Tailhades Julien, Takizawa Hotake, Gait Michael J, Wellings Don A, Wade John D, Aoki Yoshitsugu, Shabanpoor Fazel
The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, Australia.
Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.
Front Chem. 2017 Oct 17;5:81. doi: 10.3389/fchem.2017.00081. eCollection 2017.
Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2'--methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here, we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce "on-resin" aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.
随着2016年末美国食品药品监督管理局（FDA）批准依特立生（一种基于吗啉代磷酰胺的反义寡核苷酸）和诺西那生钠（2'-甲氧基乙基硫代磷酸酯），基于反义寡核苷酸（ASO）的药物开发正获得显著的发展势头。它们的吸引力主要源于骨架修饰，这种修饰改善了寡核苷酸药物的特性。另一类基于肽核酸（PNA）化学的ASO，作为针对各种疾病的基因特异性治疗开发平台也越来越受欢迎。然而，更长的、更具靶点特异性的PNA的化学合成仍然是一个持续存在的挑战。大多数报道的PNA固相合成方法存在偶联效率低的问题，这限制了小于15个残基的短PNA序列的生产。在这里，我们研究了用Hmb（2-羟基-4-甲氧基苄基）和Dmb（2,4-二甲氧基苄基）进行骨架修饰对改善困难偶联和减少“树脂上”聚集的影响。我们首先合成了一个包含Hmb或Dmb的PNA二聚体文库，并确定Hmb在去除的难易程度方面优于Dmb。随后，我们使用Hmb骨架修饰来合成一个靶向抗肌萎缩蛋白RNA剪接的22聚体富含嘌呤的PNA，而标准偶联方法无法合成该PNA。Hmb骨架修饰使这种困难的PNA得以合成，并且能够在同一固相载体上继续连接一个细胞穿透肽。这种方法为简便地固相合成困难的富含嘌呤的PNA序列提供了一种新颖且直接的策略。
