A novel cell-impermeable fluorescent zinc sensor containing poly(ethylene glycol) chain

Available online at www.sciencedirect.comCHINESEScienceDirectC HEMICALL .ETTERSELSEVIERChinese Chemical Letters 22 (2011) 863 866wwelsevier.com/ocatelccletA novel cell-impermeable fluorescent zincsensor containing poly(ethylene glycol) chainNing Zhang4*, Yi Su", Miao Yu a,b●Department of Chemistry and Chemical Engineering, Huanghuai University, Zhumadian 463000, Chinab Department of Chemistry, Zhengzhou Universiy, Zhengzhou 450001, ChinaReceived 26 October 2010Available online 16 April 2011AbstractA novel ellimpermeable zine sensor was synthesized by incorporating poly(ethylene glycol) (PEG) to N-(8-quinoly)p-aminobenzenesulfonamide (HQAS) group. The polymeric zinc sensor combines both valuable features of HQAS and PEG. TheHQAS of the sensor has the similar functions to TSQ, and exhibits a good fuorescence response to Zn2+ but poor fuorescenceresponses to other metal ions. The PEG chain can prevent the sensor to permeate healthy cell membrane. The stained experimentswith the yeast cells as model showed that the sensor cannot stain the healthy yeast cells,s but only the damaged or died yeast cells.These results indicated the novel zinc probe was a typical ell-impermeable zinc sensor.◎2011 Ning Zhang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.Keywornds: Zinc ion; Sensor; Yeast; Poly(ethylene glycol); FluorescenceZinc is an essential element to life, which exists in some tssues or cells with a relatively high concentration. Zincions handle some important biological processes such as gene transcription, metalloenzyme regulation, neural signaltransmission and others [1]. So the detection of Zn2+ in vivo has attracted increasing attention. The fluorescenceimaging technology has been used as the most effective way to detect cellular zinc with the Zn2+ fuorescent probes.Generally, there are two forms of Zn2+ probes named cell-permeable and cell-impermeable probes [2]. The former canpass through the cell membrane and has been widely used to detect the intracellular Zn2+ in living cells [3,4]. The latteris designed to hold an ionic charge which dictates the probes to be excluded from the contiguous membrane of bealthycells and has been also successfully used in monitoring the cell exocytosis [5], measuring the cell viability [6],indicating the cell apoptosis [7], etc. However, ionic cell-impermeable probes, i.e. zinquin acid, have a tendency topartition into the cell membrane under physiological conditions, which will interfere the detection of cell apoptosis orcell exocytosis [5]. In a preliminary work, we demonstrated that a large hydrophilic group conjugated to TSQ (6-methoxy-(8-p-toluenesulfonamido) quinoline, a typical zinc sensor) could prohibit TSQ permeating the cellmembrane [8,9]. Poly(ethylene glycol) (PEG) is a hydrophilic polyether, due to its low interfacial free energy, whichcan approach to cell surface. Meanwhile, the steric repulsion and hydrophilic effects of PEG chain forbid it beingembedded within cell membrane [10]. Herein, we report a novel cell-impemmeable probe, which was synthesized by中国煤化工●Corresponding author.E-mail adress: ningdzhang@ 126.com (N. Zhang).MHCNMHG1001-8417/$- see front matter◎2011 Ning Zhang. Publisbhed by Elsevier B.V. on behalf of Chinese Cbemical Society. All rights reserved.doi:10.1016j.cet2011.01.006864N. Dhang et al./Chinese Chemical Ltters 22 (2011) 863 -866NH28PEG1000 (HOBT, DCC)O2SDMF, 0°C, 6h; RT,12hHN、HN~OH。