SPECIFIC SYNTHESIS OF ADENOSINE (5')TETRAPHOSPHO(5')NUCLEOSIDE AND ADENOSINE(5')OLIGOPHOSPHO(5')ADENOSINE (N-GREATER-THAN-4) CATALYZED BY FIREFLY LUCIVERASE

Luciferase catalyzes the preferential synthesis of adenosine(5')tetraphospho(5')nucleoside (Ap4N) in the presence of luciferin (LH-2), adenosine 5'-[gamma-thio]triphosphate (ATP[gammaS]) and NTP (other than ATP), with very low, or undetectable synthesis of Ap4A or Np4N, because ATP[gammaS] is a good adenylyl donor for the formation of the E-LH2-AMP complex, but a poor adenylyl acceptor from the complex, and NTP, other than ATP, are bad nucleotidyl donors, but good acceptors of the AMP moiety of the E-LH2-AMP complex. Synthesis of the corresponding Ap4N (or Ap5G in the case of p4G were obtained in the presence of ATP[gammaS] and GTP, UTP, CTP, XTP, dTTP, ITP, dGTP, dCTP, dTTP, epsilonATP (epsilonA, N6-ethenoadenosine) or p4G. The yield of synthesis of Ap4N was at least 50% of that theoretically expected. The process can be easily scaled-up, which allows synthesis of at least 1-5 mumol Ap4N. Further evidence for the synthesis of Ap4G from ATP[gammaS] and GTP was obtained by H-1-NMR and P-31-NMR spectroscopy. Synthesis of ApN, in yields lower than those above, can also be obtained in the presence of ADP and NTP; synthesis is due to the presence in commercial luciferase of enzymes (adenylate kinase and NDP kinase) that catalyze the synthesis of ATP from ADP and NTP. In the presence of ATP and polyphosphates, luciferase catalyzes the synthesis of a variety of compounds of adenosine 5'-polyphosphates (p(n)A; n = 3 - 20 and Ap(n)A; n = 4-16). In the presence of P3 or P4, preferential synthesis of p4A and Ap5A or p5A and ApA were obtained, respectively, showing that both polyphosphates accept the adenylyl moiety of the E-LH2-AMP complex. Polyphosphates of chain length 5, 15 and 35 elicited the synthesis of a variety of P(n)A and Ap(n)A. Ap4A is also split by luciferase in the presence of P3 or P4 (but not in the presence of P5) yielding preferential synthesis Of P4A and Ap5A, or p5A and Ap6A, respectively.