A turn-on fluorescent probe for selective detection of glutathione using trimethyl lock strategy

Accepted Manuscript Title: A turn-on fluorescent probe for selective detection of glutathione using trimethyl lock strategy Authors: Junliang Zhou, Jian Zhang, Hang Ren, Xiaochun Dong, Xing Zheng, Weili Zhao PII: DOI: Reference:

S1010-6030(17)30850-X https://doi.org/10.1016/j.jphotochem.2017.10.040 JPC 10965

To appear in:

Journal of Photochemistry and Photobiology A: Chemistry

Received date: Revised date: Accepted date:

17-6-2017 25-9-2017 21-10-2017

Please cite this article as: Junliang Zhou, Jian Zhang, Hang Ren, Xiaochun Dong, Xing Zheng, Weili Zhao, A turn-on fluorescent probe for selective detection of glutathione using trimethyl lock strategy, Journal of Photochemistry and Photobiology A: Chemistry https://doi.org/10.1016/j.jphotochem.2017.10.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A turn-on fluorescent probe for selective detection of glutathione using trimethyl lock strategy Junliang Zhou,a,b,1 Jian Zhang,a,c,1 Hang Ren,a Xiaochun Dong,*a Xing Zheng*b and Weili Zhao*a,c a

b

School of Pharmacy, Fudan University, Shanghai, 201203, P. R. China.

Institute of Pharmacy and Pharmacology, Hunan Province Cooperative Innovation Center

for Molecular Target New Drug Study, University of South China, Hengyang, 421001, P. R. China. c

Key Laboratory for Special Functional Materials of the Ministry of Education, Henan University, Kaifeng, 475004, P. R. China.

* Corresponding author: Xiaochun Dong,*a Xing Zheng*b and Weili Zhao*a,c Tel: +86-21-51980111 Fax: +86-21-51980111 E-mail address: [email protected]; [email protected]; [email protected]. 1

These authors contributed equally.

Dedicated to professor Chen-Ho Tung on the occasion of his 80th Birthday for his research accomplishments and contributions to photochemistry Graphical Abstract

1

Highlights

 1) A novel off-on fluorescent probe for GSH was synthesized;  2) The probe displayed sensitive and selective turn-on response to GSH; 

3) The probe is able to detect the GSH in living cells.

Abstract A novel turn-on fluorescent probe based on trimethyl lock with acrylate recognition moiety and latent fluorophore of 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD-NH2) for the detection of glutathione has been developed. The probe can selectively and sensitively detect glutathione in solution, and the limit of detection was calculated to be 26 nM. Furthermore,

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the probe has been successfully used to detect GSH in living cells. The interesting preference of GSH was attributed to the steric repulsion of trimethyl which retarded Cys, Hcy to reach Bürgi-Dunitz angle and allowed more flexible side chain of GSH to attack carbonyl functionality. Keywords: fluorescent probe; glutathione; trimethyl lock; imaging 1. Introduction Biological thiols (biothiols), such as glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), play crucial roles in various physiological and pathological processes [1, 2]. Cys and Hcy are involved in cellular growth, and GSH plays a key role in redox homeostasis [3, 4]. The lack of Cys could cause many diseases, like decreased hematopoiesis, leucocyte loss, psoriasis, neurotoxicity, edema, liver damage, and Parkinson’s disease [5-7]. Elevated Hcy in the blood was known to be involved in cardiovascular and Alzheimer’s disease [8, 9]. GSH is the most abundant among the small intracellular molecular thiols [10]. It is regarded as an essential endogenous antioxidant that plays a central role in cellular defence against toxins and free radicals. GSH deficiency was observed to link with many diseases such as liver damage, aging, cancer, AIDS and neurodegenerative diseases [11]. Therefore, selective detection of various biothiols is of great interest in biochemistry and biomedicine fields. The discrimination of these biothiols, however, is not a trivial task to achieve due to their similarity in the functional groups. Currently, fluorescent chemodosimeters are widely developed because of operational simplicity and high sensitivity [12-17]. To date, a wide variety of fluorescent probes for biothiols based on various mechanisms have been exploited, including cleavage reaction by thiols [18-21], cyclization reaction with aldehyde [22-26], Michael addition [27-31], Michael addition-intramolecular cyclization [32-41] and others [42-50]. These developments greatly advanced the research on the optical detection of biothiols, however, many of probes reported suffered from long response time [19, 24, 45], low sensitivity [18, 42]. Recently, Strongin et al. developed an ingenious method, where acrylate moiety was used to

