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A new fluorescein isothiocyanate-based screening method for the rapiddiscovery of electrophilic compounds

Xiaoyu Zhang and Zhongjun Ma*

Received 10th May 2010, Accepted 27th July 2010

DOI: 10.1039/c0ay00305k

In the present study, a simple fluorescein isothiocyanate (FITC)-based screening method was

established for the rapid discovery of electrophilic compounds from natural products. The test sample

is firstly allowed to alkylate a certain proportion of glutathione (GSH) under basic conditions (pH 8.0),

then FITC is added to conjugate with the remaining GSH from the first step. By analyzing the

fluorescence intensity of the reaction solution under acidic conditions (pH 3.0), it could be determined

whether the test sample contained electrophilic compounds. A botanical sample, the ethyl acetate

extract of Radix Salvia miltiorrhiza, was tested using this screening assay and we successfully discovered

four electrophilic compounds from it, which were miltirone and its three derivatives (1-oxomiltirone,

4-methylenemiltirone, and 1,2-didehydromiltirone). The findings indicate that the screening method is

effective and suitable for high-throughput screening (HTS) assays in the future.

1. Introduction

Electrophilic compounds are considered as redox-active

compounds because of their electron-deficient centers.1 In cells,

electrophilic compounds can activate or inhibit specific signal

transduction pathways as a consequence of reacting with nucle-

ophiles, including protein thiols, glutathione (GSH) or guanine

bases in DNA.2,3 Thus they often manifest two disparate actions in

the life processes: a toxic or a preventive effect. Some electrophilic

compounds can attack macromolecules such as proteins and

DNA3 or deplete the cellular level of GSH (e.g. menadione4

and doxorubicin5), resulting in inhibition of the transcription and

replication of DNA or abrogation of the reducing capability of the

cell. In contrast, numerous electrophilic compounds can be used

as antitumor6–8 and neuroprotective agents.9–11 For example,

miltirone and tert-butyl hydroquinone (tBHQ) have been proven

to possess preventive effects because of their activities of inducing

NAD(P)H: quinone oxidoreductase 1 (NQO1).12,13

Fluorescein isothiocyanate (FITC) is a common fluorophore

with high fluorescence. It plays a particularly important role in

biosciences because of its reactive properties towards amine and

sulfhydryl groups. Previous studies have indicated that FITC

could be used in the labeling and imaging of proteins14,15 and

DNA primers.16 It could also be used as a labeling reagent in

separation of chiral amino acids through laser-induced fluores-

cence detection techniques.17,18

In this study, a simple FITC-based screening method was

established for the rapid discovery of electrophilic compounds

from natural products based on the initial finding that the fluo-

rescence intensities of FITC and FITC-GSH conjugate were

different under acidic conditions (pH 3.0). As shown in Fig. 1, the

screening method includes three main steps. First, the test sample

alkylates a certain proportion of GSH under basic conditions (pH

School of Pharmaceutical Sciences, Zhejiang University, ZijingangCampus, No. 388 Yuhangtang Rd, Hangzhou, 310058, P. R. China.E-mail: mazj@zju.edu.cn; Fax: +86-571-88208428; Tel: +86-571-88208427

1472 | Anal. Methods, 2010, 2, 1472–1478

8.0). Then FITC is added to conjugate with the remaining GSH

that did not react with the test sample in the first step. Finally, the

pH value of the solution is adjusted to 3.0 and the fluorescence

intensity is measured with excitation at 485 nm and emission at 535

nm. Compared with the control group, electrophilic compounds

can reduce the formation of FITC-GSH conjugates in the last step,

resulting in a lower fluorescence intensity of the reaction solution.

