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A novel fluorescein-based fluorescent probe for detecting HS and its real applications in blood plasma and biological imaging Xilang Jin, Shaoping Wu, Mengyao She, Yifan Jia, Likai Hao, Bing Yin, Lanying Wang, Martin Obst, Yehua Shen, Yongmin Zhang, and Jianli Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04087 • Publication Date (Web): 26 Oct 2016 Downloaded from http://pubs.acs.org on October 27, 2016

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Analytical Chemistry

A novel fluorescein-based fluorescent probe for detecting H2S and its real applications in blood plasma and biological imaging Xilang Jin†, ‡,§, Shaoping Wu‖, §, Mengyao She†, Yifan Jia†, Likai Hao¶, Bing Yin†, , Lanying Wang†, Martin Obst┴, Yehua Shen†, Yongmin Zhang‖, Jianli Li†,* †

Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi'an, Shaanxi 710127, P. R. China ‡ School of Materials and Chemical Engineering, Xi'an Technological University, Xi'an 710032, Shaanxi, P. R. China ‖ Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest University), Ministry of Education; Biomedicine Key Laboratory of Shaanxi Province, Northwest University, Xi'an, Shaanxi 710069, P. R. China ¶ Center for Applied Geoscience, Institute for Geoscience, Eberhard-Karls University Tübingen, Hölderlinstr. 12, Tübingen 72074, Germany ┴ Bayreuth Center for Ecology and Environmental Research (BayCEER), University of Bayreuth, Dr.-Hans-Frisch-Str. 1-3, Bayreuth 95448, Germany. Corresponding Authors *Tel.: +86 029 81535026; Fax: +86 029 88308396. E_mail: [email protected] ABSTRACT: A broad-spectrum fluorescent probe, which can be applied to monitoring H 2S in various biological systems, has been rationally designed and synthesized. This specific probe was applied to localize the endogenous H2S in living Raw264.7 macrophages cells, HepG2 cells and H9C2 cells. On the same time, the probe have successfully visualized CBS- and CSE-induced endogenous H2S production and monitored CBS and CSE activity in H9C2 cells. This probe could serve as a powerful molecular imaging tool to further explore the physiological function and the molecular mechanisms of endogenous H 2S in living animal systems. Keywords: hydrogen sulfide, fluorescent probes, endogenous, flow cytometer, enzyme activity

Hydrogen sulfide (H2S) plays a key role in biological systems, and has recently been recognized as the third endogenous gaseous signaling molecule besides nitric oxide (NO) and carbon monoxide (CO)1-3. In mammalian systems, it is produced endogenously from L-cysteine and catalyzed by two pyridoxal-5′-phosphate-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE)4,5. Endogenous hydrogen sulfide has a variety of biological effects in the cardiovascular system, such as vasorelaxation6, inhibiting vascular smooth muscle cell proliferation 7, and showing myocardial negative inotropic effects8. It also plays a pathophysiological role in numerous cardiovascular diseases, such as atherosclerosis9, hypertension10 and cardiac ischemia disease11 amongst others. Thus, direct evidence for H2S bioaccumulation or transformation from in situ (confocal) fluorescence microscopic imaging12-17 is expected to gain a better understanding of the molecular mechanisms. To date, several fluorescent probes18-21 for H2S have been reported, with different reaction mechanisms to trap H2S, such as azide reduction16,17,22-27, copper sulfide precipitation28-30 and/or nucleophilic addition13, 31-35. However, while there is an urgent requirement for a broad-spectrum H2S detective probe in the field of biological detection in interfered biological systems, the establishment of such a probe serves as a

breakthrough to those biological detections via H2S as the messenger. Normally, any reaction-based H2S probe was designed relies on modifying a fluorescent reporter with a reactive masking moiety, which was deprotected by interacting with H2S. These masking moieties are necessarily highly specific to H2S over other relevant sulfur species (Figure 1).

Figure 1. Selected sulfur-sensitive masking groups utilized in fluorescent probe design.

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Based on our previous work14,15, we present a simple and new fluorescein-based probe whose esters are able to maintain the cell membrane permeability efficiently16. Besides the rapid and specific response of exogenous and endogenous H2S in living myocardial cells, this probe can also be applied to monitoring CBS and CSE activity with confocal laser scanning microscopy and flow cytometry. We believe that the development of this probe is a significant breakthrough for visualizing endogenous H2S within living biological samples and opens up further opportunities to study its cellular biochemistry 36.

