Fluorescent molecular rotor probe with high quantum yield as well as preparation method and application of fluorescent molecular rotor probe

Abstract

The invention discloses a fluorescent molecular rotor probe with high quantum yield as well as a preparation method and application thereof, and belongs to the technical field of fluorescent probes. The fluorescent compound with an electron donor-electron acceptor structure is obtained by carrying out novenger condensation reaction on an aldehyde group derivative and an electron withdrawing group, and the finally prepared red fluorescent molecular rotor probe shows remarkable superiority in optical property, is suitable for fluorescence imaging in a scattering medium, and has a wide application prospect. The fluorescent powder can effectively penetrate through biological tissues, avoids interference from biological self-luminescence, and is particularly suitable for application scenes with requirements on red shift emission, high efficiency and high signal-to-noise ratio in fluorescence imaging and spectral analysis. Different functional groups are used for modifying the CCNJ to further realize the diversification of the functions of the CCNJ, and the obtained CCNJ-FA, CCNJ-TPP and CCNJ-EuK also have targeting property, excellent pharmacokinetic property and wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a fluorescent molecular rotor probe with high quantum yield, a preparation method and an application thereof. Background Art

[0002] Fluorescent molecular rotors are a special class of fluorescent molecules whose fluorescence quantum yield is closely related to the viscosity of the environment. This property makes them an important tool for studying viscosity changes in complex biological environments, especially in the measurement of cell membrane fluidity and microviscosity. Under different viscosity conditions, fluorescent molecular rotors exhibit different fluorescence intensities, which can provide strong support for many studies in biophysics, cell biology and materials science. The working principle of fluorescent molecular rotors is based on the dynamic behavior of their structure, especially their intramolecular rotation in the excited state. This rotation involves a phenomenon called twisted intramolecular charge transfer (TICT). In low viscosity environments, molecules can rotate freely, while in high viscosity environments, rotation is hindered. This rotation has a direct impact on the fluorescence quantum yield of the molecule (that is, the number of fluorescent photons emitted). The most commercially available molecular rotors include 9-(dicyanovinyl)-julolidine (DCVJ) and 9-(2-carboxy-2-cyano)vinyl julolidine (CCVJ), which are widely used in viscosity measurement, biomacromolecule interaction research and living cell imaging. However, as green fluorescent dyes, the excitation and emission wavelengths of DCVJ and CCVJ (usually in the range of 450-550nm) overlap with the autofluorescence of many biological tissues, especially flavin compounds and chlorophyll in tissues, which produce natural fluorescence in this band. This autofluorescence interferes with signal detection and increases background noise. In addition, the ability of green fluorescence to penetrate biological tissues is limited, making it difficult to apply to living organisms. In addition, the quantum yield of DCVJ and CCVJ is low, and a long exposure time is often required. The excitation light also causes greater photodamage to biological samples (especially living cells), which is not conducive to cell and living body detection, limiting its application in high-sensitivity fluorescence detection. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a fluorescent molecular rotor probe with high quantum yield and a preparation method and application thereof, so as to solve the technical problems of short wavelength and low quantum yield of existing fluorescent molecular rotors.

[0004] To achieve the above object, the technical solution adopted by the present invention is: to provide a method for preparing a fluorescent molecular rotor probe with high quantum yield, comprising the following steps: S1, 1-bromo-3-methyl-2-butene, 6-bromo-2-aminonaphthalene and a catalyst are mixed in a molar ratio of 12-15:2.5-3.5:25-35, and then reacted for 2-4 hours under a protective atmosphere to obtain an intermediate 1; S2, intermediate 1 and acid solution are mixed in a ratio of 8-9 mmol: 15-20 mL, followed by reaction at -15--5°C for 1.5-2.5 h. After the reaction, the pH of the reaction solution is adjusted to neutral to obtain intermediate 2; S3, reacting the intermediate 2 and the alkaline substance at -75--80°C for 30-60 minutes, then adding DMF and continuing the reaction for 20-40 minutes to obtain the intermediate 3; the molar ratio of the intermediate 2, the alkaline substance and DMF is 5-10:8-13:9-14; S4, the intermediate 3 and the electron acceptor are mixed in a molar ratio of 0.2-0.7:0.2-3.1, and then refluxed at 50-90°C for 6-12h to obtain a fluorescent molecular rotor probe with high quantum yield; the electron acceptor is , , , or .

