7-Aminocoumarinyldisulfide as a ratiometric fluorescent probe for biothiols in water

Sensors and Actuators B 185 (2013) 720–724

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7-Aminocoumarinyldisulfide as a ratiometric fluorescent probe for biothiols in water Soo-Yeon Lim, Min-Jeong Na, Hae-Jo Kim ∗ Department of Chemistry, Hankuk University of Foreign Studies, Yongin 449-791, Republic of Korea

a r t i c l e

i n f o

Article history: Received 15 March 2013 Received in revised form 14 May 2013 Accepted 14 May 2013 Available online 27 May 2013

a b s t r a c t We report an aminocoumarine-based fluorescent probe (1) possessing a disulfide group to detect biothiols in HEPES buffer. Prominent ratiometric fluorescence changes of the probe are induced by the disulfide bond cleavage of 1 upon the addition of biothiols. The ratiometric probe (1) exhibits both high sensitivity and selectivity toward amino acids containing a thiol group over other various amino acids in water. © 2013 Elsevier B.V. All rights reserved.

Keywords: Biothiol Coumarin Disulfide bond Fluorescence Ratiometric

1. Introduction

2. Experimental

Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are playing essential roles in biological processes such as cellular growth and redox homeostasis [1]. Alternation in the level of the cellular biothiols has been also associated with many human diseases, such as lethargy, liver damage, loss of muscle and fat [2], Alzheimer’s disease [3], disorders in xenobiotic metabolism and gene regulation [4]. Therefore, the detection of biothiols is of growing interest. Among the various detection methods, fluorescent probes are widely developed due to their high sensitivity, simplicity and low detection limit. To date, though a number of fluorescent probes were developed for thiols, [5] most of them have shown fluorescence intensity changes on their binding with biothiols. These intensity changes were, however, liably disrupted by many factors, such as variations in excitation intensity and emission collection efficiency, and artifacts associated with probe concentration and the environment [6]. In this regard, ratiometric fluorescent probes were reported to have advantage over the intensity-based probes [5b,6]. Herein, we report an aminocoumarinyldisulfide probe (1) which displays a ratiometric fluorescence change to afford the quantitative detection of biothiols by a thiol-induced disulfide cleavage reaction.

2.1. General All reagents and solvent were purchased from commercial sources and used without further purification, unless otherwise stated. All reactions were magnetically stirred and monitored by thin-layer chromatography (TLC). The compounds were separated by column chromatography on silica gel 60 (230–400 mesh). Absorption and fluorescence spectra were taken on an Agilent 8453 spectrophotometer and a JASCO FP-6500 fluorescence spectrometer, respectively. For fluorescence detection including competitive experiments, appropriate amounts of each amino acid (1000 equiv.) was added to a stock solution of 1 (10 mM in DMSO) and the mixture was diluted with HEPES buffer (0.10 M, pH 7.4) to afford the final concentration of 20 ␮M of 1. The spectra were monitored after saturation, 10 h. NMR measurements were performed with 200 or 300 MHz (1 H) and a 50 or 75 MHz (13 C) spectrometer. The solvent for the NMR measurements was dimethyl sulfoxide (DMSO-d6 ). All peaks were given as ı in ppm and were related to the signals of residual nondeuterated solvent peaks. The following abbreviations were used to explain the multiplicities: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. 2.2. Preparation of 1

∗ Corresponding author. Tel.: +82 31 3304703; fax: +82 31 3304566. E-mail address: [email protected] (H.-J. Kim). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.05.053

S.-Y. Lim et al. / Sensors and Actuators B 185 (2013) 720–724

2-Hydroxyethyl disulfide (154 mg, 1.00 mmol), tert-butyl dimethylsilyl chloride (TBDMSCl, 150 mg, 1.00 mmol) and imidazole (68 mg, 1.0 mmol) were dissolved in tetrahydrofuran (THF, 10 ml) at 0 ◦ C. The reaction mixture was stirred for 1 h. After completion of the reaction, the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silicagel using ethylacetate and hexane (1:5, v/v) as an eluent, to give compound 2 as a colorless liquid (120 mg) in 45% yield. 1 H NMR (CDCl , 200 MHz): ı 3.91 (q, 3 J = 6.4 Hz, 2H), 3.87(t, 3 3 J = 6.6 Hz, 2H), 2.86 (t, 3 J = 5.4 Hz, 2H), 2.84 (t, 3 J = 6.5 Hz, 2H), 2.03 (t, 3 J = 6.4 Hz, 1H), 0.90 (s, 9H), 0.08 (s, 6H).

