Recent Advances in Modified Cap Analogs: Synthesis, Biochemical Properties, and mRNA Based Vaccines

Abstract

The recent FDA approval of the mRNA vaccine for severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) emphasizes the importance of mRNA as a powerful tool for therapeutic applications. The chemically modified mRNA cap analogs contain a unique cap structure, m 7 G[5′]ppp[5′]N (where N=G, A, C or U), present at the 5′‐end of many eukaryotic cellular and viral RNAs and several non‐coding RNAs. The chemical modifications on cap analog influence orientation's nature, translational efficiency, nuclear stability, and binding affinity. The recent invention of a trinucleotide cap analog provides groundbreaking research in the area of mRNA analogs. Notably, trinucleotide cap analogs outweigh dinucleotide cap analogs in terms of capping efficiency and translational properties. This review focuses on the recent development in the synthesis of various dinucleotide cap analogs such as dinucleotide containing a triazole moiety, phosphorothiolate cap, biotinylated cap, cap analog containing N1 modification, cap analog containing N2 modification, dinucleotide containing fluorescence probe and TAT, bacterial caps, and trinucleotide cap analogs. In addition, the biological applications of these novel cap analogs are delineated.

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

“…Various cap analogs with different structural features have been developed to prepare capped mRNAs by IVT 18 . First to mention are the dinucleotide cap analogs ARCAs 20,21,37,54,56 , which were designed to control the direction of introduction of the dinucleotide cap analog m 7 G(5')ppp(5')G. It was reported that methylation of the 2' or 3' hydroxyl group completely controlled the direction of strand elongation, resulting in an approximately 2-3-fold increase in mRNA translation activity in cultured cells 54,56 .…”

“…The key step in synthesizing cap analogs is synthesizing the corresponding diphosphate by phosphorylation of guanosine derivatives. For the synthesis of cap analogs known as the ARCA derivatives, protocols consisting of monophosphorylation by the Yoshikawa method using phosphoryl chloride, activation of monophosphate thorough phosphorimidazolide formation, and diphosphate formation by reaction with alkylammonium phosphate were applied 18 . However, this method is complicated because it is based on the stepwise introduction of phosphate groups and requires multiple purifications with aqueous solvents, such as ion exchange and reversed-phase chromatography.…”

“…In the enzymatic capping, transcribed mRNA is treated with a capping enzyme 5,17 , often followed by methyltransferase treatment to convert the Cap-0 structure to the Cap-1 5 . In co-transcriptional capping, a synthetic cap analog, i.e., m 7 G-containing nucleotide, is added to the in vitro transcription (IVT) reaction 18,19 . The first generation dinucleotide cap analog m 7 G(5')ppp(5')G had the problem of the chain-elongation starting from the 3' hydroxyl group of the m 7 G. The unintended elongation results in 30-50% of the reversely capped RNA inactive for translation 19 .…”

Efficient and Improved Solution-Phase Synthesis of Modified RNA Dinucleotides: Versatile Synthons in Cap 1 mRNA Therapeutics

“…9 It is well known in the literature that the chemically modified capped mRNA plays a major role in terms of binding affinity, orientation's nature, nuclear stability, and translational efficiency. 3−7 The employment of the synthetic m 7 GpppNderived dinucleotide cap analogue lacks the 2′-OMe moiety at the N2 nucleotide, whereas synthetic trinucleotide cap analogues have the built-in 2′-OMe moiety at the N2 nucleotide, and the resulting in vitro transcribed mRNAs using these two analogues will have a Cap 1 structure. 7 Extraordinarily, the trinucleotide cap analogue outclasses the dinucleotide cap analogue in terms of capping efficiency and translational properties.…”

“…3−7 The employment of the synthetic m 7 GpppNderived dinucleotide cap analogue lacks the 2′-OMe moiety at the N2 nucleotide, whereas synthetic trinucleotide cap analogues have the built-in 2′-OMe moiety at the N2 nucleotide, and the resulting in vitro transcribed mRNAs using these two analogues will have a Cap 1 structure. 7 Extraordinarily, the trinucleotide cap analogue outclasses the dinucleotide cap analogue in terms of capping efficiency and translational properties. 10,11 Additionally, the presence of the 2′-OMe group in cap 1 has the strong ability to distinguish between self RNA in the innate immune system and nonself RNA that assists to effectively suppress pathogenesis and viral replication.…”