zebrafish based on coordination-mediated supramolecular assembling

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1 Supporting Information Bioinspired peptide for imaging Hg 2+ distribution in living cells and zebrafish based on coordination-mediated supramolecular assembling Shilang Gui,, Yanyan Huang,*,, Fang Hu,, Yulong Jin,, Guanxin Zhang,, Deqing Zhang,, and Rui Zhao*,, Beijing National Research Center for Molecular Sciences, CAS Key Laboratories of Analytical Chemistry for Living Biosystems and Organic Solids, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing, , China University of Chinese Academy of Sciences, Beijing, , China * yyhuang@iccas.ac.cn, zhaorui@iccas.ac.cn Table of Content 1. Experimental... S-2 2. Synthesis of Pep1-TPE ~ Pep5-TPE... S-4 3. HPLC and MS Characterization of Pep1-TPE ~ Pep5-TPE... S-5 4. Binding kinetics of Pep2-TPE with Hg S-7 5. The effect from ph to the fluorescence response... S-7 6. Estimation of the detection limit... S-8 7. Hg 2+ -mediated self-assembly behavior... S-9 8. Absorption spectrum of Pep2-TPE... S-9 9. Job s Plot... S Determination of association constant... S FT-IR characterization of the coordination complex... S Effect of biothiols on the fluorescence response of Pep2-TPE to Hg S Competitive binding assay in cells... S Stability assay of Pep2-TPE... S Fluorescence behavior of the oxidized product of Pep2-TPE... S Evaluation of cytotoxicity of Pep2-TPE... S-18 S-1

2 1. Experimental Materials. Metal salts (AlCl 3 6H 2 O, Cu(NO 3 ) 2 3H 2 O, Pb(NO 3 ) 2, FeCl 2 4H 2 O, NiCl 2 6H 2 O, LiCl, MgCl 2 6H 2 O, CoCl 2 6H 2 O, CaCl 2, CrCl 3 6H 2 O, KCl, NaCl, FeCl 3 6H 2 O, BaCl 2 2H 2 O, CdCl 2 2(1/2)H 2 O, ZnCl 2, MnCl 2 4H 2 O, AgNO 3, and HgCl 2 ) were all of analytical grade and used as received (Beijing Chemicals Ltd., Beijing, China). Acetonitrile (HPLC grade) was from Fisher Scientific (Hampton, USA). 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and FMOC-Glu-OtBu (CAS ) were purchased from GL Biochem (shanghai) Ltd. FMOC-Cys(Trt)-OH and FMOC-Glu(OtBu)-OH were from Advanced ChemTech (USA). FMOC-Gly-Wang resin was obtained from SIAM (USA). 1,2-ethanedithiol (EDT) and 4-methylmorpholine were from Sigma-Aldrich Co. LLC. N,N-dimethylformamide was from BoMaiJie Technology Co., Ltd (Beijing, China). Ultra-pure water from a MilliQ water purification system (18.2 M cm, Millipore, Bedford, MA, USA) was used throughout. All other chemicals were of analytical grade and used without further disposal. 2-(4-(1,2,2-triphenylvinyl)phenoxy)acetic acid (TPE-COOH) was synthesized as described in our previous work. 1 Triisopropylsilane (TIPS) was from ThermoFisher Scientific (Acros Organics, Belgium). Apparatus. A Hitachi F-4600 Fluorescence Spectrophotometer (Tokyo, Japan) was used for recording fluorescence spectra. Fourier transform infrared (FT-IR) spectra were recorded with a Perkin Elmer spotlight 200 spectrometer (Massachusetts, USA) over a range of cm -1. The HPLC-ESI-IT-MS were performed on an Ultimate 3000 UHPLC system equipped with a diode-array detector (ThermoFisher, San Jose, CA, USA). High-resolution mass spectra were recorded on a Solarix 9.4T AS FTICR MS equipped with a MALDI source (Bruker Daltonics, Bremen, Germany). Dynamic light scattering (DLS) experiments were carried out with a Zetasizer Nano ZS ZEN3600 analyzer (Malvern Instruments Ltd, UK). Transmission electron microscopy (TEM) was characterized by JEM-2100 (UHR) transmission electron microscope (JEOL, Tokyo, Japan). Scanning electron microscopy (SEM) was taken by JSM-6700F scanning electron microscope (JEOL, Tokyo, Japan). Fluorescence imaging was performed on an Olympus IX83 Fluorescence microscope. Characterization of coordination complex with high-resolution mass spectrometry (HR MS). To identify the composition of the coordination complex formed by Pep2-TPE and Hg 2+, high resolution mass spectrometry (a Solarix 9.4T AS FTICR MS equipped with an ESI source) was used. After the addition of Hg 2+ to the Pep2-TPE solution, two new peaks appeared in the HR MS spectrum (Figure 3b in the main text). The peak with m/z of is from a singly-charged ion, and can be ascribed to the 2:1 complex of Pep2-TPE and Hg 2+ [(2L+Hg-3H) -, L= Pep2-TPE] (calculated m/z ). The S-2

