Chinese Chemical Letters  2015, Vol.26 Issue (07):877-880   PDF    
Tetraphenylethene based zinc complexes as fluorescent chemosensors for pyrophosphate sensing
Hao-Ran Xu, Kun Li , Shu-Yan Jiao, Ling-Ling Li, Sheng-Lin Pan, Xiao-Qi Yu     
Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, China
Abstract: We described a serious of zinc complexes that exhibit characteristic fluorescence responses toward pyrophosphate (PPi) and adenosine triphosphate (ATP) in aqueous media. These novel probes exploited tetraphenylethene (TPE) as fluorophore and macrocycle-polyamine (including 1,4,7,10-tetraazacyclododecane and 1,4,7-triazacyclononane) Zn(II) complexes as binding group. These “OFF-ON” type probes exhibited promising selectivity and sensitivity to PPi and ATP via a restriction of intramolecular rotation (RIR) mechanism. The detection limit for PPi was found within nmol/L range.
Key words: Tetraphenylethene     Cyclen     Pyrophosphates     Zinc complex    
1. Introduction

Phosphate-based inorganic and organic molecules play important roles in biological processes such as energy storage,signal transduction and DNA sequencing [1, 2, 3, 4, 5]. Therefore,developing synthetic receptors for recognition and sensing of such anions have drawn much attention. Among the phosphates,adenosine triphosphate (ATP) and inorganic pyrophosphates (PPi) have been of particular interest [6, 7],which are involved in energy transduction in organisms and control metabolic processes through participation in various enzymatic reactions [8, 9]. In the past decades,a number of zinc-complex based fluorescent chemosensors for PPi sensing have been reported [10, 11],including several classic ligands such as bis(2-pyridylmethyl) amine (DPA) [12, 13],terpyridine (tpy) [14],and amide [15, 16]. However,most of them exhibited low sensitivity (in the mmol/L range) [17]. It is necessary to develop novel chemosensors for phosphate sensing with high selectivity and sensitivity,especially those could respond in nmol/L range.

1,4,7,10-Tetraazacyclododecane (cyclen) and 1,4,7-triazacyclononane (TACN) are cyclic organic compounds. Both of them are versatile metal chelator with excellent water-solubility [18],and their Zn(Ⅱ) complexes have been developed for biological polyphosphate anions detection [19]. Tetraphenylethene (TPE) is a kind of propeller-shaped fluorophore,which could affect the fluorescence intensity according to the restriction of intramolecular rotation (RIR) mechanism [20, 21]. Because the energy is dissipated by the intramolecular rotation,non-emission is found when the probe is dissolved in solution; while in aggregation state or somehow else,the intramolecular rotation could be restricted that induced to emit efficiently. Previously,we reported a dicyclen-TPE Zn(Ⅱ) complex as a fluorescent ensemble for PPi in water with a detection limit of 22.8 nmol/L [22]. To continue our work,herein,we synthesized a serious of cyclen- or TACN-TPE Zinc complexes,and investigated their performance for biologically important phosphate anions detection in aqueous solution.

2. Experimental

Mass spectrometer (ESI-MS) and high resolution mass spectrometer (HRMS) data were recorded on a Finnigan LCQDECA and a Bruker Daltonics Bio TOF mass spectrometer,respectively. The 1H NMR and 13C NMR spectra measured on a Bruker AM400 NMR spectrometer and the δ scale in ppm referenced to residual solvent peaks or internal tetramethylsilane (TMS). Absorption spectra recorded on Hitachi U1900 spectrophotometer at 298 K. Fluorescence emission spectra were obtained using FluoroMax-4 Spectrofluoro-photometer (HORIBA Jobin Yvon) at 298 K. Unless otherwise indicated,all syntheses and manipulations were carried out under N2 atmosphere. All the solvents were dried according to the standard methods prior to use. All of the solvents were either HPLC or spectroscopic grade in the optical spectroscopic studies.

Compounds 1,2,3,4,5 were prepared according to the literature procedures [23, 24, 25]. For dCT and dCT·Zn,they were prepared as discussed on our previous paper [22].

Synthesis and characterization of 1-(4-(1,2,2-triphenylvinyl)- benzyl)-1,4,7,10-tetraazacyclododecane (CT): A mixture of 1 (300 mg,0.708 mmol),4 (500 mg,1.06 mmol) and K2CO3 (500 mg,3.62 mmol) in acetonitrile was refluxed overnight. After the solution was concentrated,the residue was added to dichloromethane (100 mL),and the organic phase was washed with water and brine followed by drying over Na2SO4. The solvent was removed under reduced pressure; the crude product was purified by silica gel column eluting with EtOAc/PE (2:1,v/v) to give the product as a pale yellow solid (500 mg),yield 57.9%. 1H NMR (400 MHz,CDCl3): δ 7.13-6.98 (m,19H),3.64 (s,2H),3.57 (s,4H),3.34-3.15 (m,8H),2.65 (s,4H),1.50-1.46 (br,27H); 13C NMR (100 MHz,CDCl3): δ 143.7,143.6,143.5,143.0,141.1,140.6,131.3,129.6,127.7,126.5,126.4,79.3,60.4,57.9,57.0,53.4,49.9,48.6,47.4,47.0,28.5.

