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A lysosome-targeted turn-on fluorescent probe for endogenous formaldehyde in living cells Yonghe Tang, Xiuqi Kong, Zhan-Rong Liu, An Xu, and Weiying Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02879 • Publication Date (Web): 21 Sep 2016 Downloaded from http://pubs.acs.org on September 21, 2016

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Analytical Chemistry

A lysosome-targeted turn-on fluorescent probe for endogenous formaldehyde in living cells Yonghe Tang,‡ Xiuqi Kong,‡ Zhan-Rong Liu, An Xu, and Weiying Lin

*

Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Biological Science and Technology, University of Jinan, Jinan, Shandong 250022, P. R. China. *Fax: + 86-531-82769031. E-mail: [email protected].

As one of the simplest reactive carbonyl species, formaldehyde is implicated in nervous system diseases and cancer. Organelles play crucial roles in various physiological processes in living cells. Accordingly, the detection of endogenous formaldehyde at the subcellular level is of high interest. We herein describe the development of the first organelle-targeted fluorescent formaldehyde probe (Na-FA-Lyso). The new probe exhibits favorable features including a large fluorescence enhancement (about 350-fold) and a fast response to formaldehyde. Significantly, the novel probe Na-FA-Lyso was employed to visualize the endogenous formaldehyde in the lysosomes in the living cells for the first time.

As the simplest aldehyde compound, formaldehyde (FA) has found widespread use in many fields such as cosmetics,1 plastics,2 and drugs.3 In addition, FA has also attracted great attentions as the third largest indoor chemical pollutant. Exposed to FA may lead to multitudinous diseases such as the injury of memory,4 cancer,5-7 and spontaneous abortion.8 The course of histone demethylation,9 or methylation of DNA10 may produce FA in living systems. FA exists in all cells and play an important role in the carbon cycle of the metabolism.10 The normal level of FA closely relates to spatial memory and cognitive ability,11-13 nevertheless, the physiological function of FA is still not well-defined. Therefore, the tracking of the endogenous FA in living systems is of great significance. Fluorescence imaging has become a powerful tool to monitor biomolecules in living systems as it offers high sensitivity and selectivity with minimal invasiveness to the test specimens.14-25 Organelles assume an important role in the metabolism process of living cells. For example, the synergistic effects of the organelles are closely related to cellular apoptosis process.26-27 The previous studies have suggested that the generation of FA is associated with many organelles including lysosomes, endoplasmic reticulum, and golgi apparatus.28-30 Although a few FA fluorescent probes have been reported very recently,27-34 to our best knowledge, no organelletargeted fluorescent FA probes has been revealed up to date. Therefore, it is urgent to develop organelle-targeted FA fluorescent probes. Lysosomes are a type of the important organelles in most of the eukaryotic cells, playing significant roles in the metabolism processes of living cells, including cellular apoptosis, immunological stress, and enzyme processing.35-36 The biological molecules are delivered to lysosomes by the endocytic pathways and biosynthesis.37-38 So far, the lysosome-targeted fluorescent probes have been reported for detecting bioenzyme, lysosomal viscosity, reactive sulphur species (RSS),

the lysosome pH in living cells, and determining the apoptosis process of cells.39-45 However, due to lack of the lysosomespecific fluorescent probes, the detection of the endogenous FA in lysosomes is still very challenging. Herein, we report Na-FA-Lyso as the first lysosometargeted fluorescent FA probe, which used 1,8-naphthalimide as the fluorescent chromophore and hydrazine as the interaction site. The morpholine moiety has a pKa of 5-6, thus it is protonated only in lysosomes (pH 4.5−5.5). 39-42 Based on this feature, the morpholine unit is incorporated into the probe as a lysosome-targeting group. We anticipated that the new probe Na-FA-Lyso may display almost no fluorescence by a photoinduced electron transfer (PET) pathway. However, after the introduction of FA, the PET pathway is suppressed, and thus a significant turn-on signal can be monitored (Scheme 1).28 Scheme 1. The fluorescence response mechanism of the lysosome-targetable FA probe Na-FA-Lyso.