EXPERIMENTAL SECTION Materials and Measurements. Fluorescence spectra were measured on a Hitachi F-4500 fluorescence spectrophotometer equipped with a xenon discharge lamp, in a 1 cm quartz cell. Mass spectra were measured using a BrukermicroTOF-QⅡ ESI-Q-TOF LC/MS/MS Spectrometer. NMR spectra were recorded on a Varian INOVA-400 MHz spectrometer (at 400MHz for 1H NMR and 100MHz for 13C NMR) using tetramethylsilane (TMS) as internal standard. X-ray crystallography data were collected on a Bruker Smart APEX II CCD diffractometer. Bioimaging was performed using a Leica SPE confocal laser scanning microscope with an excitation wavelength of 488 nm14,15. Reagents and Chemicals. Fluorescein and 2thiophenecarbonyl chloride were obtained from J&K Scientific Ltd (Shanghai China). Analytical thin layer chromatography was performed using Merck 60 GF254 silica gel (pre-coated sheets, 0.25 mm thickness). The solutions of various testing species were prepared from NaHCO3, NaCl, NaBr, KI, NaCN, NaNO3, MgCl2,CaCl2, ZnCl2, Na2SO3, Na2S2O3, Na2SO4, KSCN, H2O2, NaClO, O2-(was generated from xanthine and xanthine oxidase), NO(was generated from SNP (Sodium Nitroferri-cyanide(III) Dihydrate). Twicedistilled water was used throughout all experiments. General Procedures for Fluorescence and UV-Visible Measurements14,15. Probes 1 stock solution (1 mM) was prepared in acetone. The solutions of various testing species stock solutions (1 mM) were prepared in distilled water. During the titration experiments, different amounts of Na2S and 0.10mL of 1 mM probes were mixed and filled up with PBS to 10 mL in volumetric tubes. During the interference experiments, 50µM Na2S, 0.10mL of 1 mM probe 1 and 1mL of 10mM testing species were mixed and filled up with PBS to 10 mL in volumetric tubes. 1 mL aliquots were pipetted into a 1 cm cuvette for spectral measurements. 5 nm bandpasses were used for both excitation and emission wavelengths. An excitation wavelength of 450 nm was used for the acquisition of emission spectra. Crystal growth and conditions. White single crystals of the probe was obtained at room temperature from the mixed solvents of acetonitrile-trichloromethane solution by slow evaporation and then mounted on the goniometer of single crystal diffractometer. The crystal data have been collected at 293 K by using Mo Kα radiation (λ = 0.710713 Å) by using φ /ω scan mode and collected for Lorentz and polarization effect (SADABS). The structure was solved using the direct method and refined by full-matrix least-squares fitting on F2 by SHELX-97.

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Synthesis of Probe 1. In a 250 mL flask containing a suspension of fluorescein (8.0 g, 24.1 mmol) in 100 mL THF, 2-thiophencarbony chloride (7.1 mL, 50.0 mmol) and Et3N (5.0 mL) was added. The reaction mixture was heated to reflux for 1 h with stirring under N2 atmosphere. Upon completion of the reaction, the aqueous suspension was filtered and the filtrate was evaporated by rotary evaporator. The residue was dissolved in 100 mL water and extracted with CHCl3, dried over Na2SO4, filtered and evaporated. Finally, the crude product was purified by recrystallization from ethanol to give 10.10 g of 1 as an off-white solid (75.9 %).1H NMR (400 MHz, CDCl3) δ = 8.13 (d, J=4.9, 2H), 8.08 (dd, J=10.2, 5.7, 3H), 7.86 (t, J=7.5, 1H), 7.79 (t, J=7.4, 1H), 7.51 (d, J=1.9, 2H), 7.47 (d, J=7.6, 1H), 7.36 – 7.30 (m, 2H), 7.13 (dd, J=8.6, 1.9, 2H), 6.96 (d, J=8.6, 2H).13C NMR (100 MHz, DMSO) δ = 173.5, 164.7, 157.3, 156.8, 155.9, 141.0, 140.7, 136.5, 135.7, 134.2, 130.5, 130.2, 129.3, 123.8, 121.6, 115.8, 86.1 ppm. MS(ESI) calcd. for C30H16O7S2 [M +H]+: 553.0416, found: 553.0402.