2. Experimental

2.1 Materials and chemicals

FITC, menadione and GSH were purchased from Sigma-Aldrich

(St. Louis, MO, USA). The roots of Salvia miltiorrhiza were

collected at Shanxi Province, People’s Republic of China, in

December 2007. The plant material was identified by the authors

and a voucher specimen (No. SM071211) has been deposited in the

herbarium of the School of Pharmaceutical Sciences, Zhejiang

University. Ten tanshinones including Miltirone, 1-oxomiltirone,

4-methylenemiltirone, 1,2-didehydromiltirone, 15,16-dihydrotan-

shinone I, 1,2-dihydrotanshinquinone, 17-hydroxycryptotan-

shinone, Tanshinone IIB, Tanshinone I and Methyl

dihydronortanshinonate were isolated from the ethyl acetate extract

of root of S. miltiorrhiza, the procedures of extraction and isolation

were described previously.12 The structures of ten tanshinones were

elucidated by NMR spectroscopic analysis and their purities were

all greater than 95% according to HPLC analysis. HPLC-grade

acetonitrile (Merck, Darmstadt, Germany), methanol (Merck,

Darmstadt, Germany) and formic acid (Tedia, Fairfield, OH, USA)

were utilized for the HPLC analysis. Deionized water was prepared

using a Milli-Q system (Millipore, Bedford, MA, USA). All other

chemicals and solvents were of analytical-reagent grade.

2.2 Liquid chromatography-tandem mass spectrometry

(LC-MS) analysis of FITC-GSH conjugate

The stock solution of FITC (2 mM) was prepared in HPLC-

grade acetonitrile. Briefly, FITC was incubated with 1 mM GSH

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Fig. 1 Schematic of the FITC-based screening method for the discovery of electrophilic compounds.

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in a total volume of 100 mL of 25 mM Tris-HCl buffer solution

(pH 8.0) for 30 min at room temperature (the final concentration

of FITC was 100 mM). The reaction solution was analyzed using

a Thermo Finnigan LCQ Deca XPplus ESI ion trap mass spec-

trometer (San Jose, CA, USA) in positive ion mode equipped

with an Agilent 1100 HPLC system (Waldbronn, Germany) and

a Zorbax SB–C18 column (4.6 mm � 250 mm, 5 mm, Agilent

Technologies, USA). The mobile phase consisted of A (HCOOH:

H2O ¼ 0.2 : 100) and B (HCOOH: CH3CN ¼ 0.2 : 100).

A 30 min gradient was used from 90% A to 100% B and

Fig. 2 The reaction between FITC and GSH. (A) The HPLC chromatograms

UVl¼ 280 nm. (B) Positive ion ESI mass spectrum of FITC (upper graph) and

FITC under basic conditions (pH 8.0).

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maintained at 100% B during the next 6 min. The flow rate was

0.5 mL min�1 and the column temperature was set at 30 �C. The

ultraviolet (UV) spectra were recorded at 280 nm. The MS

operating parameters were as follows: collision gas, ultra-high-

purity helium (He); nebulizing gas, high-purity nitrogen (N2); ion

spray voltage, �4.5 kV; sheath gas (N2), 5 arbitrary units;

capillary temperature, 275 �C; capillary voltage,�15 V; tube lens

offset voltage, �30 V. The collision energy for collision-induced

dissociation (CID) was between 30% and 45%, and the isolation

width of precursor ions was 3.0 Th.

before (upper graph) and after (lower graph) FITC incubation with GSH,

FITC-GSH conjugate (lower graph). (C) Proposed reaction of GSH with

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2.3 LC-MS analysis of FITC-CH3OH conjugate

FITC was dissolved in methanol (the final concentration of

FITC was 2 mM) for 1 h at room temperature. The reaction

solution was analyzed using a Thermo Finnigan LCQ Deca

XPplus ESI ion trap mass spectrometer in positive ion mode

equipped with an Agilent 1100 HPLC system and a Zorbax

SB–C18 column (4.6 mm � 250 mm, 5 mm). The mobile phase,

gradient program and MS operating parameters were the same as

described above.

2.4 Fluorescence assay of FITC and FITC-GSH conjugate in

different pH environments

The assay was performed in 96-well plates (Corning Costar).