3 appearance of the other new peak (m/z ) which is from a doubly-charged ion [(2L+Hg-4H) 2- ], further confirm the formation of 2:1 complex of Pep2-TPE (calculated m/z ). A MS peak with m/z of was detected for free Pep2-TPE. These results demonstrate the formation of 2:1 coordination complex between Pep2-TPE and Hg 2+. Competitive binding assays with biothiols. Competitive binding assays with biothiols (cysteine, homo-cysteine and -GSH) were carried out both individually and in mixed solution. For individual competitive binding assays, one biothiol species (12 L, 1mM in PBS) was mixed with Hg 2+ (12 L, 0.25 mm in PBS), respectively. Pep2-TPE (12 L, 1 mm in DMSO) was then added. PBS was added to the above solution to a final volume of 600 L. After incubated for 20 min, the fluorescence spectra were recorded with fluorescence spectrometer. To investigate the effect from higher concentration, the same experimental procedure was carried out except for the volume of each biothiol species which was changed to 120 L (1 mm in PBS). Competitive binding assays were also performed with the co-existence of cysteine, homo-cysteine and -GSH. Three biothiol species (each 12 L, 1 mm in PBS) were mixed with Hg 2+ (12 L, 0.25 mm in PBS), respectively. Pep2-TPE (12 L, 1 mm in DMSO) was then added. PBS was added to the above solution to a final volume of 600 L. After incubated for 20 min, the fluorescence spectra were recorded with fluorescence spectrometer. The effect from higher concentration of these biothiols was examined by increasing the volume of each biothiol species to 120 L during the preparation of solution. Cells. HeLa cells were from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Dulbecco s modified eagle media (DMEM), fetal bovine serum, penicillin (100 μg/ml) and streptomycin (100 μg/ml) were obtained from Invitrogen Corporation. Cells were cultured at 37 o C and 5% CO 2 in high-glucose DMEM supplemented with 10% fetal bovine serum (Gibco, ThermoFisher Scientific, San Jose, CA, USA). Cytotoxicity assay. HeLa cells were seeded in 96-well plates at a density of 7000 cells per well. After 24 h incubation, the culture medium was replaced by Pep2-TPE solutions at various concentrations (5, 10, 20 or 50 m), respectively. The cells were further cultured at 37 o C for another 48 h. The solutions were then discarded, and freshly prepared MTT solution (100 L; 0.5 mg/ml in culture medium) was added to each well. This was followed by incubation at 37 o C for 4 h. After removal of the MTT medium solution, DMSO (100 L) was added to each well, and the plate was shaken for 10 min to dissolve the formed precipitates. The absorbance values of the wells were read with a microplate reader at 490 nm (BIO RAD, imark). The cell viability rate (VR) was calculated from the equation S-3

4 VR=A/A 0 100%, in which A is the absorbance of the probe-treated cells and A 0 is the absorbance from cells without Pep2-TPE treatment. All data were obtained from three separate experiments. 2. Synthesis of Pep1-TPE ~ Pep5-TPE Scheme S-1. Synthesis of Pep1-TPE ~ Pep5-TPE. S-4

