JACS: light activated formaldehyde donor reveals the threshold of cell migration inhibition

Chemical tools that can monitor complex biological networks are a powerful means to evaluate biological signals Formaldehyde (FA) is not only a byproduct of cell metabolism, but also a signal molecule for purine synthesis through carbon cycle The high concentration of FA is closely related to the pathological causes of aging, cancer and Alzheimer's disease However, it is still unknown how much concentration of FA can change cells from physiological state to pathological state Therefore, it is very important to develop a reasonable method to study the critical concentration Recently, Professor Jefferson Chan of the University of Illinois Urbana Champaign has assembled a series of light activated FA donors (photofads) to show the ability of FA release through fluorescence enhancement, and to select appropriate donors to evaluate the inhibition of HEK293 according to photokinetics, biocompatibility, subcellular localization and cell retention FA concentration of cell wound healing Relevant achievements were published on J am Chem SOC (DOI: 10.1021 / JACS 9b11899) under the title of "a photoactivated formaldehyde donor with fluorescence monitoring revelations threshold to arrest cell migration" Each photofads contains a photo unstable nitrobenzyl group, which is linked to silica xanthene dye by acetal bond (Figure 1a) Due to the interference of the electron push-pull system and the influence of the donor photoelectron transfer (d-PeT) of the near end nitro group, the alkylated Si based fluorophores show the fluorescence quenching phenomenon Under the irradiation of light, photofads decompose to form semi acetal intermediate, then spontaneously decompose to form FA and corresponding dyes, and generate fluorescence (Figure 1b) (source: j.am Chem SOC.) after a series of pre experiments, the author chose photofad-3 as the best compound for the follow-up experiments In this paper, siloxantrone 1 was treated with N-chlorosuccinimide to form siloxantrone 2, then the hydroxyl was protected with tert butyl dimethylchlorosilane to form silica ether in 33% yield, and the dye 4 was obtained under the action of tert butyl lithium and 1-bromo-2 (trifluoromethyl) - benzene Finally, compound 4 was alkylated with 5 - [1 - (chloromethoxy) ethyl] - 6-nitro-1,3-benzodioxazole (cndb), and photofad-3 (scheme 1) was obtained in 45% yield (source: J am Chem SOC.) prepared photofad-3, the author first carried out spectral test Photofad-3 shows fluorescence quenching in the inactive state (Figure 2a) With the increase of irradiation time, photofad-3 showed a 139 fold fluorescence enhancement (k release = 137 nm / min) (Figure 2B, 2C) In addition, we observed that photofad-3 and fluorescent dye 4 had no significant cytotoxicity (Figure 2D) in HEK293 cells, and both compounds were located in the cytoplasm (source: J am Chem SOC.) later, the authors focused on the quantification of FA released by photofad-3 HEK293 cells stained with photofad-3 were irradiated at different times and imaged with fluorescence microscope (Figure 3a) Then, the author lyses the cells and analyzes the signals quantitatively by fluorescence spectrometry On the premise that the fluorescence intensity of lysate is directly proportional to the concentration of compound 4 (Figure 3D), the concentration of FA released by a single cell at different irradiation time is calculated according to the number of cells in each sample In addition, the IVIS fluorescence instrument with a large imaging window was used for imaging experiment (Figure 3B), and the imaging results were consistent with those of fluorescence microscope The above results show that photofad-3 has application value in multiple imaging platforms (source: J am Chem SOC.) finally, the threshold value of cell detoxification to FA was determined by photofad-3 A control reagent (ctrl-photofad-3) was synthesized It does not have acetal bond, so it only releases dye and light controlled fragment, and does not produce FA HEK293 cells were used in wound healing experiments to reveal cell proliferation and migration Cells were stained with ctrl-photofad-3 and photofad-3 and irradiated for 0-180 seconds The rate of wound closure was monitored within 24 hours (Figure 4a) The activation of ctrl-photofad-3 had no significant effect on wound healing, while the activation of photofad-3 led to the decline of time-dependent wound healing (Figure 4b) In particular, when the FA concentration of cells is higher than 2.2 μ m, the wound is difficult to heal In addition, long-term irradiation makes the cells lose the ability of adhesion (Figure 4C) (source: J am Chem SOC.) in a word, the author developed the first photoactivated FA donor, which can effectively control the level of FA in interfering cells Quantitative experiments clearly show that the increase of 2.2 μ m FA is enough to inhibit the wound healing of HEK293 cells, which makes it possible to study the role of FA in stem cell aging by using photofad-3.