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discriminate Cys, Hcy, and GSH due to the different rates in intramolecular cyclization reaction between acrylate and thiols [33].

Trimethyl lock is o-hydroxycinnamic acid derivative in which unfavourable steric interactions between the three methyl groups resulted in rapid lactonization to form a hydrocoumarin and release a leaving group [51, 52]. Raines’ laboratory applied the “trimethyl lock” strategy in the design of latent fluorophore [53, 54]. One of the uniqueness of the trimethyl lock is that even amine can be released by the efficient lactonization. Our design concept was that by applying combination of two main components, an acrylate unit to protect the hydroxyl of “trimethyl lock” as the thiol reactive site, and an amine-containing masked fluorophore as the amide derivative, the amine-containing fluorophore as reporter can be released following cascade reactions initiated by biothiols, which has not been achieved in the acrylate related probes and sensors so far [12-17]. As a model system, 4-amino-7-nitrobenz-2-oxa- 1,3-diazole (NBD-NH2) as the fluorophore was selected, which constituted probe 1 as the potential thiol reactive fluorescent probe. Since recent acrylate-based fluorescent probes are reported to be mostly selective for Cys [34-41], probe 1 was initially expected to be a putative Cys-selective probe. However, as outlined in the present study, probe 1 was discovered unexpectedly to be GSH-selective fluorescent probe. 2. Experimental section 2.1. Materials and apparatus All reagents were purchased from commercial suppliers and used without further purification. Solvents used were purified by standard methods prior to use. 1H NMR spectra were recorded on a Varian Model Mercury 400 MHz spectrometer. 1H NMR chemical shifts (δ) are given in ppm (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) downfield from Me4Si, determined by chloroform (δ = 7.26 ppm). 13C NMR spectra were recorded on a Varian Model Mercury 150 MHz spectrometer. 13C NMR chemical shifts (δ) are reported in ppm with the internal CDCl3 at δ 77.0 as standard, respectively. Spectrometer UV−vis spectra

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were acquired on a Hitachi U-2900 double beam UV-visible spectrophotometer. Fluorescence spectra were measured by Hitachi F-2500 fluorescence spectrophotometer. Electrospray ionization (ESI) mass spectra were acquired with Agilent 1100 Series LC/MSD and AB SCIEX Triple TOFTM 5600+ mass spectrometer. 2.2. Synthesis and characterization The brief synthetic route for preparation of probe 1 is outlined in Scheme 1. Detailed synthesis and characterisation were described in supporting information. Briefly, probe 1 can be easily prepared by condensation of 3-[2-(acryloyloxy)-4,6-di -methylphenyl]-3methylbutanoic acid (2) with 4-amino-7-nitrobenz-2-oxa-1,3 –diazole (3) in good yield. The structure of probe 1 was characterized by NMR and HR-MS spectroscopic analysis (ESI†). 2.3. Determination of the detection limit The detection limit was calculated based on the fluorescence titration. In the absence of GSH, the fluorescence emission spectrum of probe 1 was measured five times and the standard deviation of blank measurement was achieved. To gain the slop, the fluorescence intensity at 543 nm was plotted to the concentration of GSH. So the detection limit was calculated with the following equation: Detection limit = 3σ/k Wherein σ is the standard deviation of blank measurement, k is the slop between the fluorescence intensity versus GSH concentration. 2.4. Cell culture and fluorescence imaging HeLa cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with heat inactivated 10% (v/v) fetal bovine serum, 1% penicillin/streptomycin at 37 °C in a 95% humidity atmosphere under 5% CO2 environment. The cells were seeded in 35 mm diameter glass-bottomed dishes at a density of 3 × 105 cells per dish in RPMI 1640 medium for 24 h. Probe 1 (10 μM) was added to the cells, and the cells were incubated for 30 min at 37 °C. After washing with PBS three times, the cells were imaged by using a confocal laser scanning