The synthesis of Na-FA-Lyso was shown in the Scheme S1. The product compound Na-FA-Lyso was prepared with a satisfactory yield, and its structure was well characterized by standard 1H NMR, 13C NMR and mass spectrometry. 1

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Firstly, we evaluated the absorption profile of the probe NaFA-Lyso. The new probe displays maximal absorption at around 440 nm (ε = 26,800 M-1 cm-1) in 10 mM PBS buffer (Figure S1). As designed, the free probe Na-FA-Lyso displayed almost no fluorescence in PBS buffer. Satisfactorily, when reacted with FA, a large fluorescence enhancement was observed rapidly. There was an about 350-fold fluorescence enhancement was observed when 5 µM probe was treated with 200 µM FA for 30 min (Figure 1). The detection limit of the probe Na-FA-Lyso was calculated to be 5.02×10-6 M (Figure S2). The results indicate that the probe Na-FA-Lyso is highly sensitive to FA. To support the design scheme of the probe, the reaction product of the probe Na-FA-Lyso with FA was readily isolated at room temperature. The analysis of the 1H NMR spectrum (Figure S3), 13C NMR spectrum (Figure S4) and HR-MS spectrometry of the product (Figure S5) confirms that the product is indeed the compound 2 as proposed in Scheme 1. The photophysical data of the compound 2 are compiled in Table S1. The observation that the stable product could be rapidly produced is in accordance with the fast response and the low detection limit features of the probe, which could be attributed to the high reactive nature of the probe with FA.

Figure 1. The fluorescence response of the probe Na-FA-Lyso (5 µM) to FA at varied concentrations in PBS buffer (pH 7.4, 1 % DMSO). λex = 440 nm. The spectra were recorded after treatment of the probe with FA (0-200 µM) for 30 min. Inset: Fluorescence intensity ratio (F/F0) changes at 543 nm of Na-FA-Lyso (5.0 µM) with the amount of FA. Notably, the fluorescence intensity of the free probe is 4.11 as shown in the red arrow.

Figure 2. Reaction-time profiles of the probe Na-FA-Lyso (5 µM) in the absence [ ] or presence of FA (50 µM [ ], 100 µM

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[ ] and 200 µM [ ]). The fluorescence intensities at 543 nm were continuously monitored at time intervals in PBS buffer (pH 7.4, 1 % DMSO).

The kinetic profiles of the fluorescence intensities of NaFA-Lyso (5 µM) treated with various concentration of FA (0, 50, 100, and 200 µM) in PBS buffer (pH 7.4, 1% DMSO) are displayed in Figure 2. As anticipated, a strong turn-on fluorescence signal was observed rapidly, and a maximal response was obtained in 30 min in the presence of 50 µM FA. While treated with 200 µM FA, the plateau was reached in 10 min. The rate constant (k) for the probe was determined as 0.37 min-1 under the pseudo-first-order conditions (Figure S6). Thus, the rapid response character of the probe Na-FA-Lyso suggests its potential for real-time imaging applications in living systems. The selectivity of the probe Na-FA-Lyso (5 µM) was investigated. The new probe was treated with the representative amino acids, cations, anions, reactive oxygen species, reactive nitrogen species, ketones, and aldehydes in 10 mM PBS buffer. As shown in Figure 3, the introduction of 50 µM FA caused a strong fluorescence signal for 30 min. By contrast, the other analytes including glyoxal, methylglyoxal, sodium pyruvate, p-hydroxybenzaldehyde, trichloroacetaldehyde, acetaldehyde, 4-nitro-benzaldehyde, and acetone induced very weak or almost no fluorescence response. Ketones and aldehydes are known to be able to react with hydrazine by the condensation reaction to different extents. However, the smaller size of FA renders it more reactive than other ketones and aldehydes. Thus, the steric factor could account for the high selectivity of the probe for FA over other species. These data indicate that the novel probe has specific selectivity toward FA over the other species examined. This high selectivity feature of the probe is critical to its potential biological applications.