5 3. HPLC and MS Characterization of Pep1-TPE ~ Pep5-TPE Figure S-1. HPLC analysis and HR-MS characterization of Pep1-TPE (a, b) and Pep2-TPE (c, d). (a) For Pep1-TPE, HPLC analysis was carried out on a shim-pack XR-ODS column ( mm) monitored at the wavelength of 220 nm. Gradient: 0 20 min, 20%B 80%B (A: H 2 O containing 0.1% TFA, B: acetonitrile containing 0.1% TFA). (b) HR-MS spectrum of Pep1-TPE (MALDI, positive): m/z [M] + calcd: , found: ; m/z [M+Na] + calcd: , found: ; m/z [M+K] + calcd: , found: (c) For Pep2-TPE, HPLC analysis was carried out on a Diamonsil C18(2) ( mm) monitored at the wavelength of 220 nm. Gradient: 0 20 min, 20%B 80%B (A: H 2 O containing 0.1% TFA, B: acetonitrile containing 0.1% TFA). (d) HR-MS spectrum of Pep2-TPE (MALDI, positive): m/z [M] + calcd: , found: ; m/z [M+Na] + calcd: , found: ; m/z [M+K] + calcd: , found: S-5

6 Figure S-2. HPLC analysis and HR-MS characterization of Pep3-TPE (a, b), Pep4-TPE (c, d) and Pep5-TPE (e, f). HPLC analysis was carried out on a Dikma Diamonsil C18 column ( mm) monitored at the wavelength of 220 nm. Gradient: min, 5%B 80%B 100%B 100%B (A: H 2 O containing 0.1% TFA, B: acetonitrile containing 0.1% TFA). HRMS (MALDI) was operated in positive mode. Pep3-TPE: m/z [M] + calcd: , found: ; m/z [M+Na] + calcd: , found: ; m/z [M+K]+ calcd: , found: Pep4-TPE: m/z [M] + calcd: , found: ; m/z [M+Na] + calcd: , found: ; m/z [M+K] + calcd: , found: ; Pep5-TPE: m/z [M] + calcd: , found: ; m/z [M+Na] + calcd: , found: ; m/z [M+K] + calcd: , found: S-6

7 4. Binding kinetics of Pep2-TPE with Hg 2+ Figure S-3. Monitoring the fluorescence intensity of Pep2-TPE (20 M) in the presence of Hg 2+ (5 M) at different time intervals. 5. The effect from ph to the fluorescence response To examine the effect of ph to the fluorescence response of Pep2-TPE, ph titration experiments were performed by varying the ph values of the solution containing Pep2-TPE and Hg 2+ in the range from ph 3.0 to 10. As the control, the fluorescence emission behavior of Pep2-TPE itself in physiological buffers with different ph was also recorded. As shown in Figure S-4, stronger fluorescence from Pep2-TPE and Hg 2+ was detected in acidic solution. As ph increased, the fluorescence of the mixed solution gradually decreased. However, it is noticeable that without Hg 2+, the fluorescence of Pep2-TPE is affected more significantly by ph especially in acid environment (Figure S-4a). Pep2-TPE remains non-emissive in the ph range from 5.5 to 10. When further lowered the ph, the fluorescence from Pep2-TPE dramatically increased. At ph 3.0, the fluorescence intensity from Pep2-TPE solution was as strong as that with the addition of Hg 2+. To determine the working ph of the probe, Hg 2+ -induced fluorescence enhancement ratio (I-I 0 )/I 0 was calculated and plotted to ph vales. As shown in Figure S-4b, in the ph range from , Pep2-TPE shows most intensive response to Hg 2+. Such working ph range well satisfied the application of Pep2-TPE for tracing Hg 2+ in living biosystems which feature in neutral buffered environment. S-7

