Development of Environment–Insensitive Fluorophores and Molecular Probes for Nitroreductase

Abstract

Chapter I. Development of the Phenolic Dyes Insensitive to Environmental Factors

Section I. A polarity and viscosity–insensitive, two-photon dye with giant Stokes shift Coumarins have been widely used in fluorometric sensing, but their shorter absorption and emission wavelengths cause several limitations such as photobleaching, photodamage, shallow penetration depth and interference from auto–fluorescence during fluorescence imaging of tissues. Thus, to overcome these shortcomings, I synthesized pi–extended coumarin dyes, in particular, 8–hydroxy–benzo[g]coumarins with various 3–substituents. I have established a straightforward synthetic route to the benzo[g]coumarins, characterized their photo–physical properties, and evaluated their cellular imaging capability under both one– and two–photon excitation conditions. The dyes are dipolar in nature and show large Stokes shifts. Among them, unlike other derivatives 3–pyridium–substitued dye (5h) shows insignificant solvatochromism and viscosity–independent emission behaviour. This the medium–insensitive absorption/emission behaviour of 5h makes it an ideal dye platform for the development of fluorescent probes for biological systems. Section II. Making dyes pH and polarity–insensitive through intramolecular H–bonding Biological systems built up on cell and tissue own diverse environments, particularly in terms of medium polarity and pH. Even organelles inside cells have different microenvironments. Most of the commercially available fluorophores are susceptible toward such factors, which make it difficult to predict their cellular emission behaviour from solution data remained practically impossible. Moreover, such cell–solution experimental discrepancies raise a reliability concern in interpreting the imaging data. Thus, a dye molecule for sensing and bio–imaging application needs to be insensitive towards such perturbations. We have established a strategy based on structural modification to make phenolic fluorophores insensitive towards pH and polarity perturbations. Emission properties of new dyes in cell can be easily predicted unlike common phenolic dyes. In addition, the modified dyes have larger Stokes shifts, stronger emission, and thus are advantageous over their parent analogues for biological application.

Chapter II. Development of Nitroreductase Probes

Section I. Reaction based two–photon active, ratiometric nitroreductase probe Hypoxia means lack of oxygen. Over the past years, hypoxia has been recognized as a prominent feature of many diseases such as stroke, cardiac ischemia, solid tumor, etc. It is known that hypoxia in our body leads to overexpression of several intracellular reductase enzymes including a flavin dependent protein nitroreductase (NTR). There are a number of NTR probes reported, but none with ratiometric sensing ability and two–photon imaging capability at the same time. To develop NTR probes that can be applicable to investigate hypoxia–associated biological processes I have developed a Si-pyronine based probe. Being the first two–photon active, ratiometric, and NIR–emissive probe, it has great potential for biological applications. The probe was employed to detect hypoxia in cellular condition and in solid tumor. It also allowed us to reveal that kidney is more susceptible to hypoxic perturbations compared to other organs in mice. Moreover, different levels of hypoxia in cortex and medulla were also visualized using confocal fluorescence microscopic imaging for the first time. Section II. Binding–based, red–emitting, reversible nitroreductase probe Direct correlation of nitroreductase levels and the degree of hypoxia engenders the development of various NTR probes for the possible diagnosis of pathogenic infection, tumor, etc. All the reported molecular probes detecting NTR undergo consequent reduction to the corresponding amine and results in fluorescence change. Therefore, the fluorescence readout at a certain time practically indicates the accumulated reactivity of NTR, which cannot shed light on the exact amount of NTR present. Moreover, the known probes do not allow us to track down the enzyme dynamics (as the probe becomes no longer attached with the enzymes after reduction), to visualize the enzyme in normoxic condition. In that aspect, a binding based reversible probe that can be used to monitor cellular nitroreductase activity is extremely important. Disclosed here is a binding based reversible probe (revNTR) that allow us to selectively visualizing the “active state of NTR” and also to monitor the normoxic, hypoxic and reoxygenated states of cell. Being the first binding–based probe for NTR, it will be applicable to investigate other hypoxia–associated biological phenomena. Its application in real–time monitoring and quantification of NTR in cells and tissues is an immediate research interest.

Miscellaneous

Section I. Two–Photon Probe for Ratiometric Imaging of Endogenous HOCl Hypochlorous acid (HOCl), a reactive oxygen species (ROS), is associated with several disorders such as mitochondrial permeabilization, lysosomal rupture, rheumatoid and cardiovascular diseases, inflammatory diseases, neurodegeneration, cystic fibrosis, kidney diseases, arthritis, and cancers. In spite of vast biological relevance of HOCl, little is known about its physiological distribution and pathological mechanism mainly due to the lack of reliable means for real–time monitoring. Addressing that issue I have developed a novel two–photon fluorescent probe that allows ratiometric imaging of HOCl in cells as well as in tissue. This probe emits in the longer wavelength, and suppresses the by–product formation observed in the acedan–based probe reported previously. The probe shows a very low detection limit and excellent selectivity toward HOCl over potentially competing reactive oxygen species as well as other biomolecules. Accordingly, the probe enables ratiometric imaging of endogenous HOCl in live cells (Hela cells) as well as in mouse brain tissues by two–photon microscopy.