2.9.1. In Vitro Phosphorylation

Novel nucleoside analogues were evaluated as a substrate for adenosine kinase (ADK) in an in vitro phosphorylation assay (Precice ADK Assay Kit, NovoCIB, Lyon, France). The ADK-catalyzed phosphorylation reaction of 7-deazaKR and 8-azaKR was run for 30 min at 37 °C in a reaction buffer (100 mM Tris-HCl pH 8.5, 250 mM KCl, 10 mM MgCl2, 2.5 mM NAD, 2.75 mM ATP, IMPDH 20 mU/mL, and human recombinant ADK 2.2 mU/mL). The positive control of reaction efficiency was a phosphorylation reaction with 4 mM KR as a substrate, whereas, to inhibit reaction (negative control), 5-iodotubercidin (ADK inhibitor) was added to a final concentration of 500 µM. Phosphorylated products were separated and identified on fluorescent TLC Silica Gel 60 F254 plates (20 × 20 cm, Merck Millipore, Darmstadt, Germany) by using the following separation phase: ammonia/isopropanol/water (2:7:1 v/v). After resolution, the plates were dried, and the products were detected using the Gel Doc-it Imaging System (UVP, Upland, CA, USA). The phosphorylated products were quantified using Multi Gauge V3.0 (Fujifilm, Tokyo, Japan) software for Windows.