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

Abstract

Three new low‐affinity fluorescent Ca 2+ 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 Ca 2+ binding properties via fluorimetric titrations. The in vitro dissociation constant K d measured at 21 ∘ C in 100 mM KCl buffered solution, pH 7.05, for the Ca 2+ –DPP1 complex is 10 μ M; for Ca 2+ –DPP2 and Ca 2+ –DPP3 a K d value of 20 μ M is found. All three indicators form 1: 1 complexes with Ca 2+ . The fluorescence quantum yields of the uncomplexed forms of DPP1, DPP2 and DPP3 are 1.2 × 10 −2 , 3.4 × 10 −2 and 3.6 × 10 −2 , respectively. After binding to Ca 2+ these values increase to 4.8 × 10 −2 , 5.0 × 10 −2 and 5.1 × 10 −2 , respectively.