Synthesis And Spectroscopic Characterization Of A Fluorescent Dye

Cyanine dye is a new kind of biological fluorescent dye developed in recent years with heterocyclic structure as the main structure. A modified modification can be formed with the features of soluble in water, good stability, fluorescence quantity of high rate, because the indole dye absorption and fluorescent emission are near infrared area, it can avoid interference of the biological macromolecules fluorescence of itself, improving the detection sensitivity, and so it is suitable for quantitative analysis of biological macromolecules, widely used in nucleic acids and other biological molecules labeling and testing. Fluorescence labeling reagent as a kind of the unradioactive labeling reagent are in great attention nowerdays, and obtained the rapid development, this paper studied the spectral characteristics and synthesis of Alexa hemicyenine indol dyes.The first chapter of the paper reviewed the advantages and disadvantages of common fluorescent reagents, mainly introduces the spectrum characteristics, light stability, water-soluble and synthesis methods of Cy series.Moreover,the developments in biological macromolecules labeling of new-type hemicyenine series dyes were also emphasized.The second chapter of the paper included the synthese, purification and characterization of intermediates for the dye. Firstly, propargyl alcohol was used for the raw material, after oxided by chromic anhydride,the intermediate(Propiolaldehyde) reacted with aminobenzene directly,and the final product was obtained after recrystallizate purification the crude product. Then with 2-methyl ethyl allylacetoacetate,6-bromine caproic acid ethyl ester as raw materials,7-methyl-8-carbonyl nonylphenol acid was synthesized, and reacted with 4-Hydrazinobenzenesulfonic acid,so 3-(5-carboxyl)-5-sulfonic-l-(3-sulfonic acid) was obtained,and then quaternized with 1,3-Propanesultone.in the end the dye was synthesized after reaction of the intermediates above.The third chapter of the paper studied the absorption spectroscopy,fluorescence spectra and thermostability of the Alexa hemicyenine indoledye, and the results indicated that (1) the shapes of ultraviolet absorption spectroscopy and fluorescence spectra were similar when the Alexa hemicyenine indole dye was dissolved in PBS, methyl alcohol.DMF or DMSO, but the absorption and fluorescence emission wavelength subjected red shift when it was dissolved in protic solvent and the polarity of the solvent is decreasing. (2) the absorption value of the dye at 650nm is changed in a small range when the temperature range is 30 to 65 degrees which indicated that the dye is stable at the testing temperature range.In the third chapter of the essay, the active ester derivatives of indocyanine dye was synthesized by active ester reagents N,N,N',N'-Tetramethyl-O-(N-succinimidyl) uronium tetrafluoroborate,the active ester derivatives of the dye can react with protein's amino in the aqueous phase,generate amido bond,and achieve the labelling of proteins. The impact of Buffer's pH to the D/P value was also studied, the results showed that as the pH increases, the faster of the labeling speed was, when pH= 8.5, it took only 5min to reach the D/P value of 4, and when the pH= 7.0. even after 1h, the D/P is still less than 3.