Rapid and selective detection of cysteine over homocysteine and glutathione by a simple and effective coumarin-based fluorescent probe

Abstract

A fluorescent probe, with coumarin as the fluorophore, is capable of detecting cysteine over other biothiols, such as homocysteine and glutathione.

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Biothiols, such as those found in cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play crucial roles in biological systems.¹ Cys, as the precursor of GSH and acetyl Co-A, is a source of sulfide in iron–sulfur clusters. It is noteworthy that abnormal concentration levels of Cys are associated with many human diseases such as neurotoxicity, skin lesions, edema, muscle weakness, lethargy, slow growth, and hair depigmentation.² Abnormal levels of Hcy in human plasma have been correlated with incidence of cardiovascular and Alzheimer's diseases;³ GSH, as the most abundant intracellular non-protein thiol, has key roles in the maintenance of intracellular redox activities, metabolism, intracellular signal transduction and gene regulation. Moreover, its level is directly linked to many diseases, including cancer, cardiovascular and Alzheimer's diseases.⁴ Accordingly, the detection of these small sulfur containing species in biological and environmental samples is very important. Among the various methods for detecting thiols, fluorescence is an important detection method because of its simplicity, low detection limits and ease of handling.⁵ Especially, fluorescent probes possess the ability to monitor intracellular analytes. Therefore, the development of fluorescent probe to monitor the biomolecular thiols consistently attracts a great deal of attention. Up to now, the corresponding design strategies have been developed on the basis of various mechanisms including Michael addition, cyclization reaction, conjugate addition–cyclization, cleavage reaction, thiol–halogen nucleophilic substitution, disulfide exchange, and others.^2b,6 However, although the number of probes showing a selective response toward Cys/Hcy/GSH was designed,⁷ only few reported fluorescent probe can discriminate Cys from Hcy and GSH,⁸ because of the structural similarity and comparable reactivity. Besides that, indeed, these probes still have some limitations when considering the practical applications in biological systems, such as use of surfactant, undesired spectra overlap, long response time, relatively poor selectivity and limit of detection. Thus, the development of fluorescent probe of Cys-selective detection in biological systems is still a significant challenge for the scientific communities.

Coumarin exhibits low cytotoxicity⁹ and possesses satisfactory photophysical properties such as a large Stock shift, visible excitation and emission wavelengths.¹⁰ Therefore, coumarin is a perfect fluorophore. Moreover, based on the cyclization of Cys/Hcy with acrylates, pioneered by Strongin's group,^8a the selective detection of Cys/Hcy over Hcy and GSH could be realized. Inspired by these reports, in this work, we designed the probe 1, which used acrylate moiety as the biothiol reaction site and coumarin as the fluorophore, and envision the probe 1 can selectively sense Cys over Hcy and GSH in view of the dynamic difference.¹¹ With these considerations in mind, probe 1 was synthesized (Scheme 1, for details and characterization see the ESI†) and confirmed by NMR and HRMS (ESI).

Scheme 1 Synthesis of probe 1.

Keeping the probe 1 in mind, initially, we investigated the photophysical properties of the probe 1 in aqueous solution (pH 7.4 PBS, containing 1% DMSO). The detection properties of probe 1 can be ascertained by the UV-vis absorption and emission spectroscopy. Subsequently, the probe 1 (10 µM) was screened with the different amino acids (L-Cys, N-acety-L-Cys, Hcy, GSH, Lys, Leu, Glu, Arg, Pro, Thr, Ile, Ser, Phe, Ala, Gly, Asp, Val, His, L-Met, DL-Met, 100 µM) in aqueous solution (pH 7.4 PBS, containing 1% DMSO). When the solution of probe 1 was incubated with amino acids for 5 min at room temperature, a fluorescence emission peak was observed, ranging from 380 nm to 580 nm (Fig. 1A). Interestingly, the fluorescence intensity of the probe 1 was enhanced significantly with the Cys, but a slight change only were observed with Hcy (F/F₀ = 2.5), GSH (F/F₀ = 2.8) and other amino acids. Encouraged by the results, we determined quantitatively the fluorescence intensity with Cys. A dramatic 42-fold enhancement in fluorescence intensity was quantified for Cys, compared to the starting probe fluorescence. These results indicated that influence of Hcy/GSH is less than that of previous reports in the detection of Cys. Hence, it's obvious that probe 1 detected selectively Cys over Hcy and GSH. Subsequently, we further explored properties of the probe 1. With the increase of Cys content from 1 equiv. to 10 equiv., the fluorescence intensity was enhanced obviously when the probe solution was incubated for 5 min (Fig. 1B). Moreover, we investigated the whole process of fluorescence intensity change, when the solution of probe 1 was acted by Cys. The result showed the increase of fluorescence intensity reached the plateau stage over the course of 10 min (Fig. 2A). On this same basis, the limit of detection (3σ/m, n = 20)¹² of Cys was estimated to be 30 nm (Fig. 2B), which made the limit of detection into nanomolar concentrations. Thus, probe 1 provides the convenience for detection of low-concentration Cys in biological systems. Additionally, to confirm the selectivity, sensibility and practicability of the probe 1, on the one hand, we carried out the competition experiment. The results elucidated that fluorescence intensity of probe 1 reacted with Cys was decreased less than 23% with addition of other amino acids (Fig. 3). Thus, these results ensured the practicability of the probe 1 in the biological system. On the other hand, when the action time was increased to 30 min, we found that probe 1 did not induce any significant change by itself, which meant the background hydrolysis of probe 1 was suppressed under these reaction conditions. Meanwhile, Cys showed so rapid saturation kinetics with probe 1 that the reaction completed within 10 min, whereas Hcy and GSH exhibited much slower reaction rates (Fig. S1†). Consequently, all of these results displayed the probe 1 is able to detect effectively the Cys in the biological system.

