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

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A novel metallobridged bis(b-cyclodextrin)s fluorescentprobe for the determination of glutathioneBo Tang, Fang Liu, Kehua Xu and Lili TongCollege of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, ChinaReduced glutathione (GSH: c-Glu-Cys-Gly), a princi-pal non-protein thiol compound, plays an importantrole in many biological processes such as transport,protein synthesis, catabolism and metabolism [1]. Itcan also protect cells against reactive oxygen speciesand help them maintain an adequate intracellularredox status [2]. Thus, the quantitative detection ofGSH 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. Althoughthese methods are currently available for GSH deter-mination, most of them are complicated and inconve-nient to operate. The determination of GSH in plasmais particularly challenging because redox conditionschange rapidly after blood collection [9,10]. Therefore,a rapid and simple method for the analysis of GSH inplasma is needed.Cyclodextrins (CDs) are a class of cyclic oligosac-charides with six to eight d-glucose units linked bya-1,4-glucose bonds. They possess a hydrophobiccavity capable of including a variety of hydrophobiccompounds via host–guest complexation [11]. They arealso widely used as a solubilizer because of theirhydrophilic exterior [12,13]. Among various functionalCDs, bridged bisCDs, which comprise two CD cavitieslinked by a functional bridged has received greatattention [14]. In comparison with native CDs andmono-modified CDs, bridged bisCDs exhibit signifi-cant high-binding ability and molecular recognitionthrough the cooperative binding of two adjacent CDunits [15]. Furthermore, metallobridged bis(b-CD)s canafford more stable inclusion complexes with guestmolecules through the cooperative binding of twob-CD cavities and the additional interactions betweenthe coordinated metal and the guest molecule [16].Keywordscompetitive complexation; glutathione;metallobridged bis(b-cyclodextrin)s;molecular recognition; spectrofluorimetryCorrespondenceB. Tang, College of Chemistry, ChemicalEngineering and Materials Science,Engineering Research Center of Pesticideand Medicine Intermediate CleanProduction, Ministry of Education, KeyLaboratory of Molecular and Nano Probes,Ministry of Education, Shandong NormalUniversity, Jinan 250014, ChinaFax: +86 531 8618 0017Tel: +86 531 8618 0010E-mail: tangb@sdnu.edu.cn(Received 30 November 2007, revised 13January 2008, accepted 25 January 2008)doi:10.1111/j.1742-4658.2008.06310.xA novel metallobridged bis( b-cyclodextrin)s 2 [bis(b-CD)s 2] was synthesizedand characterized by means of1H NMR, IR, element analysis and redoxiodometric titration. The fluorescence of metallobridged bis(b-CD)s 2was weak compared with bis(b-CD)s 1 because of the paramagnetism ofcopper (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 thebis(b-CD)s. Therefore, a rapid and simple spectrofluorimetric method wasdeveloped for the determination of glutathione. The analytical applicationfor 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 limitof 63.8 nmolÆL)1. There was no interference from the plasma constituents.The proposed method had been successfully used to determine glutathione inhuman plasma.Abbreviationsbis(b-CD), bis(b-cyclodextrin); CD, cyclodextrin; GSH, glutathione.1510 FEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBSIn this study, we synthesized a novel fluorescentbis(b-CD)s 1 containing two metal-binding sites andtwo naphthyl fluorophores (Scheme 1). The compoundshowed satisfactory water solubility because of the twob-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, theaddition of GSH to 2 induced a recovery of fluores-cence (Scheme 2). Based on this principle, we devel-oped a rapid and simple spectrofluorimetric methodfor the analysis of GSH. The proposed method hasbeen successfully applied to the determination of GSHin human plasma.Results and DiscussionMetal coordination and stoichiometryJob’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 ofbis(b-CD)s 1 with copper (II) chlorate is shown inFig. 