Spectroscopic studies on the interaction of terpyridine-CuCl₂ with cysteine

Abstract

Interaction of the classical coordination complex TpyCuCl₂ (Tpy = terpyridine) with cysteine was studied. Addition of cysteine to the solution of TpyCuCl₂ was found to give greatly enhanced fluorescence. UV-vis absorption and mass spectroscopic analyses were conducted to investigate this reaction under various conditions. These studies indicate that in a pure water solution (pH = 6.10–6.55), cysteine can reduce Cu(II) to generate TpyCuSCH₂CH(NH₂)CO₂H with greatly enhanced fluorescence. In a HEPES buffer solution (pH = 7.40), the Tpy ligand can be further displaced off the copper center, contributing to the enhanced fluorescence.

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Introduction

The fluorescence-based molecular recognition of amino acids has received extensive attention because of the potential application of these molecular sensors in biological analysis.¹ Recently, we discovered that a classical metal complex TpyCuCl₂ (Tpy = 2,2′:6′,2′′-terpyridine)^2–4 can be used as a fluorescent sensor for the recognition of both histidine and cysteine.⁵ When an aqueous solution (HEPES buffer) of this nonfluorescent complex was treated with histidine or cysteine, large fluorescent enhancement was observed. All the other natural amino acids cannot turn on the fluorescence of this complex. Because cysteine can be easily oxidized by using H₂O₂ and NaI under very mild conditions to form the disulfide compound cystine which has only very small effect on the fluorescence of TpyCuCl₂, introduction of this oxidation step allows cysteine to be distinguished from histidine by using this fluorescent sensor.

A number of fluorescent sensors for cysteine were reported before.^6–9 These sensors can be classified into two categories: one utilizes the nucleophilicity of the thiol group of cysteine to generate fluorescent responses by reacting with the aldehyde or acrylate groups of the sensors;^6e,7a,8 the other category utilizes the coordinating ability of cysteine to interact with metal complexes such as Cd(II), Ag(I) and Cu(II) complexes to cause fluorescent response.^6b,c,9a,b In most cases a receptor for cysteine needs to be connected to a fluorophore in order to output the fluorescent signal and thus often lengthy synthetic steps are involved to prepare these sensors. In contrast, the TpyCuCl₂-based fluorescent sensor found in our laboratory requires no ligand synthesis which makes its use very practical. Recently, we reported a detailed investigation on the interaction of TpyCuCl₂ with histidine.⁵ In this paper, we present our study on the fluorescent response of TpyCuCl₂ toward cysteine. Through a variety of spectroscopic analyses, we have demonstrated that TpyCuCl₂ exhibits a very different response mechanism in its fluorescent recognition of cysteine.

Results and discussion

Previously, we have shown that when Tpy (2.0 × 10⁻⁵ M in 25 mM HEPES buffer solution) is treated with 1 equiv. CuCl₂, its fluorescence is completely quenched.⁵ This could be attributed to either energy or electron transfer from the coordinated Tpy to the Cu(II) center. When the isolated TpyCuCl₂ (2.0 × 10⁻⁵ M) was dissolved in 25 mM HEPES buffer solution (pH = 7.40), addition of cysteine greatly enhanced its fluorescence (Fig. 1a). Fig. 1b plots the fluorescent enhancement of TpyCuCl₂ at 352 nm versus the concentration of cysteine. In this reaction, the solution of TpyCuCl₂ was colorless which did not change with the addition of cysteine.

Fig. 1 (a) Fluorescent responses of TpyCuCl₂ (2.0 × 10⁻⁵ M) toward cysteine (0–20 equiv.) in 25 mM HEPES buffer solution (pH = 7.40) (λ_exc = 298 nm, slit: 5 nm/5 nm). (b) The fluorescence response of TpyCuCl₂ (2.0 × 10⁻⁵ M) at λ = 352 nm versus the concentration of cysteine concentration (CPS: counts per second).

