Novel Two-photon Fluorescent ESIPT Probes For The Detection Of Nitrorerease And Carboxylesterase

Nitroreductase is a class of cytoplasmic enzymes that rely on flavin adenine dinucleotides or flavin mononucleotides and are widely found in various cells.Tumor cell hypoxia can cause intracellular levels of nitro reductase.In the presence of an electron donor such as reduced nicotinamide adenine dinucleotide,the nitroreductase is capable of efficiently reducing the nitro group-containing aromatic compound to the corresponding amino compound.This reduction reaction can be applied to the biodegradation of nitro-containing aromatic compounds and the activation of drugs.At the same time,the reduction reaction can also be used to design nitro-containing fluorescent probe and detect the hypoxia of solid tumor cells.Carboxylesterase belongs to the hydrolase superfamily,which catalyzes the hydrolysis of carboxylates.These enzymes are widely used in organic synthesis and industrial production,are important drugs(digestive diseases and pancreatic diseases)targets and prodrug activators.Carboxylesterase is mainly located in different cell endoplasmic reticulum and cytosol sol,and in many drugs detoxification and metabolism play an important role.Therefore,the detection of carboxylesterase activity is important for understanding its biological function.This thesis mainly completed the following work:1.A nitroreductase probe based on ESIPT(Excited-state Intramolecular Proton Transfer)was designed and synthesized using 4-diethylamino salicylaldehyde,4-nitrobenzyl bromide and hydrazine hydrate as raw materials.TP-NTR-1,and its structure was characterized by 1H NMR,13C NMR and MS.At the excitation wavelength of 413 nm excitation light,the probe fluorescence is weak,there is no obvious emission peak;the probe and nitroreductase co-incubated for 30 minutes,at the same excitation wavelength,the emission of bright yellow Green fluorescence,the maximum emission wavelength was 520 nm.With the increase of nitro reductase concentration,the fluorescence intensity of the probe was gradually increased,the linear response range was 0-8 μg/mL,and the detection limit was 0.05 μg/mL.Conventional interference test showed that the probe had good selectivity to nitroreductase.The probe was stable in the range of pH 6.5-8.0,indicating that the probe was suitable for the detection of nitroreductase under physiological conditions.It was found that the probe had a strong two-photon effect on the fluorophore released after the response of the nitroreductase,and could emit yellow-green fluorescence(maximum emission wavelength:520 nm)at 750 nm two-photon excitation.The probe TP-NTR-1 was successfully used for two-photon cell imaging of nitroreductase.2.The carboxylate esterase probe TP-CE-1 based on ESIPTwas designed and synthesized by using p-diethylamino salicylaldehyde,hydrazine hydrate and acetic anhydride as raw materials.1H NMR,13C NMR and MS were used to characterize the structure.At the excitation wavelength of 413 nm excitation light,the probe fluorescence is weak,there is no obvious emission peak;the probe and co-incubation for 30 minutes,at the same excitation wavelength,the emission of bright yellow fluorescence,the largest The emission wavelength is 520 nm.With the increase of carboxylesterase concentration,the fluorescence intensity of the probe was gradually increased,the linear response range was 0-4 μg/mL,and the detection limit was 0.025μg/mL.Conventional interference test showed that the probe had good selectivity to carboxylesterase.pH test showed that the probe TP-CE-1 had a stable fluorescence response at pH 6.0-9.0,indicating that the probe was suitable for the detection of carboxylesterase under physiological conditions.Based on the two-photon properties of the corresponding fluorophore TP-OH-1,’ the probe TP-CE-1 was successfully used for two-photon cell imaging of carboxylesterase.