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Optimized Ratiometric Fluorescent Probes by Peptide Self-assembly Yanbin Cai, Jie Zhan, Haosheng Shen, Duo Mao, Shenglu Ji, Ruihua Liu, Bing Yang, Deling Kong, Ling Wang, and Zhimou Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02955 • Publication Date (Web): 02 Dec 2015 Downloaded from http://pubs.acs.org on December 10, 2015
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Analytical Chemistry
Optimized Ratiometric Fluorescent Probes by Peptide Selfassembly Yanbin Cai,†,‡ Jie Zhan,† Haosheng Shen,‡ Duo Mao,‡ Shenglu Ji,‡ Ruihua Liu,† Bing Yang,‡ Deling Kong,‡ Ling Wang,*,† Zhimou Yang*,†,‡ State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, and Tianjin Key Laboratory of Molecular Drug Research, ‡Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, P. R. China †
ABSTRACT: We report in this study on optimized ratiometric fluorescent probes by peptide self-assembly. The resulting selfassembled nanoprobes show extraordinary stability in aqueous solutions and extremely low background fluorescence in buffer solutions. Our optimized probes with much bigger ratiometric fluorescence ratios also show an enhanced cellular uptake, lower background noise, and much brighter fluorescence signal in the cell experiment. Our study provides a versatile and very useful strategy to design and produce fluorescent probes with better performance.
Introduction Fluorescent probes have been extensively investigated in last decades due to their advantages such as noninvasiveness, high sensitivity and specificity, and high spatiotemporal resolution.1-6 They have been widely applied for the detection of environmentally and biologically important species.7-16 One of consensuses to design fluorescent probes is to lower their background fluorescence and amplify their turn on effects.3, 1719 In order to achieve this goal, several design principles and sensing mechanisms have been reported, including photoinduced electron transfer (PET),20-22 aggregation-induced emission (AIE),23-26 aggregation caused quenching (ACQ),27-29 fluorescence resonance energy transfer (FRET),6, 30 and intramolecular charge transfer (ICT),31-34 etc. Though great successes have been achieved for the development of fluorescent probes,13, 35-37 there are still increasing needs to develop novel design strategies for fluorescent probes with better performances (e.g. with enhanced imaging contrast). Self-assembling peptides have been widely studied and they can assemble into well-defined and well-ordered nanostructures.38-41 We recently demonstrated that the wellarranged molecular packing in the nanostructures of peptides could enhance the quenching effect of a quencher,41 and the hydrophobic pocket in the nanostructures could make the environment-sensitive fluorophore emit more brightly.43-44 In this study, we show that taken the advantage of peptide selfassembly, we can make optimized ratiometric fluorescent probes with much lower short wavelength fluorescence and higher long wavelength fluorescence signals. A novel kind of fluorescent probe called ratiometric one is recently reported and widely studied.45-53 Since ratiometric fluorescent probes possess two ultraviolet absorptions, they can emit two wavelengths of fluorescence upon excitation. They have been applied for the rapid, specific, and quantitative detection of important analytes through the calculation of fluorescence ratio of two emissions. For instance, they have been used to detect both small molecules (e.g. H2S, CN-, F-, etc.)54-63 and biomacromolecules (e. g.
enzymes).49-50, 64-69 We speculate that the short-wavelength emission of the ratiometric probes maybe lowered at assembled stages due to the ACQ effect, and the longwavelength emission maybe enhanced due to the possible environment sensitive property similar to the environmentsensitive fluorophore. If so, the incorporation of selfassembling peptides in ratiometric fluorescent probes would result in optimized probes with not only lower shortwavelength fluorescence but also enhanced long-wavelength fluorescence, which would be beneficial for the detection of analytes in cells with enhanced imaging contrast. Results and Discussion
Scheme 1. Chemical structures of probes and their transformation by H2S
Molecular design of a probe for hydrogen sulfide detection. We firstly choose the 4-N3-1,8-naphthalimide to test our hypothesis because its azide group could be reduced to amine group by hydrogen sulfide (H2S), resulting in ratiometric fluorescence changes.70 We modified the 4-N3naphthalic anhydride with a β-alanine to obtain a derivative of 4-N3-1,8-naphthalimide (N3-NTI, 2a in Scheme 1) with a carboxylic acid that could be used for solid phase peptide synthesis (SPPS). We then used it as an aromatic capping
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group71-76 for peptides to generate possible self-assembling peptides. The obtained compound N3-NTI-FFY (n=0) was insoluble in phosphate buffer saline (PBS, pH = 7.4) at the concentration of 1.0 mg/mL, and the N3-NTI-FFYE2 was very well soluble in PBS even at the concentration of 5.0 mg/mL (Figure S-15). Only N3-NTI-FFYE (1a) exhibited distinguish self-assembly property, which was showed by the light beam from its solution upon irradiation by a laser pointer (Figure S15). We then focused on the characterization and application of 1a. We studied the self-assembly property of 1a firstly by dynamic light scattering (DLS). The result in Figure S-11 showed that its critical micelle concentration (CMC) was about 80.5 µM. The transmission electron microscopy (TEM) image exhibited that 1a self-assembled into nanofibers at 100 µM in PBS buffer (Figure 1A). The nanofiber dispersion of 1a was highly stable for more than 1 month, while compound 2a was insoluble in pure PBS at 100 µM (Figure S-15). In order to obtain a clear solution of 2a, it needed to be firstly dissolved in DMSO and then diluted in PBS (5% of DMSO). Even in the presence of DMSO, compound 2a would still precipitate out from the solution after 24h. The ultra-stable nanofiber dispersion of our self-assembled probe and no need of organic solvents suggested its advantages for the detection applications in biological environments. Fluorescence properties and ratio changes. We then monitored the fluorescence change of 1a and 2a by adding different concentrations of H2S. As shown in Figure 1C, we observed typical ratiometric fluorescence spectra of 2a. Upon excitation at 340 nm, there were two emission peaks appeared at 425 and 550 nm. As the concentration of H2S increased, the peak at 425 nm decreased and the peak at 550 nm gradually increased in intensity. Upon irradiation by a UV lamp at 330 nm, we observed the color of fluorescence changing from blue to yellowish green (Figure 1C inserts). The fluorescent spectra of 1a with increasing concentrations of H2S were shown in Figure 1B. We found that the intensity of peak at 425 nm of 1a was significantly lower than that of 2a at the same concentration, indicating that the self-assembly property could quench the