The yellow solid achieved above (450 mg,0.551 mmol) was dissolved in a saturated of HCl-methanol solution (100 mL) and stirred overnight at room temperature. The solvent and excess HCl were removed under reduced pressure,giving a yellow solid. The solid was dissolved in water and added pretreated anion-exchange resin until the solution is alkaline. Freeze-dried the solution to remove water and obtained the product as a pale yellow cottonlike solid (260 mg),yield 91%. 1H NMR (400 MHz,CDCl3): δ 7.12- 7.09 (m,9H),7.07-7.03 (m,8H),7.00-6.98 (m,2H),3.56 (s,2H),2.80 (t,4H,J = 8.0 Hz),2.66 (t,4H,J = 8.0 Hz),2.57 (t,8H,J = 8.0 Hz); 13C NMR (100 MHz,CDCl3): δ 143.9,143.8,142.3,140.8,137.1,131.4,131.3,128.2,127.7,127.6,126.4,126.3,59.0,51.3,47.2,46.4,45.1; ESI-HRMS: m/z calcd. for C35H41N4 [M+H]+: 517.3326,found: 517.3326.

Synthesis and characterization of 1,1,2,2-tetrakis(4-((1,4,7,10- tetraazacyclododecan-1-yl)methyl)phenyl)ethane (tCT): The preparation followed the similar procedure of CT with compounds 3 and 5. Yellow solid,yield (110 mg,12.1%). 1H NMR (400 MHz,DMSO-d6): δ 7.09 (d,8H,J = 8 Hz),6.92 (d,8H,J = 8 Hz),3.64 (s,8H),2.81 (br,16H),2.68-2.61 (br,48H); 13C NMR (100 MHz,DMSO-d6): δ 142.6,131.1,129.1,51.3,47.9,46.3,45.1,44.5; ESI-HRMS: m/z calcd. for C62H101N16 [M + H]+: 1069.8390,found: 1069.8390.

Synthesis and characterization of (Z)-1,2-bis(4-((1,4,7-triazonan-1-yl)methyl)phenyl)-1,2-diphenylethene (dAT): The preparation followed the similar procedure of CT with compounds 2 and 4. Pale yellow cotton-like solid,yield (480 mg,44.8%.) 1H NMR (400 MHz,D2O): δ 6.81-6.61 (m,18H),3.49-3.37 (br,4H),3.09 (s,8H),2.72-2.52 (m,16H); 13C NMR (100 MHz,D2O): δ 143.2,142.8,140.8,140.7,135.4,131.1,129.2,127.8,126.5,58.8,48.3,43.6,42.4; ESI-HRMS: m/z calcd. for C40H51N6 [M + H]+: 615.4166,found: 615.4170.

3. Results and discussion

All heterocyclic amine ligands and their zinc complexes could be easily synthesized via simple procedures (Scheme 1). CT and tCT were cyclen-TPE based probes and dAT was TACN-TPE based probe. The Zn(Ⅱ) ions containing the fluorescent receptors were synthesized by reacting a methanol solution of Zn(NO3)·6H2O with compounds acquired before at room temperature. The structures of CT,tCT,dAT and Zn(Ⅱ) based receptor CT·Zn was characterized by multinuclear (1H and 13C) NMR and electrospray ionization mass spectrometry (ESI-MS). All the zinc complexes have good water solubility with suitable lipophilicity,which is in conformity with our previous design. The more cyclen units contained in one probe,the better water solubility it is,and tCT showed the highest solubility in aqueous media.

With these probes in hand,we firstly investigated the selectivity of CT,tCT and dAT zinc complexes. CT·Zn,tCT·Zn,dAT·Zn were prepared in situ,and all three zinc complexes have weak fluorescence under aqueous conditions (HEPES buffer,pH 7.4,10 mmol/L). After treated with various common anions including PPi,PO43-,HPO42-,H2PO4-,SO42-,Br-,F-,AcO-,I-,Cl-,SCN-,NO3-,S2-,SO32-,CO32- (Fig. 1 and Figs. S1-S3 in Supporting information),no obvious fluorescence change was found. However,when PPi was added to the solution,dramatically fluorescence enhancement was observed,and the increase was bigger than that of previous reported probe dCT·Zn. This novel phenomenon should be attributed to the water solubility of the probes. While in buffer solution,excellent water solubility of zinc complexes made the benzene ring of TPE rotate freely and the energy is dissipated by the intramolecular rotation. After it coordinated with PPi,the water solubility of the probe decreased and the complexes aggregated in buffer,which restricted the intramolecular rotation and changed the intensity of fluorescence emission. The results indicated that these three probes (CT·Zn,tCT·Zn,dAT·Zn) could distinguish PPi from other tested anions.