RESULTS AND DISCUSSION Synthesis. The probe 1 was prepared from fluorescein in a one-step reaction according to the procedure shown in Scheme 1. A five-membered electron-rich structure, with sulfur in its ortho-position, has a specific reactive response towards its CO (sp2-O) bonds when detecting H2S via bonding xanthene with its acyl. Scheme 1. Probe 1 as a fluorescent probe for H2S

Figure 2. Fluorescence spectra of probe 1 (10 µM) in PBS buffer (20 mM, pH 7.4, 1 % CH3COCH3) at 37 oC for 15 min. Excitation: 450 nm. Insert: Linear correlation of fluorescent intensity (λmax = 542 nm) toward H2S concentration in PBS buffer (20 mM, pH 7.4, 1% CH3COCH3).The data represent the average of three independent experiments.

Spectroscopic Properties15. We then evaluated the spectral properties and H2S response of the probes in phosphate-buffer saline (PBS: 20 mM, pH 7.4) containing 1% CH3COCH3. Only probe 1 showed response to H2S in solution. In its protected forms, it adopts a closed lactone conformation that is non-fluorescent and exhibits no absorption features in the


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visible region. The new probe is quite stable since no changes in their fluorescence spectra were observed after it was stored at 4 oC for two weeks. Upon addition of 70 µM Na2S (a commonly employed H2S donor13, 22), a robust enhancement in fluorescence intensity was observed (Figure 2, insert). The maximum fluorescence intensity was achieved within 15 min under these conditions, suggesting a fast fluorescence turn-on reaction. The fluorescence response of probe 1 was further evaluated by fluorescence titration with Na2S (10-200 µM) in varying concentrations. These concentrations are well within the range that has been used to elicit physiological responses (10-600 µM)37-39. As shown in Figure 2, the fluorescence intensities were indeed linearly related to the concentrations of H 2S in the concentration range of 20-100 µM. The regression equation was y=160.24x-358.23, R²=0.9827. The detection limit for H2S was found to be 1 µM. These results demonstrated that probe 1 could detect H2S both qualitatively and quantitatively.

spectroscopic changes result from the removal of the thiophenecarboxylate and thus the formation of the deprotective product which is analogous to cut-through mechanism (Scheme 2). Scheme 2. Propose H2S-sensing mechanism of probe 1.

In order to study the specific mechanism of this probe, the density functional theory (DFT) calculations based on the Fukui function f+(r) (eq. 1) were performed for the probe 1 with PBE040 functional. Basis set of double-ζ quality (631G** for C, H elements, 6-31+G* for O, S elements) was used for the geometry optimization and the following single point energy calculation. The optimized structure was proven to be the local minimum based on the results of vibrational analysis. All the calculations were performed with the Gaussian 09 program14.   (r )  f (r )      (r ) N 1   (r ) N  N 



 X

f q

N 1 X

q

N X





(1) (2)

Equation 1. Fukui function where N is the number of electrons, υ is the external potential, qX is the electronic population of atom X in a molecule.

Figure 3. Selectivity of probe 1 for H2S (10 µM) in PBS buffer (20 mM, pH 7.4, 1 % CH3COCH3) solution. The pillars in the front row represent the value in the presence of various ions. The pillars in the back row indicate the change in the emission intensity upon subsequent addition of H2S (50 µM) to the solution containing probe 1 and the respective ion of interest. For all measurements, λmax = 542 nm.

Selectivity Studies. To study the selectivity of the probe for H2S, the fluorescence properties of probe 1 were evaluated in the presence of other anions (HCO3-, Cl-, Br-, I-, CN-, NO3-, 1 mM ), metal ions (Na+, K+, Mg2+, Ca2+, Zn2+, 1 mM), inorganic reactive sulfur species (SO32-, S2O32-, SO42-, SCN-, 1 mM ), reactive oxygen and nitrigen species (H2O2, O2-, ClO-, ONOO-, NO). Most of them did not lead to any significant fluorescence intensity (Figure 3). Only the presence of ClO-, H2O2, S2O32resulted in limited fluorescence responses. However, the increase of the fluorescence intensity was far by at least probe 1 order of magnitude which was weaker than that caused by H2S. Notably, small-molecule thiols such as glutathione (GSH) at 10 mM, cysteine (Cys) at 1 mM and Lysine (Lys) at 1 mM triggered only a small fluorescene enhancement and have nearly no interference to H2S detection (Figure 3). These results suggested the high selectivity of the probe for H2S. The pH titration curve also revealed that the maximum fluorescent signal of probe 1 was observed at physiological pH (Figure S2-S3). Mechanism Studies and Density Functional Theory. The mass spectrometry analysis confirmed that the H2S-triggered