Figure 3. Fluorescence responses of Na-FA-Lyso (5 µM) in the presence of various relevant analytes. After the incubation of the probe with the analytes for 30 min, the data were obtained. The concentrations of the representative analytes are: amino acids, 5 mM; cations and anions, 5 mM; reactive oxygen and nitrogen species, 100 µM; ketones and aldehydes, 50 µM. Legend: (1) PBS; (2) glyoxal; (3) methylglyoxal; (4) sodium pyruvate; (5) phydroxybenzaldehyde; (6) trichloroacetalde-hyde; (7) acetaldehyde; (8) 4-nitro-benzaldehyde; (9) acetone; (10) FA; (11) NaClO; (12) H2O2; (13) tert-Butyl hydroperoxide; (14) TBHP; (15) NO; (16) CaCl2; (17) MgCl2; (18) Na2SO3; (19) NaNO2; (20) NaHSO3; (21) NaHS; (22) L-Arg; (23) GSH; (24) L-Cys; (25) DL-Hcy; (26) D-Phe; (27) N-Acetylglycine; and (28) N-Acetylcysteine. λex/em = 440/543 nm.

The effects of pH on the fluorescence response of Na-FALyso in the absence or presence of FA were also investigated. As shown in Figure S7, in the absence of FA, the fluorescence intensity of the free probe displays only a very minor variation 2

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in different buffer solutions with pH ranging from 4.0 to10.0, indicating that the changes of pH have little effects on the free probe. When treated with FA, the obvious fluorescent signal change was noted in the pH range of 4.0-8.0, suggesting that FA can be detected by the probe at both the physiological pH of 7.4 and the lysosomal pH of 4.0-6.0. We then carried out the inhibitory experiment of FA by using NaHSO3 as the inhibitor, which can react effectively with FA to destroy the central carbonyl group. As shown in Figure S8, 5 µM probe treated with 50 µM FA displays a strong emission peak. However, 50 µM FA pre-treated with 200 µM NaHSO3 and subsequently incubated with 5 µM probe produced almost no fluorescence. This indicates that, as expected, FA can be effectively inhibited by NaHSO3 in PBS. Fluorescent probes as imaging agents should have the feature of low cytotoxicity. Therefore, the potential toxicity of probe Na-FA-Lyso against the HeLa cells was investigated. The living cells were incubated with various concentrations (0-20 µM) of Na-FA-Lyso for 24 h, and then the cell viability was determined by the standard 3-(4,5-dimethylthiazol-2-yl)2,5- diphenyltetrazolium bromide (MTT) assays. The results indicate that at concentrations below 20 µM Na-FA-Lyso has no marked cytotoxicity (Figure S9). Taken together, the prominent features of Na-FA-Lyso include high sensitivity and selectivity, fast onset in response to FA, functioning well in physiological and lysosomal pH, and low cytotoxicity. These desirable characteristics stimulated us to further investigate the suitability of Na-FA-Lyso for monitoring FA in living systems. The effectiveness of probe Na-FA-Lyso for fluorescence imaging of exogenous FA in living cells was examined. In the control experiments, only the HeLa cells (Figure S10b) and the HeLa cells treated with FA (Figure S10e) exhibit no significant fluorescence. By sharp contrast, the HeLa cells incubated with 50 µM FA for 30 min, and then treated with the probe Na-FA-Lyso (5 µM) for another 30 min display strong fluorescence. These data demonstrate that the probe Na-FA-Lyso is capable of responding to exogenous FA in the living cells by a fluorescence turn-on signal. Owing to the above prospective results, we further studied the feasibility of the probe Na-FA-Lyso to detect endogenous FA in living cells. We carried out the inhibitory experiment of FA by using NaHSO3 as the inhibitor. The living HeLa cells incubated with only NaHSO3 (200 µM) exhibited essentially no fluorescence (Figure 4b). However, the HeLa cells treated with the probe Na-FA-Lyso (5 µM) display a dramatic enhancement in the green emission (Figure 4e). By comparison, the HeLa cells pre-treated with NaHSO3 (200 µM) and subsequently incubated with the probe Na-FA-Lyso (5 µM) exhibit almost no fluorescence (Figure 4h). These data indicate that the probe Na-FA-Lyso can be used to sense the endogenous FA in the living HeLa cells. Finally, the subcellular distribution of Na-FA-Lyso in HeLa cells was studied. The HeLa cells were co-incubated with the probe and the lysosome indicator, the Lyso-Tracker Deep Red, to examine the feasibility of the probe Na-FA-Lyso to detect endogenous FA in lysosomes. As shown in the Figure 5, the cells display significant fluorescence in the green channel due to sensing of the endogenous FA (Figure 5b). Meanwhile, strong red fluorescence in the cells due to the staining of the