十uHQAS acidCompound 1Scbeme 1. Synthesis route to the PEG grafted zinc sensor (Compound 1).covalently linking PEG to a typical zinc sensor HQAS acid (as shown in Scheme 1). The stained experiments for yeastcells showed compound 1 had no response to healthy yeast cells, but to the damaged or died yeast cells. Therefore,compound 1 has the potential application to detect the cell exocytosis or the cell apoptosis.HQAS acid was prepared according to the literature [9]. Compound 1 was synthesized by the reaction of HQASacid with 5 equiv. of PEG 1000 (Scheme 1) [11]. The ESI mass spectra of compound 1 are shown in Fig.1. The signal(m/z) 1434.7 could be assigned as M (n = 23) + Na*, which was coincided with the corresponding calculated value(1434.8). The polydispersity of compound 1 (m/z: 1038.9- 1831.8) could also be assigned as M (n= 14 32) + Na*.Owing to the unique soluble properties of PEG moiety in compound 1, compound 1 was soluble in the polar andnonpolar solvents, i.e. CH2Cl2, CHCl3, MeOH, acetone, etc. Even in water, compound 1 was sparingly soluble.An important and desirable property of zinquin family is its fuorescence sensing ability to Zn*. The emissionspectra of compound 1 with the addition of Zn2+ are shown in Fig.2. When excited at 356 nm, the emission intensity ofcompound 1 continuously increased upon the stepwise addition of Zn2*. The uptrend of emission intensity becameslower as the concentration of Zn2+ up to 0.5 equiv. mole of compound 1, which indicated that compound 1 form 2:1complex with Zn2+. The coordination stoichiometry between compound 1 and Zn2+ was also verified by the molarratio method using UV- vis spectrometry. Meanwhile, the fuorescence spectrum underwent a red shift from 506 nm to515 nm upon Zn2+ binding, and exhibited an obvious enhancement (ca. 4.3 times) of the emission intensity.The fuorescence response of compound 1 to other metal ions including Na*, K*, Cat+, Mg?*, Fe2+, Co2*, Mn2*,Cu2+ and Ni2+ was tested (Fig. 3). These cations, which exist at high concentration in living cells, ie. Na*, K*,Ca2+and Mg2+ did not observably enhance the fuorescence intensity even at high concentration (5 X 10- molL). Theseresults were presumably due to the poor complexation of alkaline metals or alkaline earth metals with HQAS group ofcompound 1. Among first-row transition metal cations, ie. Fe2+, Co4+, and Mn2+ induced a slight change of thefuorescence intensity. Although Cu2+ and Ni'+ quenched the ftuorescence of compound 1 and interfered the Zn^+-induced fuorescence enhancement, the concentration ofCu2+ or Ni2+ in the eflux from the cell is much lower thanthat of Zn2*. Hence, Cu2+ or Ni2+ would not significantly affect the detection of Zn2+ in practice.In order to verify cell-impermeability of compound 1, we used yeast cells as model cell to investigate the stainingeffect of compound 1. Yeast cells were cultured according to the literature [8]. In our experiments, the pre-culturedyeast cells were transferred to a 3% DMSO solution of compound1 (5 x 10 5 mol/L) with or without the addition ofZnSO4 (2 x 10-5 mol/L). The fuorescence microscopic images were recorded after ca. 30 min. At the initial stage,14M中国煤化工MYHCNM H Gom”1400mFig. 1. MS (ESD spectra of compound 1. Peak marked with ◆crresponds to n= 23.N, Zhang et al./Chinese Chemical Letters 22 (2011) 863- 866865100kZn*50 t400500600λ/ nmFig. 2. Emission specra of compound 1 (2 x 10 5 molL) with the adtion ofZn2+ (Zn?