Briefly, 95 mL/well of a series of concentrations ranging from 0 to

1 mM GSH in 25 mM Tris-HCl buffer solution (pH 8.0) was

added, followed by 5 mL/well FITC stock solution (the final

concentration of FITC was 0.1 mM). For the basic condition,

after 30 min incubation at room temperature, the fluorescence

was measured using infinite F200 scanning fluorescence plate

reader (Tecan, Mannedorf, Switzerland) with excitation at

485 nm and emission at 535 nm. For the acidic condition, after

30 min incubation at room temperature, 15 mL 1% HCl was

Fig. 3 The fluorescence spectra and intensities of FITC and FITC-GSH conj

fluorescence emission (Em) spectra of FITC and FITC-GSH conjugate a

FITC ¼ 0.1 mM) and 3.0 (concentration of FITC ¼ 1 mM). (B) 0.1 mM FITC

1 mM for 30 min and the fluorescence intensities were measured with excita

(pH 3.0) conditions, respectively.

1474 | Anal. Methods, 2010, 2, 1472–1478

added to each well and the fluorescence was measured using

infinite F200 scanning fluorescence plate reader with excitation

at 485 nm and emission at 535 nm.

2.5 Fluorescence assay for test sample

The assay was performed in 96-well plates. Typically, 90 mL/well

1 mM GSH in 25 mM Tris-HCl buffer solution (pH 8.0) was

added, followed by 5 mL/well test sample (dissolved in acetoni-

trile). After 30 min incubation at room temperature, 5 mL/well

FITC solution (dissolved in acetonitrile) was added and incu-

bated for another 30 min, the final concentration of FITC was

0.1 mM. Then 15 mL/well 1% HCl was added into each well.

The fluorescence was measured using infinite F200 scanning

fluorescence plate reader with excitation at 485 nm and emission

at 535 nm.

2.6 LC-MS analysis of GSH conjugation with the ethyl acetate

extract of S. miltiorrhiza

The ethyl acetate extract of S. miltiorrhiza was incubated with

5 mM GSH for 1 h at 37 �C in a total volume of 200 mL of 25 mM

Tris-HCl buffer solution (pH 8.0) (The final concentration of the

ethyl acetate extract of S. miltiorrhiza was 2 mg mL�1). A control

ugate in different pH environments. (A) Fluorescence excitation (Ex) and

t pH 8.0 (concentration of FITC ¼ 0.1 mM), 5.0 (concentration of

was incubated with a series of concentrations of GSH ranging from 0 to

tion at 485 nm and emission at 535 nm under basic (pH 8.0) and acidic

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Fig. 4 Assay development and optimization. (A) Time course of the reaction at room temperature. (B) The HPLC chromatogram and corresponding

tandem mass spectrum of FITC in methanol for 1 h.

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experiment was carried out in which the ethyl acetate extract of

S. miltiorrhiza was incubated with 25 mM Tris–HCl buffer

solution (pH 8.0) only. The reaction solution was analyzed using

a Thermo Finnigan LCQ Deca XPplus ESI ion trap mass

Fig. 5 Screening assay for a known electrophilic compound, menadione. (A

reaction solution decreased as the concentration of menadione increased.

concentrations of menadione (0–2.5 mM) were incubated with GSH (1 mM) fo

30 min. The reaction solution was analyzed by LC-MS. (C) Tandem mass spec

FITC-GSH conjugate in positive ion mode (lower graph).

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spectrometer in positive ion mode equipped with an Agilent 1100

HPLC system and a Zorbax SB–C18 column (4.6 mm � 250 mm,

5 mm). The mobile phase consisted of A (HCOOH: H2O ¼0.03 : 100) and B (CH3CN). A gradient program was used

) Concentration course of the reaction. The fluorescence intensity of the

(B) The HPLC chromatograms of the reaction solution. A series of

r 30 min, and then FITC (0.1 mM) was added and incubated for another

tra of menadione-GSH conjugate in negative ion mode (upper graph) and

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according to the following profile: 0–90 min, 20–100% B and

maintained at 100% B during the next 10 min. The MS operating

parameters were the same as described above.