We studied the UV-vis absorption response of TpyCuCl₂ toward cysteine in the HEPES buffer solution under the same conditions as the fluorescent study. As shown in Fig. 2a, when TpyCuCl₂ was treated with 1–8 equiv. of cysteine, the UV absorption spectrum underwent only small changes mainly with a decrease of the absorption at λ_max = 340 nm. However, when the concentration of cysteine increased from 10–18 equiv., free Tpy was generated as shown in Fig. 2b and c. Thus, under these conditions, cysteine can displace Tpy off the metal center.

Fig. 2 UV spectra of Tpy and TpyCuCl₂ (2.0 × 10⁻⁵ M in 20 mM HEPES buffer solution, pH = 7.4) with 0–7 (a), 8–18 (b), and 18 equiv. of L-cysteine (c). (The absorptions of the stoichiometric amount of L-cysteine were subtracted from the corresponding plots.)

In order to gain additional information for the reaction of TpyCuCl₂ with cysteine, we also studied the fluorescent response of TpyCuCl₂ toward cysteine in a pure water solution without the buffer. The concentrated solution of TpyCuCl₂ in water (4.0 × 10⁻³ M) had a bluish color with no fluorescence. Addition of cysteine to this concentrated solution changed its color to light purple with enhanced emission at much longer wavelengths as shown in Fig. 3.

Fig. 3 Fluorescence spectra of TpyCuCl₂ in a concentrated water solution (4.0 × 10⁻³ M) treated with 0, 1, 5, 10, 20 equiv. cysteine (λ_exc = 298 nm, slit = 5 nm/5 nm).

When the concentrated bluish TpyCuCl₂ water solution (4.0 × 10⁻³ M) was diluted to 2.0 × 10⁻⁵ M, it became colorless and remained colorless with the addition of various amounts of cysteine. As shown in Fig. 4, when TpyCuCl₂ (2.0 × 10⁻⁵ M in H₂O) was treated with 2–20 equiv. of cysteine, large fluorescent enhancement at the short wavelength was observed similar to that observed in the HEPES buffer solution. Thus the colors at high concentration and the subsequent long wavelength emission upon treatment with cysteine indicate strong intermolecular interactions of TpyCuCl₂ in the ground state and/or excited state at the high concentration.

Fig. 4 Fluorescence spectra of TpyCuCl₂ (2.0 × 10⁻⁵ M in water) treated with 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 equiv. cysteine (λ_exc = 298 nm, slit = 5 nm/5 nm).

The UV spectra of TpyCuCl₂ (2.0 × 10⁻⁵ M) in water upon treatment with various amounts of cysteine were studied. As shown in Fig. 5, these solutions gave only decrease of the absorption at λ_max = 340 nm without the formation of free Tpy. This is similar to that shown in Fig. 2a at lower equiv. of cysteine for the reaction with TpyCuCl₂ in the HEPES buffer, but very different from that shown in Fig. 2b at higher concentrations of cysteine where free Tpy was generated.

Fig. 5 UV-vis absorption spectra of TpyCuCl₂ (2.0 × 10⁻⁵ M in water) treated with 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 equiv. cysteine (a), and 0 and 20 equiv. cysteine compared with Tpy (b). (The absorptions of the stoichiometric amount of L-cysteine were subtracted from the corresponding plots.)

We conducted a mass spectroscopic analysis for the reaction mixture of TpyCuCl₂ with cysteine in water and the results were summarized in Table 1. When TpyCuCl₂ (4.0 × 10⁻³ M in water) was treated with various equiv. of solid cysteine, the resulting solution, after centrifugal removal of the undissolved cysteine, was subjected to a TOF-ES⁺ mass analysis within 24 h. When 1 equiv. cysteine was used, the mass spectrum showed a base peak at m/z = 331 (100) which is assigned to TpyCuCl⁺ (Fig. S2 in ESI†). A peak at m/z = 296 (7.5) is assigned to TpyCu⁺. These signals are similar to those of TpyCuCl₂ in the absence of cysteine (Fig. S1 in ESI†). When TpyCuCl₂ was treated with 10 equiv. cysteine, the resulting product gave additional mass peaks besides the main peaks of TpyCuCl⁺ and TpyCu⁺ (Fig. S3 in ESI†). One peak at m/z = 416 (8.2) can be assigned to the cysteine coordinated complex 1⁺ (Fig. 6) and another peak at m/z = 400 (17.0) can be assigned to (1-NH₂)⁺. In addition, a peak at m/z = 241 (47.8) can be assigned for (cystine + H)⁺, the oxidized product of cysteine. The signal of (cysteine + H)⁺ is observed at m/z = 122 (22.9).