Figure 1. A) TEM image of solution of 1a (100 µM), B) and C) Fluorescence spectra of solution of 1a and 2a treated with different concentrations of H2S (5 to 200 µM), respectively, insets in B and C) images of solutions of 1a and 2a before and after adding H2S and irradiated by a hand-held UV lamp (330 nm channel), D) Fluorescence intensity ratio (F550/F425) of 1a and 2a with increasing concentrations of H2S (5, 10, 20, 30, 40, 50, 60,
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80, 100, and 200 µM, respectively. Excitation wavelength = 340 nm, acquired 2h after the addition of analyte at 37 0C). (1a in PBS buffer, 100 µM, PH=7.4, 2a in PBS buffer with 5% DMSO at 100µM)
short-wavelength fluorescence probably due to ACQ effect. However, the peak at 550 nm of 1a was higher than that of 2a after adding the same concentration of H2S. For example, the intensity was about 2,000 for 2a, while it was about 3,000 for 1a in the presence of 200 µM of H2S. The results indicated that peptide self-assembly could indeed increase the longwavelength fluorescence of the probe. Upon the irradiation of a UV lamp at 330 nm, the solution of 1a exhibited extremely low blue background fluorescence (Figure 1B insets). Meanwhile, the solution emitted bright yellowish green fluorescence in the presence of H2S. We also calculated the ratio of fluorescence intensities of 1a and 2a before and after adding H2S (F550/F425). The ratio of 1a was from 0.76 to 24.54, while the ratio of 2a was only from 0.36 to 8.29 with the concentration of H2S from 5 to 200 µM (Figure 1D). The much bigger enhanced values of the ratio of fluorescence suggested the better sensitivity and performance of our selfassembled probes.
Figure 2. A) Chemical structures of 3a, 4a, 3b, 4b, B) TEM image of solution of 3a, C) and D) Fluorescence spectra of solutions of 3a and 4a treated with different concentrations of hydrazine (10 to 500 µM), insets in C and D: top) Fluorescence color changes of 3a and 4a treated with hydrazine observed using a hand-held UV lamp with an excitation at 330 nm, down) visual color changes of 3a and 4a treated with hydrazine, E) Fluorescence intensity ratio (F550/F475) of 3a and 4a with increasing concentrations of hydrazine (0, 5, 10, 20, 30, 40, 50, 100, 200, 300, and 500 µM, respectively. Excitation wavelength = 400 nm, spectrum was acquired 2h after addition of analyte at 37 0 C. 3a was dissolved in PBS buffer at 100 µM and 4a in PBS buffer with 5% DMSO at 100 µM.)
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Analytical Chemistry
Molecular design of a probe for Hydrazine detection. The amine group in 1b or 2b could be protected to phthalimide, which would react with hydrazine and therefore be converted back to the amine group.77 Probes with this conversion had already been used for the detection of hydrazine.61 In order to validate the generality of our strategy for the development of optimized ratiometric probes, we synthesized another two probes with phthalimide group for the detection of hydrazine (3a and 4a in Figure 2A). Similar to 1a, the CMC of 3a was 85.2 µM (Figure S-17), and it also self-assembled into nanofibers at 100 µM in PBS buffer (Figure 2B). We then compared the UV-vis and fluorescence spectra of 3a and 4a before and after adding hydrazine. The compound 3a had weaker absorbance at 340 nm than 4a at the same concentration (Figure S-18), which was consistent with the observation that the solution of 3a was nearly colorless while solution of 4a was yellowish. The intensity of the shortwavelength fluorescence at 475 nm of 3a was also dramatically lower than that of 4b. Upon the irradiation by a UV lamp at 330 nm, the solution of 3a was nearly dark while the solution of 4a emitted strong green fluorescence (Figure 2C and Figure 2D inserts). After the addition of hydrazine, a new absorption peak at 430 nm appeared for both solutions of 3a and 4a. However, the absorption at 430 nm of 3a was stronger than that of 4a after the addition of hydrazine. The color of 3a was also more yellowish than that of 4a after the addition of hydrazine. For their fluorescence changes after the addition of different concentrations of hydrazine, the selfassembled probe 3a also exhibited much better performance. As shown in Figures 2C and 2D both probes showed fluorescence changes to the addition of hydrazine with a decrease in short-wavelength emission at 475 nm and an increase in long-wavelength emission at 550 nm. However, such change was more obvious for 3a and the resulting intensity of fluorescence peak at long-wavelength (550 nm) was stronger for 3a than 4a. Upon the irradiation by a UV lamp at 330 nm, the solution of 3a was yellowish while the solution of 4a was blue to light green (Figure 2B and Figure 2C inserts). We also calculated the ratio of emission intensities (F550/F475) for both probes (Figure 2E). With the concentration of hydrazine from 5 to 500 µM, the ratio of 3a was from 0.25 to 17.35, while the value of 4a was only from 0.17 to only 0.95, about 70-fold and 5.5-fold ratiometric enhancement, respectively. The fluorescence change of 3a was quite specific to hydrazine, and it showed little fluorescence changes in the presence of other amines or metal ions (Figure S-19, S-20). These observations clearly indicated that peptide selfassembly could also be applied for the development of optimized probes for the detection of hydrazine. In order to verify the role of peptide self-assembly in optimizing the fluorescence property, we connected 4a with a peptide GGGE to obtain compound 3c (Scheme S-3) without self-assembling property. Compound 3c was very soluble in PBS buffer, CMC and TEM results indicated that it had poor self-assembly capacity at 100 µM (Figure S-22). From monitoring the fluorescence change of 3c by adding different concentrations of hydrazine, we observed that the probe 3c neither exhibited lower background fluorescence nor enhanced fluorescence signal like 1a and 3a in aqueous solution (Figure S-23). These observations demonstrated that self-assembly played an important role in optimizing the fluorescence performance of the self-assembled probes.