N,N-Diisopropylethylamine (DIPEA, 174.0 ␮L, 1.00 mmol) was added to a solution of 2 (268 mg, 1.00 mmol) in THF (0.50 mL). Triphosgene (178 mg, 0.50 equiv.) dissolved in THF (0.50 mL) was added slowly over 10 min. The formation of the chloroformate was monitored by TLC. 7-Amino-4-methylcoumarin (175 mg, 1.00 mmol) and DIPEA (356 ␮L, 2.0 mmol) were dissolved in dry dimethylformamide (DMF, 1.0 mL) at 0 ◦ C. The crude chloroformate was added slowly over 10 min to the aminocoumarin solution. The reaction mixture was stirred at room temperature for 3 h. After completion of the reaction, all the volatiles were removed under reduced pressure. The crude compound was purified by column chromatograph on silicagel using ethylacetate/hexane (1:1, v/v)) as an eluent, to give compound 3 as a white solid (120 mg) in 26% yield. 1 H NMR (CDCl , 200 MHz): ı 7.58 (d, 3 J = 8.6 Hz, 1H), 7.47 (d, 3 4 J = 2 Hz, 1H), 7.40 (dd, 3 J = 5.4 Hz, 4 J = 2 Hz, 1H), 6.94 (s, 1H), 6.22 (d, 4 J = 1.2 Hz, 1H), 4.50 (t, 3 J = 6.4 Hz, 2H) 3.90 (t, 3 J = 6.6 Hz, 2H), 3.01 (t, 3 J = 6.4 Hz, 2H), 2.88 (t, 3 J = 6.6 Hz, 2H), 2.44 (d, 4 J = 1.2 Hz, 3H), 0.93 (s, 9H), 0.11 (s, 6H).

Triethylamine trihydrofluoride (0.30 mL, 5 equiv.) was added to a solution of compound 3 (120 mg, 250 ␮mol) dissolved in dichloromethane (DCM, 1.0 mL) at 0 ◦ C. The reaction mixture was stirred to obtain precipitates at room temperature for 3 h. The precipitates were filtered and washed by DCM several times. Product 1 was obtained as a white solid in 80% yield. 1 H NMR (DMSO-d , 200 MHz): ı 10.27 (s, 1H), 7.72 (d, 3 J = 8.8 Hz, 6 1H), 7.56 (d, 4 J = 2 Hz, 1H), 7.45 (dd, 3 J = 8.8 Hz, 4 J = 2 Hz, 1H), 6.25 (d, 4 J = 1.2, Hz 1H), 4.91 (s, 1H), 4.40 (t, 3 J = 6.2 Hz, 2H) 3.67 (t, 3 J = 6.4 Hz, 2H), 3.07 (t, 3 J = 6.2 Hz, 2H), 2.86 (t, 3 J = 6.4 Hz, 2H), 2.39 (d, 4 J = 1.2 Hz, 3H). 13 C NMR (DMSO-d , 75 MHz): ␦ 160.49, 154.27, 153.63, 153.53, 6 143.11, 126.47, 114.88, 114.77, 112.41, 104.99, 62.99, 59.88, 41.56, 37.17, 18.47 (15 carbon peaks). HRMS (MALDI+ , DHB): m/z obsd 378.0446 ([M+Na]+ , calcd 378.0440 for C15 H17 NO5 S2 Na).

