Báo cáo khoa học: A novel metallobridged bis(b-cyclodextrin)s fluorescent probe for the determination of glutathione doc

A novel metallobridged bisb-cyclodextrins fluorescent probe for the determination of glutathione Bo Tang, Fang Liu, Kehua Xu and Lili Tong College of Chemistry, Chemical Engineering and

A novel metallobridged bis(b-cyclodextrin)s fluorescent probe for the determination of glutathione

Bo Tang, Fang Liu, Kehua Xu and Lili Tong

College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, China

Reduced glutathione (GSH: c-Glu-Cys-Gly), a

princi-pal non-protein thiol compound, plays an important

role in many biological processes such as transport,

protein synthesis, catabolism and metabolism [1] It

can also protect cells against reactive oxygen species

and help them maintain an adequate intracellular

redox status [2] Thus, the quantitative detection of

GSH is very important for investigating biological

pro-cesses Many methods have been developed to

deter-mine GSH, including HPLC [3,4], electrochemistry

[5,6], spectrofluorimetry [7,8] and so on Although

these methods are currently available for GSH

deter-mination, most of them are complicated and

inconve-nient to operate The determination of GSH in plasma

is particularly challenging because redox conditions

change rapidly after blood collection [9,10] Therefore,

a rapid and simple method for the analysis of GSH in

plasma is needed

Cyclodextrins (CDs) are a class of cyclic oligosac-charides with six to eight d-glucose units linked by a-1,4-glucose bonds They possess a hydrophobic cavity capable of including a variety of hydrophobic compounds via host–guest complexation [11] They are also widely used as a solubilizer because of their hydrophilic exterior [12,13] Among various functional CDs, bridged bisCDs, which comprise two CD cavities linked by a functional bridged has received great attention [14] In comparison with native CDs and mono-modified CDs, bridged bisCDs exhibit signifi-cant high-binding ability and molecular recognition through the cooperative binding of two adjacent CD units [15] Furthermore, metallobridged bis(b-CD)s can afford more stable inclusion complexes with guest molecules through the cooperative binding of two b-CD cavities and the additional interactions between the coordinated metal and the guest molecule [16]

Keywords

competitive complexation; glutathione;

metallobridged bis(b-cyclodextrin)s;

molecular recognition; spectrofluorimetry

Correspondence

B Tang, College of Chemistry, Chemical

Engineering and Materials Science,

Engineering Research Center of Pesticide

and Medicine Intermediate Clean

Production, Ministry of Education, Key

Laboratory of Molecular and Nano Probes,

Ministry of Education, Shandong Normal

University, Jinan 250014, China

Fax: +86 531 8618 0017

Tel: +86 531 8618 0010

E-mail: tangb@sdnu.edu.cn

(Received 30 November 2007, revised 13

January 2008, accepted 25 January 2008)

doi:10.1111/j.1742-4658.2008.06310.x

A novel metallobridged bis(b-cyclodextrin)s 2 [bis(b-CD)s 2] was synthesized and characterized by means of 1H NMR, IR, element analysis and redox iodometric titration The fluorescence of metallobridged bis(b-CD)s 2 was weak compared with bis(b-CD)s 1 because of the paramagnetism of copper (II) ions Glutathione was able to form complexes with copper (II) derived from the metallobridged bis(b-CD)s 2 This competitive complexa-tion with copper (II) may lead to a significant fluorescence recovery of the bis(b-CD)s Therefore, a rapid and simple spectrofluorimetric method was developed for the determination of glutathione The analytical application for glutathione was investigated in NaCl⁄ Pi(pH 6.00) at room temperature The linear range of the method was 0.30–20.0 lmolÆL)1with a detection limit

of 63.8 nmolÆL)1 There was no interference from the plasma constituents The proposed method had been successfully used to determine glutathione in human plasma

Abbreviations

bis(b-CD), bis(b-cyclodextrin); CD, cyclodextrin; GSH, glutathione.

In this study, we synthesized a novel fluorescent

bis(b-CD)s 1 containing two metal-binding sites and

two naphthyl fluorophores (Scheme 1) The compound

showed satisfactory water solubility because of the two

b-CDs Complexes 2 were formed when copper (II)

ions were added to bis(b-CD)s 1, at the same time,

flu-orescence quenching was discovered Afterwards, the

addition of GSH to 2 induced a recovery of

fluores-cence (Scheme 2) Based on this principle, we

devel-oped a rapid and simple spectrofluorimetric method

for the analysis of GSH The proposed method has

been successfully applied to the determination of GSH

in human plasma

Results and Discussion

Metal coordination and stoichiometry

Job’s experiments were performed to explore the

coor-dination stoichiometry of the bis(b-CD)s 1–copper (II)

complex in aqueous solution as described previously

[16] A representative Job’s plot for the coordination of

bis(b-CD)s 1 with copper (II) chlorate is shown in Fig 1 The plot for the 1⁄ Cu2+ system showed a maximum at 0.67 which corresponded to a 1⁄ Cu2+

stoichiometry of 1 : 2 This indicates that one

Scheme 1 Synthesis of the novel

metallo-bridged bis(b-CD)s.

