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

The production of GTP was achieved in a two-step, one pot reaction. Specifically-labeled ribose and guanine were combined in the presence of phosphoribosyl pyrophosphate synthetase (PRPPS), ribokinase (RK), and xanthine-guanine phosphoribosyl transferase (XGPRT), with a dATP regeneration system. The dATP regeneration system was composed of myokinase and creatine kinase, with creatine phosphate acting as the high energy phosphate donor. The formation of GMP was monitored by FPLC and NMR (Supplementary Figure S2) . However, due to the low solubility of guanine (0.01 mM) FPLC was unsuitable to track its disappearance, thus making it difficult to monitor the progression of the reaction. However, by NMR spectroscopy, the resonance chemical shift between the labeled 13 C-1 position of unreacted ribose and newly formed GMP was used to determine the completion of the first step of the reaction (Supplementary Figure S2A) . When the majority e52 Nucleic Acids Research, 2016, Vol. 44, No. 6 PAGE 4 OF 10 of guanine was converted to GMP, in approximately 4-5 h, guanylate kinase was added to the reaction. Guanylate kinase phosphorylates GMP to GDP. GDP is phosphorylated to GTP by creatine kinase which acts promiscuously to convert NDPs to NTPs. This was unexpected, as CK is said to be highly specific (78, 79) . GMP is completely converted to GTP in an additional 3 h. We confirmed by FPLC that conversion is complete and further validate this observation by 31 P NMR (Supplementary Figure S2B) .