開發新型化學修飾反義寡核苷酸以增強基因沉默功效

Abstract

本論文旨在合成具有高立體選擇性、高反應性並適用五價磷 (P(V)) 固相合成的反義寡核苷酸 (antisense oligonucleotides, ASO) 建構單元 (building blocks)，以提升固相核酸合成的反應效率與鏡像純度。其中，ASO可專一且有效的與目標信使核糖核酸 (messenger RNA, mRNA) 結合，並透過數種不同的機制調節目標蛋白質的表現活性，近期主要被應用於RNA層級來治療罕見遺傳疾病、免疫疾病與部分癌症等，屬於近年興起的新穎藥物開發領域。為了提升ASO專一且有效的結合力並增強ASO對核酸酶 (nuclease) 的耐受性，執行多項結構修飾策略。在天然含氮鹼基源的基礎上，亦引入了在互補配對時可多額外提供一組氫鍵的2-胺基腺嘌呤 (2-aminoadenine, Z) 以及具有免疫逃脫 (immune evasion) 效果的甲基胞嘧啶 (5-methylcytosine, 5MeC) 做為含氮鹼基，同時也在核糖醣體2’位引入甲氧基乙基 (Methoxyethyl, MOE) 取代，亦將磷酸酯 (phosphate, PO) 骨架更換為磷硫酯 (phosphorothioate, PS) 骨架。 根據各含氮鹼基的結構相似性與反應性，可將其分為四類。第一類為胸腺嘧啶 (T) 與尿嘧啶 (U) 作為含氮鹼基；當醣體為核醣骨架時，可利用核醣作為起始物，先對其四個羥基 (hydroxyl group) 進行乙醯基 (Acetyl, Ac) 保護，隨後與三甲基矽 (trimethylsilyl, TMS) 活化的含氮鹼基進行反應來建立醣苷鍵，接著去除乙醯基保護，再利用分子內脫水合環反應 (intramolecular dehydration ring closure reaction) 與路易士酸促進開環取代反應 (Lewis acid-promoted ring-opening substitution reaction)，於核醣2’位引入與天然核醣立體組態一致的MOE取代，隨後對其5’位進行二甲氧基三苯基甲基 (dimethoxytrityl, DMTr) 保護，並在3’位引入掌性輔助基團 (chiral auxiliary)，即可得對應建構單元；而在以去氧核醣為骨架的例子中，僅需引入5’位DMTr保護與3’掌性輔助基團即可得對應建構單元。第二類則是以胞嘧啶 (C) 與5-甲基胞嘧啶 (5MeC) 作為含氮鹼基時，不論是核醣骨架或是去氧核醣骨架，皆可將已進行5’位DMTr保護的尿嘧啶或胸腺嘧啶的化合物直接進行芳香環親核取代反應 (nucleophilic aromatic substitution reaction，SNAr)，將其含氮鹼基上的氧元素置換為氮元素，隨後對氮元素進行苯甲醯基 (benzoyl, Bz) 保護後，於3’位引入掌性輔助基團，即可得對應建構單元。 第三類則為以腺嘌呤 (A) 與2-胺基腺嘌呤 (Z) 為含氮鹼基；當醣體為核醣骨架時，同樣可利用核醣作為起始物，先對其四個羥基進行Ac保護，隨後與三甲基矽活化的6-氯嘌呤 (6-chloropurine, 6-C-P) 或6-氯鳥糞嘌呤 (6-Chloroguanosine, 6-C-G) 進行反應來建立醣苷鍵，接著再將6-氯元素取代為6-氮元素，隨後於核醣2’位引入MOE取代，接著對含氮鹼基的氮元素進行Bz保護後，再對其5’位進行DMTr保護，隨後於3’位引入掌性輔助基團，即可得對應建構單元；而在去氧核醣為骨架的例子中，僅需直接引入含氮鹼基氮的Bz保護基與核醣5’位保護基，隨後於3’建立掌性輔助基團即可得對應建構單元。最後一類為以鳥糞嘌呤作為含氮鹼基時，不論是核醣骨架或是去氧核醣骨架，皆能利用市售可得的起始物，直接對其核醣5’位進行DMTr保護，並於3’位引入掌性輔助基團，即可得對應建構單元。 綜合上述，成功地建立一套ASO的建構單元合成平台，後續研究者可利用此平台配合目標序列進行客製化的組裝，製備高專一性的ASO藥物分子，實現更高度的精準靶向治療。此平台的建立亦可回應我國邁入超高齡社會所面臨的重大醫療挑戰，其服務範圍涵蓋多項神經退化性疾病，如家族性澱粉樣多發性神經病變與帕金森氏症，以及各類罕見疾病，期能做出實質社會貢獻，進一步增進全民健康福祉。

