Chem. SCI.: endoplasmic reticulum targeted fluorescence probe for zinc ion imaging

Endoplasmic reticulum (ER) is a vesicular, vesicular and tubular structure formed by a single unit membrane, which can form a continuous reticulum system and play a role in protein synthesis and transport, protein folding, carbohydrate metabolism, etc However, the synthesis and accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can cause endoplasmic reticulum stress and lead to inflammation, diabetes and neurodegenerative diseases In addition, endoplasmic reticulum acts as a storage site for biological media (including zinc) in cells A large number of reports have shown that zinc transporter depletion and zinc deficiency can cause ER stress and activate UPR Therefore, it is very important to study the new molecules for subcellular zinc imaging Recently, Professor Michael Watkinson of kil University, UK, designed and synthesized an ER targeting group with cyclohexylsulfonylurea as a different Zn2 + receptor, which is used to detect the Zn2 + change induced by tunicamycin or carotene under endoplasmic reticulum stress The probe has good fluorescence conversion, high selectivity and low toxicity to Zn2 +, which helps researchers better understand the reaction mechanism of endoplasmic reticulum to zinc homeostasis imbalance and the role of zinc homeostasis in the occurrence and development of diseases Relevant achievements were published on Chemical Science (DOI: 10.1039 / c9sc04300d) under the title of "endpoint targeting flexible probes to image mobilezn 2 +" In order to synthesize the target probe, the ER target group 6 was synthesized from 4 - (2-aminoethyl) benzenesulfonamide according to the literature method, and then the azide 7 was obtained by a series of reactions between it and 4-bromo-1,8-naphthalenic anhydride The simple and efficient click reaction between azide 7 and alkyne 8 or 10 resulted in compounds 9 and 11 After deprotection by TFA, compound 12 (scheme 1) was obtained The above compounds were characterized by 1H, 13C, IR and HRMS (source: Chemical Science) then, the photophysical properties of target probes 9 and 12 in solution were studied The fluorescence titration experiment was carried out with Zn2 +, and it was found that the two probes had "on" response to Zn2 + With the increase of Zn2 + equivalent, the fluorescence of probes 9 and 12 increased 16 and 4 times, respectively (Figure 2a) After TPEN treatment, the fluorescence of compound 9 was completely quenched and part of compound 12 was quenched Subsequently, the pH dependent fluorescence response of the two probes was tested The results showed that the probes had a good fluorescence response to Zn2 + in the pH range (3.0-10.0) related to physiology, and the fluorescence of the two probes was enhanced under acid conditions (Figure 2b) In addition, the competitive selectivity of the two probes for other cation probes was investigated In addition to the stereoelectron CD 2 +, the addition of 5 equivalent ions did not make compound 9 fluorescent, but CD 2 + almost did not exist in the biological environment, so there was no effect on the application of the probe Then, the author added Zn2 + to competitive cations, and found that in most cases, the fluorescence was restored, but for CO2 +, Cu2 + and Ni2 +, the fluorescence was not restored (Figure 2C), which was irrelevant because it was mainly in the form of complexation rather than free ions in biology (source: Chemical Science) based on the good spectral characteristics of compounds 9 and 12, the author explored their imaging ability for Zn2 + in cells After confirming its low toxicity to cells, the authors evaluated the targeting ability of the probe by CO location experiment In HeLa cells, er tracker, Mito tracker and lyso tracker were used to incubate with probe 9 The results showed that the fluorescence of probe 9 overlapped with ER tracker The Pearson correlation coefficient was 0.92 The correlation coefficient of Mito tracker and lyso tracker was only 0.92 0.52 and 0.58 (Figure 3) Probe 12 showed similar results (source: Chemical Science) because probes 9 and 12 have good ER localization ability, the author explored their imaging ability for free Zn 2 + level in cells The fluorescence intensity of HeLa cells incubated with probe 9 increased significantly after the addition of zinc picolinate (a membrane permeable zinc source), while the fluorescence quenching (Figure 4a) of HeLa cells incubated with TPEN showed the change clearly (Figure 4b) Similarly, similar results were obtained in MCF-7, ec23 and HepG2 cell lines (source: Chemical Science) based on the good ER localization ability and Zn2 + response ability of probe 9, the author explored its imaging ability for endoplasmic reticulum stress induced by tunicamycin and Dauricine After treatment with two inducers, the fluorescence intensity of HepG2 cells decreased significantly, indicating that the level of free Zn 2 + decreased during endoplasmic reticulum stress (Figure 5) (source: Chemical Science) in a word, based on the simple sulfonylurea targeting group, the author developed two fluorescent probes for imaging free Zn 2 + in ER The probe showed a good fluorescence response to Zn2 +, a good selectivity to its residual cation, a universal applicability to pH, a low toxicity to cells, er targeting and Zn2 + imaging ability, which is conducive to a better understanding of the role of Zn2 + in the occurrence and development of different diseases.