Chimeric RNA Oligonucleotides with Triazole and Phosphate Linkages: Synthesis and RNA Interference

Abstract

Chimeric RNA oligonucleotides with an artificial triazole linker were synthesized using solution-phase click chemistry and solid-phase automated synthesis. Scalable synthesis methods for jointing units for the chimeric structure have been developed, and after click-coupling of the jointing units with triazole linkers, a series of chimeric oligonucleotides was prepared by utilizing the well-established phosphoramidite method for the elongation. The series of chimeric 21-mer oligonucleotides that possessed the triazole linker at different strands and positions allowed for a screening study of the RNA interference to clarify the preference of the triazole modifications in small-interfering RNA molecules.

Modified Nucleosides, Nucleotides and Nucleic Acids via Click Azide-Alkyne Cycloaddition for Pharmacological Applications

“…Interestingly, the TL DNA formed a stable double strand with a natural complementary DNA strand, paving the way for a new class of oligonucleotide analogues with inertness toward nuclease hydrolysis. More recently, a series of chimeric 21-mer RNA oligonucleotides that possessed the triazole linker at different positions were synthesized using solution-phase click-chemistry and solid-phase automated synthesis ( Scheme 17 ) [ 59 ]. Two different jointing units ( 71 and 72 ) were prepared and coupled in solution phase by a copper-catalyzed cycloaddition reaction to produce the triazole-linked dinucleotides UtU , CtU and CtC subsequently converted into the corresponding phosphoroamidites with moderate to good yields for the two steps (77–87%).…”

Chimeric RNA Oligonucleotides Incorporating Triazole-Linked Trinucleotides: Synthesis and Function as mRNA in Cell-Free Translation Reactions

“…The synthesis routes for monomer units (4, 5, 6) from D-xylose were previously established, 9 and we prepared pyrimidine TL RNA dinucleotides using a combination of the pyrimidinebearing 5′-and 3′-jointing units (4 and 5) from the previous study for siRNA. 9 The next steps for synthetic developments involved: (1) inclusion of purine nucleobases and (2) inclusion of elongation units (6). We thus investigated the appropriate reaction conditions to combine all three monomer units and explored a synthetic route to utilize the phosphoramidite protocol for chimeric assembly.…”

“…The TL RNA dinucleotides were then furnished with 5′jointing units. Although a protocol for similar coupling reactions has been investigated in our previous studies, 9 we faced the issue of reproducibility of the synthesis. For instance, the yields of 12UUC from a coupling reaction between 11UC and 4U varied from 51% to 99% (Table 2).…”

“…The Journal of Organic Chemistry solid-phase synthesis on an automated synthesizer (M-2-MX, Nihon Techno Service). 9 After elongation of a 6-mer RNA oligonucleotide on CPG beads from the 3′-end, the TL RNA trinucleotide 13UUC was incorporated, and subsequent elongation of four nucleotides afforded the target chimeric RNA oligonucleotide 19. The yield was determined by a standard protocol to quantify the removed trityl cation from 17 using a UV−vis spectrometer, which showed a total yield of 60% for the 10 elongation reactions.…”

“…We thus investigated chimeric RNA oligonucleotides to combine triazole linkages with phosphate linkages and developed a method to introduce a single triazole linker in chimeric RNA oligonucleotides ( 3 , Figure ). This method assembled a 5′-jointing unit ( 4 , with 5′-OH and 3′-azide) and a 3′-jointing unit ( 5 , with 5′-alkyne and 3′-OH) by solution-phase click synthesis and utilized a phosphoramidite protocol to synthesize chimeric RNA oligomers with a single triazole linker. To further expand the scope of the synthesis and the potential applications of the chimeric RNA, we herein explore a synthesis method to introduce multiple triazole linkers in chimeric RNA analogues.…”