The interest in multi-enzyme cascades for the synthesis of pharmaceutically relevant active ingredients has increased in recent years. Through a smart selection of enzymes, cascades enable multi-step synthesis in a one-pot reaction without the purification of intermediates. In this study, a five-enzyme cascade for the formation of cyclic 2′3′-GMP-AMP (2′3′-cGAMP) from adenosine and guanosine in seven reaction steps was successfully developed. First, the substrate scope of kinases for the phosphorylation of nucleosides and nucleotides was investigated, which were then combined in an enzyme cascade for 2′3′-cGAMP formation from adenosine, guanosine, and polyphosphate. An overall conversion of 57% of the substrates into 2′3′-cGAMP was achieved in relation to the initial guanosine concentration.