Intracellular imaging of hydrazine. Since hydrazine can cause serious damage to the liver, lungs, kidneys, and the nervous system, several probes have been reported for imaging hydrazine in living cells.62, 78-81 we also compared the application of probes 3a and 4a in living cells for the detection of hydrazine. HeLa cells were firstly incubated with 3a and 4a (100 µM) for 4h at 37 0C (4a was firstly dissolved in DMSO
Figure 3. Confocal fluorescence images of HeLa cells incubated with 100 µM of probe A) 3a and B) 4a for 4h (blue channel, λabs=450±40nm) and subsequently with 200 µM of hydrazine C) and D) for another 2h (yellow channel, λabs=550±40nm). (Scale bars represent 25µm, 40×)
and then diluted by culture medium, final DMSO solution was 0.5%). The confocal images in Figure 3A and 3B showed that cells treated with 3a had nearly no intracellular fluorescence, while those treated with 4a had blue fluorescence. We then added hydrazine (200 µM) to the cells, which were then incubated for another 2h. We observed highly strong yellow fluorescence in cells treated with 3a (Figure 3C), while much weaker yellow fluorescence in those treated with 4a (Figure 3D). The much stronger yellow fluorescence in cells treated with 3a then those treated with 4a was due to the higher cellular uptake of 3a by cells (Figure S-21). However, the different emission profiles of compounds 3a and 4a might also contribute to the lower contrast of our self-assembled probes. We also tested whether our probes could respond to lower concentrations of hydrazine. The results indicated that our self-assembled probe exhibited much better performance at low concentrations such as 10 µM in cells, where we still could observe yellow fluorescence for probe 3a, while we could not observed it for probe 4a (Figure S-27). Moreover, the MTT assay results indicated that about 90% of cell viability was observed at 100 µM of compound 3a, and compound 3a showed better compatibility than compound 4a (Figure S-26). These observations clearly indicated that our optimized probes exhibited better performance in cell experiments.
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Conclusion In summary, we have introduced a versatile and very useful strategy to design optimized ratiometric fluorescent probes by peptide self-assembly. The self-assembled nanoprobes have excellent stabilities in pure aqueous solutions, while general probes need organic solvents to help their dispersions. Nanoprobes also exhibit very low blue background fluorescence and slightly higher yellow signal fluorescence than the conventional probes after the addition of analytes. The optimized probes show enhanced cellular uptakes and much better performance in cell experiments. However, one of shortcomings of our strategy was that several factors such as pH value and temperature might affect the self-assembly property of the probes, thus leading to the variation of the fluorescence intensity.42 Overall, our strategy may be developed into a general and very useful one for the design of novel fluorescence probes. Experimental Section Chemicals and Materials. Fmoc-amino acids were obtained from GL Biochem. (Shanghai, China). All the other starting materials were obtained from Alfa (Beijing, China). Commercially available reagents and solvents were used without further purification, unless noted otherwise. The synthesized compounds were characterized by 1H NMR (Bruker ARX-300) using DMSO-d6 as the solvent. HPLC was conducted at LUMTECH HPLC (Germany) system using a C18 RP column with MeOH (0.05% of TFA) and water (0.05% of TFA) as the eluents. LC-MS was conducted at the LCMS20AD (Shimadzu) system. HR-MS was performed at the Agilent 6520 Q-TOF LC/MS using ESI-L low concentration tuning mix (Lot No. LB60116 from the Agilent Tech.). Peptide synthesis. The peptide derivative was prepared by solid phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and the corresponding N-Fmoc protected amino acids with side chains properly protected by a tert-butyl group. 20% piperidine in anhydrous N,N’-dimethylformamide (DMF) was used during deprotection of Fmoc group. Then the next Fmoc-protected amino acid was coupled to the free amino group using O-(Benzotriazol-1-yl)-N,N,N’,N’tetramethyluroniumhexafluorophosphate (HBTU) as the coupling reagent. The growth of the peptide chain was according to the established Fmoc SPPS protocol. After the last coupling step, excessive reagents were removed by a single DMF wash for 5 minutes (5 mL per gram of resin), followed by five steps of washing using DCM for 2 min (5 mL per gram of resin). The peptide derivative was cleaved using 95% of trifluoroacetic acid with 2.5% of trimethylsilane (TMS) and 2.5% of H2O for 30 minutes. 20 mL per gram of resin of ice-cold diethylether was then added to cleavage reagent. The resulting precipitate was centrifuged for 10 min at 4 0C at 10,000 rpm. Afterward the supernatant was decanted and the resulting solid was dissolved in DMSO for HPLC separation using MeOH and H2O containing 0.1% of TFA as eluents. Characterization of the compounds. The 1H NMR and HR-MS spectra were in the ESI.
Synthesis of 3: To a suspension of 4-bromo-1,8-naphthalic anhydride 4 (2.77g, 10mmol) in DMF (50 mL) at room temperature was added a suspension of sodium azide (0.975g, 15mmol) in water (2ml) .The mixture was stirred vigorously
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for 5h at room temperature, and the solution was poured into ice water. The precipitated yellow solid was filtered to give 3 (2.17g, 90.79% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.63 (t, J = 8.0 Hz, 2H), 8.38 (d, J = 7.9 Hz, 1H), 8.28 (d, J = 7.9 Hz, 1H), 8.08 – 8.02 (m, 1H). LC-MS: calc. M+= 239, obsvd. (M+H)+ =240.
