Isolation Of N - Carboxybenzylindole Cyanine Dyes And Labeling Of Proteins

Squaraine dye is constantly expanding its application range as a fluorescent function dye, especially the widely application in marking biological macromolecules. Squaraine dye is featured with a wide spectrum range, large molar extinction coefficient, higher fluorescence quantum yield and high sensitivity, so it is gradually becoming a new generation of probe dyes.In this paper,2,3,3-trimethyl-3H-indol-5-sulfonic acid which is obtained from the synthesis of 3-methyl-2-butanone and hydrazino benzenesulfonic acid under the catalytic effect of glacial acetic acid, is reacted with N-alkylating reagent, so as to get indole quaternary ammonium salt to substitute N-carboxylic benzyl. Then one-pot two-step methodology is adopted. The mixing solution of n-butanol and toluene is taken as the solvent, and squaraine and indole quaternary ammonium salt are condensed to get water-soluble symmetric indol-squaraine dye of N-carboxylic benzyl. C-18 reversed-phase column chromatography method, acetone recrystallization and normal phase silica gel column chromatography method are used respectively to separate and purify the prepared water-soluble indol-squaraine dye, and nuclear magnetic resonance hydrogen spectrum (1HNMR) is adopted to characterize the structure of the compound, thereby determining that the optimal separation and purification method is silica gel column chromatography self-made by silica gel (FCP) for normal phase chromatography. Hence, using the product for the first time in the neutral mixture of barbitone and pentobarbital sodium as a buffer level of solvents in the electrophoresis tank for cellulose acetate membrane chromatography, to realise the protein directional probe marking.