Dinucleotide analogs of the messenger RNA cap (m7GpppN) are useful research tools and have potential applications as translational inhibitors or reagents for modification of in vitro transcribed mRNAs. It has been previously reported that replacing the methyl group at the N7-position with benzyl (Bn) produces a dinucleotide cap with superior properties. Here, we followed up on this finding by synthesizing 17 novel Bn7GpppG analogs and determining their structure–activity relationship regarding translation and translational inhibition. The compounds were prepared in two steps, including selective N7-alkylation of guanosine 5′-monophosphate by arylmethyl bromide followed by coupling with imidazole-activated GDP, with total yields varying from 22% to 62%. The compounds were then evaluated by determining their affinity for eukaryotic translation initiation factor 4E (eIF4E), testing their susceptibility to decapping pyrophosphatase, DcpS—which is most likely the major cellular enzyme targeting this type of compound—and determining their translation inhibitory properties in vitro. We also synthesized mRNAs capped with the evaluated compounds and tested their translational properties in A549 cells. Our studies identified N7-(4-halogenbenzyl) substituents as promising modifications in the contexts of either mRNA translation or translational inhibition. Finally, to gain more insight into the consequences at the molecular level of N7-benzylation of the mRNA cap, we determined the crystal structures of three compounds with eIF4E.

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.

Cytosolic nucleotidases (cNs) catalyze dephosphorylation of nucleoside 5’-monophosphates and thereby contribute to the regulation of nucleotide levels in cells. cNs have also been shown to dephosphorylate several therapeutically relevant nucleotide analogues. cN-IIIB has shown in vitro a distinctive activity towards 7-mehtylguanosine monophosphate (m7GMP), which is one key metabolites of mRNA cap. Consequently, it has been proposed that cN-IIIB participates in mRNA cap turnover and prevents undesired accumulation and salvage of m7GMP. Here, we sought to develop molecular tools enabling more advanced studies on the cellular role of cN-IIIB. To that end, we performed substrate and inhibitor property profiling using a library of 41 substrate analogs. The most potent hit compounds (identified among m7GMP analogs) were used as a starting point for structure–activity relationship studies. As a result, we identified several 7-benzylguanosine 5’-monophosphate (Bn7GMP) derivatives as potent, unhydrolyzable cN-IIIB inhibitors. The mechanism of inhibition was elucidated using X-ray crystallography and molecular docking. Finally, we showed that compounds that potently inhibit recombinant cN-IIIB have the ability to inhibit m7GMP decay in cell lysates.

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