8 Figure S-4. (a) Effect of ph on the fluorescence intensity of Pep2-TPE (20 M) with and without the presence of Hg 2+ (5 M). (b)fluorescence response of Pep2-TPE (20 M) to Hg 2+ (5 M) under different ph. 6. Estimation of the detection limit The detection limit was estimated according to the equation LOD = 3D/S, where D is the standard derivation of the blank Pep2-TPE solution and S represents the slope of the calibration curve (Figure S-5). The standard derivation was determined by measuring the fluorescence spectra of blank Pep2-TPE solutions for 11 times. Figure S-5. Linear fitting of the fluorescence intensity of Pep2-TPE (20 M) versus Hg 2+ concentration. S-8

9 7. Hg 2+ -mediated self-assembly behavior Figure S-6. (a) TEM image of Pep2-TPE without the addition of Hg 2+. (b) Fluorescence microscopic images of Pep2-TPE without the addition of Hg 2+. Figure S-7. TEM image of Pep3-TPE (a), Pep4-TPE (b) and Pep5-TPE (c) in the presence of Hg Absorption spectrum of Pep2-TPE Figure S-8. Absorption spectrum of Pep2-TPE (100 M). S-9

10 9. Job s Plot Fluorescence titration was carried out to obtain the equilibrium complex between Pep2-TPE and Hg 2+. In a solution of Pep2-TPE (20 M), Hg 2+ was added continuously. Job s plot, a classical method for determining the stoichiometry of a binding event, was plotted by relating the fluorescence intensity at 470 nm to the mole fraction of Hg 2+ in the Hg 2+ /Pep2-TPE solution (Figure S-9). At the beginning of the titration, the continuous increase in Hg 2+ mole fraction led to the increase in fluorescence intensity. The plot reached the maximum when the mole fraction of Hg 2+ was The maximum of the plot represents the binding equilibrium and the stoichiometry of the two species in the solution. Hence, the 2:1 complex between Pep2-TPE and Hg 2+ can be confirmed. Figure S-9. Job s plot for the binding of Hg 2+ with Pep2-TPE. Fluorescence intensity at 470 nm was plotted as a function of the molar ratio of [Hg 2+ ]/[Hg 2+ +Pep2-TPE]. S-10

11 10. Determination of association constant The association constant (K a ) between Pep2-TPE and Hg 2+ was determined based on the titration curve. Following equation was used for the nonlinear fitting of the data. 2,3 Y = x b x 2 a (1 x) 2 + b 2 where x is (I-I 0 )/(I max -I 0 ), Y is the concentration of Hg 2+, a represents K a (the association constant), and b is the concentration of Pep2-TPE. As shown in Figure S-10, the curve fitting gave a K a value of M -2, which reveals the strong binding between Pep2-TPE and Hg 2+. Figure S-10. Nonlinear curve fitting for the evaluation of association constant (K a ) between Pep2-TPE and Hg 2+. S-11

12 11. FT-IR characterization of the coordination complex From microscopic FT-IR characterization (Figure S-11), the S-H bond vibration (2561 cm -1 ) of Pep2-TPE disappeared in the presence of Hg 2+, providing direct evidence for the coordination of SH with Hg 2+. The shifts of carboxylic C=O (1723 cm -1 ) and C-O (1245 cm -1 ) stretching vibrations towards lower wavenumbers (1712 cm -1 and 1235 cm -1 respectively) confirms the involvement of carboxyl group in Pep2-TPE for Hg 2+ chelating (Figure S-11). Moreover, with the addition of Hg 2+, shift of vibration of amide bond from 1664 cm -1 to 1646 cm -1 was also observed, which can be ascribed to the formation of hydrogen bonds during supermolecular assembly. The hydrogen bonds brings together the amide groups in the backbone of the molecules leading to the stacking of the coordination complex, thus the nanofibers. The above FT-IR results provide strong evidence and insights into the formation of coordination complex and supermolecular assembly. Figure S-11. FT-IR chemical imaging microscopic characterization of Pep2-TPE (20 M) with and without the existence of Hg 2+ (10 M). S-12