This thesis aims to develop a synthetic platform for constructing antisense oligonucleotide (ASO) building blocks with high stereoselectivity, enhanced reactivity, and compatibility with phosphorus(V)-based (P(V)-based) solid-phase synthesis. These building blocks are designed to improve the reaction efficiency and diastereomeric purity in oligonucleotide assembly. ASOs specifically and effectively hybridize with target messenger RNA (mRNA) and regulate gene expression through various mechanisms. In recent years, ASOs have gained recognition as a promising class of RNA therapeutics, with expanding applications in the treatment of rare genetic disorders, immune-related diseases, and certain cancers. Several structural modifications were introduced to enhance ASO's binding specificity and nuclease resistance. These include the incorporation of 2-aminoadenine (Z), which provides an additional hydrogen bond within Watson-Crick base pairing, and 5-methylcytosine (5MeC), a nucleobase associated with immune evasion. Furthermore, a 2'-O-methoxyethyl (MOE) group was introduced at the sugar moiety to enhance duplex stability by raising the melting temperature (Tₘ), and the phosphate backbone was substituted with a phosphorothioate (PS) linkage to improve nuclease resistance. The building blocks were categorized into four types based on nucleobase structure and reactivity. The first category includes thymine (T) and uracil (U) derivatives. Utilizing ribose as the sugar scaffold, all four hydroxyl groups were protected with acetyl (Ac) groups prior to glycosylation with trimethylsilyl (TMS)-activated nucleobases. Following deprotection, an intramolecular dehydration ring-closure reaction and a Lewis acid-promoted ring-opening substitution were employed to introduce 2'-OMOE in the natural ribo-configuration. Subsequently, 5'-dimethoxytrityl (DMTr) protection and 3'-chiral auxiliary installation yielded the final building blocks. In the case of deoxyribose scaffolds, only 5'-DMTr protection and 3'-auxiliary incorporation were required. The second category encompasses cytosine (C) and 5MeC nucleobases. In both ribose and deoxyribose series, the corresponding uracil or thymine intermediates bearing 5'-DMTr were subjected to nucleophilic aromatic substitution (SNAr) to facilitate oxygen-to-nitrogen replacement, followed by benzoyl (Bz) protection and 3'-chiral auxiliary installation, resulting in the corresponding final building blocks. The third category includes adenine (A) and 2-aminoadenine (Z). Ribose derivatives were prepared by Ac-protecting ribose, followed by glycosylation with 6-chloropurine (6-C-P) or 6-chloroguanosine (6-C-G). Subsequently, SNAr reaction at the C6 position with an amino group was followed by MOE introduction at the 2'-position, N-benzoylation, 5'-DMTr protection, and 3'-chiral auxiliary attachment. For deoxyribose synthesis, the process necessitated only N-benzoylation protection, 5'-DMTr protection, and 3'-auxiliary installation, which were needed in the deoxyribose series. The fourth category comprises guanine derivatives, for which ribose and deoxyribose scaffolds were derived from commercially available starting materials. These were converted to the desired building blocks via standard 5'-DMTr protection and 3'-auxiliary attachment. In conclusion, a modular synthesis platform for constructing ASOs has been successfully established. This platform enables subsequent researchers to assemble highly sequence-specific ASO drug molecules in a customizable manner according to target sequences, thereby facilitating the development of more precise and effective targeted therapies. The establishment of this platform also addresses critical healthcare challenges posed by Taiwan’s rapidly aging society. Its applications encompass a range of neurodegenerative diseases, such as familial amyloid polyneuropathy and Parkinson’s disease, as well as various rare diseases. It is anticipated that this platform will contribute meaningfully to society by advancing public health and improving overall well-being.