3. Results and discussion

3.1 The fluorescence intensities of FITC and FITC-GSH

conjugate in different pH environments

We first investigated the reaction between GSH and FITC. In the

HPLC chromatogram, for which the wavelength of detection

was 280 nm, the FITC peak eluting at 26.97 min diminished after

Fig. 6 Screening assay for the ethyl acetate extract of S. miltiorrhiza and ten

acetate extract of S. miltiorrhiza (0.5 mg mL�1, 0.25 mg mL�1, 0.1 mg mL�1, 0.

alkylating capabilities using the screening assay. (B) The HPLC chromatogram

with GSH, UVl ¼ 254 nm. (C) Chemical structures of ten tanshinones isola

electron-deficient centers. (D and E) Screening assay for ten isolated tanshino

1476 | Anal. Methods, 2010, 2, 1472–1478

incubation with GSH, and one extra peak formed, eluting at

15.93 min (Fig. 2A). In the corresponding tandem mass spec-

trum, the [M + H]+ ion at m/z 697 was identified as FITC-GSH

conjugate (Fig. 2B). Considering the chemical structure of FITC,

the carbon of the isothiocyanate group is electron-deficient

because its electron density is drawn to the sulfur beside it, thus

making FITC more reactive for the alkylation of the thiol group

of GSH (Fig. 2C).

It is known that the fluorescence intensity of FITC is depen-

dent on the pH value of the assay buffer.19 Thus we investigated

the fluorescence spectra of FITC and FITC-GSH conjugate in

different pH environments. FITC (0.1 mM) was allowed to

tanshinones isolated from it. (A) A series of concentrations of the ethyl

05 mg mL�1, 0.025 mg mL�1 and 0.01 mg mL�1) were selected to test their

s before and after incubation of the ethyl acetate extract of S. miltiorrhiza

ted from the ethyl acetate extract of S. miltiorrhiza. Asterisks represent

nes.

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Table 1 The fluorescence intensities of test samples and their conju-gating capabilities with GSH

Compound

Fluorescenceintensity of control(%) at theconcentration2.5 mM

GSH conjugatingrate (%) asliteraturereports12

Miltirone 51.0 � 1.9 1001-oxomiltirone 46.8 � 3.5 1004-methylenemiltirone 45.2 � 6.6 1001,2-didehydromiltirone 49.6 � 3.1 10015,16-dihydrotanshinone I 95.1 � 2.2 01,2-dihydrotanshinquinone 99.5 � 2.9 017-hydroxycryptotanshinone 95.9 � 0.9 0Tanshinone IIB 97.2 � 1.8 0Tanshinone I 95.2 � 3.4 0Methyl dihydronortanshinonate 101.2 � 1.2 0

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incubate with GSH (1 mM) and then the pH value of the reaction

solution was adjusted to 8.0, 5.0 and 3.0, respectively. We might

consider the fluorescence intensity of the reaction solution in

which 0.1 mM FITC was incubated with 1 mM GSH represented

the fluorescence intensity of FITC-GSH conjugate because FITC

formed FITC-GSH completely (as shown in Fig. 2A). When the

pH values were 8.0 and 5.0, the fluorescence excitation and

fluorescence emission spectra of FITC and FITC-GSH conjugate

were almost the same (Fig. 3A). However, when the pH value

was adjusted to 3.0, the fluorescence excitation and fluorescence

emission spectra of FITC and FITC-GSH conjugate were

different (Fig. 3A). We noticed that in the fluorescence excitation

spectra of FITC and FITC-GSH conjugate at pH 3.0, there were

two excitation peaks at 441 nm and 485 nm. It was known that

there were often some fluorescent products in the botanical

sample (e.g., coumarins) that could interfere with the results if

441 nm was chosen as the excitation wavelength, thus excitation

at 485 nm was selected for further experiments. Subsequently, we

chose a series of concentrations of GSH ranging from 0 to 1 mM

for incubation with 0.1 mM FITC and measured the fluorescence

intensity of the reaction solution under basic (pH 8.0) and acidic

(pH 3.0) conditions, respectively. Compared with the control

group, after 30 min incubation of GSH with FITC, the fluores-

cence intensity of the reaction solution increased in a dose-

dependent manner under acidic conditions (pH 3.0) but was

almost the same under basic conditions (pH 8.0) (Fig. 3B).