13 12. Effect of biothiols on the fluorescence response of Pep2-TPE to Hg 2+ Considering the intracellular existence of biothiols such as cysteine (Cys), homo-cysteine (Homo-cys) and -GSH and their possible coordination to Hg 2+, the competition of biothiols to the binding of Pep2-TPE with Hg 2+ was examined. The interference from different concentrations (20 M and 200 M) of Cys, Homo-cys and -GSH were investigated both individually and in the mixture. As shown in Figure S-12, the fluorescence response of Pep2-TPE to Hg 2+ was not affected by the presence of cysteine, Homo-cys or -GSH at the concentration of 20 M. With the presence of 200 M Cys, Homo-cys or -GSH, the fluorescence intensity of Pep2-TPE responding to Hg 2+ maintained 80%, 86% and 83% of that without the addition of biothiols. Only a little sacrifice in the fluorescence signal was detected. Furthermore, competitive binding assay with the co-existence of Cys, homo-cys and -GSH was also carried out. Even the concentration of each biothiol (200 M) is 10-times of that of Pep2-TPE, the fluorescence from the binding of Pep2-TPE with Hg 2+ was not significantly affected by co-existence of Cys, homo-cys and -GSH. The loss in fluorescence signal is 19%. These results demonstrated the high affinity of Pep2-TPE towards Hg 2+. The presence of biothiols in biosystems cannot significantly interfere their binding and emission. Pep2-TPE can be applied for detection and monitoring of Hg 2+ in living biosystems. Figure S-12. Effect of different concentrations of biothiols (Cys, Homo-cys and -GSH) on the fluorescence response of Pep2-TPE (20 M) to Hg 2+ (5 M). Normalization was calculated by using the fluorescence intensity from the mixture of Pep2-TPE (20 M) and Hg 2+ (5 M) as 100%. S-13

14 13. Competitive binding assay in cells Competitive binding assays employing a classic Hg 2+ chelator dimercaptosuccinic acid (DMSA) as the competitor were carried out to confirm the binding of Pep2-TPE to Hg 2+ in cells. After loaded with Hg 2+, HeLa cells were treated with DMSA, and then Pep2-TPE. As shown in Figure S-13a, no fluorescence can be observed inside cells after the addition of Pep2-TPE. This phenomenon can be attributed to the pre-occupation of Hg 2+ by DMSA which blocked the binding from Pep2-TPE. In comparison, without DMSA, bright fluorescence emitted from the probe in Hg 2+ -loaded cells. The competitive assay was also carried out in a reversed mode. The Hg 2+ -loaded cells were firstly treated with Pep2-TPE, and then incubated with DMSA (Figure S-13b). The addition of Pep2-TPE to Hg 2+ -loaded cells led to blue fluorescence. However, after further addition of DMSA, the blue fluorescence disappeared. The fluorescence turn-off phenomenon confirms the competition between Pep2-TPE and DMSA for the chelating with Hg 2+. The competitive binding results clearly demonstrate the fluorescence signal was caused by the specific binding of Pep2-TPE with Hg 2+, which provides the foundation for tracing Hg 2+ biodistribution in living cells with the probe. Figure S-13. Competitive binding assays using DMSA as the chelator. (a) Competitive assay 1: the cells were treated with Hg 2+, DMSA and Pep2-TPE sequentially. After treatment, the cells were observed with CLSM (upper panel). Hg 2+ -loaded cells only treated with Pep2-TPE were imaged as the control (lower panel). (b) Competitive assay 2: the cells were firstly imaged after the treatment with Hg 2+ and Pep2-TPE (upper panel). Afterward, further treatment with DMSA was carried out (lower panel). Scale bar: 10 m. S-14

15 14. Stability assay of Pep2-TPE The stability of Pep2-TPE in the working solvent (PBS with 2% DMSO, v/v) has been studied. As analyzed with HPLC, Pep2-TPE remains stable without oxidation at least for 48 h (Figure S-14). No oxidized product was detected. Figure S-14. HPLC analysis of Pep2-TPE solution (20 M, in PBS added with 2% DMSO). Upper panel: freshly prepared; middle panel: Pep2-TPE solution left for 48 hours after preparation; lower panel: solvent (PBS with 2% DMSO). The peak areas of Pep2-TPE kept the same and no new peaks appeared after 48 hours. These results suggest the stability of Pep2-TPE and absence of oxidation product. 15. Fluorescence behavior of the oxidized product of Pep2-TPE To examine the possible oxidation of the probe, the oxidized product (Pep2-TPE-S-S-Pep2-TPE) was synthesized and its fluorescence behavior to Hg 2+ was characterized. Pep2-TPE was oxidized with iodine for 24 hours to form Pep2-TPE-S-S-Pep2-TPE (Figure S-15). After purification, the resultant product was characterized with HPLC and high-resolution mass spectrometer (Figure S-15). S-15