Synthesis of 2a: The reaction mixture of 4-N3-1,8naphthalic anhydride (2.39g, 10mmol), β-alanine (1.16g, 13mmol), DMAP (0.122g, 1mmol) and ethanol (100mL) was refluxed for 4h. The mixture was filtered, and the solid was washed with ethanol, and then dried in the oven to give 2a (2.65g, 85.2% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 7.1 Hz, 1H), 8.48 (d, J = 8.0 Hz, 1H), 8.44 (d, J = 8.4 Hz, 1H), 7.87 (t, J = 7.8 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 4.29 – 4.21 (m, 2H), 2.61 – 2.55 (m, 2H). LC-MS: calc. M+= 310, obsvd. (M+H)+ =311. Synthesis of 6: A mixture of 7 (1.215g, 5mmol) and BetaAlanine (1.090g, 6mmol) in ethanol (100 mL) in a single necked flask and was heated to reflex for 4 hours. After cooling to room temperature, the solvent was removed under reduced pressure and purification by flash column chromatography as a yellow solid (1.56g, 91.49%). 1H NMR (400 MHz, DMSO-d6) δ 8.72 (d, J = 8.7 Hz, 1H), 8.67 – 8.59 (m, 2H), 8.56 (d, J = 8.2 Hz, 1H), 8.10 (t, J = 7.7 Hz, 1H), 4.32 – 4.21 (m, 2H), 2.65 – 2.55 (m, 2H). LC-MS: calc. M+= 314, obsvd. (M+H)+ =315. Synthesis of 5: A mixture of 6 (1.364g, 4mmol) dissolved in ethanol (100 mL) with suspended the Pd/C (0.15g) catalyst in a single necked flask, after that the reactor was closed, purged before with nitrogen and then with hydrogen. The reactor was heated to reflux for 20h at atmospheric pressure. After cooling to room temperature, the reaction liquid was filtered to remove Pd/C, and then removed the solvent under reduced pressure, 1.275g brownish red solid was obtained (yield 95.14%). 1H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 8.3 Hz, 1H), 8.43 (d, J = 7.2 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.47 (s, 2H), 6.84 (d, J = 8.4 Hz, 1H), 4.22 (t, J = 7.3 Hz, 2H), 2.54 (d, J = 7.4 Hz, 2H). LC-MS: calc. M+= 284, obsvd. (M+H)+ =285.. Synthesis of 4a: A mixture of 5 (1.023g, 3mmol) and phthalic anhydride (666 mg, 4.5mmol) in acetic acid (50 mL) then heated to reflex and stirred for 4 hours. The solvent was removed under reduced pressure. Purification by flash column chromatography (silica gel, 1:4 hexane/EtOAc) gave 1 as a faint yellow solid (903 mg, 72.7 % yield) and it was used directly for solid phase peptide synthesis. 1H NMR (400 MHz, DMSO-d6) δ 8.64 (d, J = 7.8 Hz, 1H), 8.57 (d, J = 7.1 Hz, 1H), 8.42 (d, J = 8.4 Hz, 1H), 8.12 – 7.93 (m, 5H), 7.90 – 7.84 (m, 1H), 4.34 – 4.25 (m, 2H), 2.66 – 2.59 (m, 2H).HR-MS: calc. M+= 414.3671, obsvd. (M+H)+ =415.0931. N3-NTI-FFY, N3-NTI-FFYE, N3-NTI-FFYEE 3a and 3c: These compounds were obtained by SPPS using peptide synthesizer with an average yield of 50%, afterward they were purified by HPLC. N3-NTI-FFY: HR-MS: calc. M+= 767.2704, obsvd. (M+H)+ =768.2776. N3-NTI-FFYE: HRMS: calc. M+= 896.3130, obsvd. (M+H)+ =897.3199. N3-NTIFFYEE: HR-MS: calc. M+=896.3130, obsvd. (M+H)+ =897.3199. Compound 3a: HR-MS: calc. M+= 1000.3279, obsvd. (M+H)+ =1001.3336. Compound 3c: HR-MS: calc. M+=896.3130, obsvd. (M+H)+ =897.3199.
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Analytical Chemistry
Critical micelle concentration (CMC). The CMC values of peptides in water solution (pH = 7.4) was determined by dynamic light scattering (DLS). Solutions containing different concentration of peptides were tested and the light scattering intensity was recorded for each concentration analyzed. Dynamic Light Scattering (DLS) was performed on a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (BI-9000AT) at 532 nm under room temperature (22-25 0C). Transmission electron microscopy. TEM samples (100 µM 1a and 3a) were prepared at 25 °C. A micropipette was used to load 5 µL of sample solution to a carbon coated copper grid. The excess solution was removed by apiece of filter paper. The samples were dried overnight in a desiccator and then conducted on a Tecnai G2 F20 system, operating at 200 kV. Laser scanning confocal microscopy for imaging Hydrazine in HeLa cells: HeLa cells were incubated in Class Bottom Cell Culture Dish at a density of 20,000 cells per dish. After incubating for 24h, the DMEM solution containing 100 µM of 3a and 4a was then added to the HeLa cells. 4h later, the DMEM solution was removed and cells were washed for 3 times with PBS, we recorded the images by a laser scanning confocal microscopy at blue channel, λabs=450±40nm.Subsequently, the DMEM solution containing 200 µM of hydrazine added for incubating additional 2h, after we removed the DMEM and washed the cells for 3 times with PBS, the images were taken by a laser scanning confocal microscopy another time at yellow channel, λabs=550±40 nm. All images were taken by a laser scanning confocal microscopy (Leica TSC SP8) at same voltage. Determination of Peptide Concentration in HeLa Cells by LC-MS. HeLa cells were incubated in 24-well plates at a density of 2×106 cells per-well for 24 h. A stock solution containing 1 mg mL-1 of 3a and 4a was prepared (sodium carbonate was used to adjust the pH value to 7.4, 4a stock solution with 5% DMSO). 20 µL of stock solution and 980 µL DMEM with 10% FBS were added to cells and the cells were then incubated at 37 0C or 4 0C (final concentration of 3a and 4a was 0.2 mg mL-1, final DMSO solution was 0.5% in 4a). 4 hours later, DMEM containing 3a and 4a was removed and cells were washed with PBS for 3 times. 500 µL of DMSO was added to each well to dissolve compounds in cells. After being treated with sonication for 15 min, the solutions were collected and centrifuged at 1,570g for 10 min. The amount of 3a and 4a in the HeLa cells was determinedby LC-MS.
for Changjiang Scholars and Innovative Research Team in University (IRT13023). We thank Ms Nannan Xiao for her kind help with confocal fluorescence microscopy measurement.
