Development of Fluorogens for the Detection of Physiological Analytes

The phenomenon of fluorescence has enabled us to gain a deeper understanding of biological structures and processes. Labeling of biomolecules using fluorescence tools such as fluorescent proteins, organic fluorophores, inorganic complexes, and quantum dots allows for these biomolecules to be visualized spatiotemporally for increasing our understanding of their function. To augment our understanding of biological processes there is a growing interest in the detection and quantification of the concentrations of various cellular analytes as they play a key role in the regulation of cellular behavior. Thisthesis focusses on the functionalization of existing fluorescence tools to enable them to detect and quantify various cellular analytes. The fluorescence tool described here is the Fluorogen Activating Protein (FAP) technology as it provides spatiotemporal resolution and ease of labeling both at the cellular and organismal levels. This technology is able to activate the fluorescence of a dark (non-fluorescent) dye molecule upon non-covalent binding to a cognate, targeted protein partner that can be linked to a protein of interest. The fluorogenic dyes can be modified to obtain desired properties, but are limited, therefore a tandem dye approach was initiated. This thesis summarizes the outcomes of the development of a new tandem dye and demonstration of its localization ability and potential in analyte detection. This tandem dye is a coumarin-Malachite Green (MG) conjugate (donor-acceptor), in which the MG binds to a FAP, dL5**, and emits in the farred region where autofluorescence of cells is limited. 7-hydroxycoumarins were coumarins of choice since there is a vast literature to show that the fluorescence of 7-hydroxycoumarin depends on whether it has been capped or is in its phenolate form. As a first step, to demonstrate the ability of 7-hydroxycoumarins to act as donors for MG dL5**, we rationally selected two coumarins and determined that a coumarin containing a hydroxy group in the 7th position and a carboxylic acid in the 3rd position are importantfor any energy transfer to take place in the tandem dye. Further, we concluded that it was essential for the 7-hydroxy group to have a low pKa for the coumarin to be in its phenolate form under physiological conditions. Pacific blue was determined to be a suitable donor and the cell-permeable property of Pacific Blue-Malachite Green (BluR2) tandem dye has been used in conjunction with a cell-excluded tandem dye, HCM, to visualize anddistinguish protein pools inside and outside the cell using different colors. To demonstrate the ability of the 7-hydroxycoumarin-MG tandem dye as a functional probe, a 7- hydroxycoumarin analogous to Pacific Blue was conjugated to MG to enable the detection of pH (hydrogen ion concentration) changes in cells. 6-chloro 7-hydroxycoumarin-3- carboxylic (6ClC) is structurally and spectroscopically similar to Pacific Blue, except thatthe 7-hydroxy group on the former has a lower pKa compared to the latter since it is flanked by one weak-electron withdrawing chlorine (in 6ClC) instead of two strongelectron-withdrawing fluorines (in Pacific Blue). This difference elevated the pKa from 3.7 to 6.3 and the elevated pKa displayed a systematic enhancement in the coumarinexcitation cross-section and the energy transfer between 6ClC to MG-dL5** with an increase in alkalinity of buffers from pH 4.0 to 7.7. This in vitro experiment is significantas this systematic change in fluorescence can be visualized during pH changes in cellular processes like lysosomal trafficking and exocytosis. The availability of a FAP targeted to the lysosomal protein makes this measurement possible, thus making 6ClC-MG (BluRpH) a genetically targeted, far-red, ratiometric pH sensor. To expand the repertoire of analyte detecting tandem dyes with MG-dL5** as an acceptor, we chose to develop new tandem dye probes to detect reactive oxygen species. As a first step, we chose to convert the Cy3-MG tandem dye pair into Cy3[H]MG, as reduced Cy3[H], also known as hydrocyanines. Hydrocyanines are well-characterized superoxide anion sensors and display superior selectivity to superoxide anion compared to existing commercial probes like hydroethidine and mitosox. Also, the modularity of synthesis allowed us tofunctionalize hydrocyanines to label another genetically encodable tag, a Halo tag, by conjugating a suitable linker that can be recognized by the haloalkane dehalogenaseenzyme (Halo tag). In pursuance of our original goal of developing more analyte-detecting tandem dyes using the BluR platform – coumarin-MG tandem dyes, building blocks were developed for the detection of hydrogen peroxides. For this, a 7-hydroxycoumarin-3-ethyl ester was converted into 7-Bpincoumarin-3-ethyl ester, which can then be conjugated to MG. The coumarin will become fluorescent and transfer energy when it detects hydrogen peroxide as it converts Bpin into a hydroxy group. This mechanism is well-established using other fluorophores containing a phenol as part of their structure. In conclusion, this thesis has shown that the FAP platform can be functionalized to detect small molecule analytes in the cell. The combined use of tandem dyes and a genetically targetable tag (FAP) is a wonderful recipe provides many advantages for unravelling the mysteries of biology.