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microscope (λex =488 nm, Carl Zeiss LSM 710). In the first control experiment, the cells were pretreated with N-ethylmaleimide (NEM, 5 mM) at 37 °C for 40 min to remove endogenous biothiols, after washing with PBS three times and further incubated with the probe 1 (10 μM) in PBS at 37 °C for 30 min. The fluorescence images of cells were taken after being washed with PBS three times. In the second control experiments, the cells were pretreated with NEM (5 mM) at 37 °C for 40 min, washed with PBS three times, then the cells were further incubated with GSH (200 μM) for 20 min, followed by washing with PBS and incubated with the probe 1 (10 μM) in PBS at 37 °C for 30 min. After being washed with PBS three times to remove free probe, the fluorescence images of cells were taken.

3. Results and discussion 3.1. Probe preparation Probe 1 was efficiently prepared under standard coupling conditions using 1-ethyl3-(3-dimethylaminopropyl)carbodiimide

(EDCI)

as

activating

reagent,

4-

dimethylaminopyridine (DMAP) as catalyst by condensation of 3-[2-(acryloyloxy)4,6-dimethylphenyl]-3-methylbutanoic acid (2) with 4-amino-7-nitrobenz-2-oxa- 1,3diazole (3) in good yield as outlined in Scheme 1. 3.2. Spectroscopic properties and responses to biothiols To verify the response of probe 1 to various analytes, the absorbance and fluorescence studies were firstly carried out. Probe 1 absorbed in the UV-vis region with absorption maximum at 395 nm (Fig. 1). However, the fluorescence of probe 1 was considerably diminished upon introducing electron withdrawing acyl group on NBD-NH2, which resulted in efficiently decreased electron density of NBD-NH2 and thus low fluorescence intensity of probe 1 compared to NBD-NH2. Probe 1 was originally designed as latent Cys-selective turn-on fluorescent probe based on many recent reported on acrylate-based fluorescent dyes as Cys selective probes [34-41]. Thus, initial investigations started on the response of probe 1 to various biothiol. Time-dependent fluorescence behaviours of probe 1 (10 μM) in the presence 6

of biothiols (Cys, Hcy, GSH, 1 mM) in DMSO/PBS buffer (1:1, v/v, 10 mM, pH 7.4) at 37 °C were studied (Fig. 2). When treated with GSH to probe 1, a steady increment of fluorescence with extended time was noted (Fig. 2a) and the fluorescence intensity reached a plateau within 25 min (Fig. 2b). When Cys was tested for probe 1, to our surprise a much weaker fluorescence enhancement was noticed and a relatively slow responsive rate was observed (Fig. 2b). Moreover, Hcy performed slightly faster rate than Cys and displayed stronger fluorescence enhancement than Cys, however, much weaker than GSH in the current situation. Such observations implied interesting sensing property of probe 1. The general trend observed herein for Cys, Hcy, and GSH was completely reversed compared with other publications with acrylate recognition functionality wherein Michael addition-intramolecular cyclization mechanism operates [32-41]. The proposed reaction mechanism was illustrated in Scheme 2. Among acrylate-functionalised probes for thiols, addition-cyclization pathway which favorite Cys over Hcy or GSH was a well-known process [32]. The peculiar trend to favourite GSH for probe 1 was believed to be induced by the unique trimethyl lock moiety. A plausible explanation is that although the addition of thiol to acrylate is the fastest for Cys, the trimethyl lock may retard the subsequent intramolecular nucleophilic attack of the amino group to the carbonyl to reach Bürgi-Dunitz angle [55]. Similar retardation of cyclization is expected to be encountered for Hcy even though fast Michael addition happens. In contrast, though GSH is the least reactive in Michael addition stage, the longer and flexible chain may allow the subsequent nucleophilic attack more effective by evading the steric repulsion of the trimethyl lock. Once the free phenol was released, the following cyclization step to release the fluorophore is known to be extremely fast [53]. Thus the overall rate-limiting step is most possibly the nucleophilic attack of amino group and the most flexible chain in GSH results in the fastest response. As a consequence, approximately 39-fold fluorescence enhancement was observed for GSH in response to probe 1 as shown in the inset of Scheme 2. 3.3. Selectivity of probe 1 7

