A depropargylation-triggered spontaneous cyclization based fluorescent “turn-on” chemodosimeter for the detection of palladium ions and its application in live-cell imaging

Abstract

A novel depropargylation-triggered spontaneous cyclization reaction based fluorescent turn-on chemodosimeter for the detection of palladium ions has been rationally designed and developed. Based on the specific reactivity of the palladium promoted hydrolysis reaction, the probe exhibited a high selectivity and sensitivity for palladium ions. Furthermore, the probe was successfully used for fluorescence imaging of Pd²⁺ in living cells.

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Palladium is widely distributed in the environment due to its use in alloys, jewellery, dental crowns, fuel cells, chemical catalysts and especially in automobile catalytic converters.¹ Palladium is not biodegradable, and hence can be concentrated through the food chain. Excess palladium accumulation may result in degradation of DNA and cell mitochondria, allergic reactions, and also enzyme inhibition.² Therefore, the determination of palladium in environmental and biological samples is crucial both to the monitoring of environmental pollution and to the diagnosis of clinical disorders.

Whereas conventional techniques used for quantification of palladium species, such as atomic absorption spectroscopy, inductively coupled plasma atomic emission spectroscopy, and solid-phase microextraction high-performance liquid chromatography, usually suffer from the expensive and sophisticated instrumentation, and/or complicated sample preparation, and are therefore not suitable for real-time and in situ analysis.³ In comparison with those conventional methods for palladium species, fluorescent probe techniques display apparent advantages because of their ease of application in solution as well as their high sensitivity to and selectivity for trace analytes with spatial and temporal resolution.⁴

Over the past several years, considerable efforts have been made to develop fluorescent probe for palladium ions based on the coordination of Pd²⁺ to heteroatom-based ligands, Pd²⁺ catalyzed ring-opening reaction, Pd²⁺ catalyzed oxidative cyclization reaction, and palladium catalyzed depropargylation and deallylation reaction (Fig. 1).⁵ However, many of them still have limitations such as interference from other coexisting metal ions, long response time, and need additional reagents. Therefore, for practical applications, it is still strongly desirable to develop novel fluorescent probes with high sensitivity, and quick response for real-time detection of palladium ions.

Fig. 1 Some reported palladium probes.

Herein, the aim of this work is to develop a new fluorescent probe with novel response mechanism for palladium ions. As shown in Scheme 1, probe SPd1 was developed with coumarin precursor derivative and a terminal propargyl ether moiety. It is well known that the terminal propargyl ether can be cleaved by palladium-catalyzed hydrolysis reaction to generate the corresponding free hydroxyl group.^5d,5m We envisioned that the fluorescent intensity of the SPd1 is greatly reduced due to the effect of intramolecular rotation. However, the deprotection of the propargyl ether group of SPd1 by palladium ions promoted hydrolysis reaction would releases the hydroxy intermediate, which wills readily spontaneous cyclize to form a highly fluorescent coumarin derivative 4 (Scheme 1). To the best of our knowledge, this is the first example of depropargylation-triggered spontaneous cyclization based fluorescent chemodosimeter for the detection of palladium ions. Furthermore, SPd1 can be successfully applied for Pd²⁺ imaging in living cells.

Scheme 1 The “deprotection–cyclization” strategy for the design of SPd1.

As shown in Scheme 2, SPd1 can be readily prepared in two convenient steps under facile conditions with high yield starting with commercially available 4-(diethylamino)-2-hydroxybenzaldehyde. The product (SPd1) was well characterized by ¹H, ¹³C NMR, and HR-MS (ESI†).

Scheme 2 Synthesis of SPd1: (a) 3-bromopropyne/K₂CO₃, acetone, reflux, 12 h, 93%; (b) Et₃N, EtOH, rt, 12 h, 65%; (c) PdCl₂, THF–H₂O (1 : 1), rt, 3 h, 72%.

We firstly assessed the UV-vis spectroscopic properties of SPd1 in PBS buffer solution (10 mM, pH 7.4, containing 50% EtOH). SPd1 (20.0 μM) displayed a moderate UV-vis absorption around 474 nm. Upon incremental addition of Pd²⁺ (0–10.0 equiv.), the peak at 474 nm slightly decreased, and the absorption at 350 and 555 nm increased instantly with two clear isosbestic points at 452 and 502 nm, respectively, indicating that compound 4 was formed in the present of Pd²⁺ (Fig. 2). Furthermore, a good linear relationship was observed between the changes in the absorbance at 452 and 520 nm with Pd²⁺ in the range of 0–10.0 equiv. (Fig. S1, ESI†).

Fig. 2 Absorption spectra of SPd1 (20.0 μM) in PBS buffer solution (10 mM, pH 7.4, containing 50% EtOH) in the presence of different concentrations of Pd²⁺ (0–10.0 equiv.).

The emission spectra of SPd1 and its fluorescent titration with Pd²⁺ were recorded in PBS buffer solution (10 mM, pH 7.4, containing 50% EtOH) (Fig. S2, ESI†). As expected, SPd1 alone is almost non-fluorescent (λ_ex = 510 nm, Φ = 0.004, Table S1, ESI†) due to the effect of intramolecular rotation (Scheme 1). However, upon progressive addition of Pd²⁺, the emission band at 542 nm rapidly increased (Fig. 3), which was attributed to the cleavage of propargyl ether group by palladium ions-promoted hydrolysis followed by spontaneous cyclization reaction to form the highly fluorescent coumarin derivative 4 (Scheme 1). Moreover, the fluorescence titration curve revealed that the fluorescence intensity at 542 nm increased linearly with increasing concentration of Pd²⁺ (R² = 0.99213) (Fig. S3 and S4, ESI†) and further smoothly increased until a maximum was reached up to 100.0 μM Pd²⁺ (λ_ex = 510 nm, Φ = 0.023, Table S1, ESI†). Based on these results, the detection limit of SPd1 for Pd²⁺ was calculated to be 9.3 × 10⁻⁸ M.⁶ Owing to the specific reactivity of palladium ions-promoted hydrolysis reaction, SPd1 displayed a high sensitivity toward Pd²⁺.