16 Figure S-15. (a) Synthesis of the oxidized product Pep2-TPE-S-S-Pep2-TPE. (b, c) HPLC analysis and HR-MS characterization of the oxidized product Pep2-TPE-S-S-Pep2-TPE. (b) HPLC analysis was carried out on a Diamonsil C18 column ( mm) monitored at the wavelength of 220 nm. Gradient: 0 20 min, 60%B 100%B (A: H 2 O containing 0.1% TFA, B: acetonitrile containing 0.1% TFA). (c) HR-MS spectrum of Pep2-TPE-S-S-Pep2-TPE. (MALDI, positive): m/z [M + Na] + calcd: , found: ; m/z [M + K] + calcd: , found: As shown in Figure S-16, without Hg 2+, Pep2-TPE-S-S-Pep2-TPE itself (20 M) is highly emissive. After the addition of Hg 2+, no further fluorescence enhancement was detected. With lower concentration of Pep2-TPE-S-S-Pep2-TPE (10 M), similar results were obtained (Figure S-16b). These results demonstrate the poor solubility and poor response of Pep2-TPE-S-S-Pep2-TPE to Hg 2+. In comparison, the probe Pep2-TPE alone is non-emissive in the same solvent, while emits bright fluorescence upon the addition of Hg 2+. Such obviously different fluorescence behavior suggests the absence of oxidation during its application for Hg 2+ detection. S-16

17 Figure S-16. (a) Fluorescence spectra of Pep2-TPE-S-S-Pep2-TPE (20 M) with and without the presence of Hg 2+ (5 M). (b) Fluorescence response of Pep2-TPE-S-S-Pep2-TPE to Hg 2+ (5 M) under different concentrations. Insert: photos of corresponding Pep2-TPE-S-S-Pep2-TPE with and without the presence of Hg 2+ under UV light. Cell imaging experiments were also performed after the incubation with Pep2-TPE-S-S-Pep2-TPE. As shown in Figure S-17, blue fluorescence was emitted from HeLa cells only treated with Pep2-TPE-S-S-Pep2-TPE. For HeLa cells preloaded with Hg 2+, fluorescence with similar intensity was detected after the incubation with Pep2-TPE-S-S-Pep2-TPE. The results demonstrate the disability of Pep2-TPE-S-S-Pep2-TPE for discriminating Hg 2+ in living cells. It is worth noting that Pep2-TPE-S-S-Pep2-TPE nonspecifically located on cell membrane and in cytosol, but was almost absent from cell nucleus (Figure S-17). Such subcellular localization is different from Pep2-TPE which can trace the distribution of Hg 2+ in nucleus. Figure S-17. Fluorescence microscopic imaging of HeLa cells treated with Pep2-TPE-S-S-Pep2-TPE (10 μm) with and without the presence of Hg 2+ (5 M). Scale bar: 10 m. S-17

18 17. Evaluation of cytotoxicity of Pep2-TPE Figure S-18. Viability of HeLa cells after incubation with Pep2-TPE at four concentrations (5, 10, 20, or 50 M) for 24 hours based on MTT assays. References (1) Gui, S.; Huang, Y.; Hu, F.; Jin, Y.; Zhang, G.; Yan, L.; Zhang, D.; Zhao, R. Anal. Chem., 2015, 87, (2) Neupane, L. N.; Oh, E.-T.; Park, H. J.; Lee, K.-H. Anal. Chem., 2016, 88, (3) Kubo, Y.; Kato, M.; Misawa, Y.; Tokita, S. Tetrahedron Lett. 2004, 45, S-18