General materials and instrumentation
All chemicals were purchased from commercial sources. Hydrogen-nuclear magnetic resonance (1H NMR) spectra were acquired on a Bruker 400 MHz magnetic resonance spectrometer. Data for 1H NMR spectra are reported as follows: chemical shifts are reported as δ in units of parts per million (ppm) relative to chloroform-d (δ 7.26, s), multiplicities are reported as: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m (multiplet), or br (broadened); coupling constants are reported as a J value in Hertz (Hz); the number of protons (n) for a given resonance is indicated nH, and based on the spectral integration values.
Mass spectra were recorded on a Micromass Quattro II triple-quadrupole mass spectrometer or Synapt G2-Si mass spectrometer using electrospray ionization with a MassLynx operating system (Water, USA). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) spectrometric analyses were performed on an Applied Biosystems 4700 MALDI TOF mass spectrometer. Ultraviolet–visible (UV–Vis) absorption spectra were measured on a PekinElmer Lambda 25 UV–Vis spectrophotometer. Fluorescence spectra were measured on a Fluorog-3 spectrofluorometer (Horiba Jobin Yvon, France) and the fluorescence emitting within the NIR-II region were measured by a Cary Eclipse fluorophotometer. The laser of 808 nm wavelength was purchased from Beijing Hi-Tech Optoelectronic (China). DJO-2776 sonicator was used to generate ultrasound during the treatment. Hydrodynamic diameter was measured using a Malvern Zetasizer Nano ZS. The analysis and purification of probes was performed on High-Performance Liquid Chromatograph with UV–Vis Detector within a reversed-phase C18 column (Kromasil, 4.6 mm *150 mm). TEM images were recorded on JEM-1400 plus. SYH1-3 probe solution were dropped onto copper grids, and the measurements were performed with transmission electron microscopy (TEM) operated at 200 kV after they were dried naturely. Small animal in vivo imaging system was purchased from Perkin Elmer. The NIR-II in vivo system and Full-Spectrum Fluorescence Microscope were purchased from Suzhou NIR-Optics Technologies Co., Ltd.
Synthesis and characterization
All intermediates and final products were identified through NMR spectroscopy and MALDI-TOF-MS analysis (Figures S3–S15, Supporting Information).
Synthetic of compound 1–2
Compound 1–1 (2.66 g, 18.72 mmol) was added into a 100 mL round-bottomed flask, pumped three times for anhydrous and anaerobic nitrogen protection, and then placed in a low-temperature reactor at – 78 ℃, into which tetrahydrofuran (THF) solution of n-butyl lithium (15.21 mL, 24.34 mmol) was added, and slowly rose to room temperature for 2 h. Then at – 78 ℃, tributyltin chloride (7.92 g, 24.34 mmol) was slowly added to the reactor and allowed to react overnight at room temperature. After the reaction was complete, the reaction mixture was extracted with ethyl acetate and dried with anhydrous sodium sulfate. The yellow oil-like liquid product 1–2 was 6.16 g, and the yield was 75.9%. The crude product can be used directly for the next reaction without purification.
Synthetic of compound 1–4
Compound 1–2 (6.16 g, 14.21 mmol), compound 1–3 (2.72 g, 7.11 mmol) and palladium dichloride (0.997 g, 1.42 mmol) were added into a 50 mL round-bottomed flask, and pumped three times with anhydrous and anaerobic nitrogen for protection (50 mL). Then the THF solution is added, heat the mixture to reflux and stir it overnight. After cooling, add saturated potassium fluoride aqueous solution (40 mL) and stir at room temperature for 1 h. The reaction mixture is filtered through diatomaceous earth pads and the cake was washed with methylene chloride. The organic layer was extracted with saturated sodium chloride solution, dried with anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the crude product. Then, compounds 1–4 were recrystallized from ethyl acetate to obtain 2.25 g red powder with a yield of 90.9%.