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2.3. General UV–vis and fluorescence spectral measurements A stock solution (10 mM) of 1 in DMSO was prepared and used by dilution in 0.10 M HEPES buffer (pH 7.4). For UV–vis measurement, sample solutions were obtained by mixing appropriate amount of stock solution of 1 (10 mM in DMSO) with appropriate amount of each amino acid (AA) and finally diluted with 0.10 M HEPES buffer solution (pH 7.4) to afford desired concentrations of probe 1 and AA in HEPES buffer. The fluorescence spectroscopy was measured similarly with a slit width of 3 nm × 3 nm.

2.4. Binding stoichiometry The reaction stoichiometry of 1 with 2-mercaptoethanol (ME) was determined by using Job’s plot [7]. For the Job’s plot analysis, a series of solutions with varying mole fraction of ME were prepared by maintaining the total 1 and ME concentrations constant (20 ␮M). The fluorescence emission was measured for each sample by exciting at 340 nm and the spectra were measured from 350 to 650 nm. For Job’s plot, fluorescence at 440 nm was monitored for each solution and its intensity (I) were plotted against the X1 (mole fraction of 1) of final solution.

3. Results and discussion The synthesis of ratiometric fluorescent probe 1 began on the partial protection of the hydroxyl groups of 2,2 dithiodiethanol by TBDMS chloride (Scheme 1). The unprotected

hydroxyl group of compound 2 was activated as chloroformate with triphosgene and treated with 7-amino-4-methylcoumarin to afford carbamate 3. The TBDMS group of 3 was removed using triethylamine trihydrofluoride to afford final product 1. Fig. 1 shows the time-dependent fluorescence spectral changes of 1 (20 ␮M) on the addition of GSH (20 mM) and IgG (0.10 mM) in HEPES buffer (0.10 M, pH 7.4). When excited at ex 345 nm, probe 1 displayed an initial maximum emission at 400 nm. Upon the addition of GSH, the maximum peak underwent a red shift to 440 nm with a clear isoemissive point at 413 nm, whereas oxidized gluthathione (GSSG) and immunoglobulin G (IgG), a model protein, did not induce any fluorescence changes. The ratio of fluorescence intensities (F440 /F400 ) changes from 0.50 to 9.3, showing a prominent ratiometric fluorescence for GSH. The reaction rate of 1 was linearly increased by the addition of GSH (Fig. 2). The initial rate analysis showed that the reaction of 1

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S.-Y. Lim et al. / Sensors and Actuators B 185 (2013) 720–724

S

HO

TBDMSCl

OH

S

S

HO

OTBDMS

S

2 O

1) Triphosgene, DIPEA O

2) 7-Amino-4-methylcoumarin

O

N H

S

O

S

OTBDMS

3 O

3HF TEA O

O

N H

S

O

S

OH

1

Scheme 1. Synthesis of 1.

R(F440/F400)

60

Intensity (a.u.)

1+GSH

9

80

1+IgG

6 3 0 0

120

240

360

480

600

Time/min

40

20

0 450

350

550

λ /nm Fig. 1. Time-dependent fluorescence spectra of 1 (20 ␮M) upon the addition of GSH (20 mM) in HEPES buffer (0.10 M, pH 7.4). Inset: ratiometric fluorescence kinetics for GSH (20 mM) and IgG (0.10 mM).

was moderate to give a second order reaction kinetics with GSH, k2 = 6.5 × 10−3 M−1 s−1 . The reaction stoichiometry between 1 and thiol was determined by the Job’s plot [7], which showed one to one stoichiometry between probe 1 and GSH (Fig. 3). In order to estimate the selectivity of probe 1 toward thiolcontaining amino acids, we screened several natural amino acids (AAs) possessing neutral, basic and acidic side chains. Probe 1 exhibited a selective ratiometric fluorescence response to AAs with

