Synthesis of 4′‐Thiomodified c‐di‐AMP Analogs

Abstract

AbstractCyclic diadenosine monophosphate (c‐di‐AMP) is a bacterial cyclic dinucleotide (CDN) comprising two adenosine monophosphates covalently linked by two 3′,5′‐phosphodiester bonds. c‐di‐AMP works as a second messenger, regulating many biological processes in bacteria such as cell wall homeostasis, DNA integrity, and sporulation via specific protein and/or RNA receptors. Moreover, c‐di‐AMP can function as an immunomodulatory agent in eukaryote cells via the stimulator of interferon genes (STING) signaling pathway.This protocol describes the chemical synthesis of two c‐di‐AMP analogs with a sulfur atom at the 4′‐position of the furanose ring instead of an oxygen atom: c‐di‐4′‐thioAMP (1) and cAMP‐4′‐thioAMP (2). Analogs 1 and 2 have resistance to phosphodiesterase‐mediated degradation and are therefore useful for understanding the diverse biological phenomena regulated by c‐di‐AMP. In this protocol, two 4′‐thioadenosine monomers are initially prepared via a Pummerer‐like reaction assisted by hypervalent iodine. The CDN skeleton is then constructed through two key reactions based on phosphoramidite chemistry: dimerization of two appropriately protected nucleoside monomers to produce a linear dinucleotide, followed by macrocyclization of the resulting linear dinucleotide to form the CDN skeleton. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Preparation of 4′‐thioadenosine monomers 13 and 14Basic Protocol 2: Preparation of c‐di‐4′‐thioAMP (1) and cAMP‐4′‐thioAMP (2)