lysosomes by Lyso-Tracker Deep Red was observed (Figure 5c). The merged image indicates that the green fluorescence overlaps well with the red fluorescence (Figure 5d). Moreover, as shown in Figure 5e, the intensity scatter plot of the green channel and red channel displays good correlation with a high Mander's overlap coefficient of 0.9 in the region of interest (ROI) Figure 5d. These results demonstrate that, as designed, the new probe could exhibit an excellent lysosome-targeted property and image endogenous FA in the lysosomes.

Figure 4. Fluorescence imaging of the endogenous FA in the HeLa cells. a) The bright-field image of the HeLa cells treated with NaHSO3 (200 µM); b) The fluorescence image of a; c) The merged image of a and b; d) The bright-field image of the HeLa cells treated with the probe Na-FA-Lyso (5 µM); e) The fluorescence image of d; f) The merged image of d and e; g) The brightfield image of the HeLa cells treated with NaHSO3 (200 µM) and the probe Na-FA-Lyso (5 µM); h) The fluorescence image of g; i) The merged image of g and h. Excitation was at 488 nm and emission collection was from 500 - 550 nm. Scale bar: 20 µm.

Figure 5. The images of the living HeLa cells co-incubated with the probe Na-FA-Lyso (5 µM) and Lyso-Tracker Deep Red. a) Bright-field image of the HeLa cells treated with Na-FA-Lyso (5 µM) and Lyso-Tracker Deep Red; b) The fluorescence image of the green channel; c) The fluorescence image of the red channel; d) The merged image of a, b, and c. e) Intensity scatter plot of the green and red channels in the ROI of d. Scale bar: 20 µm. 3

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In conclusion, we have rationally designed and synthesized the first organelle-targeted FA fluorescent probe, Na-FALyso. The novel probe Na-FA-Lyso shows a significant fluorescence enhancement (about 350-fold) and a very fast onset in response to FA. Importantly, we have demonstrated that the unique probe Na-FA-Lyso possesses the desirable lysosometargeted feature. We expect that the new probe will be a powerful molecular tool for studying both the physiological and pathological roles of FA in the context of the subcellular environments, lysosomes. Further development of various types of organelle-targeted FA fluorescent probes for the investigation of FA in other subcellular environments is under progress.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Synthesis of the probes, absorption and fluorescence spectra, imaging assays, 1H NMR and 13 C NMR spectra. (PDF)

AUTHOR INFORMATION Corresponding Author * Fax: + 86-531-82769031. E-mail: [email protected]

Author Contributions ‡ These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by NSFC (21172063, 21472067), Taishan Scholar Foundation (TS 201511041), and the startup fund of the University of Jinan.

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