Quick Access to Nucleobase-Modified Phosphoramidites for the Synthesis of Oligoribonucleotides Containing Post-Transcriptional Modifications and Epitranscriptomic Marks

Abstract

Herein, we report a straightforward one-step procedure for modifying N -nucleophilic groups in the nucleobases of commercially available nucleoside phosphoramidites. This method involves the deprotonation of amide groups under phase-transfer conditions and subsequent reaction with electrophilic molecules such as alkyl halides or organic isocyanates. Using this approach, we obtained 10 different classes of modified nucleoside phosphoramidites suitable for the synthesis of oligonucleotides, including several noncanonical nucleotides found in natural RNA or DNA (e.g., m 6 A, i 6 A, m 1 A, g 6 A, m 3 C, m 4 C, m 3 U, m 1 G, and m 2 G). Such modification of nucleobases is a common mechanism for post-transcriptional regulation of RNA stability and translational activity in various organisms. To better understand this process, relevant cellular recognition partners (e.g., proteins) must be identified and characterized. However, this step has been impeded by limited access to molecular tools containing such modified nucleotides.

Chemical Synthesis of Modified RNA

“…Notably, rapid access to methylated nucleoside phosphoramidites (e.g., m 6 A, m 1 A, m 3 C, m 4 C, m 3 U, m 1 G, and m 2 G) has been reported by directly transforming commercially available standard nucleoside phosphoramidites in a one-step procedure to modify N-nucleophilic groups in their nucleobases using phase-transfer catalysis. [64] Finally, we note that-to the best of our knowledge-no phosphoramidite building block for 7-methylguanosine has been described in the literature, probably due to the inherent instability of m 7 G under and basic and also under acidic conditions, the latter seems incompatible with the detritylation step of the RNA solid-phase elongation cycle. [65]…”

Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo

“…The synthetic pathway leading to trinucleotide cap analogue 1 included the solid-phase synthesis of dinucleotide 5′-monophosphate 3 , followed by its activation into P -imidazolide 4 and ZnCl 2 -mediated coupling reaction with 7-methylguanosine 5′-diphosphate (m 7 GDP) in solution (Scheme ). The N 6-benzyladenosine phosphoramidite for solid-phase synthesis was prepared by one-step alkylation of commercially available N 6-phenoxyacetyl-2′- O -methyladenosine phosphoramidite in the presence of a base and phase-transfer catalyst. , The dinucleotide 5′-phosphate 3 was cleaved from the solid support, deprotected using standard protocols, and then isolated by ion-exchange chromatography on DEAE Sephadex to give a triethylammonium salt suitable for further activation. The P -imidazolide 4 was prepared as described previously for mononucleotides, precipitated as a sodium salt and reacted with m 7 GDP in the presence of excess ZnCl 2 to give trinucleotide cap analogue 1 .…”

Trinucleotide mRNA cap analog N6-benzylated at the site of posttranscriptionalm6Am mark facilitates mRNA purification and confers superior translational properties in vitro and in vivo

“…The N 6- benzyladenosine phosphoramidite for solid-phase synthesis was prepared by one-step alkylation of commercially available N 6-phenoxyacetyl-2′- O -methyladenosine phosphoramidite in the presence of a base and phase-transfer catalyst. 17, 36 The dinucleotide 5′-phosphate 3 was cleaved from the solid support and deprotected using standard protocols and then isolated by ion-exchange chromatography on DEAE Sephadex to give a triethylammonium salt suitable for further activation. The P -imidazolide 4 was prepared as described previously for mononucleotides, 37 precipitated as a sodium salt and reacted with m 7 GDP in the presence of excess ZnCl2 to give trinucleotide cap analog 1 .…”