f+(r) has been successfully used to describe the reactivity concerning nucleophilic attack14,41, similar as the H2S in this work. According to NBO analysis42, the condensed Fukui function (Figure 4) of ester carbon (C30: 0.075; C29: 0.064) should be the better active site to react with H2S.

Figure 4. (a) 3D representation and condensed Fukui function f+(r) of the iso-value of 0.003 a.u. (positive in red colour and negative in green colour). (b) HOMO distribution of probe 1. (c) LUMO distribution of probe 1.



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Figure 5. Theoretical wavelength (λ), excitation energy, oscillator strength (f), relevant frontier MOs (3D distribution and orbital energy), and corresponding CI coefficient of the absorption and emission of the deprotective product in water.

The spatial distributions and orbital energies of HOMO and LUMO of probe 1 and fluorescein were also determined. As shown in Figure 4, the HOMO was almost entirely distributed within the xanthene moiety while the LUMO was found within the protective group. The difference of energy between the HOMO-LUMO levels was found to be 4.58 eV. In addition, we also performed time-dependent density function theory (TD-DFT) calculations according to the optimized structure of the ground S0 state and the first excited S1 state for the deprotected product. It gave a calculated emission band at 517 nm belonging to the S1-S0 energy state (Figure 5). This is consistent with the emission band at 542 nm obtained experimentally. Visualizing Na2S in Living Cells. Due to its high sensitivity, we blithely hope to put the probe into the detections in biological system. For living cell imaging, biocompatibility is always the first consideration. The MTT assay in HepG2 cells (Figure S9) was performed to assess the cytotoxicity of the probe 1. The results showed that cell viability is not affected even at concentrations of probe 1 in the culture medium as high as 20 µM. In term of the biocompatibility of the probe 1, we pursued the progress of the broad-spectrum detection of H2S in complicatedly interfered biological systems such as in immunocytes, in oncocytes and in ordinary cells. To further establish the utility of probe 1 for the determination of H2S in biological samples, probe 1 (10 µM) was evaluated in commercially available bovine plasma. Upon addition of H2S, the probe 1 in serum showed significant fluorescence intensity increases, and the reaction completed within 15 min at room temperature (Figure S4). As shown in Figure S5, we obtained a standard curve between fluorescence intensity and the concentration of H2S. We observed that the fluorescence intensity response in plasma was lower than the signal obtained in pure buffer solutions. This is possibly due to the fast metabolism and volatile nature of hydrogen sulfide in biological systems. Therefore, the fast response and excellent linear relationship provided a real-time quantitative detection method for hydrogen sulfide in complex biological systems such as bovine plasma. T cells and natural killer (NK) cells are lymphocytes that play complementary activities in cell-mediated immunity. We used the CLSM to reveal the cellular morphological changes under exogenous H2S stress. As shown in Figure 6, H2S dispersed in the cytoplasmic space with nucleic acid and the whole cell was wrapped with polysaccharides; while exogenous H2S did not lead to shape change of Jurkat T cells, most of the H2S were distributed in the cytoplasmic space with nucleic acid and more hotspots also were found on the cellular surface; normal NK-92 cells accumulated intracellular H2S near the nucleus area, whereas polysaccharides were distributed homogeneously around cellular surface. Exogenous H2S led NK-92 cells to apoptosis associated with lysis of the nuclei and morphological changes; most of the H 2S were then dispersed in the cytoplasmic space and only few hotspots of H2S were formed with the nucleic acid, while polysaccharides formed hotspots on the cell surface. These different

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distribution patterns of natural H2S in Jurkat T cell and NK-92 cell also indicated that this novel probe was suitable to establish fluorescent probe based on microscopic fingerprint of lymphocytes to explore the cellular morphological changes associated with certain diseases and infections, which may be helpful for next clinical diagnosis and further medical treatment.