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ASSOCIATED CONTENT Supporting Information. 1H NMR and HR-MS spectra, CMC of peptides, cell inhibition curves, confocal images of 3a and 4a with different concentration analytes, LC-MS spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
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Corresponding Author
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Z. Yang: [email protected] L. Wang: [email protected]
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ACKNOWLEDGMENT
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This work is supported by International S&T Cooperation Program of China (ISTCP, 2015DFA50310), NSFC (31370964 and 51373079), Tianjin MSTC (15JCZDJC38100) and Program
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Article
Optimized Ratiometric Fluorescent Probes by Peptide Self-assembly Yanbin Cai, Jie Zhan, Haosheng Shen, Duo Mao, Shenglu Ji, Ruihua Liu, Bing Yang, Deling Kong, Ling Wang, and Zhimou Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02955 • Publication Date (Web): 02 Dec 2015 Downloaded from http://pubs.acs.org on December 10, 2015
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Analytical Chemistry
Optimized Ratiometric Fluorescent Probes by Peptide Selfassembly Yanbin Cai,†,‡ Jie Zhan,† Haosheng Shen,‡ Duo Mao,‡ Shenglu Ji,‡ Ruihua Liu,† Bing Yang,‡ Deling Kong,‡ Ling Wang,*,† Zhimou Yang*,†,‡ State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, and Tianjin Key Laboratory of Molecular Drug Research, ‡Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, P. R. China †
ABSTRACT: We report in this study on optimized ratiometric fluorescent probes by peptide self-assembly. The resulting selfassembled nanoprobes show extraordinary stability in aqueous solutions and extremely low background fluorescence in buffer solutions. Our optimized probes with much bigger ratiometric fluorescence ratios also show an enhanced cellular uptake, lower background noise, and much brighter fluorescence signal in the cell experiment. Our study provides a versatile and very useful strategy to design and produce fluorescent probes with better performance.
Introduction Fluorescent probes have been extensively investigated in last decades due to their advantages such as noninvasiveness, high sensitivity and specificity, and high spatiotemporal resolution.1-6 They have been widely applied for the detection of environmentally and biologically important species.7-16 One of consensuses to design fluorescent probes is to lower their background fluorescence and amplify their turn on effects.3, 1719 In order to achieve this goal, several design principles and sensing mechanisms have been reported, including photoinduced electron transfer (PET),20-22 aggregation-induced emission (AIE),23-26 aggregation caused quenching (ACQ),27-29 fluorescence resonance energy transfer (FRET),6, 30 and intramolecular charge transfer (ICT),31-34 etc. Though great successes have been achieved for the development of fluorescent probes,13, 35-37 there are still increasing needs to develop novel design strategies for fluorescent probes with better performances (e.g. with enhanced imaging contrast). Self-assembling peptides have been widely studied and they can assemble into well-defined and well-ordered nanostructures.38-41 We recently demonstrated that the wellarranged molecular packing in the nanostructures of peptides could enhance the quenching effect of a quencher,41 and the hydrophobic pocket in the nanostructures could make the environment-sensitive fluorophore emit more brightly.43-44 In this study, we show that taken the advantage of peptide selfassembly, we can make optimized ratiometric fluorescent probes with much lower short wavelength fluorescence and higher long wavelength fluorescence signals. A novel kind of fluorescent probe called ratiometric one is recently reported and widely studied.45-53 Since ratiometric fluorescent probes possess two ultraviolet absorptions, they can emit two wavelengths of fluorescence upon excitation. They have been applied for the rapid, specific, and quantitative detection of important analytes through the calculation of fluorescence ratio of two emissions. For instance, they have been used to detect both small molecules (e.g. H2S, CN-, F-, etc.)54-63 and biomacromolecules (e. g.
enzymes).49-50, 64-69 We speculate that the short-wavelength emission of the ratiometric probes maybe lowered at assembled stages due to the ACQ effect, and the longwavelength emission maybe enhanced due to the possible environment sensitive property similar to the environmentsensitive fluorophore. If so, the incorporation of selfassembling peptides in ratiometric fluorescent probes would result in optimized probes with not only lower shortwavelength fluorescence but also enhanced long-wavelength fluorescence, which would be beneficial for the detection of analytes in cells with enhanced imaging contrast. Results and Discussion
Scheme 1. Chemical structures of probes and their transformation by H2S
Molecular design of a probe for hydrogen sulfide detection. We firstly choose the 4-N3-1,8-naphthalimide to test our hypothesis because its azide group could be reduced to amine group by hydrogen sulfide (H2S), resulting in ratiometric fluorescence changes.70 We modified the 4-N3naphthalic anhydride with a β-alanine to obtain a derivative of 4-N3-1,8-naphthalimide (N3-NTI, 2a in Scheme 1) with a carboxylic acid that could be used for solid phase peptide synthesis (SPPS). We then used it as an aromatic capping
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group71-76 for peptides to generate possible self-assembling peptides. The obtained compound N3-NTI-FFY (n=0) was insoluble in phosphate buffer saline (PBS, pH = 7.4) at the concentration of 1.0 mg/mL, and the N3-NTI-FFYE2 was very well soluble in PBS even at the concentration of 5.0 mg/mL (Figure S-15). Only N3-NTI-FFYE (1a) exhibited distinguish self-assembly property, which was showed by the light beam from its solution upon irradiation by a laser pointer (Figure S15). We then focused on the characterization and application of 1a. We studied the self-assembly property of 1a firstly by dynamic light scattering (DLS). The result in Figure S-11 showed that its critical micelle concentration (CMC) was about 80.5 µM. The transmission electron microscopy (TEM) image exhibited that 1a self-assembled into nanofibers at 100 µM in PBS buffer (Figure 1A). The nanofiber dispersion of 1a was highly stable for more than 1 month, while compound 2a was insoluble in pure PBS at 100 µM (Figure S-15). In order to obtain a clear solution of 2a, it needed to be firstly dissolved in DMSO and then diluted in PBS (5% of DMSO). Even in the presence of DMSO, compound 2a would still precipitate out from the solution after 24h. The ultra-stable nanofiber dispersion of our self-assembled probe and no need of organic solvents suggested its advantages for the detection applications in biological environments. Fluorescence properties and ratio changes. We then monitored the fluorescence change of 1a and 2a by adding different concentrations of H2S. As shown in Figure 1C, we observed typical ratiometric fluorescence spectra of 2a. Upon excitation at 340 nm, there were two emission peaks appeared at 425 and 550 nm. As the concentration of H2S increased, the peak at 425 nm decreased and the peak at 550 nm gradually increased in intensity. Upon irradiation by a UV lamp at 330 nm, we observed the color of fluorescence changing from blue to yellowish green (Figure 1C inserts). The fluorescent spectra of 1a with increasing concentrations of H2S were shown in Figure 1B. We found that the intensity of peak at 425 nm of 1a was significantly lower than that of 2a at the same concentration, indicating that the self-assembly property could quench the
