Figure 4.

Enzyme cascade for the synthesis of the cyclic dinucleotide MK-1454. Engineered adenylate kinase (AK) and guanylate kinase (GK) were used to convert the 2′-fluoro modified α-thio-monophosphates to 2′F-thio-ADP and 2′F-thio-GDP, respectively, using 2′F-thio-ATP as a phosphate donor. Acetate kinase (AcK) catalyzed the final phosphorylation step using acetylphosphate to provide the α-thiotriphosphates, which were subsequently coupled using an engineered cyclic guanosine-adenosine synthase (cGAS).