a thiol side chain such as Cys, Hcy, and GSH, whereas it did not induce any significant fluorescence changes with other AAs (Pro, Ala, Val, His, Gly, Gln, Glu, Ser, Asn, Asp, Lys, Arg) (Fig. 4A). To confirm the selective response of 1 toward the biothiols, competitive assays were performed by adding GSH (20 mM) to 1 (20 ␮M) in the presence of other AAs (20 mM) in HEPES buffer (0.10 M, pH 7.4) (Fig. 4B). The addition of GSH to the mixture of 1 and AA induced as much ratiometric fluorescence as that of 1-GSH conjugate except some AAs containing an acidic side chain (Glu, Asp). The acidic AA might interrupt the disulfide bond cleavage reaction as observed in pH profile. To investigate the effect of pH on the fluorescence response of 1 to thiols, the fluorescence intensity of 1 was measured at various pHs in the presence and absence of GSH. While 1 itself showed a weak fluorescence intensity over a wide range of pH, strong fluorescence of 1 in the presence of GSH was detectable at basic pH (pH 8–13) (Fig. 5). This indicated that probe 1 could be used to detect GSH around biological pH condition. The ratiometric fluorescence changes of 1 were also observable by the naked eye. Upon the addition of GSH to 1 in HEPES buffer (0.10 M, pH 7.4), the solution displayed a strong blue fluorescence, while other AAs did not induce any detectable changes under a portable UV spectroscope (Fig. 6). To determine the low limit of detection of 1 for biothiol, the ratiometric fluorescence of 1 was measured by incremental addition of GSH (0–20 mM) in HEPES buffer. The limit of detection (LOD) for GSH was determined to be 6.0 ␮M at 3/m, where  is the standard deviation of blank measurements and m the slope obtained from the linear plot of 1 against GSH (Fig. 7) [8]. 60

30

20

F 4440

k/10-5ssec-1

y = 0.647x + 0.537 R² = 0.995

30

10

0

0 0

10

20

30

40

[GSH]/mM Fig. 2. Second-order rate constant of 1 (20 ␮M) upon the addition of various amounts of GSH in HEPES buffer (0.10 M, pH 7.4).

0

0.3

0.6

0.9

[GSH]/([1]+[GSH]) [GSH]/([1]+[GSH]) Fig. 3. Job’s plot of the reaction between 1 (ex = 345 nm) and GSH in HEPES (0.10 M, pH 7.4) where total concentrations of 1 and ME was kept constant at 20 ␮M.

S.-Y. Lim et al. / Sensors and Actuators B 185 (2013) 720–724

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Fig. 4. (A) Fluorescence response of 1 (20 ␮M, ex = 345 nm, 10 h) with various amino acids(AA, 20 mM) and GSH (20 mM) in HEPES buffer (0.10 M, pH 7.4) (B) The competitive assay of 1 +AA by the addition of GSH (20 mM). 18 1

5

1 + GSH

F440/F4000

4 F440/F400

12

y = 0.0371 x + 0.3793 R² = 00.9993 9993

3 2

6

1 0

0 3

5

7

9

11

13

pH

0

50

100

GSH/[μM]

Fig. 5. The fluorescence response of 1 (ex 345 nm) in the presence and absence of GSH under different pH in HEPES (0.10 M, pH 7.4) ([1] = 20 ␮M, [GSH] = 20 mM).

Fig. 7. Linear plot of 1 (20 ␮M, ex = 365 nm) upon the addition of various amounts of GSH (0–20 mM) in HEPES (0.10 M, pH 7.4).

To elucidate the reaction mechanism, we investigated 1 H NMR spectra in DMSO-d6 after the addition of ME, an organic soluble model compound of biothiol and compared it with that of 1 itself (Fig. 8). Upon the addition of ME, NH proton of 1 disappeared, while the aromatic protons of 1 were significantly upfield shifted. The resulting aromatic protons were coincident with the spectrum of 7aminocoumarin. These spectral changes indicated that ME readily reacted with the relatively electron-poor species(1) to induce a

reactive intermediate (Int), which was cyclized to finally afford the electron-rich 7-aminocoumarine. Taking together the experimental evidence, we propose a reaction mechanism of 1 with GSH (Scheme 2). The reaction of 1 with GSH triggered cleavage of a disulfide bond through the disulfide exchange reaction, and a subsequent intramolecular cyclization gave free 7-aminocoumarin to afford ratiometric fluorescence response.

