Development of Two-Photon Absorbing Benzocoumarin Dyes and Fluorescent Probes

Abstract

Section I. Development of pyridinium-benzocoumarin dyes and fluorescent probes Developing the far-red emitting pyridinium-benzocoumarin dyes Fluorescence detection methods are particularly useful for monitoring the biological process owing to several advantageous features such as non-invasiveness, safe detection, accessibility. Along with an increase of interest in fluorescence detection methods, two-photon microscopy (TPM) which uses the two-photon absorption (TPA) dyes as materials has been attracted because of several advantages such as the use of half energy for excitation, deep tissue imaging, reduced photodamage, and reduced photobleach. Therefore, the developments of various TPA dyes with desirable photophysical properties are highly required. For that purpose, several dipolar dyes which have molecular weight of several hundreds and short maximum absorption and emission wavelengths (~370 nm and ~500 nm in EtOH respectively) are widely used for TPM materials. However, autofluorescence from intrinsic biomolecules becomes a serious issue in tissue imaging using the dyes which emit in the short wavelength region (< 650 nm). I have developed a new class of far-red emitting, TP absorbing pyridinium-benzocoumarin (Py+BC) dyes, with which I was able to minimize autofluorescence in tissue imaging by TPM. The new Py+BC dyes are highly soluble in aqueous media, highly photostable, and very bright inside cells as well as in tissue. A ratiometric H2O2 probe based on pyridyl-benzocoumarin For monitoring a specific analyte in the biological system, ratiometric fluorescent probes are useful. Since we use the emission intensity ratios between that from probe and that from product (in case of reaction based sensing), we can exclude those factors that influence on fluorescence intensity, such as microenvironment around probe, local concentration of probe, instrumental signal fluctuation, etc. I have designed a new ratiometric sensing system based on the pyridyl-benzcoumarin dyes. As an example, I have synthesized a ratiometric probe for H2O2, which selectively responded to H2O2 in solution as well as in cell. The probe has potential in studying disease assoiated H2O2 levels. A FRET hNQO1 probe based on pyridinium-benzocoumarin The ratiometric fluorescent probes are classified into two types: ICT-based and FRET-based probe. Some ICT-type ratiometric probes, however, have a limitation in measuring fluorescence intensity ratios due to serious spectral before and after interaction with specific analytes. Such an issue can be with FRET-type ratiometric probes, which are composed of two fluorophores with well-separated emission peaks. However, development of FRET probes with suitable photophysical properties remains challenging. I have studied a new FRET sensing system based on the pyridinium-benzocoumarin dyes. As an example, I have developed an hNQO1 FRET probe. All the known hNQO1 probes are a ‘turn-on’ type, due to the quenching effect of the quinone moiety (tlq) that is a substrate for hNQO1. This quenching problem can be solved by a FRET probe. I developed the new FRET system and hNQO1 probes. Section II. Development of other benzocoumarin derivatives and fluorescent probes Imidazole-benzocoumarin dyes that sense extremely acidic pHs The TP absorbing pyridinium-benzocoumarin dyes have relatively low quantum yields due to the pyridinium moiety. To reduce the free-rotation, instead of the pyridinium ring I introduced an imidazole ring that is expected to have intramolecular hydrogen bond with the lactone carbonyl oxygen. Indeed, the imidazole-benzocoumarin dyes showed huge enhancement in the quantum yield. Furthermore, they showed different emission behavior depending on pH. In neutral pHs, the dyes showed little noticeable changes in their maximum emission wavelengths. However, they showed ratiometric emission changes in extremely acidic pHs, as the electron donor amine is protonated to cause a reduction in the intramolecular charge transfer (ICT). The probe may be applied to image the pH change in stomach. The fluorescent probes for H2S/Amyloid-β The reactive oxygen species level is known to be higher in the Alzheimer’s disease (AD) brain than in the normal brain. As the biological redox states tend to be tightly regulated, it is of importance to know the hydrogen sulfide level in the AD brain compared to that in the normal brain. Furthermore, it is of interest to correlate the hydrogen sulfide level with the progress of AD, which can be followed by amyloid-β plaques (Aβ). Fluorescent probes that can be used to co-monitor H2S and Aβ are not known. For the dual detection of H2S and Aβ, I have designed the probe IBC-H2S, which contains a para-azido benzyl ether as the reactive site with H2S: Once it is cleaved, a florescent benzocouarin dye IBC 2 is produced, which in turn can bind with Aβ with further fluorescence enhancement. I have found that the dicyanovinyl also reacts with H2S, which interferes the desired reaction between the azido group with hydrogen sulfide. To solve the issue, I have designed the probe BtBC-H2S that contains a benzothiazole instead of the dicyanovinyl group. The probe responded to H2S, with turn-on fluorescence change, and the resulting product, a benzothiazole benzocoumarin dye, upon addition of Aβ exhibited further fluorescence enhancement. Therefore, the probe has potential for co-monitoring of H2S and Aβ, by following the different fluorescence changes. In the above, I noted that the dicyanovinyl group interacts with hydrogen sulfide. It is an electron-withdrawing group, which is widely used in dipolar dye-derived fluorescent probes for bisulfite and hypochlorite. I have thus further evaluated reactivity of the dicyanovinyl group and structurally modified electron-acceptors toward those analytes. The results show that we can realize high selectivity among the analytes by choosing suitable acceptors.