Preparation ofanalogues ofNAD+assubstrates forasensitive fluorimetric assay ofnucleotide pyrophosphatase

synthesized,characterizedandtestedassubstratesforafluorimetricassayofnucleotidepyrophos-phatase(EC3.6.1.9). Uponcleavageoftheirpyrophosphatebond,thefluorescenceofpyridine-1,N6-ethenoadenine dinucleotide (ePdAD+) and of4-hydrazinocarbonyl-pyridine-1,N6-ethenoadenine dinucleotide (shy4PdAD+)increased respectively 15-and 73-fold, at pH7.4. This property allows a convenient steady-state assay ofnucleotide pyrophosphatase by continuous monitoring of reaction progress. Bothcompoundsweregoodsubstratesofthisclassofenzyme.Therelativeinsensitivity ofthe fluorescence ofePdAD+ and shy4PdAD+ to pHchanges allowed assays underconditions preserving cellular integrity. ePdAD