Synthetic of compound 1–5 (2–6)
Compounds 1–4 (500 mg, 0.988 mmol) were dissolved in a mixture of dimethyl formamide (DMF) (15 mL) and acetonitrile (7.5 mL). The solution was heated to 65℃ and N-Bromo succinimide (NBS) (200 mg, 1.12 mmol) was added in batches. The reaction was stirred in the dark. Hydrobromic acid (four drops, 48% water solution) is then added to the reaction. After 3 h, thin layer chromatography (TLC) analysis revealed three spots. Another portion of NBS was added (200 mg, 1.12 mmol) and the reaction was continued for another 2 h. When a total of 700 mg of NBS (3.93 mmol) was added, TLC analysis showed that the feedstock had been completely transformed. After cooling, the reaction mixture was acidified with hydrochloric acid (150 mL, 2 M) and stirred at room temperature for 2 h. The precipitate was collected by filtration and washed with water and methanol, and the red powder compounds 1–5 were 0.394 g, with a yield of 78.8%.
Synthetic of compound 2–3
Under the protection of argon, 2-bromocarbazole (2.40 g, 9.75 mmol) and potassium iodide (161.9 mg, 0.975 mmol) were dissolved in dimethyl sulfoxide (20 mL). Methyl 3-bromopropionate (2.17 mL, 3.257 g, 19.5 mmol) was added to the reaction mixture. Then potassium hydroxide (2.736 g, 48.76 mmol) was added to the solution in 10 parts. The reaction solution was heated to 9 ℃ for 24 h and quenched with water. The mixture was acidified to pH = 5 with 2 M hydrochloric acid aqueous solution. After extraction with ethyl acetate, anhydrous magnesium sulfate was dried and concentrated to obtain white solid compounds 2–3 (2.29 g), with a yield of 74.1%. The crude product was directly used in the next step without purification. 1H NMR (400 MHz, DMSO-d6) δ 8.18 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 8.3 Hz, 1H), 7.93 (d, J = 1.2 Hz, 1H), 7.65 (d, J = 8.2 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.35 (dd, J = 8.3, 1.4 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 4.62 (t, J = 6.9 Hz, 2H), 2.71 (t, J = 6.9 Hz, 2H).13C NMR (101 MHz, DMSO) δ 173.37, 141.19, 140.41, 126.71, 122.64–121.96 (m), 121.78, 120.88, 119.86, 119.05, 112.89, 110.18, 34.64.
Synthetic of compound 2–4
Compound 2–3 (2.29 g, 7.22 mmol) was dissolved in dichloromethane (24 mL) under the protection of argon gas. 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride (EDCI) (2.22 g, 11.6 mmol) and 4-dimethylaminopyridine (DMAP) (0.088 g, 0.722 mmol) were added in a single dose. After reaction for 30 min, 2-trimethyl-silicyl ethanol (1.657 mL, 1.367 g, 11.6 mmol) was added. The reaction mixture was stirred at room temperature for 12 h. Dilute with methylene chloride (300 mL), the organic layer was extracted with saturated ammonium chloride solution (100 mL), water (4 × 100 mL) and saturated brine (100 mL) and dried with anhydrous magnesium sulfate. The organic layer was concentrated and purified by silica gel column (oleyl ether: ethyl acetate = 32:1, v/v) to obtain yellow oily liquid compounds 2–4 (2.26 g, 75.1% yield). 1H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 7.7 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.60 (s, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 7.28 (t, J = 7.4 Hz, 1H), 4.54 (t, J = 7.0 Hz, 2H), 4.19–4.12 (m, 2H), 2.81 (t, J = 7.0 Hz, 2H), 0.94–0.87 (m, 2H), 0.06 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 171.29, 140.84, 140.13, 126.28, 122.58, 122.38, 122.06, 121.51, 120.42, 119.80, 119.41, 111.88, 108.94, 63.33, 38.89, 33.74, 17.24, 1.45.
