Fluorescent Probes Based On 2-(Azidemethyl)arylbenzoate:Synthesis And The Application

Hydrogen sulfide?H₂S? has been recently recognized as the third gaseous transmitter after nitric oxide and carbon monoxide, playing an important role in the pathological and physiological aspects of organisms. Hydrogen peroxide plays an important role in the normal activities of organisms as an important oxide. Accordingly, tracking and detection of them in vivo is very important. Compared with the traditional detection methods, fluorescence spectroscopy has highly sensitivity and selectivity and can achieve in situ detection. Therefore, it is very important to design and study of fluorescent probes for hydrogen sulfide and hydrogen peroxide in vivo.Aiming at the problem of poor selectivity, low sensitivity and slow response of traditional hydrogen sulfide fluorescent probes and basing on the principle that the hydrogen sulfide could reduce the azide group of the 2-?azidomethyl?-4-nitrobenzoate and occur intramolecular reaction to release of the fluorescent group, a hydrogen sulfide fluorescent probe ANR was synthesized using Rhodol as the fluorophore. ANR could rapidly respond to H₂ S and offered highly sensitivity and selectivity. The reaction time was as short as 4 minutes to reach a satisfied fluorescent intensity. The detection limit of ANR was determined to be 0.4327 ?M. MTT assay showed that ANR had almost no toxicity to cells. Excellent imaging of probe to H₂ S in cells illustrated that ANR could be applied to biological testing as a high performance H₂ S fluorescent probe.During the screening of probe ANR, we found that the molecular ACR synthesized by 7-Cl-Rhodol and 2-?azidemethyl? benzoate showed no response to hydrogen sulfide, but had excellent selectivity to hydrogen peroxide. Further tests showed that ACR had rapid response and high sensitivity to hydrogen peroxide. The low cytotoxicity and good imaging of probe ACR to hydrogen peroxide in cells showed that ACR could be well applied to the detection of hydrogen peroxide in vivo.Based on the study of ACR, another hydrogen peroxide fluorescent probe ACF was synthesized from 2',7'-dichlorodihydrofluorescein and 2-?azidemethyl? benzoate. When treated with H₂O₂, ACF showed extremely rapid response. Only in 5 min, the fluorescent intensity was increased by 118-fold. ACF had excellent selectivity and anti interference for hydrogen peroxide. Specifically, the detection limit of H₂O₂ was determined to be 6.5 nM. The fluorescence intensity of probe ACF increased almost linearly with the concentration of H₂O₂ in the range of 50- 400 ?M. The good linear correlation indicated that probe ACF was able to qualitatively and quantitatively determine the level of H₂O₂. ACF showed almost no toxicity to cells and was observed excellent imaging for hydrogen peroxide in cells, implying that the probe is suitable for bioimaging of H₂O₂ in living cells.This paper designed and synthesized H₂ S fluorescent probe ANR and H₂O₂ fluorescent probes ACR and ACF using 2-?azidemethyl?aryl benzoate as the reaction sites. Structural characterizations of those three kinds of probes were carried out through high resolution mass nuclear magnetic resonance?¹H NMR, 13 C NMR?, spectrometry?HRMS? and infrared spectroscopy?FTIR?. Moreover, their optical properties were analysised and studied through UV-vis spectroscopy and fluorescence emission spectra. Finally, they were successfully applied in cell testing. The test results illustrated that ANR, ACR and ACF had excellent properties, and could be used for the detection of H₂ S and H₂O₂ in environment or organisms, having important significance whatever in theory research and practical application.