Ratiometric Fluorescent Probe for the Detection of Glutathione in Living Cells†

doi:10.7503/cjcu20150725

Abstract

Abstract:

A fluorescent probe(4) for the detection of biothiols was designed and synthesized and its properties for labeling glutathione was investigated. After reaction with glutathione in 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid(HEPES) buffer, the color of solution changed from light yellow to pink, which could be detected by naked eyes. Meanwhile, the fluorescence was enhanced at 608 nm. Probe 4 was useful for measuring glutathione at concentrations ranging from 1.6×10^-5 to 2×10^-4 mol/L with a detection limit of 8.9×10^-7 mol/L. Besides, probe 4 was sensitive to glutathione in MCF-7 cells, which emitted red fluorescence when it was incubated with MCF-7 cells.

Key words: Fluorescent probe, Glutathione, Intracellular imaging

CLC Number:

O657
O625.75

TrendMD:

YANG Runjie, TANG Yao, ZHU Weiping. Ratiometric Fluorescent Probe for the Detection of Glutathione in Living Cells^†[J]. Chem. J. Chinese Universities, 2016, 37(4): 643.

Figures/Tables 8

Scheme 1 Synthetic routes of probe 4

Table 1 Appearance, melting points, yields, HRMS and elemental analysis data of compounds 3 and 4

Compd.	Appearance	m. p./℃	Yield(%)	HRMS(ESI) [M-H]^-(calcd.), m/z	Elemental analysis(%, calcd.)
Compd.	Appearance	m. p./℃	Yield(%)	HRMS(ESI) [M-H]^-(calcd.), m/z	C	H	N
3	Red solid	293.6—294.2	53	302.0930(302.0934)	70.87(71.29)	4.18(4.29)	13.72(13.86)
4	Yellow solid	﹥300	40	532.0564(532.0563)	53.03(54.03)	2.94(2.81)	13.06(13.13)

Table 2 1H NMR and 13C NMR data of compounds 3 and 4

Compd.	¹H NMR(DMSO-d₆, 400MHz), δ	¹³C NMR(DMSO-d₆, 100 MHz), δ
3	10.63(s, 1H, OH), 7.91(d, 1H, CH═C), 7.81(d, 2H, ArH), 7.02(d, 1H, CH═C), 6.9(d, 2H, ArH), 1.77(s, 6H, CH₃)	177.3, 175.8, 162.3, 148.3, 132.3, 125.7, 116.4, 112.9, 112.1, 111.7, 111.2, 99.0, 96.6, 53.2, 25.3
4	9.14(s, 1H, ArH),8.61(dd, 1H, ArH), 8.27(d, 1H, ArH), 8.00(d, 2H, ArH), 7.89(d, 1H, CH═C), 7.34(d, 2H, ArH), 7.23(d, 1H, CH═C), 1.79(s, 6H, CH₃)	176.9, 174.6, 151.6, 150.3, 148.1, 144.9, 134.4, 133.6, 131.4, 130.5, 127.5, 122.8, 121.1, 116.9, 112.5, 111.7, 110.6, 100.8, 99.5, 54.9, 54.9, 25.0

Fig.1 UV-Vis absorption(A) and fluorescence spectra(B) of probe 4(10 μmol/L) before(a) and after(b) reaction with glutathione(200 μmol/L) in HEPES buffer(0.1 mol/L, pH=7.4, 1% DMSO)Inset of (A): color changes of solution before(a) and after(b) reaction.

Fig.2 UV-Vis absorption spectra of probe 4(10 μmol/L) with different concentrations of GSH(A) and the linear relationship of A568/A394 vs. the concentrations of GSH over 0—200 μmol/L(B)c(GSH)/(μmol·L-1): 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200.

Fig.3 UV-Vis absorption(A) and fluorescence spectra(B) changes of probe 4(10 μmol/L) to glutathione(200 μmol/L) in 15 min

Fig.4 Fluorescence emission changes of probe 4(10 μmol/L) in HEPES upon addition of different amino acids(200 μmol/L)

Fig.5 Bright-field(A, C) and fluorescence(B, D) images of MCF-7 cells(A, B) and MCF-7 cells incubated with 50 μmol/L probe 4 for 30 min(C, D)

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