Figure 6. Map the distribution of natural H2S and exogenous H2S by probe 1 in T cell incubated with 40 μM Na2S for 1 h at 25 °C. The cells were simultaneously incubated with 20 μM probe 1 (b), fluorescent nucleic acid stain (a) and lectin conjugate (c) for 1 h at 25 °C, T cells outline are visualized by their reflection signal (d) (λex = 405 nm, 488 nm, 635 nm). The overlay image of (a) (b) (c) and (d) is shown in (e). Brighter colour indicates higher concentrations. Scale bar: 10 µm.

Inspired by these promising results, we further evaluated whether probe 1 could be used to monitor endogenous H2S in living cells. CSE is distributed in smooth muscle cells, liver, heart and pancreas, whereas CBS is found in the brain, liver, kidney and pancreas 43. HepG2 cells (high expression of CBS and CSE) were chosen as bioassay models for endogenous H2S. When stimulated by pyridoxal-5′-phosphate (PLP) enzymes, HepG2 cells may produce endogenous H2S. The living HepG2 cells loaded with only probe 1 (10 μM) displayed a weak fluorescence (Figure 7a), indicating that probe 1 could capture endogenous H2S in HepG2 cells. However, stimulation with PLP in the presence of probe 1 led to a pronounced increase in fluorescence intensity (Figure 7b). Then the cells were further incubated with 0.2 mM DLpropargylglycine (PAG, a CSE inhibitor) or 0.05 mM hydroxylamine (HA, a CBS inhibitor), resulting in much weaker fluorescence (Figure 7c-d). Similar results were also generated on Raw264.7 macrophage cells (Figure 8). These results demonstrated that probe 1 had successfully visualized CBS- and CSE-induced endogenous H2S production and monitored CBS and CSE activity in HepG2 cells and Raw264.7 macrophage cells.

Figure 7. Probe 1 responds to endogenous H2S in living HepG2 cells (a1-d1) and living Raw264.7 macrophage cells (a2-d2). (a) The cells were treated with 10 μM probe 1. (b) Cells were incubated with 2 mM L-Cys +0.5 mM PLP before being stained with probe 1. (c) Cells were incubated with 2 mM L-Cys+0.5 mM PLP +0.2 mM PAG before being stained with probe 1. (d) Cells were incubated with 2 mM L-Cys+0.5 mM PLP +0.05 mM HA before being stained with probe 1.


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Encouraged by the results of the HepG2 cells, we decided to further examine the suitability of probe 1 to detect endogenously produced H2S in living H9C2 cells (low expression of CSE). To localize H2S in the cells, fluorescent nucleic acid stain (such as hochest dyes 33258) was applied simultaneously with probe 1 (Figure 8). When the cells were treated with 2 mM L-Cys and 0.5 mM PLP for 24 h and then stained with probe 1 (10 µM) for 30 min, the fluorescence intensity increased significantly compared with that of the control. Moreover, treatment of H9C2 cells with DLpropargylglycine (PAG), a CSE inhibitor, also attenuated the probe 1 turn-on response. Confocal microscopy offers high spatial resolution but with limited statistical sampling. To test the feasibility of using probe 1 for high-throughput analysis, we employed flow cytometry (FCM)44,45 for the H9C2 cells (Figure S15-S20). In comparison to non-stained control cells, after addition of 2 mM L-Cys and 0.5 mM PLP, significant emission signals were predominantly derived from the probe-stained cells. However, addition of HA or PAG triggered a decrease in the emission signal of the stained cells. These results indicate that probe 1 could be used to visualize H2S in living cell under physiological conditions to avoid artifacts from sample preparation. The ability to monitor changes and distributions of endogenous levels of H2S within living biological samples opens up further opportunities to study the physiological function and the molecular mechanisms of endogenous H 2S in the cardiovascular systems.

Figure 9. Bioluminescence imaging of exogenous H2S in Kunming Mice acquired at 0, 1, 5, 10, 20 min. Left: the mice were treated with fluorescein. Middle: the mice were treated with 5 mM probe 1. Right: the mice were treated with 5 mM probe 1 and 5mM Na2S.