Figure 1. A) TEM image of solution of 1a (100 µM), B) and C) Fluorescence spectra of solution of 1a and 2a treated with different concentrations of H2S (5 to 200 µM), respectively, insets in B and C) images of solutions of 1a and 2a before and after adding H2S and irradiated by a hand-held UV lamp (330 nm channel), D) Fluorescence intensity ratio (F550/F425) of 1a and 2a with increasing concentrations of H2S (5, 10, 20, 30, 40, 50, 60,
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80, 100, and 200 µM, respectively. Excitation wavelength = 340 nm, acquired 2h after the addition of analyte at 37 0C). (1a in PBS buffer, 100 µM, PH=7.4, 2a in PBS buffer with 5% DMSO at 100µM)
short-wavelength fluorescence probably due to ACQ effect. However, the peak at 550 nm of 1a was higher than that of 2a after adding the same concentration of H2S. For example, the intensity was about 2,000 for 2a, while it was about 3,000 for 1a in the presence of 200 µM of H2S. The results indicated that peptide self-assembly could indeed increase the longwavelength fluorescence of the probe. Upon the irradiation of a UV lamp at 330 nm, the solution of 1a exhibited extremely low blue background fluorescence (Figure 1B insets). Meanwhile, the solution emitted bright yellowish green fluorescence in the presence of H2S. We also calculated the ratio of fluorescence intensities of 1a and 2a before and after adding H2S (F550/F425). The ratio of 1a was from 0.76 to 24.54, while the ratio of 2a was only from 0.36 to 8.29 with the concentration of H2S from 5 to 200 µM (Figure 1D). The much bigger enhanced values of the ratio of fluorescence suggested the better sensitivity and performance of our selfassembled probes.
Figure 2. A) Chemical structures of 3a, 4a, 3b, 4b, B) TEM image of solution of 3a, C) and D) Fluorescence spectra of solutions of 3a and 4a treated with different concentrations of hydrazine (10 to 500 µM), insets in C and D: top) Fluorescence color changes of 3a and 4a treated with hydrazine observed using a hand-held UV lamp with an excitation at 330 nm, down) visual color changes of 3a and 4a treated with hydrazine, E) Fluorescence intensity ratio (F550/F475) of 3a and 4a with increasing concentrations of hydrazine (0, 5, 10, 20, 30, 40, 50, 100, 200, 300, and 500 µM, respectively. Excitation wavelength = 400 nm, spectrum was acquired 2h after addition of analyte at 37 0 C. 3a was dissolved in PBS buffer at 100 µM and 4a in PBS buffer with 5% DMSO at 100 µM.)
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Analytical Chemistry
Molecular design of a probe for Hydrazine detection. The amine group in 1b or 2b could be protected to phthalimide, which would react with hydrazine and therefore be converted back to the amine group.77 Probes with this conversion had already been used for the detection of hydrazine.61 In order to validate the generality of our strategy for the development of optimized ratiometric probes, we synthesized another two probes with phthalimide group for the detection of hydrazine (3a and 4a in Figure 2A). Similar to 1a, the CMC of 3a was 85.2 µM (Figure S-17), and it also self-assembled into nanofibers at 100 µM in PBS buffer (Figure 2B). We then compared the UV-vis and fluorescence spectra of 3a and 4a before and after adding hydrazine. The compound 3a had weaker absorbance at 340 nm than 4a at the same concentration (Figure S-18), which was consistent with the observation that the solution of 3a was nearly colorless while solution of 4a was yellowish. The intensity of the shortwavelength fluorescence at 475 nm of 3a was also dramatically lower than that of 4b. Upon the irradiation by a UV lamp at 330 nm, the solution of 3a was nearly dark while the solution of 4a emitted strong green fluorescence (Figure 2C and Figure 2D inserts). After the addition of hydrazine, a new absorption peak at 430 nm appeared for both solutions of 3a and 4a. However, the absorption at 430 nm of 3a was stronger than that of 4a after the addition of hydrazine. The color of 3a was also more yellowish than that of 4a after the addition of hydrazine. For their fluorescence changes after the addition of different concentrations of hydrazine, the selfassembled probe 3a also exhibited much better performance. As shown in Figures 2C and 2D both probes showed fluorescence changes to the addition of hydrazine with a decrease in short-wavelength emission at 475 nm and an increase in long-wavelength emission at 550 nm. However, such change was more obvious for 3a and the resulting intensity of fluorescence peak at long-wavelength (550 nm) was stronger for 3a than 4a. Upon the irradiation by a UV lamp at 330 nm, the solution of 3a was yellowish while the solution of 4a was blue to light green (Figure 2B and Figure 2C inserts). We also calculated the ratio of emission intensities (F550/F475) for both probes (Figure 2E). With the concentration of hydrazine from 5 to 500 µM, the ratio of 3a was from 0.25 to 17.35, while the value of 4a was only from 0.17 to only 0.95, about 70-fold and 5.5-fold ratiometric enhancement, respectively. The fluorescence change of 3a was quite specific to hydrazine, and it showed little fluorescence changes in the presence of other amines or metal ions (Figure S-19, S-20). These observations clearly indicated that peptide selfassembly could also be applied for the development of optimized probes for the detection of hydrazine. In order to verify the role of peptide self-assembly in optimizing the fluorescence property, we connected 4a with a peptide GGGE to obtain compound 3c (Scheme S-3) without self-assembling property. Compound 3c was very soluble in PBS buffer, CMC and TEM results indicated that it had poor self-assembly capacity at 100 µM (Figure S-22). From monitoring the fluorescence change of 3c by adding different concentrations of hydrazine, we observed that the probe 3c neither exhibited lower background fluorescence nor enhanced fluorescence signal like 1a and 3a in aqueous solution (Figure S-23). These observations demonstrated that self-assembly played an important role in optimizing the fluorescence performance of the self-assembled probes.
