Quest for high-performance phenolic fluorophores: synthesis, photophysical properties and practical Hg(II) sensing applications of a rigidified phenolo-merocyanine dye

Among the strategies currently available to improve photophysical properties of organic-based fluorophores intended for imaging and sensing, structural modifications allowing for scaffold rigidification are particularly effective for enhancing brightness and photostability while generally causing a moderate bathochromic shift in the corresponding absorption/emission spectra. In order to expand the range of high-performance phenolic fluorophores usable as effective analytical molecular tools, we have explored the synthesis of a rigidified analog of flexible Brooker's merocyanine popularized by Eastman-Kodak company in the 1950s. We had the idea of applying a rigidification approach previously devised to assemble conformationally restrained polymethine indocyanine dyes. Thus, the implementation of an efficient acid-catalyzed domino double cyclization process allowed the synthesis of unprecedented fluorescent phenol KR306. A comprehensive photophysical study has highlighted its outstanding optical properties in neutral aq. buffered conditions; a brightness of 71 500 M-1 cm-1 at pH 7.5 for a yellow emission centered at ca. 560 nm, was obtained in particular. As with more traditional phenolic fluorophores, the hydroxyl group of KR306 can be modified to easily regulate its fluorescence properties. This feature has been demonstrated through the preparation of chromo-fluorogenic thiophosphinate and thiophosphonate esters suitable for mercuric cations Hg(II) sensing in neat aq. buffers. The validation of these Hg(II)-responsive chemodosimeters of using a robust analytical methodology based on fluorescence assays and HPLC-fluorescence/-MS analyses, also enabled to highlight good sensing performances of thiophosphinate-based chemodosimeter 5. Furthermore, its ability to be readily activated by microorganisms in kefir grains doped with Hg(II) ions, was confirmed by simply observing a color change at the naked eye. This discovery paves the way for wider use of this novel biological platform for validating small molecule fluorescent probes.