Development of Two-Photon π-extended Fluorescent Sensing System for Redox Species and Bio-phosphates

DoctorSection I: Introduction of microscopic imaging and small molecular fluorescent probes Fluorescence microscopic imaging aided with molecular probes is indispensable in studying biological systems. Besides the conventional laser scanning confocal microscopy (LSCM) that operates under one-photon excitation, two-photon microscopy offers several advantages in tissue imaging. Accordingly, two-photon fluorescent probes for biological analytes have received considerable attention recently, as they allow detection and monitoring of the target analytes beyond the cellular level. For the reliable and quantitative analysis of biological analytes, furthermore, ratiometric probes that enable ratiometric imaging are highly demanded. I have made efforts in the development of two-photon ratiometric probes for several biological analytes, as described below. Section II: Development of two-photon fluorescent probes for redox species Bisulfite. The reduction and oxidation (redox) processes are essential in living systems; accordingly, fluorescent probes for those molecules involved in the biological redox processes are of great importance. Thus, two-photon probes for various “redox species” such as biothiols (glutathione, cysteine, hydrogen sulfide, etc.) and reactive oxygen/nitrogen species have attracted recent attention. Sulfur dioxide, equilibrating with bisulfite and sulfite in aqueous media, is generated from biothiols in the cell. Detection of these sulfur species is focused on the aqueous equilibrium species, in particular, bisulfite. At its elevated level, bisulfite can cause several physiological disorder and diseases. To study the “bisulfite biology” in cell and tissue, two-photon probes with ratiometric imaging capability are in demand. To this end, we investigated an aryl aldehyde-type probe derived from a dipolar benzocoumarin dye. The probe sensed bisulfite through the formation of the corresponding aldehyde–bisulfite adduct in aqueous buffer at pH 7.4, with excellent selectivity over other biologically relevant species and with well-resolved ratiometric fluorescence changes from 625 nm to 552 nm. The high sensitivity (LOD = 0.08 μM) and reactivity (within 30 s) of the probe towards bisulfite enabled us to quantify an endogenous bisulfite level present in human serum samples. The promising in vitro ratiometric behavior, however, was deteriorated in the cellular environment, owing to large red-edge and blue-shift effects observed for both the probe and its bisulfite adduct, respectively, which occurred into the unfavorable direction. Thus, the cellular imaging with the probe was confined to observe the bisulfite level changes, using one-photon as well as two-photon microscopy. To overcome the small Stokes shift observed in the above sensing scheme of aryl aldehdye–bisulfte adduct formation, I have investigated a benzopyronin dye system that reacts with bisulfite in the 1,6-addition mode which causes deconjugation and thus induces significant Stokes shift of 192 nm. In this case, I introduced a morpholine group that directs the probe to lysosomes. Lysosomes, known as the scavenger of living cells, play a crucial role in the metabolic process, and bisulfite is often produced inside the lysosomes. Therefore, detection of bisulfite in lysosomes is a subject of significant interest. The probe is two-photon excitable, emits in near-infrared (NIR) wavelength region, and provides ratiometric signaling with complete spectral separation. The probe also shows excellent selectivity toward bisulfite over other biologically relevant species. Cysteine. Cysteine (Cys) is an important biothiol and involved in various biological processes; hence, many fluorescent probes, including two-photon excitable, are reported. However, none of the reported probes are capable of organelle targeting live-cell imaging and quantification of Cys dynamics. I have developed a two-photon ratiometric probe that emits in the NIR region and senses lysosomal Cys with a large Stokes shift of 175 nm. The probe BXan, a benzoxanthene dye, enables ratiometric (I510/I685) imaging of Cys in lysosomes within seconds against live cell by two-photon microscopy for the first time. Further, the Cys detection can be reversed by addition of hydrogen peroxide, which seems to oxidize the Cys in the adduct and release it. Such a probe has potential to study a lysosomal Cys fluctuation dependent on hydrogen peroxide, an important reactive oxygen species. Section III: Development of two-photon fluorescent probes for bio-phosphates Biophosphates such as ATP and other nucelosides are essential biological components. For example, ATP is the key energy currency of cells and required by almost all the living organisms: therefore, fluorescent probes for measuring subcellular ATP fluctuation are crucial to understand various ATP associated molecular mechanisms. Previously, our group reported the first two-photon probe for ATP, which is based on an acedan dye containing Zn(II)–2,2′-dipicolylamine [Zn(II)DPA] complex. To develop ATP probes emitting at the longer wavelengths, I have investigated a series of benzocoumarin (BC) dyes containing the phosphate binding motif, Zn(II)DPA. The BC-Zn(II)DPA compounds senses ATP with different levels of fluorescence enhancement depending on the structural changes: They show turn-on and ratiometric fluorescence responses but, in the latter, case, the signal intensity is not sufficient enough for bioimaging application. A two-photon ATP probe that emits in the far-red/NIR region with ratiometric imaging capability still remains as a challenge. Interestingly, one of the ATP probes, NucATP, localizes in nuclei and visualize ATP molecules there. This is the first two-photon probe that sense ATP in nuclei