Synthetic of compound 2–5
The compound 2–4 (2.26 g, 5.42 mmol), diphenalborate (2.477 g, 9.75 mmol), potassium acetate (1.276 g, 13.0 mmol) and bis (triphenyl phosphorus) palladium (II) dichloroethylene complex (380.0 mg, 0.542 mmol) dissolved in N, n-dimethylformamide (30 mL) and pumped anhydrous and oxygen-free argon gas for protection. Then the reaction liquid was reacted at 80℃ for 12 h. After the reaction is complete, ethyl acetate (60 mL) is added and solids are removed by filtration. The solution was diluted with water (120 mL) and the organic phase was extracted with methylene chloride (3 × 60 mL). After extraction with water (4 × 60 mL) and saturated brine (150 mL), dry with anhydrous magnesium sulfate. The organic phase was concentrated and purified by silica gel column (petroleum ether: ethyl acetate = 16:1, v/v) to obtain colorless oil liquid compounds 2–5 (2.01 g, 79.8% yield). 1H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 7.7 Hz, 2H), 7.93 (s, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.52 (s, 2H), 7.32–7.20 (m, 1H), 4.72 (t, J = 7.3 Hz, 2H), 4.24–4.11 (m, 2H), 2.89 (t, J = 7.3 Hz, 2H), 1.30 (s, 12H), 0.95–0.86 (m, 3H), 0.05 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 171.57, 140.56, 139.50, 126.44, 125.69, 125.49, 122.85, 120.85, 119.73, 119.19, 114.87, 108.89, 63.18, 38.71, 33.80, 24.99, 17.19, 1.48.
Synthetic of compound 2–7
Argon gas was vented into 12 mL tetrahydrofuran solution of compounds 2–5 (204 mg, 0.438 mmol) and 1–5 (130.6 mg, 0.196 mmol) for 5 min. Under the protection of argon gas, potassium carbonate (76.1 mg, 0.548 mmol) aqueous (3.0 mL) solution and 1, 1, -bis (diphenylphosphoyl) ferrocene Palladium (II) dichloromethane complex (8.96 mg, 0.011 mmol) were added to the reaction mixture above. The mixture was heated in an oil bath at 75 ℃ for 8 h. After cooling to room temperature, the solvent was removed by vacuum. The residue was extracted with water and ethyl acetate and dried with anhydrous magnesium sulfate. The crude products were purified by silica gel column (petroleum ether: ethyl acetate = 10:1, v/v) to obtain blue solid compounds 2–7 (126 mg, 54.3% yield). 1H NMR (400 MHz, CDCl3) δ 8.12 (s, 4H), 7.91 (s, 2H), 7.71 (s, 2H), 7.51 (s, 6H), 4.73 (s, 4H), 4.46 (s, 4H), 4.36 (s, 4H), 4.17 (s, 4H), 2.91 (s, 4H), 1.29 (s, 15H), 0.02 (s, 25H). 13C NMR (101 MHz, CDCl3) δ 171.58, 152.68, 143.21, 140.25, 129.67, 128.77, 126.37, 122.93, 120.59, 119.55, 118.38, 108.84, 106.79, 102.82, 77.36, 77.15, 76.73, 64.60, 63.28, 38.86, 33.72, 17.19, 1.04.
Synthetic of compound SYH-TMS
Zinc powder (332.08 mg, 5.10 mmol) and ammonium chloride (81.49 mg, 1.50 mmol) were added to the mixed solution of dichloromethane (10 mL), and 90% methanol (9 mL) of compound 2–7 (50 mg, 0.042 mmol) at room temperature. After stirring for 4 h at room temperature under argon, the solution was filtered through a diatomite pad, diluted with dichloromethane, extracted with saturated aqueous sodium bicarbonate solution and dried with anhydrous magnesium sulfate to obtain a yellow solid, which was directly used in the next reaction without purification.