Intracellular imaging of hydrazine. Since hydrazine can cause serious damage to the liver, lungs, kidneys, and the nervous system, several probes have been reported for imaging hydrazine in living cells.62, 78-81 we also compared the application of probes 3a and 4a in living cells for the detection of hydrazine. HeLa cells were firstly incubated with 3a and 4a (100 µM) for 4h at 37 0C (4a was firstly dissolved in DMSO
Figure 3. Confocal fluorescence images of HeLa cells incubated with 100 µM of probe A) 3a and B) 4a for 4h (blue channel, λabs=450±40nm) and subsequently with 200 µM of hydrazine C) and D) for another 2h (yellow channel, λabs=550±40nm). (Scale bars represent 25µm, 40×)
and then diluted by culture medium, final DMSO solution was 0.5%). The confocal images in Figure 3A and 3B showed that cells treated with 3a had nearly no intracellular fluorescence, while those treated with 4a had blue fluorescence. We then added hydrazine (200 µM) to the cells, which were then incubated for another 2h. We observed highly strong yellow fluorescence in cells treated with 3a (Figure 3C), while much weaker yellow fluorescence in those treated with 4a (Figure 3D). The much stronger yellow fluorescence in cells treated with 3a then those treated with 4a was due to the higher cellular uptake of 3a by cells (Figure S-21). However, the different emission profiles of compounds 3a and 4a might also contribute to the lower contrast of our self-assembled probes. We also tested whether our probes could respond to lower concentrations of hydrazine. The results indicated that our self-assembled probe exhibited much better performance at low concentrations such as 10 µM in cells, where we still could observe yellow fluorescence for probe 3a, while we could not observed it for probe 4a (Figure S-27). Moreover, the MTT assay results indicated that about 90% of cell viability was observed at 100 µM of compound 3a, and compound 3a showed better compatibility than compound 4a (Figure S-26). These observations clearly indicated that our optimized probes exhibited better performance in cell experiments.
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Conclusion In summary, we have introduced a versatile and very useful strategy to design optimized ratiometric fluorescent probes by peptide self-assembly. The self-assembled nanoprobes have excellent stabilities in pure aqueous solutions, while general probes need organic solvents to help their dispersions. Nanoprobes also exhibit very low blue background fluorescence and slightly higher yellow signal fluorescence than the conventional probes after the addition of analytes. The optimized probes show enhanced cellular uptakes and much better performance in cell experiments. However, one of shortcomings of our strategy was that several factors such as pH value and temperature might affect the self-assembly property of the probes, thus leading to the variation of the fluorescence intensity.42 Overall, our strategy may be developed into a general and very useful one for the design of novel fluorescence probes. Experimental Section Chemicals and Materials. Fmoc-amino acids were obtained from GL Biochem. (Shanghai, China). All the other starting materials were obtained from Alfa (Beijing, China). Commercially available reagents and solvents were used without further purification, unless noted otherwise. The synthesized compounds were characterized by 1H NMR (Bruker ARX-300) using DMSO-d6 as the solvent. HPLC was conducted at LUMTECH HPLC (Germany) system using a C18 RP column with MeOH (0.05% of TFA) and water (0.05% of TFA) as the eluents. LC-MS was conducted at the LCMS20AD (Shimadzu) system. HR-MS was performed at the Agilent 6520 Q-TOF LC/MS using ESI-L low concentration tuning mix (Lot No. LB60116 from the Agilent Tech.). Peptide synthesis. The peptide derivative was prepared by solid phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and the corresponding N-Fmoc protected amino acids with side chains properly protected by a tert-butyl group. 20% piperidine in anhydrous N,N’-dimethylformamide (DMF) was used during deprotection of Fmoc group. Then the next Fmoc-protected amino acid was coupled to the free amino group using O-(Benzotriazol-1-yl)-N,N,N’,N’tetramethyluroniumhexafluorophosphate (HBTU) as the coupling reagent. The growth of the peptide chain was according to the established Fmoc SPPS protocol. After the last coupling step, excessive reagents were removed by a single DMF wash for 5 minutes (5 mL per gram of resin), followed by five steps of washing using DCM for 2 min (5 mL per gram of resin). The peptide derivative was cleaved using 95% of trifluoroacetic acid with 2.5% of trimethylsilane (TMS) and 2.5% of H2O for 30 minutes. 20 mL per gram of resin of ice-cold diethylether was then added to cleavage reagent. The resulting precipitate was centrifuged for 10 min at 4 0C at 10,000 rpm. Afterward the supernatant was decanted and the resulting solid was dissolved in DMSO for HPLC separation using MeOH and H2O containing 0.1% of TFA as eluents. Characterization of the compounds. The 1H NMR and HR-MS spectra were in the ESI.
Synthesis of 3: To a suspension of 4-bromo-1,8-naphthalic anhydride 4 (2.77g, 10mmol) in DMF (50 mL) at room temperature was added a suspension of sodium azide (0.975g, 15mmol) in water (2ml) .The mixture was stirred vigorously
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for 5h at room temperature, and the solution was poured into ice water. The precipitated yellow solid was filtered to give 3 (2.17g, 90.79% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.63 (t, J = 8.0 Hz, 2H), 8.38 (d, J = 7.9 Hz, 1H), 8.28 (d, J = 7.9 Hz, 1H), 8.08 – 8.02 (m, 1H). LC-MS: calc. M+= 239, obsvd. (M+H)+ =240.