Fig. 1.Fluorescence response of various anions with dAT·Zn (blue bars), CT·Zn (red bars) and tCT·Zn (black bars) in HEPES (pH 7.4, 10 mmol/L). F is the fluorescence intensity measured in the experiment and F0 is the fluorescence intensity of sensor without adding any anions. 1. Blank, 2. CO32-, 3. NO3-, 4. SO42-, 5. Br-, 6. Cl-, 7. H2PO4-, 8. HPO42-, 9. PO43-, 10. PPi. λex = 329 nm.

Fluorescence titration experiment was then conducted by gradually increasing concentrations of PPi (Fig. 2). The results showed that fluorescence intensity enhanced gradually with the increase of PPi. Meanwhile,the solution of CT·Zn and tCT·Zn with PPi showed the formation of a 2:1 stoichiometric complex (Figs. S4 and S5 in Supporting information). However,dAT·Zn showed a 1:1 bonding mode with PPi (Fig. S6). Job’s plot of sensors and PPi also confirmed the stoichiometric coefficient (Figs. S7-S9 in Supporting information). The different ligancy indicate that the type of heterocyclic amine ligands could affect the interaction between zinc complexes and PPi. It is four-coordinated when a zinc complex with a cyclen,but three-coordinated toward a TACN. Hence every zinc in dAT·Zn could coordinate more oxygen atoms from PPi which led to the special dAT·Zn·PPi bonding mode.

Fig. 2.Fluorescence titration of dAT·Zn, CT·Zn and tCT·Zn (10 μmol/L) with PPi (0–1.0 eq.) in HEPES (pH = 7.4, 10 mmol/L).

To further investigate the non-covalently binding between zinc complex and PPi,we researched CT·Zn in depth as the most simple complex. The binding constant was calculated as KCT·Zn = 168.63 (mol/L)-2 through the fluorescent titration spectrum (Fig. S10 in Supporting Information). Its linear fitting constant reaches 0.993 which is further confirmed the formation of a 2:1 stoichiometric complex. The quantum yield of CT·Zn increased from 0.735% to 7.796% (fluorescence of the quinine sulphate solution as reference [26],Qr = 0.560). The detection limit of CT·Zn for PPi was found to be 5.8 nmol/L which is suitable for PPi detection in biological systems. To the best of our knowledge,there is only one example reported a lower detection limit [14]. To inspect the excellent detection limit,titration of CT·Zn with trace quantities of PPi (100 nmol/L) was investigated (Fig. S11 in Supporting information),and a good linear relationship was achieved in the nmol/L range. Through the above investigation,CT·Zn was served as lightup fluorescent sensors for PPi with high sensitivity. As to dAT·Zn and tCT·Zn,the binding constants were calculated as KdAT·Zn = 5.27 × 105 (mol/L)-1 and KtCT·Zn = 162.60 (mol/L)-2 (Figs. S12 and S13 in Supporting information) respectively,and their detection limit as well as quantum yield were listed in Table 1.

Table 1
Binding constant, detection limit and fluorescence quantum yield of CT·Zn, tCT·Zn and dAT·Zn.

As in situ prepared chemosensors has been found to recognize PPi among the anions,the studies were extended to organic as well as biorelevant molecules bearing phosphate moiety,viz.,ATP,ADP,AMP (Fig. 3). Among these,ATP could lead to fluorescence enhancement as strong as PPi; however,the ADP and AMP did not induce the change of the fluorescence intensity. These result suggested that the enhancement is dependent both on the bulkiness of the organic moiety and on the number of phosphate moieties present. ATP and ADP have the same binding sites as PPi,oxygen atoms from pyrophosphate part are exposed outside to coordinate with zinc. Unlike dCT·Zn,it could not form suitable inside cavity structure to prevent the base part of the ATP or ADP,so that biological phosphates could coordinate with zinc complexes and restrict the intramolecular rotation in a similar way.

Fig. 3.Relative fluorescence intensity of dCT·Zn, tCT·Zn, CT·Zn and dAT·Zn to various biological phosphates including PPi, ATP, ADP, AMP and Pi in HEPES buffer (10 mmol/L, pH 7.4). The results of dCT·Zn were based on Ref. [18].
4. Conclusion

We have designed and developed a series simple but efficient TPE and nitrogen heterocycles based fluorescence sensors,which exhibited ‘‘turn-on’’ fluorescence response toward PPi and ATP with high selectivity and sensitivity in a 100% aqueous medium. When ATP and PPi bind to the zinc complexes,it will lead to fluorescence enhancement due to the restriction of intramolecular rotation (RIR) mechanism. The detection limit of the probes reaches in the nmol/L range,which is low enough for the detection of a trace quantity of PPi in biological and environmental samples. The biological applications of these probes are undergoing.


This work was financially supported by the National Program on Key Basic Research Project of China (973 Program,Nos. 2012CB720603,2013CB328900),the National Science Foundation of China (Nos. 21232005,21321061,J1310008and J1103315),and the Specialized Research Fund for the Doctoral Program of Higher Education in China (No. 20120181130006).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at

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