Under argon, N-sulfinyl aniline (0.1 mL, 0.085 mmol, 11.78 mg) and trimethylchlorosilane (0.2 mL, 0.076 mmol, 8.276 mg) were added to the dark yellow solution of anhydrous pyridine (2 mL). The mixture was heated in an oil bath at 80℃ for 20 h. The reaction mixture was cooled to room temperature, poured into ice water and extracted with dichloromethane. The combined organic layers were washed with water and saturated brine, dried with anhydrous magnesium sulfate and concentrated in vacuum. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1), and the product SYH-TMS was obtained as a green solid (23 mg, two-step yield 47.3%). 1H NMR (400 MHz, CDCl3) δ 8.15–8.07 (m, 4H), 7.98–7.75 (m, 4H), 7.51 (d, J = 3.5 Hz, 4H), 7.27 (d, J = 5.1 Hz, 1H), 4.74 (t, J = 7.2 Hz, 4H), 4.58 (s, 3H), 4.44 (s, 4H), 4.22–4.15 (m, 4H), 2.92 (t, J = 7.2 Hz, 4H), 0.89 (s, 4H), 0.03 (s, 19H). MS(MALDI-TOF) m/z: calcd for: C58H56N6O8S3SeSi2+: Calcd 1196.44, found: 1197.155.
Synthetic of compound CCNU1020
The SYH-TMS was added into the mixture of 1:1 ratio of DCM/Trifluoroacetic acid and then shacked for 3–5 h. After the solution was dried over N2 and washed with CH3CN for two times. The resulting crude compounds CCNU1020 could be directly used.
The preparation of SYH1-3
CCNU1020 (1 mg), o-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (HBTU) (6 mg), and PEGX-NH2 (x = 3, 10, 16) (2.1 mg, 4 mmol) were dissolved in 1 mL of DMF. Then 5 μL of N, N-diisopropylethylamine (DIPEA) was added into the solution, and reacted overnight at room temperature. After completion of the reaction, the crude product was purified with high performance liquid chromatography (HPLC). Subsequently, HPLC-purified compounds were lyophilized to obtain the solid product SYH1-3. Dissolve 2 mmol in deionized water (2 mL) with continuous bath sonication for 10 min to obtain the nanoform stock solution of SYH1-SYH3 (1 mol/L).
Absorption and photoluminescence excitation (PLE) spectral studies
The UV–Vis-NIR absorbance spectrum of SYH1-3 was recorded on a PerkinElmer Lambda 25 UV–Vis spectrophotometer. PLE spectra of SYH1-3 solutions were obtained using a Cary Eclipse fluorophotometer.
In vitro NIR-II imaging
Dilute the SYH1-3 stock solution to 100 µM with ultra-pure water at Eppendorf tube, then set the imaging conditions on the NIR-II in vivo system as follows: exposure time of 100 ms, LP1000, and proceed with imaging.
Fluorescence imaging for leafs
Diluted the SYH1-3 stock solution to 100 µM at Eppendorf tube with ultra-pure water, then immersed the petiole part of the leaf into the probe solution, and performed NIR-II fluorescence imaging on the leaf at different time points on the NIR-II in vivo system (100 ms, LP1000).
NIR-II fluorescence imaging for Arabidopsis plant
The NIR-II fluorescent images of the root, rhizome, and leaf on living Arabidopsis thaliana plants were captured by an Full-Spectrum Fluorescence Microscope (Suzhou NIR-Optics, China). 100 µM aqueous solutions of ICG and SYH3 were prepared respectively, then the whole Arabidopsis plants were immersed in the probe solution for 3 h. Afterward, the plants were rinsed with purified water, and then imaging of root, rhizome, and leaf were performed using an Full-Spectrum Fluorescence Microscope (Suzhou NIR-Optics, China).
In vivo imaging of Arabidopsis thaliana infected with TMV
Inoculate Arabidopsis plants with the GFP-labeled TMV virus, then cultivate them in soil in an incubator for three days. Set the incubator parameters: humidity at 40%, temperature at 22 °C, and maintain light exposure every 6 h for 1 h each time. Uproot the successfully inoculated plants, clean the roots, and immerse them in SYH3 probe solution (100 µM) for 12 h. Rinse the roots with water to remove any residual probe solution, and then image them using both the NIR-II in vivo system with exposure time 100 ms and filter LP1000 and small animal in vivo imaging system with exposure time 15 s.