P2Y nucleotide receptors (P2Y-Rs) play important physiological roles. However, most of the P2Y-R subtypes are still lacking potent and selective agonists and antagonists. Based on data mining analysis of binding interactions in 44 protein-uridine nucleos(t)ides complexes, we designed uracil nucleotides, substituted at the C5/C6 position. All C6-substituted derivatives were inactive at the P2Y(2,4,6)-Rs, while out of the C5-substituted analogues, only 5-OMe-UD(T)P showed activity. To rationalize the data, the ionization and conformation of these analogues were evaluated. The pK(a) values of most analogues substituted at the C5/C6 positions were unaltered compared to UTP (pK(a) 9.42), except for 5-F-UTP nucleotide (pK(a) 7.85). C6-substituted analogues adopt the syn or high-syn conformations, which are disfavored by the receptors, while 5-OMe-UD(T)P adopt the favored anti conformation. Furthermore, 5-OMe-UDP adopts the S sugar puckering, which is the conformation preferred by the P2Y(6)-R, but not the P2Y(2)- or P2Y(4)-Rs. 5-OMe-UDP fulfills the conformational and H-bonding requirements of P2Y(6)-R, thus, making a potent P2Y(6)-R agonist (EC(50) 0.08 microM), more than UDP (EC(50) 0.14 microM).