Table 1 Mass spectroscopic data for the reaction mixture of TpyCuCl₂ (4.0 × 10⁻³ M in water) with cysteine

	Observed m/z (relative intensity)	Peak assignment (calculated mass)
TpyCuCl2	330.98 (100)	TpyCuCl^+ (331.00)
	296.03 (7.5)	TpyCu^+ (296.03)
+1 eq. Cys	330.98 (100)	TpyCuCl^+ (331.00)
	296.03 (7.5)	TpyCu^+ (296.03)
+10 eq. Cys	330.99 (100)	TpyCuCl^+ (331.00)
	296.03 (7.5),	TpyCu^+ (296.03)
	416.03 (8.2)	Complex 1^+ (461.05)
	400.01 (17.0)	(1-NH2)^+ (400.03)
	241.05 (47.8)	(Cystine + H)^+ (241.03)
	122.03 (22.9)	(Cysteine + H)^+ (122.03)
+20 eq. Cys	330.99 (16.2)	TpyCuCl^+ (331.00)
	241.03 (35.2),	(Cystine + H)^+ (241.03)
	263.01 (6.6)	(Cystine + Na)^+ (263.01)
	362.05 (13.7)	[(Cysteine + cysteine) + H]^+ (362.05)
	481.08 (7.6)	[(Cystine)2 + H]^+ (481.06)
	416.03 (11.7)	Complex 1^+ (461.05)
	122.03 (41.3)	(Cysteine + H)^+ (122.03)
	243.05 (100)	[(Cysteine)2 + H]^+ (243.05)
	265.03 (11.3)	[(Cysteine)2 + Na]^+ (265.03)
	364.06 (15.9)	[(Cysteine)3 + H]^+ (364.07)
	485.09 (9.6)	[(Cysteine)4 + H]^+ (485.09)
+20 eq. Cys (pH = 7.4 with NaOH)	256.08 (100)	(Tpy + Na)^+ (256.09)
	263.01 (19)	(Cystine + Na)^+ (263.01)
	489.17 (17)	[(Tpy)2 + Na]^+ (489.18)
	525.01 (17)	[(Cystine)2 + Cu − H2O]^+ (524.98)
	547.00 (16.5)	[(Cysteine)4 + Cu]^+ (547.02)

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Fig. 6 Structures and exact mass data of the TpyCu-based complexes, cysteine and cystine.

As shown in Fig. S4 in ESI,† further increasing the amount of cysteine to 20 equiv. for the reaction with TpyCuCl₂ greatly decreases the relative intensity of the peak of TpyCuCl⁺ at m/z = 331 (16.2) with significantly increased signals for the cysteine oxidation products at m/z = 241 (35.2) for (cystine + H)⁺, at m/z = 263 (6.6) for (cystine + Na)⁺, at m/z = 362 (13.7) for [(cysteine + cysteine) + H]⁺, and at m/z = 481 for [(cystine)₂ + H]⁺. The peak for complex 1⁺ at m/z = 416 (11.7) was observed. The following peaks for cysteine were also observed: at m/z = 122 (41.3) for (cysteine + H)⁺, at m/z = 243 (100) for [(cysteine)₂ + H]⁺, at m/z = 265 (11.3) for [(cysteine)₂ + Na]⁺, at m/z = 364 (15.9) for [(cysteine)₃ + H]⁺, and at m/z = 485 (9.6) for [(cysteine)₄ + H]⁺. In all of the mass spectra, however, only trace amount of free Tpy signal was observed. This indicates that displacement of Tpy from TpyCuCl₂ by cysteine in pure water solution is not significant which is consistent with that shown in the UV-vis absorption spectra of Fig. 5. This is in contrast to that occurred in the HEPES buffer solution as shown by Fig. 2b and c.

