MODIFICATION OF XANTHENE AND SILICON ANALOGUES OF XANTHENE FLUOROPHORES FOR CHEMICAL PROBES DEVELOPMENT

Author

Chuangjun Liu, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2016

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

scott, colleen

Abstract

Fluorescence techniques have been widely used in chemistry and many areas of biology due to its high sensitivity, simplicity, fast response, and capability of spatial imaging. Currently there are six major classes of small-molecular fluorophores-coumarins, boron dipyrromethene (BODIPY) dyes, fluoresceins, rhodamines, oxazines, and cyanines. This dissertation mainly focuses on modifications of fluoresceins and development of pH probes based on rhodamines. We have modified the xanthene core of fluorescein by replacing the xanthene oxygen with a dimethylsilyl group which shows 90 nm red-shift relative to fluorescein (λabs/λem =582 nm/598 nm). These fluorophores have advantages over their oxygen counterparts because longer-wavelength allows deeper penetration of the light, causing less damage to the cells and less interference from the biological medium. Based on this scaffold, we have tried to develop copper, mercury, and hydrogen peroxide probes by functionalizing the hydroxyl group of the fluorophore with different groups. Although the results weren’t satisfactory, we have gained some new insights about this scaffold. The hydroxyl group of the fluorophore is not very reactive and this scaffold is not stable under strong basic conditions and its electronic configuration is very different with the regular fluorescein. We have synthesized and characterized Si-rhodol fluorophore, the hybrid structure of Si-fluorescein and Si-rhodamine. The preliminary investigation of Si-rhodol, UV-vis and fluorescence, has been performed. It shows that the absorption and fluorescence emission of Si-rhodol are around 30 nm red-shift compared to Si-fluorescein (λabs/λem =617 nm/630 nm). And Si-rhodol is more fluorescent than Si-fluorescein (pKa = 6.8) at pH 7.4 (biological condition) due to its lower pKa (5.3). Despite the excellent photophysical properties of Si-rhodol, there are actually limited examples about its application. Among these examples, none of them gave the detailed information about functionalization of the hydroxyl group of Si-rhodol. We have found a method to functionalize the hydroxyl group of Si-rhodol. We have developed a series of pH probes through modification of rhodamines which possess either an aniline-methyl or cyclic ring moiety. These analogues maintain a coloress, non-fluorescent spirocyclic structure at high pH and the fluorescence goes up while the pH decreases. The substituent effects on anilinomethylrhodamines (AnMR) system follows Hammett’s equation. The effect of cycloalkane ring size on the acid/base properties of the AMR system can be predicted by Baeyer’s ring strain theory. The pKa of these rhodamine analogues can be tuned from around 3 to 8. We have also investigated aminomethylfluoroscein (AMF) system. In this system, we found that once the phenolic hydroxyl group was functionalized at one side, the fluorescence was off at pH>3. In addition, variation of the substituents on aminomethyl moiety didn't affect the pKa of this system significantly. Therefore, we were set out to develop mitochondria-targetable hydrogen peroxide probe. However, in the presence of triphenylphosphine cation, the conversion from the OTf group to the boronate group didn't work.