[0005] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the catalyst is potassium iodide or triethylamine.

[0006] Furthermore, the protective atmosphere is made of nitrogen, argon or helium.

[0007] Furthermore, the acidic solution is a concentrated sulfuric acid solution.

[0008] Furthermore, the reagent used to adjust the pH of the reaction solution in S2 is sodium hydroxide solution, potassium hydroxide solution or ammonia water.

[0009] Furthermore, the alkaline substance is n-butyllithium.

[0010] Furthermore, S4 can also mix the intermediate 3, the electron acceptor and the piperidine in the ratio of 0.2-0.7 mmol: 0.2-3.1 mmol: 20-26 μL, and then reflux for reaction.

[0011] Further, the method further comprises the following steps: A1, mixing the fluorescent molecular rotor probe, tetrafluorophenol and activator at a molar ratio of 0.5-0.7:0.7-0.9:0.7-0.9 for 11-13 hours to obtain intermediate 4; A2. The intermediate 4, EuK and the catalyst are mixed and reacted for 2-4 hours, and then purified. The purified solution is mixed with the composite solution and reacted for 5-7 hours to obtain a high quantum yield fluorescent molecular rotor probe CCNJ-EuK; the ratio of intermediate 4, EuK, triethylamine and composite solution is 0.1-0.3 mmol: 0.1-0.3 mmol: 2.5-3 mmol: 5-7 mL.

[0012] Furthermore, the activator is N,N'-dicyclohexylcarbodiimide.

[0013] Furthermore, the composite liquid is obtained by mixing trifluoroacetic acid, dichloromethane and triisopropylsilane in a volume ratio of 0.8-1.2:0.8-1.2:0.3-0.7.

[0014] The invention also discloses a fluorescent molecular rotor probe with high quantum yield prepared by the preparation method.

[0015] On the basis of the above technical solution, the present invention can also be improved as follows: Furthermore, the general structural formula of the fluorescent molecular rotor probe is: , Wherein, R1 is an electron acceptor compound having electron-withdrawing property.

[0016] Furthermore, in the fluorescent molecular rotor probe, R1 is an electron acceptor derivative containing a carboxyl group, an amino group, an azide group, an alkynyl group or a hydroxyl group.

[0017] Furthermore, the structural formula of R1 in the fluorescent molecular rotor probe is: .

[0018] The invention also discloses the application of the fluorescent molecular rotor probe with high quantum yield in fluorescence detection.

[0019] The beneficial effects of the present invention are: 1. The fluorescent molecular rotor probe prepared by the present invention exhibits significant superiority in optical properties, and both the absorption spectrum and the emission spectrum are red-shifted to the long-wave direction (fluorescence excitation wavelength is 471-505nm, and fluorescence emission wavelength is 584-610nm), which helps to avoid background fluorescence interference of biological samples in fluorescence imaging. The red fluorescent molecular rotors CCNJ and DCNJ both exhibit good imaging capabilities, are suitable for fluorescence imaging in scattering media, can effectively penetrate biological tissues, avoid interference from biological autoluminescence, and have in vivo application value.

[0020] 2. Compared with the parent nucleus CCNJ, the modified probes CCNJ-FA, CCNJ-TPP and CCNJ-EuK maintain or even slightly improve their optical properties (such as fluorescence wavelength, quantum yield, brightness, etc.), and can effectively respond to different viscosities. CCNJ-FA can specifically target the cell membrane and can locate the cell membrane; CCNJ-TPP can achieve efficient targeting of the mitochondria of living cells; CCNJ-EuK obtained by coupling and modifying CCNJ and the prostate cancer targeting group EuK can achieve efficient targeting of prostate tumor cells.

