Synthesis and Evaluation of Light Triggerable Redox-Active Species Generators

Abstract

A majority of essential biomolecules or scaffolds are based upon carbon, oxygen, sulfur and nitrogen. These molecules exist in a flux and are constantly broken down and resynthesized. Some derivatives of O, S and N form highly reactive species, which can undergo redox processes in cells and are termed as redox-active species. Based on the reactive atom(s), these species are classified as reactive oxygen species (ROS), reactive sulfur species (RSS) and reactive nitrogen species (RNS). Amongst these, ROS are oxygen-derived species, primarily generated by NAD(P)H oxidase and the leakage of electrons in electron transport chain (ETC) during oxidative phosphorylation in mitochondria. When present in an optimum concentration, ROS are known to regulate several essential signaling pathways. However, at elevated levels, ROS damage essential biomolecules which, in turn, leads to cell death. To counter ROS-mediated damage, cells have evolved antioxidant machinery (which regulates ROS levels), such as superoxide dismutase (SOD), catalases, thiols etc. Apart from ROS, RSS such as glutathione (GSH), cysteine, sulfur dioxide (SO2) and hydrogen sulfide (H2S) are also important redox species in cellular metabolism. For instance, they play vital roles in signaling and antioxidant mechanisms. In addition, RNS – nitric oxide-based molecules – are endogenously synthesized and perform important modulation for certain signaling mechanisms. Therefore, it becomes essential to have tools to probe into redox-mediated cellular pathways. In this attempt, several molecules have been developed which are either triggered by one of several metabolic stimuli (such as cellular thiols, hydrogen peroxide and enzymes) to generate the aforementioned reactive species or are spontaneous generators of the same. Here, the triggerable approach provides control over the release of these reactive species. However, due to the wide prevalence of the metabolic stimuli, reactive species are produced in nearly all cells and achieving spatio-temporal control over release becomes challenging. Alternatively, light was sought as a stimulus to attain spatio-temporally controlled release of reactive species. As an external stimulus, it can provide better handle over localization and rate of cleavage can be tuned by varying intensity of light. To this end, a masked ROS generator was synthesized wherein the Diels-Alder adduct of 1,3-cyclohexadiene and juglone was attached with a 2-nitrobenzyl-based photo-responsive linker. The designed molecule released ROS only upon UV light irradiation, and was highly potent in inhibiting cancer cell growth which was not observed in absence of light. However, few limitations associated with UV light as stimulus include marginal increase in cellular ROS levels and low tissue penetration which may hinder the usefulness of this strategy in studying cellular processes. To address the above shortcomings, a linker was to be chosen such that it would cleave upon visible light irradiation. Herein, the visible light activatable group in the form of BODIPY was linked with a hydroquinone-based ROS generator. This molecule cleaved upon visible light irradiation to release ROS. BODIPY-based photo-responsive group was further utilized to mask hydrogen sulfide (H2S), a gasotransmitter, in the form of COS which was subsequently hydrolyzed by carbonic anhydrase to generate H2S. This molecule was further evaluated for cytoprotection against ROS-induced damage. In this thesis, the presented light-triggered tools may find an application to study redox-mediated signaling pathways in a spatio-temporally controlled manner.