Chemical probes are important tools for investigating molecular processes as they are routinely used to image biological structures, detect specific biomarkers, and explore protein-ligand interactions. Nicotinamide adenine dinucleotide (NAD+), a key cofactor in cellular metabolism, can be enzymatically cleaved into ADP-ribose and its cyclic variants, facilitating post-translational modifications and cell signaling. Cyclic ADP-ribose (cADPR), a well-known secondary messenger in calcium signaling, is produced from NAD+ by ADP-ribosyl cyclases like CD38. Our group has recently identified two novel cADPR variants, 2′cADPR and 3′cADPR, which form O-glycosidic bonds between the two ribose moieties. While 3′cADPR has been recognized as an activator of bacterial anti-phage defence and a modulator of plant immunity, neither the functions of 2′cADPR in bacteria and plants nor the roles of both 2′cADPR and 3′cADPR in humans/animals are well understood. To elucidate these functions, we have designed analogues of 2′cADPR and 3′cADPR and developed chemoenzymatic methods to produce modified NAD+, 2′cADPR, and 3′cADPR. These analogues are functionalized through cross-coupling reactions, enabling the attachment of affinity handles, such as biotin and photoactivable groups. These probes will be used in proteomics studies to identify their protein interaction partners in bacterial, plant, and mammalian systems, shedding light on the biological roles of 2′cADPR and 3′cADPR across kingdoms of life.