1. The plot for the 1 ⁄ Cu2+system showed amaximum at 0.67 which corresponded to a 1 ⁄ Cu2+stoichiometry of 1 : 2. This indicates that oneScheme 1. Synthesis of the novel metallo-bridged bis(b-CD)s.Scheme 2. The detection mechanism.0.2 0.4 0.6 0.8 absorbance (a.u.)[Cu2+] / ([Cu2+]+[1])Fig. 1. Job’s plot of the 1 ⁄ Cu2+system at 349 nm.[1] + [Cu2+] = 1.00 · 10)4molÆL)1; pH 6.00.B. Tang et al. Glutathione determination using bis(b-CD)sFEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBS 1511bis(b-CD)s 1 could bind two copper (II) ions, as illus-trated in Scheme 1.Excitation and emission spectraFollowing 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 wasweak compared with bis(b-CD)s 1. A significant recov-ery of fluorescence was observed when GSH wasadded to metallobridged bis(b-CD)s 2 in this analyticalsystem.Influence of pHBecause of the instability of CD and the amido bondat very low pH, the use of strongly acidic solution wasavoided [17]. Moreover, copper (II) will deposit inalkali solution. Thus the optimal pH of the system wasin the range 4.00–9.00. The results are shown in Fig. 3.As can be seen, the fluorescence intensity was relativelyhigh and remained almost constant over the pH range5.00–6.50. Therefore, a pH of 6.00 was fixed usingNaCl ⁄ Pibuffer.The effect of the buffer is lost if too small a quantityis 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 volume ofbuffer added (1.00–3.00 mL) had little effect on thefluorescence intensity, 2.00 mL of buffer was chosen insubsequent experiments.Influence of the concentration of metallobridgedbis(b-CD)s 2The influence of the concentration of 2 on fluorescenceintensity is shown in Fig. 4. As can be seen, as theconcentration of 2 increased, the fluorescence intensityof the system also increased slightly. We therefore used2.00 mL of 2.00 · 10)4molÆL)1metallobridged bis(b-CD)s 2.Influence of reaction timeThe effect of reaction time was studied, the result(Fig. 5) showed that the fluorescence intensity reacheda maximum after the reagents had been added for300 320 340 360 380400420 440 460 480 500 520 540 560 580 6000500100015002000250030003500400045003322111:bis(β-CD)s12:metallobridged bis(β-CD)s23:metallobridged bis(β-CD)s2+GSHFluorescence intensity (a.u.)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 6.00.150200250300350Relative fluorescence intensity (a.u.)4.005.00 6.00 7.00 8.00 9.00pHFig. 3. Influence of pH on the fluorescence intensity.[GSH] = 2.00 · 10)6molÆL)1;[2] = 4.00 · 10)5molÆL)1.1601802002202402602803003203401.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00Relative fluorescence intensity (a.u.)[metallobridged bis(β-CD)s 2] (10–5mol·L–1)Fig. 4. Influence of the concentration of 2 on fluorescence inten-sity. [GSH] = 2.00 · 10)6molÆL)1; pH 6.00.Glutathione determination using bis(b-CD)s B. Tang et al.1512 FEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBS$ 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 interferenceThe influence of the main constituents of plasma onthe determination of 2.00 · 10)6molÆL)1GSH werestudied. The criterion for interference was fixed at a± 5.0% variation in the average fluorescence intensitycalculated for the established level of GSH. A 3000-fold mass excess of plama over 2.00 · 10)6molÆL)1GSH was tested first. If interference occurred, the ratiowas gradually reduced until interference ceased. Theresults are shown in Table 1 and it can be seen thatthe determination was free from interference by theconstituents of plasma.MechanismThe novel fluorescent bis(b-CD)s 1 contained twostrong coordination sites for copper (II) ions and twonaphthyl fluorophores. The compound could be dis-solved in aqueous solution and showed high bindingability because of the two adjacent bis(b-CD)s.Because of the conformation of the linker of 1, thenitrogen atoms and amido bond formed two chelaterings