Novel N7-Arylmethyl Substituted Dinucleotide mRNA 5′ cap Analogs: Synthesis and Evaluation as Modulators of Translation

Abstract

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.

Cap analogs with a hydrophobic photocleavable tag enable facile purification of fully capped mRNA with various cap structures.

“…Synthesis of functional cap analogs that can create labeled translatable mRNA by introducing fluorescent groups or biotin into the 2' and 3' hydroxyl groups of m 7 G was also reported 61,62 . The introduction of a benzyl group at the N 2 position of m 7 G or the substitution of a 7-methyl group with an arylmethyl group was reported to enhance the translational activity of mRNA in human cultured cells 54,63,64 . Some cap analogs have been developed for on/off control of the mRNA translation 30,65,66,67 .…”

Substrate-Based Design of Cytosolic Nucleotidase IIIB Inhibitors and Structural Insights into Inhibition Mechanism

“…The N7-benzyl-substituted GMP analogs were synthesized according to a procedure previously described [ 28 ]. Triethylammonium salt of guanosine 5’-monophosphate (1 equiv.)…”

The Pivotal Role of Chemical Modifications in mRNA Therapeutics

“…A slew of other analogs have been explored as well, including phosphorothiolate ( Wojtczak et al, 2018 ), phosphoroselenoate ( Kowalska et al, 2009 ), boranophosphate ( Kowalska et al, 2014 ), imidodiphosphate modified caps ( Rydzik et al, 2012 ), etc. ( Warminski et al, 2013 ; Shanmugasundaram et al, 2016 ; Dülmen et al, 2021 ; Wojcik et al, 2021 ) Locked nucleic acid (LNA) caps have also been investigated, in which the ribose is locked in an C3′-endo conformation by a bridging methylene group between the 2′ oxygen and 4′ carbon ( Kore et al, 2009 ). Although LNAs have primarily been used in oligonucleotides, mRNAs capped by an LNA analog have recently been demonstrated to have increased translational efficiency and stability ( Senthilvelan et al, 2021 ).…”