After the establishment of probe 1 responded to biothiols, the selectivity of probe 1 to various analytes was subsequently evaluated. Probe 1 (10 μM) was screened with 10 equiv. of biothiols (Cys, Hcy, GSH) and other amino acids (Pro, Thr, Leu, Trp, Ile, Tyr, Ala, His, Glu, Lys, Met, Asp, Phe, Gly, Arg, Gln, Ser) in DMSO/PBS buffer (1:1, v/v, 10 mM, pH 7.4) at 37°C and incubated for 25 min. Probe 1 exhibited weak emission (Φ = 0.04) centered at 571 nm upon excitation at 470 nm. However, upon addition of GSH, dramatic increase up to 39-fold enhancement in fluorescent intensity (Φ = 0.20) at the peak of 543 nm was observed (Fig. 3). In comparison, only 12.7-fold enhancement for Hcy and 7.2-fold enhancement for Cys were noticed for probe 1. No measurable fluorescence enhancement could be triggered by the treatment of other nonbiothiol analytes. To further validate the selectivity and practical utility of the probe, the competition experiments were also carried out by addition of 100 μM of GSH to the solution of probe 1 in the presence of 100 μM of other amino acids (Glu, Leu, Gly, Ile, Phe, Ala, Thr, Gln, Asn, Met, Ser, Pro, Try, Lys, Arg, His, Tyr, Cys, Hcy) (Fig. S1). The coexistence of non-thiol analytes did not have obvious impact on the detection of GSH. Although Cys and Hcy resulted in a slight decrease in fluorescence intensity, probe 1 could still detect GSH effectively. Therefore, probe 1 responded preferably for GSH. 3.4. Detection limit To investigate the detection limit of probe 1 for GSH, probe 1 (10 μM) was treated with various concentrations of GSH (0100 μM). Fluorescence spectra of solutions containing probe 1 and increasing concentrations of GSH are shown in Fig. 4. The fluorescence intensity at 543 nm increases with enhanced GSH concentration. The observed fluorescence intensity is nearly proportional to the GSH concentration up to 8 μM with a detection limit to be 26 nM (3σ/k, wherein σ is the standard deviation of blank measurement, k is the slope between the fluorescence intensity versus GSH concentration). The results indicated that promising utility of the probe for detection of GSH in biological systems. 8

3.5. pH dependence To verify whether the probe is suitable for physiological detection, we evaluated the effect of pH on the fluorescence of probe 1. As shown in Fig. S2, the probe was stable within the pH range of 6.08.0, after addition of GSH, the fluorescence of probe 1 increased with increasing pH value till pH of 7.6 and then slightly declined at pH of 8.0. Overall, probe 1 displays obvious response for GSH in the region of 6.88.0. The above study suggested that the designed probe was suitable to be applied in physiological pH for the sensing of GSH. 3.6. Cytotoxicity assay of probe 1 and imaging GSH in living cells To explore the capacity for live cell imaging of GSH, the cytotoxicities of probe 1 on HeLa cells were evaluated using standard cell viability protocols (MTT assay). The results showed that cell viability was over 90% even under treatment of 20 M of probe 1 (Fig. S3), indicating the low cytotoxicity and good biocompatibility of our probe. In living cells, the plausible disturbance of Cys and Hcy can be neglected due to fact that GSH is the most abundant intracellular thiol species, ranging from 1 to 10 mM (Cys: 30200 μM) [56, 57]. The preferable response of probe 1 to GSH and the natural abundance of GSH in living cells may allow probe 1 to be applied as selective fluorescence probe for GSH in cell imaging. To demonstrate the practical utility of probe 1 in biological samples, the cell imaging experiments for intracellular biothiols were carried out in living Hela cells. As shown in Fig. 5, when Hela cells were incubated with probe 1, the cells started to show strong green fluorescence (Fig. 5A1). In contrast, when Hela cells were pre-treated with NEM (a known thiol trapping reagent) prior to incubation with probe 1, only weak fluorescence was observed (Fig. 5B1). Moreover, when HeLa cells were firstly pretreated with NEM, then added GSH (200 μM) followed by incubation with probe 1, green color of fluorescence (Fig. 5C1) with nearly comparative intensity as Fig. 5A1 can be observed. Therefore, the green fluorescence observed in living cells was attributed to GSH rather than Cys or Hcy. 4. Conclusion 9

