Design, Synthesis And Application Of Fluorescent Probes For Phosphatase

Two fluorescent probes for the detection of alkaline phosphatase(ALP) have been designed and synthesized. The optical properties and the capability of detecting alkaline phosphatase were investigated. The results show that P1 is not a proper sensor for ALP, while P2 could detect ALP both in vitro and in living cells.P1 was based on BODIPY fluorophore and was synthesized using 2,4-dimethylpyrrol, 4-hydroxylbenzaldehyde and boron trifluoride etherate as starting materials. The fluorophore have desirable optical properties such as long wavelength absorption and emission in organic solvent. While the fluorescence was readily quenched in water. Thus P1 cannot work properly in water. Then a phosphate group was attached to the fluorophore and directly used in live cell imaging. And the result show that P1 was not a proper sensor for ALP because P1 and compound 2 show similar fluorescence intensity.Taking lessons from the failure in design of P1, a ratiometric fluorescent probe(P2) has been designed and synthesized, and it has been proved to be a good probe for ALP sensing. The probe incorporates a phosphate group and two electron-withdrawing cyanogroup. The intensity of intramolecular electron transfer process could be modulated by the cleavage of the phosphate group, thus resulting in spectral shift and color change of probe solution.After addition of alkaline phosphatase to buffer solution of P2, the phosphate group is cleaved and a dramatic bathochromic shift from 550 nm to 650 nm could be observed from the emission spectrum. The redshift is attributed to the intensity variation of ICT process between the cleavage site and the cyanogroup. The fluorescence intensity ratio( 650 nm versus 550 nm) displays a linear relationship with the concentration of ALP with a high correlation coefficient in the concentration range from 50 UL-1 to 200 UL-1. Therefore, P2 could quantify ALP concentration in buffer solution samples with a detection limit of 3.8 U/L. Besides, the selectivity of the probe has been studied. The results show that other analytes including lysozyme, trypsin, pepsin, acetyl cholinesterase give rise to no distinct variation to the optical properties of the probe, indicating that P2 is highly selective for ALP. With low cytotoxicity and excellent biocompatibility, P2 has also been applied to detect endogenous ALP activity in living cells. It can clearly show the distribution of ALP in living cells through a fluorescence turn-on method at red channel.