Angelw: a double reactive fluorescent probe for the detection of HSNO in cells

Thiocyanate (HSNO), as the smallest s-nitroso-mercaptan, is the key intermediate connecting H 2S and no, which can regulate the redox state of cells So far, FTIR, 15N NMR and MS have been successfully used to detect and characterize HSNO under physiological conditions However, these methods are not suitable for real-time monitoring of HSNO in biological samples Therefore, in order to better understand the role of HSNO in physiological environment, fluorescence imaging technology with high sensitivity and space-time resolution has shown great potential Recently, a fluorescence probe TAP-1 (scheme 1) for the detection of HSNO was designed and synthesized by a distinguished professor in the Department of chemistry, Washington State University The fluorescence of TAP-1 was completely quenched by hydroxyl protecting group and intramolecular spiral cyclization effect, and the background fluorescence was low In the presence of HSNO, partial nucleophilic reaction and intramolecular cyclization of 2-mercaptobenzoate release benzobisulfil and compound 2; meanwhile, HSNO reacts with o-phenylenediamine to form benzotriazole derivative 3 Subsequently, the conjugated system of the fluorophores recovered and showed strong fluorescence It is worth mentioning that the polysulfide alone only reacts with 2-mercaptobenzoate partially, and the compound 2 produced does not show fluorescence due to the intramolecular spiral cyclization and photoinduced electron transfer (PET) process Relevant achievements were published on angelw Chem Int ed (DOI: 10.1002 / anie 201908950) under the title of "rational design of a dual reactivity based fluorescent probe for visualizing intrinsic HSNO" (source: angel Chem Int ed.) first, the author explored the synthesis of TAP-1 (scheme 2) Compound 4 was used as raw material, which was coupled with n-boc-1,2-phenylenediamine, and then treated with LiOH to form compound 5 The amide bond of compound 5 was reduced by borane and oxidized by Tetrachlorobenzoquinone to compound 6 After a series of esterification, deprotection and reduction reactions, target probe TAP-1 was obtained (source: angelw Chem Int ed.) later, the author tested the responsiveness of TAP-1 to HSNO The PBS buffer (50 mm, pH 7.4) of HSNO (1 mm) and Na2S (0.3 mm) was used as the mother liquor of HSNO As expected, TAP-1 has almost no fluorescence emission in PBS (Φ = 0.01) In the presence of HSNO solution (50 μ m), TAP-1 showed an obvious fluorescence emission peak at 521 nm, and reached a stable state after 8 min (Figure 1a) The author further verified the selectivity of TAP-1 to HSNO, and found that about 16 active substances (GSH, Hcy, Cys, Na2S, ONOO - etc.) did not make TAP-1 respond and produce fluorescence, while TAP-1 showed weak fluorescence in the mixed solution of Na2S and pyrrolidine noate (Figure 1b) The authors speculate that the interaction between H 2S and no produces HSNO (source: angelw Chem Int ed.) next, the authors explored the imaging ability of TAP-1 in HSNO in cells CCK-8 showed that TAP-1 showed low cytotoxicity and good biocompatibility Based on this, HeLa cells were co incubated with TAP-1 (10 μ m) and no significant fluorescence signal was observed (Figure 2a); then cells were treated with a mixture of Na2S / S-nitrosoglutathione (GSNO) and obvious fluorescence signal was observed (Figure 2D); cells were treated with a mixture of Na2S / pyrrolidine-nooate and similar results were observed (Figure 2a) 2e) Interestingly, the cells treated only with pyrrolidine NONOate also showed a certain fluorescence signal (Figure 2f), which may be due to the endogenous H 2S and no reaction to generate HSNO However, after pretreatment with PAG, a synthetic inhibitor of H 2S, the fluorescence signal decreased significantly (Figure 2G) All the above results show that TAP-1 has good cell permeability and can be used for the detection of HSNO in cells (source: angelw Chem Int ed.) finally, the author explored the imaging effect of TAP-1 in human embryonic kidney cells (HEK293T), and further explored the efficiency of the visualization of HSNO in cells by TAP-1 Cysteine tRNA synthetase (cars2) in mitochondria contributes to the production of endogenous H 2S, and noc7 is the donor of No The results showed that the response of wild-type (WT) HEK293T cells treated with noc7 was stronger than that of cars2 knockout (KO), and the response showed a concentration dependence of noc7 The above results show that TAP-1 can react with endogenous HSNO, which is mainly produced by the reaction of H 2S and no derived from cars2 (source: angelw Chem Int ed.) in a word, the author reported a double reactive fluorescent probe TAP-1 for the detection of HSNO, and realized high selectivity and sensitivity response to HSNO in vitro and in vivo The probe is expected to become an important tool for exploring the function of HSNO in biological system.