In conclusion, we have developed a selective fluorescent probe for GSH over other biothiols including Hcy and Cys. Such a probe may follow cascade reactions of Michael addition-cyclization to differentiate GSH from Cys, Hcy probably due to the steric congestion of the trimethyl lock which favors GSH over Cys, Hcy. We have also demonstrated that the probe can be used for the fluorescent imaging of cellular GSH. Due to its good sensitivity and selectivity, the developed chemodosimeter can be used to detect GSH at physiological levels. Acknowledgements This work was supported by the National Natural Science Foundation of China (21372063). References

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Scheme 1. Synthesis of probe 1

Fig. 1 UV-vis absorption (black and red) and fluorescence spectra (blue and pink) of probe 1 (10 μM) prior to and after addition of GSH (10 equiv.) in DMSO/PBS buffer (1:1, v/v, 10 mM, pH 7.4) at 37°C.

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Scheme 2. proposed sensing mechanism

Fig. 2 (a) Time-dependent fluorescence spectral changes of probe 1 (10 μM) in the presence of GSH (10 equiv.) in DMSO/PBS buffer (1:1, v/v, 10 mM, pH 7.4) at 37°C; (b) Intensity changes of probe 1 (10 μM) at 543 nm in the presence of GSH, Hcy, Cys (10 equiv. each individually) in DMSOPBS (1:1, v/v) solution (10 mM, pH = 7.4) at 37 °C. ex= 470 nm.

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Fig. 3 (a) Fluorescence spectra changes and (b) fluorescence intensities of probe 1 (10 μM) at 543 nm in DMSO/PBS buffer (1:1, v/v, 10 mM, pH 7.4) after the addition of various amino acids (10 equiv.) at 37 °C for 25 min (ex= 470 nm). (0) None; (1) Glu; (2) Leu; (3) Gly; (4) Ile; (5) Phe; (6) Ala; (7) Thr, (8) Gln; (9) Asp; (10) Met; (11) Ser; (12) Pro; (13) Trp; (14) Lys; (15) Arg; (16) His; (17) Tyr; (18) Cys; (19) Hcy; (20) GSH.

Fig. 4 (a) Fluorescence spectra changes and (b) fluorescence intensities of probe 1 (10 μM) in the presence of increasing concentrations of GSH (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.5, 5.0, 7.5, 10.0 equiv.) in DMSO/PBS buffer (1:1, v/v, 10 mM, pH 7.4) at 37 °C.

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Fig. 5 Confocal fluorescence images of probe 1 (λex = 488 nm) to GSH in Hela cells. (A) Fluorescence image of Hela cells incubated with probe 1 (10 μM) for 30 min; (B) Fluorescence image of Hela cells pretreated with NEM (5 mM, 40 min) and then incubated with probe 1 for 30 min; (C) HeLa cells were pretreated with NEM, then with the addition of GSH (200 μM) and incubated with the probe 1 for 30 min. A1-C1 are images in green channel; A3-B3 are bright-field images; A2-C2 are overlapped images.

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