Study On Synthesis Of Fluorinated Indicator 5F-BAPTA-AM

Intracellular free calcium ion has important effects on physiological functions. It is recognized that free calcium ion is involved in cell regulation, metabolism, and may serve as a second messenger. Because of its important functions, there is increasing demand for flawless analytical techniques for measuring intracellular calcium in intact viable tissues, in which ¹9F-NMR is a non-invasive technique which allows us to examine the intracellular free calcium ion inside living tissue.Fluorinated indicators are needed in ¹9F-NMR , in which 5F-BAPTA-AM is the most widely used probe for studying cytosolic free calcium ion ,which has high selectively for free calcium ion ,is not particularly pH sensitive. In addition, determinations with 5F-BAPTA are not sensitive to cells which have a high background fluorescence.During the process of synthesis of 5F-BAPTA-AM. we synthesized 5-fluoro-2-nitrophenol from 2,4-difluoro nitrobenzene and potassium hydroxide at 45 ℃ and bromomethyl acetate was synthesized from acetyl bromide and paraformaldehyde(1:1). 5-fluoro-2-nitrophenol was converted to the corresponding sodium salt in aqueous with equal moles of sodium hydroxide. The salt was reacted with superfluous 1,2-dibromoethane in refluxing DMF. The nitro group of the product can be reduced with H₂ or by iron powder reduction. The progress of the reduction with H₂ was highly active, but complicated equipment was needed. The amine product was with superfluous methyl bromoacetate (1:6) in aqueous acetonitrile The product was puried by column chromatography and recrystallized from aqueous ethanol. The tetraester with aqueous methanol containing equivalents of KOH gave the tetrapotassium carboxylate which wassufficiently pure for the last step. The final conversion was effected by the esterification of the tetrapotassium carboxylate with superfluous bromomethy acetate (1:5) to give the acetoxymethy ester. The final compound was purified by column chromatography and the resulting syrup was then resoluted at high temperature. At -4°C, colorless needle-shaped crystal separated out, which was 5F-BAPTA-AM.The purity of the compounds was detected by HPLC spectrum , and the constitution of 5-fluoro-2-nitrophenol and 5F-BAPTA-AM was identified by El spectrum, ultraviolet spectrum and 'H-NMR spectrum ,and compared with the spectrum in the literature. In corroboration of the correctness of the process, the intermediate products were detected by El spectrum.The successful synthesis of 5F-BAPTA-AM prepared the ground for 19F-NMR in measuring intracellular calcium, and we piled up experience for synthesis of the derivatives of BAPTA.