Synthesis Of Fluorescence Ca²⁺ Indicators And Novel Chelators

Calcium is one of the most abundant elements,and it plays a critical role in regulating numerous physiological phenomena.Fluorescent indicators which were developed by Tsien and colleagues in the 1980's have been of tremendous applications to give the change of[Ca²⁺].The cell-impermeable indicators are generally in the water-soluble or potassium salt form.The cell-permeant indicator acetoxy methyl(AM)ester is hydrolyzed in cells to obtain the signals of intracellular [Ca²⁺]_i.So these reagents are predominantly used for detecting calcium ion in live cells.Most of the classical members of this group were produced by Tsien and colleagues.There are several different fluorescent Ca²⁺indicators,such as quin-2, indo-1,fura-2 and fluo-3.They can be divided into various groups based on several different criteria.One way of dividing these probes into two groups is to separate them based on whether they are ratiometric versus nonratiometric(single wavelength) indicators.The former includes indo-1 and fura-2,whereas the latter includes fluo-3 and rhod-2.Indo-1 has several advantages,including,(1)a shift in emission wavelength upon binding Ca²⁺;(2)greater selectivity for Ca²⁺over other divalent metal ion such as Mg²⁺,Mn²⁺,and Zn²⁺;and(3)increased fluorescence quantum efficiency.In this thesis,the investigations about synthesis of fluorescence Ca²⁺indicators and novel chelators include the following several aspects:1.This paper provided an improved synthetic method for fluorescence indicator indo-1 by 11 reaction steps,and also a shift in emission spectra for indo-1 as a function of free Ca²⁺was reported.Indo-1 is a highly fluorescence indicator for measuring free calcium concentration.However,works towards the synthesis of indo-1 were seldom mentioned and quite inexplicit.In this work,we improved the synthetic method of BAPTA-based chelator and the wittig reagent,and the alkaline environment of giving indo-1 was studied.2.A novel ratiometric fluorescent calcium indicator with moderately binding affinity—indo-MF was synthesized and its shift in emission wavelength upon binding Ca²⁺was about 45nm.There are some limitations on indo-1,our aim in the present study was to find indo-1 analogue with binding affinity only about one order of magnitude less than the parent indo-1,filling a needed gap between indo-1 and indo-FF.We achieved this aim by synthesizing indo-MF.3.Two novel BAPTA-based chelators of calcium fluorescent indicator were designed and synthesized.The most widely used Ca²⁺indicators,such as fura-2, indo-1 and fluo-3,were synthesized from BAPTA(1,2-bis-(2-aminophenoxy)ethane-N,N,N'N' -tetraacetic acid)by combination with a fluorescent-like structure.The BAPTA chelator part of these indicators didn't change at all.By modifying this BAPTA chelator,we obtained BAPTA-vinyl and BAPTA-EClCl,which could attach different fluorophores.They provide a new way to synthesize a variety of Ca²⁺ indicators,and will have excellent applications.