We determined the pH values for the reaction of TpyCuCl₂ (pH = 6.55) with cysteine (isoelectric point at 5.05) in pure water solution. As shown in Fig. 7, the pH of the reaction solution decreased with the addition of cysteine. At 20 equiv. cysteine, the pH of the reaction solution is 6.10 which is 20 times more acidic than that of the HEPES buffer solution at pH = 7.40. In Fig. 7, we also show that addition of cysteine to a water solution at pH = 6.55 caused very little change in the pH value in the absence of TpyCuCl₂. That is, the reaction of TpyCuCl₂ with cysteine should produce more acid.

Fig. 7 pH values for the reaction of TpyCuCl₂ (2.0 × 10⁻⁵ M) with cysteine (0–20 equiv.) in pure water solution (blue line) and those of a water solution (adjusted to 6.55 before adding cysteine) with the same amount of cysteine added without TpyCuCl₂ (red line).

In order to determine the role of pH in this reaction, we treated the water solution of TpyCuCl₂ (2.0 × 10⁻⁵ M) + 20 equiv. cysteine with 50 mM NaOH and monitored the UV-vis absorption of this solution when the pH was adjusted to 7.40. As shown in Fig. 8, when the pH was adjusted from 6.10 to 7.40, the absorption at 340 nm decreased while the characteristic absorption peaks of Tpy appeared.

Fig. 8 UV-vis absorption of TpyCuCl₂ (2.0 × 10⁻⁵ M) + cysteine (20 equiv.) in pure water solution before and after treatment with NaOH solution compared with Tpy.

The above TpyCuCl₂ (1 mM) + 20 equiv. cysteine solution at pH = 7.40 was subjected to mass spectroscopic analysis (Fig. S5†) and the spectrum shows an intense peak at m/z = 256 (100) for (Tpy + Na)⁺, indicating the displacement of Tpy from the copper complex (Table 1). The following peaks for the complexes of cysteine and cystine with copper at m/z = 547 and 525 respectively were also observed as shown in Table 1. These peaks are absent in the mass spectra of the reaction of TpyCuCl₂ with cysteine in the pure water solution. It demonstrates that at a higher pH, cysteine and cystine displace the Tpy ligand off the copper center.

We also studied the interaction of TpyCuCl₂ with cysteine in a PBS buffer (20 mM, pH = 7.40) by using fluorescence and UV-vis absorption spectroscopic analyses. Similar to that observed in the HEPES buffer, the fluorescence of TpyCuCl₂ was turned on with the addition of cysteine (Fig. 9). The UV-vis absorption spectra show the generation of free Tpy with the addition of more than 4 equiv. of cysteine (Fig. 10). These observations substantiate our hypothesis that the different UV-vis responses of TpyCuCl₂ toward cysteine in pure water and HEPES buffer are mainly due to the pH difference.

Fig. 9 (a) Fluorescent responses of TpyCuCl₂ (2.0 × 10⁻⁵ M) toward cysteine (0–20 equiv.) in 20 mM PBS buffer solution (pH = 7.40) (λ_exc = 298 nm, slit: 5 nm/5 nm). (b) The fluorescence response of TpyCuCl₂ (2.0 × 10⁻⁵ M) at λ = 352 nm versus the concentration of cysteine concentration.

Fig. 10 UV spectra of TpyCuCl₂ (2.0 × 10⁻⁵ M in 20 mM PBS buffer solution, pH = 7.4) with 0–20 equiv. of L-cysteine (a) and 0, 20 equiv. of L-cysteine compared with Tpy (b). (The absorptions of the stoichiometric amount of L-cysteine were subtracted from the corresponding plots.)

The UV-vis absorption and luminescence of Tpy have been studied in details before.¹⁰ The absorption spectra of Tpy in neutral and basic solutions show two absorption bands at around 235 and 285 nm while the acidic solution and the metal chelate compound exhibits three main absorption bands at around 230, 285 and 325 nm.^10a Both the protonated and the neutral forms of Tpy are fluorescent but the intensity in the neutral form is weaker than that in the protonated form. More specifically, the quantum yield of Tpy in pure water is 0.27 and that in 0.1 N H₂SO₄ is 0.61.^10c