Fig. 3 Fluorescence spectra of SPd1 (10.0 μM) in PBS buffer solution (10 mM, pH 7.4, containing 50% EtOH) in the presence of different concentrations of Pd²⁺ (0–150.0 μM) (λ_ex = 510 nm). Inset: cuvette images of probe SPd1 before and after addition of PdCl₂ taken under a hand held UV-lamp (λ_ex = 365 nm).

The plausible mechanism of the palladium ions induced fluorescence response is shown in Scheme 1. Efforts were then made to explore the nature of the palladium ions induced response. To this end, a comparison of fluorescent spectra between the SPd1–Pd²⁺ system and compound 4 was made to confirm the generation of 4 after treatment with Pd²⁺ (Fig. S5, ESI†). The ¹H NMR spectra of the isolated product of the SPd1–Pd²⁺ solution were also measured to support the depropargylation-triggered spontaneous cyclization of SPd1 (see ESI†).

Subsequently, the time-dependence of fluorescence was also evaluated in the presence of Pd²⁺ in PBS buffer solution (10 mM, pH 7.4, containing 50% EtOH) (Fig. 4, ESI†). The result shows that the fluorescence of the tested solutions remarkably increased to the maximum value within 70 minutes. Accordingly, the observed rate constant (k_obs) for the formation of compound 4 has been calculated to be 2.4 × 10⁻² min⁻¹ (Fig. S6, ESI†).⁷

Fig. 4 Time-dependent fluorescence intensity changes of SPd1 (10.0 μM) upon addition of Pd²⁺ (10.0 equiv.) in PBS buffer solution (10 mM, pH 7.4, containing 50% EtOH) (λ_ex = 510 nm).

Further, the fluorescence titration of SPd1 with various metal ions was conducted to examine the selectivity (Fig. 5, and S7, ESI†). Much to our delight, the examined alkali, alkaline-earth metal ions, transition metal ions, and even Hg²⁺ showed nominal changes to the fluorescence spectra of SPd1. It should be mentioned that SPd1 still responded to palladium ions sensitively even in the presence of other relevant competing ions (Fig. 5, and S8, ESI†). Therefore, these results suggest that SPd1 displays high selectivity toward palladium ions in neutral aqueous solution.

Fig. 5 Fluorescence responses of SPd1 to various metal ions (including Na⁺, K⁺, Ag⁺, Co²⁺, Mn²⁺, Al³⁺, Cd²⁺, Cr³⁺, Cs⁺, Cu²⁺, Fe²⁺, Fe³⁺, Hg²⁺, Mg²⁺, Zn²⁺, Pb²⁺, and Pd²⁺). Black bars represent the addition of 10.0 equiv. of the appropriate metal ions to a 10.0 μM solution of SPd1 (in PBS buffer solution, 10 mM, pH 7.4, containing 50% EtOH). Red bars represent the addition of 10.0 equiv. of Pd²⁺ to the solutions containing SPd1 (10.0 μM) and the appropriated metal ions (10.0 equiv.) (λ_ex = 510 nm).

Moreover, the palladium ions sensing ability of SPd1 at a wide range of pH values was investigated. As depicted in Fig. S9, ESI,† SPd1 alone was inert to pH in the range of 5.5–9.8. On the other hand, it readily reacted with palladium ions within the biologically relevant pH range (6.5–8.5). These results indicate that SPd1 could be used in living cells without interference from pH effects.

Due to the favorable properties of SPd1 in vitro, the potential utility of SPd1 in living cells was studied. HeLa cells were incubated with 10.0 μM of SPd1 for 30 min at 37 °C and exhibited only weak fluorescence (Fig. 6b). The cells were then treated with PdCl₂ (30.0 μM) for 30 min at 37 °C, which resulted in a dramatic increase of intracellular green fluorescence (Fig. 6d). These obvious changes indicated that SPd1 was cell membrane permeable and capable of image Pd²⁺ in living cells.

Fig. 6 Fluorescence image of HeLa cells incubated with SPd1 (10.0 μM) for 0.5 h, and then washed quickly with PBS for imaging (b). The cells were then treated with PdCl₂ (30.0 μM) for 0.5 h which resulted in a dramatic increase in intracellular green fluorescence (d). (a) and (c) Bright-field images of live cells in (b) and (d).

In conclusion, we have rationally developed a novel and simple depropargylation-triggered spontaneous cyclization based fluorescent chemodosimeter for the detection of palladium ions. The probe displayed a specific fluorescence response towards palladium ions under mild conditions with a low detection limit. Furthermore, fluorescence imaging of Pd²⁺ in living cells indicated that this probe might be favorable for biological applications.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (20902082), the Project Grants 521 Talents Cultivation of Zhejiang Sci-Tech University, and the Program for Innovative Research Team of Zhejiang Sci-Tech University (13060052-Y).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Experimental details, characterization of the compounds, and additional spectroscopic data. See DOI: 10.1039/c5ra23645b

‡ These authors contributed equally.

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