[0021] 3. The fluorescent molecular rotor probe prepared by the present invention has excellent in vivo metabolic properties and can be widely used in many fields such as cell membrane fluidity, protein aggregation, microviscosity measurement, in vivo imaging, tumor detection and fluorescence intraoperative navigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the fluorescence spectrum of the fluorescent molecular rotor probe CCVJ; Figure 2 is the fluorescence spectrum of the fluorescent molecular rotor probe DCVJ; Figure 3 is the fluorescence spectrum of the fluorescent molecular rotor probe CCNJ; Figure 4 is the fluorescence spectrum of the fluorescent molecular rotor probe DCNJ; Figure 5 is the fluorescence spectrum of the fluorescent molecular rotor probe CCNJ-FA; Figure 6 The fluorescence spectrum of the fluorescent molecular rotor probe CCNJ-TPP; Figure 7 is the fluorescence spectrum of the fluorescent molecular rotor probe CCNJ-EuK; Figure 8 The fluorescence emission spectra of the fluorescent molecular rotor probe CCNJ in solutions with different viscosities; Fig. 9 This is the fluorescence emission spectra of the fluorescent molecular rotor probe DCNJ in solutions with different viscosities; Fig.10 Fluorescence emission spectra of the fluorescent molecular rotor probe CCNJ-FA in solutions with different viscosities; Fig.11 The fluorescence emission spectra of the fluorescent molecular rotor probe CCNJ-TPP in solutions with different viscosities; Fig.12 The fluorescence emission spectra of the fluorescent molecular rotor probe CCNJ-EuK in solutions with different viscosities; Fig.13 is the fluorescence intensity of the fluorescent molecular rotor probe CCNJ in solutions with different viscosities; Fig.14 is the fluorescence intensity of the fluorescent molecular rotor probe DCNJ in solutions with different viscosities; Fig.15 is the fluorescence intensity of the fluorescent molecular rotor probe CCNJ-FA in solutions with different viscosities; Fig.16 is the fluorescence intensity of the fluorescent molecular rotor probe CCNJ-TPP in solutions with different viscosities; Fig.17 is the fluorescence intensity of the fluorescent molecular rotor probe CCNJ-EuK in solutions with different viscosities; Fig.18 Comparison of fluorescence signal intensities of fluorescent molecular rotor probes CCVJ, DCVJ, CCNJ and DCNJ; Fig.19 Comparison of signal-to-noise ratio (SBR) data of fluorescent molecular rotor probes CCNJ and DCNJ; Fig. 20 Comparison of the full width at half maximum (FWHM) data of the fluorescent molecular rotor probes CCNJ and DCNJ; Fig.21 The half-peak width of the fluorescence signal of the fluorescent molecular rotor probe CCNJ in scattering media at different depths; Fig. 22 The half-peak width of the fluorescence signal of the fluorescent molecular rotor probe DCNJ in scattering media at different depths; Fig.23 This is the co-localized fluorescence image of CCNJ-FA and cell membrane marker DiO; Fig.24 This is the co-localization fluorescence image of CCNJ-TPP and mitochondrial marker MTG; Fig.25 For in vivo imaging of the fluorescent molecular rotor probe CCNJ; Fig.26 The in vitro distribution of the fluorescent molecular rotor probe CCNJ; Fig. 27 The fluorescence semi-quantitative data of the fluorescent molecular rotor probe CCNJ in different organs; Fig.28 The UV absorption and fluorescence emission wavelength data of the fluorescent molecular rotor probe DA-5; Fig.29 This is the whole-body in vivo imaging of mice in the near-infrared zone II using the fluorescent molecular rotor probe DA-5; Fig.30 This is the in vivo imaging of the near-infrared II region of the mouse brain using the fluorescent molecular rotor probe DA-5; Fig.31 This is a near-infrared zone II imaging image of a mouse thigh using the fluorescent molecular rotor probe DA-5. DETAILED DESCRIPTION