Fig. 1 (A) Fluorescence spectra of probe 1 (10 µM) treated with various amino acids (L-Cys, N-acety-L-Cys, Hcy, GSH, Lys, Leu, Glu, Arg, Pro, Thr, Ile, Ser, Phe, Ala, Gly, Asp, Val, His, L-Met, DL-Met, 100 µM) in aqueous solution (pH 7.4 PBS, containing 1% DMSO). (B) Emission spectra of probe 1 (10 µM) with increase of Cys concentration (from 0 to 10 equiv.) incubated for 5 min at room temperature, λ_ex = 340 nm.

Fig. 2 (A) Time-dependent fluorescence spectra of probe 1 (10 µM) in the presence of Cys (10 equiv.). (B) Emission spectra of probe 1 (10 µM) incubated with appointed concentrations of Cys for 5 min in aqueous solution (pH 7.4 PBS, containing 1% DMSO) at room temperature, λ_ex = 340 nm.

Fig. 3 Competition analysis of probe 1 (10 µM) in the presence of various amino acids in aqueous solution (pH 7.4 PBS, containing 1% DMSO), λ_ex = 340 nm. Other amino acids (other AAS, every group) from left to right represent N-acety-L-Cys, Hcy, GSH, Lys, Leu, Glu, Arg, Pro, Thr, Ile, Ser, Phe, Ala, Gly, Asp, Val, His, L-Met, DL-Met.

In order to explore the reaction mechanism, we subsequently performed the HRMS experiment on probe 1 treated with Cys, wherein the peak at m/z = 177.0550, corresponding to product 2 was clearly observed (Fig. S2†). Thus, according to the experimental evidence, we proposed a probable reaction mechanism of probe 1 with Cys (Scheme 2). The initial nucleophilic reaction of the thiol group of Cys and subsequent intramolecular cyclization afforded the final fluorescent compound, leading to the remarkable fluorescence turn-on response of probe 1 in aqueous solution (pH 7.4 PBS, containing 1% DMSO).^8a Here, Cys and Hcy are able to be apparently distinguished in view of size of ring formation. Cys induced a rapid cyclization reaction due to the formation of a stable seven-membered ring, whose formation is kinetically favored over the possible formation of a strained eight- and twelve-membered ring induced by Hcy and GSH, respectively.

Scheme 2 Proposed reaction mechanism of probe 1 with Cys.

Before utilizing probe 1 in bioimaging, we determined the cytotoxic activity of the probe 1 in HeLa cells by a classic MTT assay.¹³ Interestingly, the probe 1 (10 µM) did not lead to cell death, when cells were incubated with probe 1 for 24 h (Fig. 4). Then, we investigated the imaging of probe 1 in HeLa cells (Fig. 5). When HeLa cells were incubated with probe 1 (10 µM) for 30 min, the fluorescence of probe 1 was moderately expressed by cellular thiols to afford blue fluorescence. However, as the cells were pretreated with NEM (N-ethyl maleimide, 1 mM) for 30 min, the blue fluorescence did not appear. Therefore, these results demonstrate that the probe 1 can be used to sense Cys in living cells with 30 min.

Fig. 4 Cytotoxicity of probe 1 for HeLa cells.

Fig. 5 Fluorescence image of probe 1 (10 µM)-loaded HeLa cells (A) blank; (B) cells were incubated with DMSO for 30 min; (C) cells were incubated with probe 1 for 30 min; (D) cells pre-incubated with NEM (1 mM) for 30 min and incubated subsequently with probe 1 for 30 min; λ_ex = 340 nm, scale bar = 50 µm.

In conclusion, in this study, with the acrylate moiety as the biothiol reaction site and dynamic difference of cyclization reaction as strategy for selective detection of Cys, we designed and prepared the probe 1. Remarkably, probe 1 exhibited a highly selective and sensitive response to Cys over Hcy/GSH and other amino acids in the test. A detection limit of 30 nm and 42-fold increase in fluorescence intensity with 5 min offered to powerful safeguard for practicability of Cys test in biological system. At the same time, the probe 1 is capable of detecting Cys in living cells. Thus, this will provide the convenience of more understanding of the roles of Cys in the biological systems.

Acknowledgements

This work was supported in part by the Natural Science Foundation of Hebei Province (No. B2016405026) and Young Elitist Foundation of Hebei Province (No. BJ2016003).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Experimental details and other data. See DOI: 10.1039/c6ra19267j

‡ Y.-F. Kang, H.-X. Qiao and Y.-L. Meng contributed equally to this work.

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