Scheme 2 The detection mechanism.

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18

Fig 1 Job’s plot of the 1 ⁄ Cu 2+ system at 349 nm [1] + [Cu 2+ ] = 1.00 · 10)4molÆL)1; pH 6.00.

bis(b-CD)s 1 could bind two copper (II) ions, as

illus-trated in Scheme 1

Excitation and emission spectra

Following the procedure described below, the

excita-tion (left) and emission (right) spectra were scanned

(Fig 2) The maximum excitation and emission

wave-lengths were 365 and 480 nm respectively The

fluores-cence intensity of metallobridged bis(b-CD)s 2 was

weak compared with bis(b-CD)s 1 A significant

recov-ery of fluorescence was observed when GSH was

added to metallobridged bis(b-CD)s 2 in this analytical

system

Influence of pH

Because of the instability of CD and the amido bond

at very low pH, the use of strongly acidic solution was

avoided [17] Moreover, copper (II) will deposit in

alkali solution Thus the optimal pH of the system was

in the range 4.00–9.00 The results are shown in Fig 3

As can be seen, the fluorescence intensity was relatively

high and remained almost constant over the pH range

5.00–6.50 Therefore, a pH of 6.00 was fixed using

NaCl⁄ Pibuffer

The effect of the buffer is lost if too small a quantity

is used Whereas if the amount of buffer is excessive,

the ionic strength is too great, which influences the

flu-orescence intensity Therefore, the influence of the

vol-ume of buffer was measured Because the volvol-ume of

buffer added (1.00–3.00 mL) had little effect on the

fluorescence intensity, 2.00 mL of buffer was chosen in

subsequent experiments

Influence of the concentration of metallobridged bis(b-CD)s 2

The influence of the concentration of 2 on fluorescence intensity is shown in Fig 4 As can be seen, as the concentration of 2 increased, the fluorescence intensity

of the system also increased slightly We therefore used 2.00 mL of 2.00· 10)4molÆL)1 metallobridged bis(b-CD)s 2

Influence of reaction time The effect of reaction time was studied, the result (Fig 5) showed that the fluorescence intensity reached

a maximum after the reagents had been added for

300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

0

500

1000

1500

2000

2500

3000

3500

4000

4500

3 3

1 1

1:bis(β-CD)s1

2:metallobridged bis(β-CD)s2

3:metallobridged bis(β-CD)s2+GSH

Wavelength (nm)

Fig 2 Excitation (left) and emission (right) spectra

[bis(b-CD) 1] = 2.00 · 10)5molÆL)1; [metallobridged bis(b-CD) 2] =

2.00 · 10)5molÆL)1; [GSH] = 5.00 · 10)6molÆL)1; pH 6.00.

150 200 250 300 350

pH

Fig 3 Influence of pH on the fluorescence intensity [GSH] = 2.00 · 10)6molÆL)1; [2] = 4.00 · 10)5molÆL)1.

160 180 200 220 240 260 280 300 320 340

Fig 4 Influence of the concentration of 2 on fluorescence inten-sity [GSH] = 2.00 · 10)6molÆL)1; pH 6.00.

 10 min and remained constant for at least 1 h.

Hence, the reaction was left to proceed for 10 min,

and the fluorescence was then measured at room

tem-perature

Influence of interference

The influence of the main constituents of plasma on

the determination of 2.00· 10)6molÆL)1 GSH were

studied The criterion for interference was fixed at a

± 5.0% variation in the average fluorescence intensity

calculated for the established level of GSH A

3000-fold mass excess of plama over 2.00· 10)6molÆL)1

GSH was tested first If interference occurred, the ratio

was gradually reduced until interference ceased The

results are shown in Table 1 and it can be seen that

the determination was free from interference by the

constituents of plasma

Mechanism

The novel fluorescent bis(b-CD)s 1 contained two

strong coordination sites for copper (II) ions and two

naphthyl fluorophores The compound could be

dis-solved in aqueous solution and showed high binding

ability because of the two adjacent bis(b-CD)s

Because of the conformation of the linker of 1, the

nitrogen atoms and amido bond formed two chelate

rings to coordinate with copper (II) ions This

coordi-nation effect and the paramagnetism of copper (II)

ions induced fluorescence quenching However, GSH

has a great propensity for forming complexes with

metal ions that have strong electrophilic characteristics

[18], such as copper (II) [19,20], mercury [21] and

cadmium [22] In this system, the quenched copper (II)

complex 2 could interact with the thiol and amino of GSH via a cooperative chelation effect [23,24], which led to recovery of the fluorescence intensity of bis(b-CD)s Based on this principle, we developed a spectro-fluorimetric method with high selectivity to determine GSH in human plasma

