Labeled adenosine(5′)tetraphospho(5′)adenosine (Ap 4A) and adenosine(5′)tetraphospho(5′)nucleoside (Ap 4N). Synthesis with firefly luciferase

Labeled dinucleoside polyphosphates are not commercially available, in spite of being important molecules in metabolic regulation. Firefly luciferase (EC 1.13.12.7) is a useful enzyme for the synthesis of adenosine(5′)tetraphospho(5′)adenosine (Ap 4A). As luciferase behaves as a nucleotidase at low ATP concentration, adequate concentrations (higher than 0.1 mM ATP) should be used to obtain a good yield of labeled Ap 4A. [ 32P]Ap 4A has also been synthesized from ATP and [ 32P]PP i. In a first step, [β,γ- 32P]ATP is generated in a ATP-[ 32P]PP i exchange reaction catalyzed by luciferase. In a second step, the reaction is supplemented with pyrophosphatase and 32P labeled Ap 4A is obtained. Radioactive adenosine(5′)tetraphospho(5′)nucleoside (Ap 4N) can also be synthesized from ATPγS and labeled NTP or from low concentrations of labeled ATP and high concentrations of cold NTP. The syntheses of radioactive Ap nA and p nA ( n > 4) can also be approached with luciferase.