New chemical tools for hydrogen peroxide detection and molecular imaging

Abstract

Hydrogen peroxide (H2O2) has been recognized as one of the most significant reactive oxygen species (ROS) in human health and disease. Due to the intrinsic low reactivity of H2O2, it is very challenging to develop small-molecule fluorescent probes to discern H2O2 selectively and sensitively in an intricate biological milieu. This thesis describes a rationally designed tandem Payne/Dakin reaction for specific H2O2 molecular recognition, and its applications in developing selective, sensitive and biocompatible fluorescent probes (Figure 1). In tandem Payne/Dakin reaction, H2O2 can be activated by its addition to trichloroacetonitrile at physiological pH to form a peroxyimidic acid (Payne reaction), which further reacts with salicylaldehyde rapidly to give a catechol (Dakin reaction). Four H2O2 probes based on this sensing strategy have been developed, including three turn-on fluorescent probes HKPerox-1, HKPerox-2, HKPerox-4, and one ratiometric fluorescent probe HKPerox-3 (Figure 2). The design, synthesis, characterization and applications of HKPerox series probes are described, and all of them show excellent selectivity and sensitivity toward H2O2, which are essential for investigations of H2O2 related biology. In particular, HKPerox-1 is a very sensitive yellow-emission H2O2 probe with a detection limit of 0.53 nM, and cellular H2O2 production in response to starvation was successfully detected by both confocal imaging and flow cytometry with HKPerox-1. HKPerox-2 is a mono-protected green-emission probe, which reacts with H2O2 10 times faster than previously reported probes based on boronate oxidation or Baeyer-Villiger type reaction. The ratiometric probe HKPerox-3 allows the quantitative detection of H2O2 by measuring the ratio of the blue and green fluorescence emission simultaneously, and therefore is a more reliable indicator with a built-in correction. Moreover, the red fluorescent probe HKPerox-4 with outstanding permeability could be used in zebrafish imaging by simple co-incubation, and rotenone induced oxidative stress was successfully visualized in zebrafish embryo. Two synthetic routes have been developed to prepare the above mentioned H2O2 probes based on tandem Payne/Dakin reaction: (1) a general coupling reaction between halogen- or triflate-substituted fluorophores and the carbamate sensing moiety; (2) a general addition reaction between isocyanate-substituted fluorophores and the benzyl alcohol sensing moiety (Figure 3). This convergent synthetic strategy will facilitate the development of next-generation H2O2 probes as powerful molecular tools in redox biology and medicine.