勹]=0,2, 4,6,8, 10, 12, 14, 16, 18, and 20x 10- mol/Lfroma to k).the yeast cells did not exhibit appreciable fuorescence. Even added extra Zn2+ to the 1-treated yeast cells, thefluorescence intensity has no observable enhancement. However, when the cells were transferred from the incubatedsolution to the mixture solution of compound 1 and Zn2+ for a long time, some cells exhibited strong fuorescence andsome cells only presented very weak green dots, but many cells were still silent (Fig. 4). These results indicatedcompound 1 was a typical cell-impermeable zinc probe. Possessing a compact and integrated cell membrane, thehealthy cells had the ability to exclude cell- impenetrable molecule [12]. Therefore, compound 1 gave no response tobealthy cells. With time prolonged, the cells would be damaged or dead due to organic solvent in solution. The deadyeast cells that had lost their membrane integrity would allow compound 1 embedded within the cell membranes. Socompound 1 only stained damaged or died yeast cells.In summary, we reported one novel cell-impermeable probe compound 1, which possess a hydrophilic PEG moietyand a Zn2+ fuorescence response probe. Due to the hydrophilic PEG can prevent the probe permeate cell membrane,the PEG grafted zinc sensor may be used as a possible probe to monitor the cell exocytosis or the cell apoptosis.之2lul罾zx8皇8起皇会中国煤化工fYHCNMHGFig. 3. Fluorescence intensities of 1 (2x 10~5 mol/L) in the presence of various metal ions and ion mixtures. (Na勹]= [K力=[Cath=[Mg*]=5x 10~3 mol/L, [Zn2]=[Cu勹= [Ni2"]=[Co2]=[Fe2]= [Mn*]=1x 10 s mol/L).866N. Zhang et al./Chinese Chemical Letters 22 (2011) 863 -866Fig. 4. Optical microscopic (ef) and fluorescence microscopic (ighl) images of 1stained yeast clls with the adition ofZn2+ (2 x 10 s molL),AcknowledgmentWe thank the National Science Foundation of China (NSFC) (No. 20973072) for financial support.References[1] P. Jiang, Z. Guo, Coord. Chem. Rev. 248 (200) 205.[2] N.C. Lim, H.C. Freake, C. Bruckner, Chem. Eur.J. 11 (2005) 38.[3] M. Taki, JL. Wolford, T.V. O'Halloran, J. Am. Chem. Soc. 126 (2004) 712.[4] M. Royzen, A. Durandin, V.G. Young, J. Am. Chem. Soc. 128 (2006) 3854.[5] J. Qian, K.R. Gee, R.T. Kennedy, Anal. Chem.75 (2003) 3468.[6] CJ. Stork, Y.V. Li, J. Neurosci. Methods 155 (2006) 180.[7] E. Kimura, s. Aoki, E. Kikuta, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 3731.[8] Y. Liu, N. Zhang, Y Chen, et al. Org. Lett. 9 (2007) 315.[9] N. Zhang, Y. Chen, M. Yu, et al. Chem. Asian J. 4 (2009) 1697.[10] N. Dan, Biochim. Biophys. Acta 1564 (2002) 343.[11] The syothesis of compound 1: a solution of HQAS acid (1 mmol, 0.4 g) in dry DMF (10 mL) was added to a solution of DCC (1.5 mmol,0.31 g) and HOBT(1.1 mmol, 0.15 g) in DMF (10 mL). The mixture solution was stired at0 °C for 1 h. A solution ofPEG 1000(ca. 5 mmol,5.0 g) in DMF (10 mL) was added to the reaction mixture, and the mixture was stired at0 °C for 6 h and then sired at room temperature for24 h. Insoluble materials were removed by filtration, the filtrate was evaporated under reduced pressure. The reside was washed three times with30 mL water and then purified by silica gel chromatography (eluent, methanolchloroform 1:8). After dried in vacuo, 0.46 g of pale yellowproduct was obtained in 32.8% yield. 'HNMR (300 MHz, CDCla): 89.28 (S, 1H), 8.78(s, lH),7.28 -8.25 (m, 10H), 4.19(s, 2H),3.23-3.95 (m,91H), 2.60 2.90 (m, 4H), 2.17.(s, 1H).[12] G. Crivat, K. Kikuchi, T, Nagano, Anal. Chem.78 (2006) 5799.中国煤化工MYHCNMHG