It was also reported that when a Cu(II) complex was treated with cysteine, there was oxidation of cysteine to cystine as well as reduction of Cu(II) to Cu(I).¹¹ On the basis of the literature precedents and our studies described above, a reaction scheme of TpyCuCl₂ with cysteine is proposed in Scheme 1. In water solution, TpyCuCl₂ can oxidize cysteine to cystine while forming the Cu(I) complex 1. In this Cu(I) complex, the reduced copper center as well as the coordination of cysteine might have inhibited the energy or electron transfer from Tpy to the copper, turning on the fluorescence. The mass spectroscopic analysis shows the molecular ion of 1, but we currently do not have direct evidence for the oxidation state of this complex. Nevertheless, the literature precedents¹¹ and our observed oxidation product cystine support the reduction of the Cu(II). This step produces two molecules of HCl and increases the acidity of the solution as shown by Fig. 8. In the HEPES buffer solution, the ammonium group of 1 should be more significantly deprotonated because of the higher pH value which should allow a better coordination of the amine group to the Cu(I) center to displace the Tpy ligand off. Thus, free Tpy was observed from the reaction in the higher pH HEPES buffer solution but not in the lower pH water solution.

Scheme 1 A proposed reaction of TpyCuCl₂ with cysteine.

Conclusions

We have demonstrated that the classical coordination complex TpyCuCl₂ exhibits large fluorescent enhancement in the presence of cysteine both in a HEPES buffer solution and a pure water solution. On the basis of the UV-vis absorption and mass spectroscopic analyses, it is proposed that in the pure water solution (pH = 6.1–6.5), cysteine can reduce Cu(II) to TpyCuSCH₂CH(NH₂)CO₂H with greatly enhanced fluorescence. In the HEPES buffer solution of a higher pH (7.40), after the reduction of Cu(II) to Cu(I) by cysteine, the Tpy ligand can be further displaced off the copper center by the amino acid, also contributing to the fluorescent enhancement. These findings should be of significant value for the development of the TpyCuCl₂-based fluorescent sensor for amino acids.

Experimental section

General data

Fluorescent emission spectra except those in Fig. 10 were obtained by using FluoroMax-4 Spectrofluorophotometer (HORIBA Jobin Yvon) at 298 K. Fluorescent emission spectra in Fig. 10 were obtained by using Cary Eclipse Fluorescence Spectrophotometer at 298 K. Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. All of the solvents used in the optical spectroscopic studies were either HPLC or spectroscopic grade. CuCl₂·2H₂O was used as the Cu²⁺ source.

Preparation of TpyCuCl₂

A THF solution (5 mL) of Tpy (233 mg, 1.0 mmol) was mixed with CuCl₂·2H₂O (205 mg, 1.2 mmol) in H₂O (20 mL) which was stirred at room temperature for 30 min. The resulting solution was then concentrated to about 5 mL to give green precipitate. The green solid was collected by filtration, washed with CH₃OH (5 mL) and dried under vacuum to give TpyCuCl₂ (305 mg, 83%).

Preparation of the solutions for UV and fluorescence spectroscopic analyses

A solution of TpyCuCl₂ (30 μL, 2 mM in water) was treated with various equivalences of cysteine (12 mM in water, 5 μL equal to 1 equiv.) in optical cuvettes which was allowed to stand at room temperature for 10 min. The resulting solution was diluted with either 3 mL HEPES buffer (20 mM, pH = 7.40) solution or 3 mL water. Measurements were then taken after 10 min. There is no precipitation or color change observed during the process.

Preparation of HEPES buffer solution

HEPES (4.766 g, 20 mmol) was dissolved in water (950 mL). The pH was adjusted to 7.40 by adding a NaOH solution and additional water was added to obtain a 1 L solution.

Preparation of PBS buffer solution

A solution of NaH₂PO₄ (3.8 mL 0.100 M) was mixed with that of Na₂HPO₄ (16.2 mL 0.100 M). The pH was adjusted to 7.40 with a HCl solution or a NaOH solution. The mixture was then diluted to 100 mL with water.

Acknowledgements

The authors gratefully acknowledge the Sichuan University High Level Talent Project and Sichuan Province 1000 Talents Plan Project for financial support.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra08234j

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