Department of Medical and Clinical Biophysics, Faculty of Medicine, P. J. Šafárik University in Košice, Trieda SNP 1, 04011 Košice, Slovakia.
Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark.
Org Biomol Chem. 2022 Nov 23;20(45):8873-8884. doi: 10.1039/d2ob01466a.
The low binding affinity of unmodified triplex-forming oligonucleotides (TFO) is the main drawback to their promising utilization in gene therapy. In the present study, we have synthesized DNA intercalator 5-(pyren-1-ylethynyl)indole Y, known as twisted intercalating nucleic acid (TINA), by a Cu-mediated Sonogashira palladium-catalyzed coupling reaction of 1-ethynylpyrene with 5-iodoindole at a high temperature under anaerobic conditions. Coupling with indole C-5 was far more preferable in obtaining stable TINA-indole than enamine site C-3, as neither hydration of the triple bond to ketones nor competitive Glaser-type homocoupling of acetylenes was observed. The insertion of the new TINA monomer Y as a bulge in the middle or at the 5'-end of the oligodeoxynucleotide sequence a flexible butane-1,2-diol linker showed extraordinary binding potential, resulting in excellent thermal stabilization of Hoogsteen-type triplex- and duplex-deoxyribonucleic acid (DNA) structures which was detected by thermal denaturation studies and supported by circular dichroism (CD). Molecular dynamics AMBER* revealed the lowest energy conformation in which a pyrenyl residue of the TINA monomer Y stacks in the dsDNA part, while an indolyl unit intercalates between the nucleobases of the TFO pattern. Overall the torsionally rigid conjugated TINA system with a decent twisting of 15.1° around acetylene is confirmed here as a requirement for the best fit inside the intercalation site of the triplex, resulting in high TFO-dsDNA affinity.
未修饰的三聚体形成寡核苷酸（TFO）的低结合亲和力是其在基因治疗中应用的主要缺点。在本研究中，我们通过铜介导的 Sonogashira 钯催化偶联反应，在无氧条件下于高温下，将 1-乙炔基芘与 5-碘吲哚偶联，合成了 DNA 插入剂 5-（芘-1-基乙炔基）吲哚 Y，即扭曲插入核酸（TINA）。与吲哚 C-5 的偶联在获得稳定的 TINA-吲哚方面远比烯胺 C-3 更可取，因为既没有观察到三键的水化生成酮，也没有观察到竞争的 Glaser 型炔烃同偶联。将新的 TINA 单体 Y 作为寡脱氧核苷酸序列中的中或 5'-端的凸起插入柔性丁烷-1,2-二醇连接子，显示出非凡的结合潜力，导致 Hoogsteen 型三聚体和双链脱氧核糖核酸（DNA）结构的极好热稳定性，这通过热变性研究得到了检测，并得到圆二色性（CD）的支持。分子动力学 AMBER*揭示了最低能量构象，其中 TINA 单体 Y 的芘基残基堆积在 dsDNA 部分，而吲哚基单元插入 TFO 模式的核碱基之间。总的来说，这里证实了具有良好扭转的刚性共轭 TINA 系统，其在乙炔周围扭转 15.1°，是最适合三聚体插入位点的要求，从而导致 TFO-dsDNA 亲和力高。