[0023] The specific embodiments of the present invention are described below to facilitate the understanding of the present invention by those skilled in the art. If the specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

[0024] Example 1 A method for preparing a fluorescent molecular rotor probe with high quantum yield, the reaction equation is as follows: , The specific steps include: S1. Dissolve 6-bromo-2-aminonaphthalene (3.0 g, 13.5 mmol) and potassium iodide (488.0 mg, 2.9 mmol) in 20.0 mL DMF, then slowly drop 1-bromo-3-methyl-2-butene (4.4 g, 29.7 mmol) into the reaction system, and then react at room temperature for 3 h under a nitrogen atmosphere to generate a brown-black liquid; remove DMF by rotary evaporation, extract, filter, and purify the filtrate by silica gel column chromatography (eluent: n-hexane, 100.0%) to obtain intermediate 1 (3.7 g, 62.0%); S2, dissolve intermediate 1 (3.7 g, 8.4 mmol) in 20 mL of dichloromethane, control the temperature to -10 ° C in a 20.0 wt% potassium chloride water salt bath, then slowly drop concentrated sulfuric acid (18.0 mL), and react at this temperature for 2 h to obtain a brown solution; adjust the pH of the reaction solution to neutral with a saturated sodium hydroxide solution, then extract and filter, and purify the filtrate by silica gel column chromatography (eluent is n-hexane: dichloromethane = 10: 1, v: v) to obtain a white waxy solid, namely intermediate 2 (2.5 g, 51.9%); S3, dissolve intermediate 2 (2.5 g, 7.0 mmol) in 20.0 mL of anhydrous THF, then slowly drop n-butyl lithium (670.0 mg, 10.5 mmol) at -78 °C, slowly drop anhydrous DMF (790.0 mg, 10.9 mmol) after reacting for 45 min, continue to react for 30 min to generate an orange liquid; quench with 1.0 M dilute hydrochloric acid, remove THF by rotary evaporation, adjust pH to 7.5 with aqueous ammonia, then extract, filter, and purify the filtrate by silica gel column chromatography (eluent is n-hexane: ethyl acetate = 10: 1, v: v) to obtain a white waxy solid, namely intermediate 3 (1.1 g, 51.0%); S4. Dissolve intermediate 3 (152.0 mg, 0.5 mmol) and cyanoacetic acid (210.3 mg, 2.5 mmol) in 10.0 mL of methanol, then add 20.0 μL of piperidine, reflux at 90 °C for 12 h, and finally purify by silica gel column chromatography (eluent: dichloromethane: methanol = 20:1, v:v) to obtain a red solid, i.e., the fluorescent molecular rotor probe CCNJ (135.0 mg, 73.6%).

