修饰siRNA的各种多GalNAc衍生物的寡核苷酸共轭物的高通量合成。
