Synthesis and in vitro evaluation of dioxopyrrolopyrroles as potential low-affinity fluorescent Ca2+ indicators

Three new low-affinity fluorescent Ca2+ indicators excitable with visible light, namely 3-phenyl-6-(4-(3-carboxymethoxy-4-(N,N-dicarboxymethylamino) phenyl) phenyl)-2,5-dicarboxymethyl-1,4-dihydropyrrolo[ 3,4- c] pyrrole-1,4-dione (DPP1), 3-phenyl-6-( 5-(3-carboxymethoxy-4-( N, N-dicarboxymethylamino) phenyl) thien-2-yl)-2,5-dicarboxymethyl-1,4- dihydropyrrolo[ 3,4- c] pyrrole-1,4- dione (DPP2) and 3( thien-2-yl)-6-( 5-(3-carboxymethoxy-4-( N, N-dicarboxymethylamino) phenyl) thien-2-yl)-2,5- dicarboxymethyl-1,4- dihydropyrrolo[ 3,4- c] pyrrole-1,4- dione (DPP3) have been synthesized and evaluated for their Ca2+ binding properties via fluorimetric titrations. The in vitro dissociation constant K-d measured at 21degreesC in 100 mM KCl buffered solution, pH 7.05, for the Ca2+ -DPP1 complex is 10 muM; for Ca2+ -DPP2 and Ca2+ - DPP3 a K-d value of 20 muM is found. All three indicators form 1 : 1 complexes with Ca2+. The fluorescence quantum yields of the uncomplexed forms of DPP1, DPP2 and DPP3 are 1.2 x 10(-2), 3.4 x 10(-2) and 3.6 x 10(-2), respectively. After binding to Ca2+ these values increase to 4.8 x 10(-2), 5.0 x 10(-2) and 5.1 x 10(-2), respectively.