3.2 Assay development and optimization

A time course study showed that the reaction was complete

within 10 min at pH 8.0 and the fluorescence intensity of FITC-

GSH conjugate was almost unchanged during the next 50 min

(Fig. 4A). So an incubation time of 30 min was selected for

further experiments.

As methanol is a commonly used solvent for compounds in

screening assays, the methanol tolerance of this assay was

examined. FITC was dissolved in methanol and analyzed by

LC-MS after 1 h. It was found that after 1 h incubation, FITC

and methanol formed an FITC-CH3OH conjugate (Fig. 4B),

which could be explained by the hydroxyl of CH3OH making

a nucleophilic attack on the isothiocyanate group of FITC.

When using acetonitrile as solvent, FITC was more stable (data

not shown), thus acetonitrile was selected as the suitable solvent

for FITC.

3.3 Screening assay for a known electrophilic compound

menadione

Based on the above findings that the fluorescence intensities of

FITC and FITC-GSH conjugate were different under acidic

conditions (pH ¼ 3.0), we established a simple screening method

for the rapid discovery of electrophilic compounds. The sche-

matic of the screening assay was shown in Fig. 1. A known

electrophilic compound, menadione, was tested in this screening

assay. Previous studies had shown that menadione could easily

alkylate GSH.20 A series of concentrations of menadione

(0–2.5 mM) were incubated with 1 mM GSH for 30 min, then

0.1 mM FITC was added and incubated for another 30 min. As

shown in Fig. 5A, the fluorescence intensity of the reaction

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solution decreased as the concentration of menadione increased.

The results of LC-MS analysis of the reaction solution indicated

that when 2.5 mM menadione was added, FITC-GSH conjugate

could not be detected in the HPLC chromatogram (Fig. 5B).

When lower concentrations of menadione were added, the FITC-

GSH conjugate signal gradually increased in a dose-dependent

manner (Fig. 5B).

3.4 Screening assay for the ethyl acetate extract of S.

miltiorrhiza and ten tanshinones isolated from it

A botanical sample, the ethyl acetate extract of S. miltiorrhiza,

was tested using this screening method. As shown in Fig. 6A, the

fluorescence intensity of the reaction solution decreased as the

concentration of the ethyl acetate extract of S. miltiorrhiza

increased, indicating there might be some electrophilic

compounds contained in it, which was verified by the subsequent

LC-MS analysis of the incubation solution of the ethyl acetate

extract of S. miltiorrhiza and GSH. As shown in Fig. 6B, after

incubation of the ethyl acetate extract of S. miltiorrhiza with

GSH, plenty of extra peaks formed in the HPLC chromatogram,

which represented the GSH conjugates. Then ten tanshinones

were isolated from the ethyl acetate extract of S. miltiorrhiza

(Fig. 6C) and tested for their alkylating capabilities. The results

indicated that miltirone and its three derivatives (1-oxomiltirone,

4-methylenemiltirone and 1,2-didehydromiltirone) were electro-

philic compounds while the other six were not (Fig. 6D and 6E),

which was in accordance with the previous report,12 in which

miltirone, 1-oxomiltirone, 4-methylenemiltirone and 1,2-dide-

hydromiltirone could react with GSH completely (Table 1). The

results were easily understood because the four electrophilic

compounds all contained electron-deficient centers (as the

asterisks indicated in Fig. 6C) and thus could be easily attacked

by the thiol group of GSH.

4. Conclusion

In this paper, a simple FITC-based screening method was

established for the rapid discovery of electrophilic compounds.

It was found that the fluorescence intensities of FITC and FITC-

GSH conjugate were different under acidic conditions (pH 3.0).

Based on these findings, we tested the ethyl acetate extract of

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S. miltiorrhiza and successfully identified four electrophilic

compounds from it. Therefore, it is a fast and effective screening

method and is suitable for high-throughput screening (HTS)

assays in the future.

Acknowledgements

The work was financially supported by Zhejiang Key Science &

Technological Program (2009C13028), Administration of

Traditional Chinese Medicine of Zhejiang Province (2010ZA049)

and Zhejiang Innovation Program for Graduates (YK2009015).

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