Phosphorodiamidate Morpholino Oligonucleotides (PMOs) have been well established in the milieu of FDA-approved oligonucleotide-based drugs in the past decade. Given their relevance in antisense therapeutics, a DNA/RNA synthesizer-compatible modular synthesis protocol of PMOs is long awaited to explore next-generation PMO chimeras with other therapeutically proven oligonucleotide backbones. Herein, we demonstrate a streamlined 5′ → 3′phosphoramidite approach for the synthesis of PMOs using tert-butyl-protected 5′-^tBu-morpholino phosphoramidites, which were synthesized from 5′–OH morpholino monomers derived from commercially available ribonucleosides. 2-Cyanoethyl (CE)-protected 5′-CE-morpholino phosphoramidites were also synthesized to generate thiophosphoramidate (TMO) and phosphoramidate (MO) morpholino oligonucleotides. Full-length PMOs and TMOs in exceptional overall yields were obtained with operational simplicity and compatibility with automated DNA/RNA synthesizers utilizing controlled pore glass (CPG) as the solid support and CH₃CN as the solvent. Importantly, this method has opened the possibility of designing various biologically relevant ASO design modalities, such as PMO-TMO and PMO-MO, which were inaccessible otherwise. Moreover, DNA nucleotides were also incorporated to generate PMO-psDNA and PMO–DNA using commercially available 5′-DNA phosphoramidites. The biophysical properties of all synthesized oligonucleotides were analyzed using UV melting and circular dichroism studies. The serum interaction profile and innate immune response of the PS-modified polythymidine oligonucleotides were analyzed and it was found that the mixed backbone oligonucleotides had a balanced profile compared to fully charged or neutral extremities. Overall, the synthesis is amenable to fast generation of various types of PMO chimeras for biological screening, which will expedite their therapeutic exploration and transition to clinic.

中文翻译：

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用于合成吗啉代寡核苷酸和多种嵌合骨架的统一亚磷酰胺平台


磷酸二酰胺吗啉代寡核苷酸 （PMO） 在过去十年中已在 FDA 批准的基于寡核苷酸的药物环境中得到广泛应用。鉴于它们在反义疗法中的相关性，人们期待与 DNA/RNA 合成器兼容的 PMO 模块化合成方案，以探索具有其他经治疗验证的寡核苷酸骨架的下一代 PMO 嵌合体。在此，我们展示了一种简化的 5' → 3' 亚磷酰胺方法，用于使用叔丁基保护的 5'-^tBu-吗啉亚磷酰胺合成 PMO，这些亚磷酰胺是由市售核糖核苷衍生的 5'-OH 吗啉单体合成的。还合成了 2-氰乙基 （CE） 保护的 5′-CE-吗啉代亚磷酰胺，以生成硫代磷酰胺 （TMO） 和亚磷酰胺 （MO） 吗啉代寡核苷酸。获得总产率极高的全长 PMO 和 TMO，操作简单，并与使用受控孔玻璃 （CPG） 作为固体支持物和 CH₃CN 作为溶剂的自动化 DNA/RNA 合成仪兼容。重要的是，这种方法为设计各种生物学相关的 ASO 设计模式提供了可能性，例如 PMO-TMO 和 PMO-MO，而这些模式是无法通过其他方式获得的。此外，还掺入了 DNA 核苷酸，以使用市售的 5′-DNA 亚磷酰胺生成 PMO-psDNA 和 PMO-DNA。使用 UV 熔解和圆二色性研究分析所有合成寡核苷酸的生物物理性质。 分析 PS 修饰的聚胸苷寡核苷酸的血清相互作用谱和先天免疫反应，发现与完全充电或中性肢体相比，混合骨架寡核苷酸具有平衡谱。总体而言，该合成适用于快速生成用于生物筛选的各种类型的 PMO 嵌合体，这将加快它们的治疗探索和向临床的过渡。