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

Cyclic 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. (c) 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)