[0025] Example 2 A method for preparing a fluorescent molecular rotor probe with high quantum yield, the reaction equation is as follows: , The specific steps include: S1. Dissolve 6-bromo-2-aminonaphthalene (3.0 g, 13.5 mmol) and potassium iodide (488.0 mg, 2.9 mmol) in 20.0 mL DMF, then slowly drop 1-bromo-3-methyl-2-butene (4.4 g, 29.7 mmol) into the reaction system, and then react at room temperature for 2 h under a nitrogen atmosphere to generate a brown-black liquid; remove DMF by rotary evaporation, extract, filter, and purify the filtrate by silica gel column chromatography (eluent: n-hexane, 100.0%) to obtain intermediate 1; S2, dissolve intermediate 1 (3.7 g, 8.4 mmol) in 20 mL of dichloromethane, control the temperature to -15 °C in a 20.0 wt% potassium chloride water salt bath, then slowly drop concentrated sulfuric acid (18.0 mL), and react at this temperature for 1.5 h to obtain a brown solution; adjust the pH of the reaction solution to neutral with a saturated sodium hydroxide solution, then extract, filter, and purify the filtrate by silica gel column chromatography (eluent is n-hexane: dichloromethane = 10:1, v:v) to obtain a white waxy solid, namely intermediate 2; S3, dissolve intermediate 2 (2.5 g, 7.0 mmol) in 20.0 mL of anhydrous THF, then slowly drop n-butyl lithium (670.0 mg, 10.5 mmol) at -78 °C, slowly drop anhydrous DMF (790.0 mg, 10.9 mmol) after reacting for 60 min, continue to react for 20 min to generate an orange liquid; quench with 1.0 M dilute hydrochloric acid, remove THF by rotary evaporation, adjust pH to 7 with ammonia water, then extract, filter, and purify the filtrate by silica gel column chromatography (eluent is n-hexane: ethyl acetate = 10:1, v:v) to obtain a white waxy solid, namely intermediate 3; S4. Dissolve intermediate 3 (194.0 mg, 0.6 mmol) and malononitrile (208.0 mg, 3.1 mmol) in 10.0 mL of methanol, then add 26.0 μL of piperidine, reflux at 90 °C for 12 h, and finally purify by silica gel column chromatography (eluent: n-hexane: dichloromethane: ethyl acetate = 20:5:1, v:v) to obtain an orange-red solid, i.e., the fluorescent molecular rotor probe DCNJ (200.7 mg, 89.6%).

[0026] Example 3 A method for preparing a fluorescent molecular rotor probe with high quantum yield, the reaction equation is as follows: , The specific steps include: S1. Dissolve 6-bromo-2-aminonaphthalene (3.0 g, 13.5 mmol) and potassium iodide (488.0 mg, 2.9 mmol) in 20.0 mL DMF, then slowly drop 1-bromo-3-methyl-2-butene (4.4 g, 29.7 mmol) into the reaction system, and then react at room temperature for 4 h under a nitrogen atmosphere to generate a brown-black liquid; remove DMF by rotary evaporation, extract, filter, and purify the filtrate by silica gel column chromatography (eluent: n-hexane, 100.0%) to obtain intermediate 1; S2, dissolve intermediate 1 (3.7 g, 8.4 mmol) in 20 mL of dichloromethane, control the temperature to -5°C in a 20.0 wt% potassium chloride water salt bath, then slowly drop concentrated sulfuric acid (18.0 mL), and react at this temperature for 2.5 h to obtain a brown solution; adjust the pH of the reaction solution to neutral with a saturated sodium hydroxide solution, then extract and filter, and purify the filtrate by silica gel column chromatography (eluent is n-hexane: dichloromethane = 10:1, v:v) to obtain a white waxy solid, namely intermediate 2; S3, dissolve intermediate 2 (2.5 g, 7.0 mmol) in 20.0 mL of anhydrous THF, then slowly drop n-butyl lithium (670.0 mg, 10.5 mmol) at -78 °C, slowly drop anhydrous DMF (790.0 mg, 10.9 mmol) after reacting for 30 min, continue to react for 40 min to generate an orange liquid; quench with 1.0 M dilute hydrochloric acid, remove THF by rotary evaporation, adjust pH to 8 with ammonia water, then extract, filter, and purify the filtrate by silica gel column chromatography (eluent is n-hexane: ethyl acetate = 10:1, v:v) to obtain a white waxy solid, namely intermediate 3; S4, intermediate 3 (158.0 mg, 0.5 mmol) and (446.0 mg, 1.5 mmol) was dissolved in 10.0 mL of dichloromethane, followed by the addition of 20.0 μL of piperidine, and the mixture was refluxed at 50 °C for 6 h. Finally, it was purified by silica gel column chromatography (eluent: n-hexane: dichloromethane: ethyl acetate = 20:5:1, v:v) to obtain a red oily liquid, namely the fluorescent molecular rotor probe CCNJ-FA (135.0 mg, 73.6%).