Fig. 6. Fluorescence image of 1 (20 ␮M, ex = 365 nm) with various AAs (20 mM, GSH, Pro, Ala, Val, His, Gly, Gln, Glu, Ser, Asn, Asp, Lys, Arg) in HEPES buffer (0.10 M, pH 7.4).

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O

O O

O

N H

S

O

S

GSH

OH

O

O

NH 2

S

O

1 O O

O

N H

O

SH

Int Scheme 2. A proposed reaction mechanism of 1 with GSH.

References

D C B A NH 10.0

9.0

8.0

7.0

6.0

Fig. 8. 1 H NMR spectra of 1 (10 mM) upon the addition of ME (1.1 equiv) in DMSO-d6 . (A) 1; (B) 1 + 1.1 equiv ME, 4 h; (C) 1d; and (D) 7-amino-4-methylcoumain.

[1] (a) Z.A. Wood, E. Schröder, J.R. Harris, L.B. Poole, Trends of Biochemical Science 28 (2003) 32; (b) S.Y. Zhang, C.-N. Ong, H.-M. Shen, Cancer Letter 208 (2004) 143. [2] S. Shahrokhian, Analytical Chemistry 73 (2001) 5972. [3] S. Seshadri, A. Beiser, J. Selhub, P.F. Jacques, I.H. Rosenberg, R.B.N. D’Agostino, New England Journal of Medicine 346 (2002) 476. [4] T.P. Dalton, H.G. Shertzer, A. Puga, Annual Review of Pharmacology and Toxicology 39 (1999) 67. [5] (a) W. Wang, O. Rusin, X. Xu, K.K. Kim, J.O. Escobedo, S.O. Fakayode, K.A. Fletcher, M. Lowry, C.M. Schowalter, C.M. Lawrence, F.R. Fronczek, I.M. Warner, R.M. Strongin, Journal of the American Chemical Society 127 (2005) 15949; (b) G.-J. Kim, K. Lee, K. Kwon, H.-J. Kim, Organic Letters 13 (2011) 2799; (c) H. Kwon, K. Lee, H.-J. Kim, Chemical Communications 47 (2011) 1773; (d) J. Bouffard, Y. Kim, T.M. Swager, R. Weisssleder, S.A. Hilderbrand, Organic Letters 10 (2008) 37. [6] K. Komatsu, Y. Urano, H. Kojima, T. Nagano, Journal of the American Chemical Society 129 (2007) 13447. [7] P. Job, Annales de Chimie 9 (1928) 113. [8] D. MacDougall, W.B. Crummett, Analytical Chemistry 52 (1980) 2242.

Biographies 4. Conclusion In conclusion, we prepared an aminocoumarin-based fluorescencent probe possessing a disulfide bond. Owing to the disulfide exchange reaction, probe 1 exhibited a highly selective and sensitive response to biothiols in HEPES buffer. Interestingly, probe 1 showed a ratiometric fluorescence change upon the addition of biothiols, which could be useful for the quantitative detection of biologically abundant GSH in living cells. Acknowledgements We thank the Hankuk University of Foreign Studies for financial support.

Soo-Yeon Lim is a master course student at the Hankuk University of Foreign Studies, Korea. Her research interest is to develop highly selective fluorescence sensors for biological thiols and reactive oxygen species for tumor imaging. Min-Jeong Na is a undergraduate course student at the Hankuk University of Foreign Studies, Korea. She joined Dr. H.-J. Kim’s laboratory for her junior research program at HUFS. Her research interest is to develop highly selective fluorescence sensors for biological thiols. Dr. Hae-Jo Kim received his B.S. degree in Chemistry Education, and M.S. and Ph.D. degrees in Chemistry from Seoul National University, Korea with Professor Jong-In Hong (2002). After completing his postdoctoral research at University of Toronto, Canada with Professor Jik Chin (2005), he joined Kyonggi University, Korea as a faculty member (2006). He is currently associate professor and chairperson of the Department of Chemistry at Hankuk University of Foreign Studies, Korea. His research focuses on fluorescent molecular probes for tumor imaging and therapy.