The mice were given an injection of the probe 1 and then injected with different amounts of Na2S (5 mM; 10 mM; 20 mM), and then the images were obtained. As shown in Figure 10, the fluorescence intensity in the mice is concentrationdependent.

Figure 10. Representative fluorescence images of the mice given an injection of the probe 1 (5 mM) and then injected with different amounts of Na2S: (a) 5 mM; (b) 10 mM; (c) 20 mM;

Figure 8. Probe 1 responds to endogenous H2S in living H9C2 cells (a-d). (a) The H9C2 cells were treated with 10 μM probe 1. (b) Cells were incubated with 2 mM L-Cys +0.5 mM PLP before being stained with probe 1. (c) Cells were incubated with 2 mM L-Cys+0.5 mM PLP +0.2 mM PAG before being stained with probe 1. (d) Cells were incubated with 2 mM L-Cys+0.5 mM PLP +0.05 mM HA before being stained with probe 1.

We further evaluated whether probe 1 could be used to monitor endogenous H2S in living mice. When stimulated by PLP, the probe 1 has led to a pronounced increase in fluorescence intensity. Then the cells were further incubated the cells with 0.2 mM PAG or 0.05 mM HA, resulting in much weaker fluorescence. Therefore, the results well suggested that the probe 1 can even detect H2S in real time in vivo, which may help us to further study the biological roles of the H 2S in the future (Figure 11).

Visualizing H2S in Living Animals. Finally, the above desirable results encouraged us the suitability of the probe 1 for monitoring H2S in the context of living animals. Kunming mice were divided into three groups. One group was given fluorescein. The second group was treated with 5 mM probe 1. The third group was treated with 5 mM probe 1 and 5 mM Na2S. Live mice injected with probe 1 for 20 min showed very weak emission. However, the living mice were injected with Na2S under the same conditions; a significant enhancement was observed with time (Figure 9).



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Figure 11. Bioluminescence imaging of endogenous H2S in Kunming Mice acquired at 0, 1, 5, 10, 20 min. In each picture: (a) The mice were treated with 5 mM probe 1. (b) The mice were incubated with 2 mM L-Cys +0.5 mM PLP before being stained with probe 1. (c) The mice were incubated with 2 mM L-Cys+0.5 mM PLP +0.2 mM PAG before being stained with probe 1. (d) The mice were incubated with 2 mM L-Cys+0.5 mM PLP +0.05 mM HA before being stained with probe 1.

CONCLUSIONS In conclusion, a novel fluorescein-based fluorescence probe for H2S was designed, synthesized and evaluated, demonstrating high selectivity and sensitivity with a linear detection range from 20-100 μM of H2S in aqueous solution and biological bovine plasma systems. The probe was successfully used to map exogenous H2S sorption in NK-cell and T-cell aggregates by confocal laser scanning microscopy. Based on the results of the fluorescence imaging of exogenous H2S, we further used it to monitor endogenous H 2S in living Raw264.7 macrophages cells, HepG2 cells and H9C2 cells. The detecting mechanism of this probe is analogous to cutthrough mechanism, which realizes an access to the broadspectrum detection of H2S while avoiding distractions from other complexes in biological systems. Furthermore, probe 1 have successfully visualized CBS- and CSE-induced endogenous H2S production and monitored CBS- and CSEactivity in H9C2 cells and living mouse model. Moreover, we expect that probe 1 would be a potentially powerful tool for studying and providing further insight into H2S chemistry in the living animal imaging. ASSOCIATED CONTENT Supporting Information Spectroscopic Property, Structure characterizations and crystal data of probe 1, data for investigation of the sensing mechanism , and more experimental results and figures as noted in the text.

AUTHOR INFORMATION Corresponding Author *Tel.: +86 029 81535026; Fax: +86 029 88308396. E-mail: [email protected] Author Contributions §X. L. J. and S. P. W. contributed equally to this work Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT The project was supported by National Natural Science Foundation of China (NSFC 21572177; 21272184; 21103137 and J1210057), Shaanxi Provincial Natural Science Fund Project (No. 2015JZ003 and 2016JZ004), the Xi'an City Science and Technology Project (No. CXY1511(3)) and the Northwest University Science Foundation for Postgraduate Students (No. YZZ14052; YZZ15040; YZZ15045 and YZZ15006) and the Emmy-Noether fellowship program of the DFG to M.O. (OB 362/1-1).

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