to coordinate with copper (II) ions. This coordi-nation effect and the paramagnetism of copper (II)ions induced fluorescence quenching. However, GSHhas a great propensity for forming complexes withmetal ions that have strong electrophilic characteristics[18], such as copper (II) [19,20], mercury [21] andcadmium [22]. In this system, the quenched copper (II)complex 2 could interact with the thiol and amino ofGSH via a cooperative chelation effect [23,24], whichled 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 determineGSH in human plasma.Analytical characteristicsUnder optimum experimental conditions, there was alinear relationship between fluorescence intensity andGSH concentration in the range 0.30–20.0 lm with acorrelation 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], wasdetermined to be 63.8 nmolÆL)1, according to theformula C = KS0⁄ S, where K = 3 (standard devia-tion = 1.36), obtained from a series of 11 reagentblanks, and S is the slope of the standard curve. Therelative standard deviation was 2.5%, obtained from aseries of 11 standards each containing 2.00 lm GSH.When the concentration of GSH exceeded that ofmetallobridged 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 byLiu et al. [16].10 20 30 40 50 601001201401601802002202402602803003203400Relative fluorescence intensity (a.u.)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 substanceConcentration(molÆL)1)Relative error(%)K+6.0 · 10)3)3.7Na+6.0 · 10)3)3.0Ca2+1.6 · 10)31.4Mg2+2.4 · 10)44.2Zn2+6.0 · 10)41.2Fe2+1.0 · 10)4)4.4Fe3+6.0 · 10)5)3.6Arginine 1.0 · 10)43.5L-Phenylalanine 2.0 · 10)54.6Lysine 2.0 · 10)53.1L-Cysteine 1.0 · 10)44.3Tyrosine 1.0 · 10)4)4.5Bilirubin 2.0 · 10)54.8Glucose 6.0 · 10)5)2.6Cholesterol 1.0 · 10)43.5Ascorbic acid 1.0 · 10)3)3.3Uric acid 4.0 · 10)44.1Gly–Leu 2.0 · 10)42.7Gly–Gly–Gly 2.0 · 10)41.5Gly–Gly 2.0 · 10)42.3Gly–Phe 8.0 · 10)54.4Gly–Pro 6.0 · 10)43.5Leu–Gly–Gly 8.0 · 10)42.9Leu–Gly 8.0 · 10)44.3B. Tang et al. Glutathione determination using bis(b-CD)sFEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBS 1513ApplicationsSample collecting and processingFasting venous blood (5.00 mL) was routinely col-lected from the author Y. Liu, and transferred to a10 mL centrifuge tube containing heparin sodium asan anticoagulant. The blood was immediately centri-fuged at 1000 g for 1 min at room temperature toremove cells and platelets [10]. Afterwards, 0.50 mLof absolute alcohol was added to the plasmawith shaking. Plasma proteins were precipitatedand removed by centrifugation. The final plasmasamples used in the determination of GSH wereobtained.Determination of GSH in plasma and accuracyassessment by recovery experimentsIn order to evaluate the applicability of the proposedmethod, fluorescence determination in plasma was per-formed according to the following procedures. Into aseries 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 of1.00 · 10)3molÆL)1metallobridged bis(b-CD) 2 and0.20 mL of 0.10 molÆL)1NaCl ⁄ Pi(pH 6.00). Theexperimental data are shown in Table 2. The mixturewas diluted to mark with ultra-pure water, shakenthoroughly and equilibrated at room temperature for10 min. The fluorescence intensity of the solution wasmeasured at 365 ⁄ 480 nm.The GSH content of the plasma was derived fromthe standard curve and the regression equation. Theaverage recovery test was made using the standardaddition method, and the RSD was generally goodwhen obtained from a series of six plasma samples.These results are also given in Table 2. Compared withpreviously reported methods (Table 3), our resultsindicate that the recovery and precision of our methodof determining GSH in plasma are satisfactory.ConclusionsWe synthesized a novel metallobridged bis(b-CD)s 2,which afforded two hydrophobic binding sites coopera-tively associating with the guest GSH and also providedadditional binding interactions between the hetero-atoms of GSH and the coordinated metal center. GSHwas able to form complexes with copper (II) derivedfrom the metallobridged bis(b-CD)s 2. This competitive024681012141618202226002800300032003400360038004000Fluorescence intensity (a.u.)