Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations

Longhini, Andrew P.; LeBlanc, Regan M.; Becette, Owen; Salguero, Carolina; Wunderlich, Christoph H.; Johnson, Bruce A.; D'Souza, Victoria M.; Kreutz, Christoph; Dayie, T. Kwaku

We have created an improved method for the synthesis of site-selective isotopically labeled ATP and GTP with increased yields and speed of synthesis. Both purine reactions proceed to completion without the need to purify intermediate species. Final yields of >90% and >75% respectively for ATP and GTP were achieved relative to starting input adenine or guanine. Both yields are better than previously reported (72) (73) (74) (75) (76) (77) . In addition to improved yields, ATP synthesis is complete in 4-5 h while GTP synthesis is complete in 7-8 h. Previously, ATP synthesis was reported to take 29-48 h and GTP synthesis 48-70 h (72-77). These improvements allow reactions to be complete in a single day. Additionally we have taken advantage of the ability of creatine kinase to act on a variety of substrates to convert NDPs to NTPs and adapted the use of dATP as the energy source in the energy regeneration system (77) (78) (79) . The use of dATP is ideal since the lack of a 2 -OH of the ribose in dATP prevents its interaction with the boronate column used to purify ATP and GTP. This offers a more robust synthesis, free of contaminants, and does not dilute the synthesized labels with unlabeled ATP. The effectiveness of these nucleotides is demonstrated by their incorporation into a number of interesting RNAs.