Chemoenzymatic Synthesis of 3-Deoxy-3,4-didehydro-cytidine triphosphate (ddhCTP)

3 & PRIME;-Deoxy-3 & PRIME;,4 & PRIME;-didehydro-cytidinetriphosphate(ddhCTP) is a novel antiviral molecule produced by the enzyme viperinduring the early stages of the innate immune response. ddhCTP hasbeen shown to act as a chain terminator of flavivirus RNA-dependentRNA polymerases. To date, synthesis of ddhCTP requires complicatedsynthetic protocols or isolation of the enzyme viperin to catalyzethe production of ddhCTP from CTP. Recombinant viperin approachespreclude the production of highly pure ddhCTP (free of contaminantssuch as CTP), whereas the chemical synthesis involves techniques orequipment not readily available to most laboratories. Herein, we describethe chemoenzymatic synthesis of ddhCTP, starting from commerciallyavailable ddhC. We utilize these methods to produce milligram quantitiesof ddhCTP, ddhCDP, and ddhCMP. Using purified semisynthetic ddhCTPand fully synthetic ddhCTP, we also show ddhCTP does not inhibit NAD(+)-dependent enzymes such as glyceraldehyde 3-phosphate dehydrogenase,malate dehydrogenase, or lactate dehydrogenase, contrary to a recentreport.