[GSH] (µM)Fig. 6. Linear plot of fluorescence intensity with increase in GSHconcentration. [2] = 4.00 · 10)5molÆL)1; pH 6.00. All spectra wereobtained under the optimum experimental conditions at365 ⁄ 480 nm and room temperature.Table 2. GSH determination in plasma samples (n =6,P = 95%).SamplesPlasma(mL)GSH added(lM)Measureda(lM)RSD(%)Recovery(%)GSH contentof plasma (lM)A 0.25 0 0.94 4.43 ) 3.77B 0.50 0 2.02 3.97 – 4.05C 0.75 0 3.66 3.56 – 4.88A¢ 0.25 9.00 10.3 2.58 104 3.77B¢ 0.50 6.00 7.94 3.47 98.7 4.05C¢ 0.75 3.00 6.80 1.62 105 4.88aMean of six determinations using the proposed method.Table 3. Analytical characteristics compared with other methods reported.Methods Linear range (lM) Limit of detection GSH in plasma (lM)HPLC [26] 0.81–13.02 0.13 lM 3.39 ± 1.04HPLC with fluorimetry [27] 0.2–20.0 14 fM 1.82 ± 0.55Proposed method 0.30–20.0 63.8 nM 4.01 ± 0.38Glutathione determination using bis(b-CD)s B. Tang et al.1514 FEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBScomplexation 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 withhigh selectivity to determine GSH. The linear range ofthe method was 0.30–20.0 lmolÆL)1with a detectionlimit of 63.8 nmolÆL)1. There was no interference fromthe plasma constituents. The proposed method was suc-cessfully used to determine GSH in human plasma.Experimental proceduresApparatus and reagentsAll spectrofluorimetric measurements were carried out withan Edinburgh FLS920 spectrofluorimeter (Edinburgh Instru-ments Ltd, Livingston, UK) equipped with a xenon lamp and1.0 cm quartz cell. Absorption spectra were obtained fromUV-1700 (Shimadzu, Kyoto, Japan) UV–visible spectroph-tometer. Infrared spectra were obtained from a PE-983G IR-spectrophotometer (Perkin-Elmer, Palm Springs, CA, USA).1H NMR spectra were recorded on a Bruker Avance 300,elemental analysis was performed on Perkin-Elmer Series PCHNS ⁄ O analyzer. pH measurements was made with a 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 stocksolution (1.00 · 10)3molÆL)1) of GSH was prepared withultra-pure water. b-CD (China Medicine Group ShanghaiChemical Reagent Corporation, Shanghai, China) was puri-fied by recrystallizing twice in ultra-pure water, followed byvacuum drying at 95 °C for 24 h. 3-Amino-2-naphthoicacid (Alfa Aesar, Word Hill, MA, USA) was usedwithout further purification. Mono(6-p-toluenesulfonyl-6-deoxy)-b-cyclodextrin was prepared by reacting p-tosylchloride with b-CD in dry pyridine as described previously[28,29]. Mono(6-p-toluenesulfonyl-6-deoxy)-b-cyclodextrinwas then converted to mono(6-aminoethylamino-6-deoxy)-b-CD with 57.1% yield upon heating in excess ethylenedi-amine at 75 °C under nitrogen for 7 h [30]. Compound 3,oxamide bis(2-naphthyl) acid, was prepared according to theprocedure reported previously [31]. Other chemicals usedwere of analytical reagent grade. The water used in this studywas purified using a Mill-Q (18.2 MWÆcm)1) water system. A100 k Nanosep filter (Pall Corp., East Hills, NY, USA) andmicoron YM—30-30000 NMWL (Millipore, Billerica, MA,USA) were used as ultra-purification instrumentation.Synthesis of the novel bis(b-CD)sSynthesis of compound 1Mono (6-aminoethylamino-6-deoxy)-b-CD (2.00 g) was dis-solved in dimethylformamide (50 mL) in the presence of asmall amount of 0.4 nm molecular sieves, and then 3(0.21 g) was added. The mixture was stirred for 24 h at70 °C under nitrogen. It was then allowed to stand for 5 huntil no further precipitate was deposited. The precipitatewas removed by filtration, and the filtrate evaporated todryness under reduced pressure. The residue was dissolvedin a minimum amount of hot water and poured into ace-tone to give an orange precipitate. The orange precipitatewas purified by three recrystallization steps in ultra-purewater. After the residue had been dried under a vacuum,pure sample 1 was obtained with a 27% yield. UV ⁄ vis kmax(H2O) ⁄ nm (log