Synthesis of 2a: The reaction mixture of 4-N3-1,8naphthalic anhydride (2.39g, 10mmol), β-alanine (1.16g, 13mmol), DMAP (0.122g, 1mmol) and ethanol (100mL) was refluxed for 4h. The mixture was filtered, and the solid was washed with ethanol, and then dried in the oven to give 2a (2.65g, 85.2% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 7.1 Hz, 1H), 8.48 (d, J = 8.0 Hz, 1H), 8.44 (d, J = 8.4 Hz, 1H), 7.87 (t, J = 7.8 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 4.29 – 4.21 (m, 2H), 2.61 – 2.55 (m, 2H). LC-MS: calc. M+= 310, obsvd. (M+H)+ =311. Synthesis of 6: A mixture of 7 (1.215g, 5mmol) and BetaAlanine (1.090g, 6mmol) in ethanol (100 mL) in a single necked flask and was heated to reflex for 4 hours. After cooling to room temperature, the solvent was removed under reduced pressure and purification by flash column chromatography as a yellow solid (1.56g, 91.49%). 1H NMR (400 MHz, DMSO-d6) δ 8.72 (d, J = 8.7 Hz, 1H), 8.67 – 8.59 (m, 2H), 8.56 (d, J = 8.2 Hz, 1H), 8.10 (t, J = 7.7 Hz, 1H), 4.32 – 4.21 (m, 2H), 2.65 – 2.55 (m, 2H). LC-MS: calc. M+= 314, obsvd. (M+H)+ =315. Synthesis of 5: A mixture of 6 (1.364g, 4mmol) dissolved in ethanol (100 mL) with suspended the Pd/C (0.15g) catalyst in a single necked flask, after that the reactor was closed, purged before with nitrogen and then with hydrogen. The reactor was heated to reflux for 20h at atmospheric pressure. After cooling to room temperature, the reaction liquid was filtered to remove Pd/C, and then removed the solvent under reduced pressure, 1.275g brownish red solid was obtained (yield 95.14%). 1H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 8.3 Hz, 1H), 8.43 (d, J = 7.2 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 7.47 (s, 2H), 6.84 (d, J = 8.4 Hz, 1H), 4.22 (t, J = 7.3 Hz, 2H), 2.54 (d, J = 7.4 Hz, 2H). LC-MS: calc. M+= 284, obsvd. (M+H)+ =285.. Synthesis of 4a: A mixture of 5 (1.023g, 3mmol) and phthalic anhydride (666 mg, 4.5mmol) in acetic acid (50 mL) then heated to reflex and stirred for 4 hours. The solvent was removed under reduced pressure. Purification by flash column chromatography (silica gel, 1:4 hexane/EtOAc) gave 1 as a faint yellow solid (903 mg, 72.7 % yield) and it was used directly for solid phase peptide synthesis. 1H NMR (400 MHz, DMSO-d6) δ 8.64 (d, J = 7.8 Hz, 1H), 8.57 (d, J = 7.1 Hz, 1H), 8.42 (d, J = 8.4 Hz, 1H), 8.12 – 7.93 (m, 5H), 7.90 – 7.84 (m, 1H), 4.34 – 4.25 (m, 2H), 2.66 – 2.59 (m, 2H).HR-MS: calc. M+= 414.3671, obsvd. (M+H)+ =415.0931. N3-NTI-FFY, N3-NTI-FFYE, N3-NTI-FFYEE 3a and 3c: These compounds were obtained by SPPS using peptide synthesizer with an average yield of 50%, afterward they were purified by HPLC. N3-NTI-FFY: HR-MS: calc. M+= 767.2704, obsvd. (M+H)+ =768.2776. N3-NTI-FFYE: HRMS: calc. M+= 896.3130, obsvd. (M+H)+ =897.3199. N3-NTIFFYEE: HR-MS: calc. M+=896.3130, obsvd. (M+H)+ =897.3199. Compound 3a: HR-MS: calc. M+= 1000.3279, obsvd. (M+H)+ =1001.3336. Compound 3c: HR-MS: calc. M+=896.3130, obsvd. (M+H)+ =897.3199.
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Analytical Chemistry
Critical micelle concentration (CMC). The CMC values of peptides in water solution (pH = 7.4) was determined by dynamic light scattering (DLS). Solutions containing different concentration of peptides were tested and the light scattering intensity was recorded for each concentration analyzed. Dynamic Light Scattering (DLS) was performed on a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (BI-9000AT) at 532 nm under room temperature (22-25 0C). Transmission electron microscopy. TEM samples (100 µM 1a and 3a) were prepared at 25 °C. A micropipette was used to load 5 µL of sample solution to a carbon coated copper grid. The excess solution was removed by apiece of filter paper. The samples were dried overnight in a desiccator and then conducted on a Tecnai G2 F20 system, operating at 200 kV. Laser scanning confocal microscopy for imaging Hydrazine in HeLa cells: HeLa cells were incubated in Class Bottom Cell Culture Dish at a density of 20,000 cells per dish. After incubating for 24h, the DMEM solution containing 100 µM of 3a and 4a was then added to the HeLa cells. 4h later, the DMEM solution was removed and cells were washed for 3 times with PBS, we recorded the images by a laser scanning confocal microscopy at blue channel, λabs=450±40nm.Subsequently, the DMEM solution containing 200 µM of hydrazine added for incubating additional 2h, after we removed the DMEM and washed the cells for 3 times with PBS, the images were taken by a laser scanning confocal microscopy another time at yellow channel, λabs=550±40 nm. All images were taken by a laser scanning confocal microscopy (Leica TSC SP8) at same voltage. Determination of Peptide Concentration in HeLa Cells by LC-MS. HeLa cells were incubated in 24-well plates at a density of 2×106 cells per-well for 24 h. A stock solution containing 1 mg mL-1 of 3a and 4a was prepared (sodium carbonate was used to adjust the pH value to 7.4, 4a stock solution with 5% DMSO). 20 µL of stock solution and 980 µL DMEM with 10% FBS were added to cells and the cells were then incubated at 37 0C or 4 0C (final concentration of 3a and 4a was 0.2 mg mL-1, final DMSO solution was 0.5% in 4a). 4 hours later, DMEM containing 3a and 4a was removed and cells were washed with PBS for 3 times. 500 µL of DMSO was added to each well to dissolve compounds in cells. After being treated with sonication for 15 min, the solutions were collected and centrifuged at 1,570g for 10 min. The amount of 3a and 4a in the HeLa cells was determinedby LC-MS.
for Changjiang Scholars and Innovative Research Team in University (IRT13023). We thank Ms Nannan Xiao for her kind help with confocal fluorescence microscopy measurement.
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ASSOCIATED CONTENT Supporting Information. 1H NMR and HR-MS spectra, CMC of peptides, cell inhibition curves, confocal images of 3a and 4a with different concentration analytes, LC-MS spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
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Corresponding Author
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Z. Yang: [email protected] L. Wang: [email protected]
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ACKNOWLEDGMENT
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This work is supported by International S&T Cooperation Program of China (ISTCP, 2015DFA50310), NSFC (31370964 and 51373079), Tianjin MSTC (15JCZDJC38100) and Program
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