Analytical characteristics Under optimum experimental conditions, there was a linear relationship between fluorescence intensity and GSH concentration in the range 0.30–20.0 lm with a correlation coefficient of 0.9976 (Fig 6) The regres-sion equation was F = 2644.17 + 63.98 [GSH] (lm) The detection limit, as defined by IUPAC [25], was determined to be 63.8 nmolÆL)1, according to the formula C = KS0⁄ S, where K = 3 (standard devia-tion = 1.36), obtained from a series of 11 reagent blanks, and S is the slope of the standard curve The relative standard deviation was 2.5%, obtained from a series of 11 standards each containing 2.00 lm GSH When the concentration of GSH exceeded that of metallobridged bis(b-CD)s 2 by as much as 100-fold,

a decrease in fluorescence intensity was discovered This is consistent with that previously reported by Liu et al [16]

100

120

140

160

180

200

220

240

260

280

300

320

340

0

Time (min)

Fig 5 Effect of reaction time on fluorescence intensity.

[GSH] = 2.00 · 10)6molÆL)1; [2] = 4.00 · 10)5molÆL)1; pH 6.00.

Table 1 Interferences of various coexisting biological substances.

Coexisting substance

Concentration (molÆL)1)

Relative error (%)

Sample collecting and processing

Fasting venous blood (5.00 mL) was routinely

col-lected from the author Y Liu, and transferred to a

10 mL centrifuge tube containing heparin sodium as

an anticoagulant The blood was immediately

centri-fuged at 1000 g for 1 min at room temperature to

remove cells and platelets [10] Afterwards, 0.50 mL

of absolute alcohol was added to the plasma

with shaking Plasma proteins were precipitated

and removed by centrifugation The final plasma

samples used in the determination of GSH were

obtained

Determination of GSH in plasma and accuracy assessment by recovery experiments

In order to evaluate the applicability of the proposed method, fluorescence determination in plasma was per-formed according to the following procedures Into a series of 1.00-mL Eppendorf microtubes were sequen-tially added different aliquots of the plasma samples, GSH stock solution (3.00 · 10)4molÆL)1), 0.04 mL of 1.00· 10)3molÆL)1 metallobridged bis(b-CD) 2 and 0.20 mL of 0.10 molÆL)1 NaCl⁄ Pi (pH 6.00) The experimental data are shown in Table 2 The mixture was diluted to mark with ultra-pure water, shaken thoroughly and equilibrated at room temperature for

10 min The fluorescence intensity of the solution was measured at 365⁄ 480 nm

The GSH content of the plasma was derived from the standard curve and the regression equation The average recovery test was made using the standard addition method, and the RSD was generally good when obtained from a series of six plasma samples These results are also given in Table 2 Compared with previously reported methods (Table 3), our results indicate that the recovery and precision of our method

of determining GSH in plasma are satisfactory

Conclusions

We synthesized a novel metallobridged bis(b-CD)s 2, which afforded two hydrophobic binding sites coopera-tively associating with the guest GSH and also provided additional binding interactions between the hetero-atoms of GSH and the coordinated metal center GSH was able to form complexes with copper (II) derived from the metallobridged bis(b-CD)s 2 This competitive

2600

2800

3000

3200

3400

3600

3800

4000

Fig 6 Linear plot of fluorescence intensity with increase in GSH

concentration [2] = 4.00 · 10)5molÆL)1; pH 6.00 All spectra were

obtained under the optimum experimental conditions at

365 ⁄ 480 nm and room temperature.

Table 2 GSH determination in plasma samples (n = 6, P = 95%).

Samples

Plasma (mL)

GSH added (l M )

Measureda (l M )

RSD (%)

Recovery (%)

GSH content

of plasma (l M )

a Mean of six determinations using the proposed method.

Table 3 Analytical characteristics compared with other methods reported.

complexation with copper (II) may lead to a

fluores-cence recovery of the bis(b-CD)s Based on this

princi-ple, we developed a spectrofluorimetric method with

high selectivity to determine GSH The linear range of

the method was 0.30–20.0 lmolÆL)1 with a detection

limit of 63.8 nmolÆL)1 There was no interference from

the plasma constituents The proposed method was

suc-cessfully used to determine GSH in human plasma

Experimental procedures

Apparatus and reagents

All spectrofluorimetric measurements were carried out with

an Edinburgh FLS920 spectrofluorimeter (Edinburgh

Instru-ments Ltd, Livingston, UK) equipped with a xenon lamp and

1.0 cm quartz cell Absorption spectra were obtained from

UV-1700 (Shimadzu, Kyoto, Japan) UV–visible

spectroph-tometer Infrared spectra were obtained from a PE-983G

IR-spectrophotometer (Perkin-Elmer, Palm Springs, CA, USA)