e): 348 (1.62).1H NMR (300 MHz, DMSO-d6, TMS ppm): d2.00–3.00 (m, 14H); 3.30–3.80 (m, 84H);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);7.90–7.97 (m, 2H); 8.30–8.35 (m, 2H). IR (KBr, cm)1):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, 706.8,579.5, 531.7. Elemental analysis calculated (%) forC112H164O72N6: C, 48.98; H, 5.98; N, 3.06. Found: C,48.77; H, 6.12; N, 3.24.Synthesis of metallobridged bis(b-CD)s 2According to the Liu et al. [16], bis(b-CD)s 1 was addeddropwise 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 waskept at 5 °C for 2 days. The solution was then evaporatedunder reduced pressure, and the precipitate formed wascollected by filtration, washed successively with a smallamount of ethanol and diethyl ether, and dried in vacuo togive complex 2 as a green solid with 63% yield. UV ⁄ viskmax(H2O) ⁄ nm (log e): 349.5(1.36). IR (KBr, cm)1): m3419.5, 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 calcu-lated (%) for C112H164O72N6Æ2CuCl2: C, 44.59; H, 5.44;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 complex 2 in 50 mL ofultra-pure water, and added 25.00 mL of the complex 2solution to a 125 mL Erlenmeyer flask. This was analyzediodometrically. Copper (II) was first reduced to Cu(I) byKI according to the following reaction:2Cu2þþ 4IÀ! 2CuIðsÞþI2and the liberated I2was titrated against thiosulfate;26.00 mL of 0.020 m Na2S2O3was required to titrate theliberated I2according to the following reaction:I2þ 2S2O2À3! 2IÀþ S4O2À6The percentage of copper in the sample was 4.41. Theresults confirmed that the mole ratio of complex 2 toB. Tang et al. Glutathione determination using bis(b-CD)sFEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBS 1515copper (II) was 1 : 2, which was consist with the Job’ s plotof the 1 ⁄ Cu2+system at 349 nm.Calibration graphInto a series of 10-mL colorimetric tube were sequenti-ally added different aliquots of GSH stock solutioncontaining 0–2.00 · 10)4molÆL)1of GSH, 2.00 mL of2.00 · 10)4molÆL)1metallobridged bis(b-CD)s 2 and2.00 mL of 0.10 mol Æ L)1NaCl ⁄ Pi(pH 6.00). The mixturewas 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 at365 ⁄ 480 nm (Fig. 7).AcknowledgementsThis study was supported by the National BasicResearch Program of China (973 Program,2007CB936000), National Natural Science Funds forDistinguished Young Scholar (No.20725518), MajorProgram of National Natural Science Foundation ofChina (No.90713019), National Natural Science Foun-dation of China (No.20575036) Important Project ofNatural Science Foundation of Shandong Province inChina (No.Z2006B09) and the Research Foundationfor the Doctoral Program of Ministry of Education(No.20060445002).References1 Meiser A & Anderson M (1983) Glutathione. Annu RevBiochem 52, 711–760.2 Sezginturk MK & Dinckaua E (2004) An amperometricinhibitor biosensor for the determination of reducedglutathione (GSH) without any derivatization in someplants. 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Accounts Chem Res 39, 681–691.460 480 500 520 540 560 58005001000150020002500300035004000450020 µM18 µM15 µM10 µM5.0 µM1.0 µM0.5 µM0 µMFluorescence intensity (a.u.)Wavelength (nm)Fig. 7. Changes in fluorescence intensity after different concen-trations of GSH were added to the analytical system underoptimal conditions at 365 ⁄ 480 nm and room temperature.[2] = 4.00 · 10)5molÆL)1; pH 6.00.Glutathione determination using bis(b-CD)s B. Tang et al.1516 FEBS Journal 275 (2008) 1510–1517 ª 2008 The Authors Journal compilation ª 2008 FEBS15 Breslow R, Halfon S & Zhang B (1995) Molecular rec-ognition by cyclodextrin dimers. Tetrahedron 51, 377–388.16 Liu Y, Zhao YL, Chen Y, Ding F & Chen GS (2004)Binding behavior of aliphatic oligopeptides by bridgedand metallobridged bis(b-cyclodextrin)s bearing anoxamido bis(2-benzoic) carboxyl linker. BioconjugateChem 15, 1236–1245.17 Vanetten RL, Sebastian JF, Clowes GA & Bender MA(1967) Acceleration of phenyl ester cleavage by cyclo-amyloses. 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