Spiropyran-based fluorescent anion probe and its application for urinary pyrophosphate detection

In recent decades, numerous spiropyran derivatives have been designed and utilized for optical sensing of metal ions. However, there is still less research on spiropyran-based anion sensors. In this work, a new spiropyran compound (L) appended with a pendant bis(2-pyridylmethyl)amine was synthesized and used in fluorescent sensing of pyrophosphate ion (PP_i) in aqueous solution. The molecular recognition and signal transduction are based on the cooperative ligation interactions and the ligation-induced structural conversion of the spiropyran, which leads to a significant change in the photophysical property of the spiropyran. In an ethanol/water solution (30:70, v/v) at pH 7.4, ligation of L with Zn²⁺ causes an intense fluorescence emission at 620 nm at the expense of the original fluorescence at 560 nm. Once PP _i was introduced, interaction between PP_i and the L-Zn²⁺ complex leads to full quenching of the 620 nm band emission which was concomitant with recovery of the 560 nm band emission, and the fluorescence intensity ratio, F₅₆₀/F₆₂₀, is proportional to the PP_i concentration. Under the optimum condition, the L-Zn ²⁺ complex responds to PP_i over a dynamic range of 1.0 × 10^-6 to 5.0 × 10^-4 M, with a detection limit of 4.0 × 10^-7 M. The fluorescence response is highly selective for PP_i over other biologically related substrates, especially the structurally similar anions, such as phosphate and adenosine triphosphate. The mechanism of interaction among L, Zn²⁺, and PP_i was primarily studied by ¹H NMR, ³¹P NMR, and HRMS. To demonstrate the analytical application of this approach, the PP_i concentration in human urine was determined. It was on the order of 3.18 × 10^-5 M, and the mean value for urinary PP_i excretion by three healthy subjects was 62.4 μmol/24 h.