pHS-3 digital pH meter (Shanghai Lei Ci Device Works, Shanghai,

China) with a combined glass-calomel electrode

Centrifuga-tion was carried out on a of Sigma 3K 15 centrifuge

Reduced glutathione (99.8%) (Sigma, Mannheim,

Ger-many) was used without further purification A stock

ultra-pure water b-CD (China Medicine Group Shanghai

Chemical Reagent Corporation, Shanghai, China) was

puri-fied by recrystallizing twice in ultra-pure water, followed by

acid (Alfa Aesar, Word Hill, MA, USA) was used

Mono(6-p-toluenesulfonyl-6-deoxy)-b-cyclodextrin was prepared by reacting p-tosyl

chloride with b-CD in dry pyridine as described previously

was then converted to

mono(6-aminoethylamino-6-deoxy)-b-CD with 57.1% yield upon heating in excess

oxamide bis(2-naphthyl) acid, was prepared according to the

procedure reported previously [31] Other chemicals used

were of analytical reagent grade The water used in this study

100 k Nanosep filter (Pall Corp., East Hills, NY, USA) and

micoron YM—30-30000 NMWL (Millipore, Billerica, MA,

USA) were used as ultra-purification instrumentation

Synthesis of the novel bis(b-CD)s

Synthesis of compound 1

Mono (6-aminoethylamino-6-deoxy)-b-CD (2.00 g) was

dis-solved in dimethylformamide (50 mL) in the presence of a

small amount of 0.4 nm molecular sieves, and then 3 (0.21 g) was added The mixture was stirred for 24 h at

until no further precipitate was deposited The precipitate was removed by filtration, and the filtrate evaporated to dryness under reduced pressure The residue was dissolved

in a minimum amount of hot water and poured into ace-tone to give an orange precipitate The orange precipitate was purified by three recrystallization steps in ultra-pure water After the residue had been dried under a vacuum,

H NMR (300 MHz,

4.00–4.95 (m, 28H); 5.50–6.00 (m, 26H); 7.01–7.08 (m, 2H); 7.23–7.29 (m, 2H); 7.40–7.48 (m, 2H); 7.60–7.70 (m, 2H);

m 3383.3, 2928.2, 2151.4, 1703.7, 1653.6, 1522.2, 1368.4, 1231.9, 1156.0, 1080.2, 1030.5, 945.7, 859.5, 755.9, 706.8,

48.77; H, 6.12; N, 3.24

Synthesis of metallobridged bis(b-CD)s 2 According to the Liu et al [16], bis(b-CD)s 1 was added dropwise to a dilute aqueous solution of slightly excess cop-per (II) chlorate in an ice-water bath Several drops of chlo-roform were further added, and the resultant solution was

under reduced pressure, and the precipitate formed was collected by filtration, washed successively with a small amount of ethanol and diethyl ether, and dried in vacuo to

3419.5, 2930.3, 2048.1, 1637.6, 1536.4, 1406.0, 1337.1, 1301.6, 1238.3, 1155.3, 1121.3, 1078.7, 1028.9, 946.5, 856.1, 755.1, 706.6, 618.1, 579.2, 531.3 Elemental analysis

N, 2.79 Found: C, 44.85; H, 5.72; N, 3.04

Redox iodometric titration of copper (II) was also per-formed to establish the coordination stoichiometry of com-plex 2 We dissolved 1.508 g of comcom-plex 2 in 50 mL of ultra-pure water, and added 25.00 mL of the complex 2 solution to a 125 mL Erlenmeyer flask This was analyzed iodometrically Copper (II) was first reduced to Cu(I) by

KI according to the following reaction:

I2þ 2S2O23 ! 2Iþ S4O26 The percentage of copper in the sample was 4.41 The results confirmed that the mole ratio of complex 2 to

copper (II) was 1 : 2, which was consist with the Job’ s plot

Calibration graph

Into a series of 10-mL colorimetric tube were

sequenti-ally added different aliquots of GSH stock solution

was diluted to mark with ultra-pure water, shaken

thor-oughly and equilibrated at room temperature for 10 min

The fluorescent intensity of the solution was measured at

Acknowledgements

This study was supported by the National Basic

Research Program of China (973 Program,

2007CB936000), National Natural Science Funds for

Distinguished Young Scholar (No.20725518), Major

Program of National Natural Science Foundation of

China (No.90713019), National Natural Science

Foun-dation of China (No.20575036) Important Project of

Natural Science Foundation of Shandong Province in

China (No.Z2006B09) and the Research Foundation

for the Doctoral Program of Ministry of Education

(No.20060445002)

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