5′‐O‐(4,4′‐Dimethoxytrityl)thymidine was attached to a pentaerythrityl‐derived core, and the resulting tetravalent nucleoside cluster and the next dendritic generations served as a soluble support for the synthesis of short oligo‐2′‐deoxyribonucleotides in solution. Couplings using a small excess (1.5 equiv. per 5′‐OH group) of the standard phosphoramidite building blocks proved efficient, and the products could be purified by quantitative precipitation from methanol. Ammonolysis released nearly homogeneous oligonucleotides (CCT, GCT, ACT, and AGCCT) in high yields.

Therapeutic oligonucleotides have emerged as a powerful paradigm with the ability to treat a wide range of the human diseases. As a result, we have witnessed more than one hundred oligonucleotides currently in active clinical trials and eight Food and Drug Administration (FDA)‐approved drugs. Until now, the demand for oligonucleotide‐based drugs has been fulfilled by conventional solid‐phase synthesis in an effective manner. However, there are products in advanced stages of clinical trials projecting a collective demand of metric ton quantities in the near future. Therefore, large‐scale manufacturing of these products has become a high priority for process chemists. This article summarizes the advances in liquid‐phase oligonucleotide synthesis (LPOS) as a possible alternative strategy to meet the scale‐up challenge. A review of the literature describing major efforts in developing LPOS technologies is presented. Gratifyingly, serious attempts are under way to develop an efficient environmentally benign green chemistry protocol that is scalable and cost effective for the manufacturing of oligonucleotides. A summary of the most innovative LPOS protocols has been included to provide a glimpse of what may be possible in the future for large‐scale production of oligonucleotides. © 2019 by John Wiley & Sons, Inc.

Novel soluble supports for oligonucleotide synthesis 11a–c have been prepared by immobilizing a 5′-O-protected 3′-O-(hex-5-ynoyl)thymidine (6 or 7) to peracetylated or permethylated 6-deoxy-6-azido-β-cyclodextrins 10a or 10b by Cu(I)-promoted 1,3-dipolar cycloaddition. The applicability of the supports to oligonucleotide synthesis by the phosphoramidite strategy has been demonstrated by assembling a 3′-TTT-5′ trimer from commercially available 5′-O-(4,4′-dimethoxytrityl)thymidine 3′-phosphoramidite. To simplify the coupling cycle, the 5′-O-(4,4′-dimethoxytrityl) protecting group has been replaced with an acetal that upon acidolytic removal yields volatile products. For this purpose, 5′-O-(1-methoxy-1-methylethyl)-protected 3′-(2-cyanoethyl-N,N-diisopropyl-phosphoramidite)s of thymidine (5a), N4-benzoyl-2′-deoxycytidine (5b) and N6-benzoyl-2′-deoxyadenosine (5c) have been synthesized and utilized in synthesis of a pentameric oligonucleotide 3′-TTCAT-5′ on the permethylated cyclodextrin support 11c.

SummaryAn effective method for the synthesis of short oligoribonucleotides in solution has been elaborated. Novel 2'-O-(2-cyanoethyl)-5'-O-(1-methoxy-1-methylethyl) protected ribonucleoside 3'-phosphoramidites have been prepared and their usefulness as building blocks in RNA synthesis on a soluble support has been demonstrated. As a proof of concept, a pentameric oligoribonucleotide, 3'-UUGCA-5', has been prepared on a precipitative tetrapodal tetrakis(4-azidomethylphenyl)pentaerythritol support. The 3'-terminal nucleoside was coupled to the support as a 3'-O-(4-pentynoyl) derivative by Cu(I) promoted 1,3-dipolar cycloaddition. Couplings were carried out with 1.5 equiv of the building block. In each coupling cycle, the small molecular reagents and byproducts were removed by two quantitative precipitations from MeOH, one after oxidation and the second after the 5'-deprotection. After completion of the chain assembly, treatment with triethylamine, ammonia and TBAF released the pentamer in high yields.

SummaryThe preparation of a disulfide-tethered precipitative soluble support and its use for solution-phase synthesis of trimeric oligodeoxyribonucleotide 3´-(2-chlorophenylphosphate) building blocks is described. To obtain the building blocks, N-acyl protected 2´-deoxy-5´-O-(4,4´-dimethoxytrityl)ribonucleosides were phosphorylated with bis(benzotriazol-1-yl) 2-chlorophenyl phosphate. The “outdated” phosphotriester strategy, based on coupling of PV building blocks in conjunction with quantitative precipitation of the oligodeoxyribonucleotide with MeOH is applied. Subsequent release of the resulting phosphate and base-protected oligodeoxyribonucleotide trimer 3’-pTpdCBzpdGibu-5’ as its 3’-(2-chlorophenyl phosphate) was achieved by reductive cleavage of the disulfide bond.