[0027] Example 4 A method for preparing a fluorescent molecular rotor probe with high quantum yield, the reaction equation is as follows: , The specific steps include: S1. Dissolve 6-bromo-2-aminonaphthalene (3.0 g, 13.5 mmol) and potassium iodide (488.0 mg, 2.9 mmol) in 20.0 mL DMF, then slowly drop 1-bromo-3-methyl-2-butene (4.4 g, 29.7 mmol) into the reaction system, and then react at room temperature for 2 h under a nitrogen atmosphere to generate a brown-black liquid; remove DMF by rotary evaporation, extract, filter, and purify the filtrate by silica gel column chromatography (eluent: n-hexane, 100.0%) to obtain intermediate 1; S2, dissolve intermediate 1 (3.7 g, 8.4 mmol) in 20 mL of dichloromethane, control the temperature to -15°C in a 20.0 wt% potassium chloride water salt bath, then slowly drop concentrated sulfuric acid (18.0 mL), and react at this temperature for 2.5 h to obtain a brown solution; adjust the pH of the reaction solution to neutral with a saturated sodium hydroxide solution, then extract and filter, and purify the filtrate by silica gel column chromatography (eluent is n-hexane: dichloromethane = 10:1, v:v) to obtain a white waxy solid, namely intermediate 2; S3, dissolve intermediate 2 (2.5 g, 7.0 mmol) in 20.0 mL of anhydrous THF, then slowly drop n-butyl lithium (670.0 mg, 10.5 mmol) at -78 °C, slowly drop anhydrous DMF (790.0 mg, 10.9 mmol) after reacting for 60 min, continue to react for 40 min to generate an orange liquid; quench with 1.0 M dilute hydrochloric acid, remove THF by rotary evaporation, adjust pH to 7 with ammonia water, then extract, filter, and purify the filtrate by silica gel column chromatography (eluent is n-hexane: ethyl acetate = 10:1, v:v) to obtain a white waxy solid, namely intermediate 3; S4, intermediate 3 (200.0 mg, 0.7 mmol) and (484.3 mg, 2.0 mmol) was dissolved in 10.0 mL of dichloromethane, followed by the addition of 20.0 μL of piperidine, and the mixture was refluxed at 50 °C for 6 h. Finally, it was purified by silica gel column chromatography (eluent: n-hexane: dichloromethane: ethyl acetate = 20:5:1, v:v) to obtain an orange-red oily liquid, namely the fluorescent molecular rotor probe CCNJ-TPP (160.0 mg, 44.4%).

[0028] Example 5 A method for preparing a fluorescent molecular rotor probe with high quantum yield, the reaction equation is as follows: , The specific steps include: S1. Dissolve 6-bromo-2-aminonaphthalene (3.0 g, 13.5 mmol) and potassium iodide (488.0 mg, 2.9 mmol) in 20.0 mL DMF, then slowly drop 1-bromo-3-methyl-2-butene (4.4 g, 29.7 mmol) into the reaction system, and then react at room temperature for 3 h under a nitrogen atmosphere to generate a brown-black liquid; remove DMF by rotary evaporation, extract, filter, and purify the filtrate by silica gel column chromatography (eluent: n-hexane, 100.0%) to obtain intermediate 1; S2, dissolve intermediate 1 (3.7 g, 8.4 mmol) in 20 mL of dichloromethane, control the temperature to -15°C in a 20.0 wt% potassium chloride water salt bath, then slowly drop concentrated sulfuric acid (18.0 mL), and react at this temperature for 2.5 h to obtain a brown solution; adjust the pH of the reaction solution to neutral with a saturated sodium hydroxide solution, then extract and filter, and purify the filtrate by silica gel column chromatography (eluent is n-hexane: dichloromethane = 10:1, v:v) to obtain a white waxy solid, namely intermediate 2; S3, dissolve intermediate 2 (2.5 g, 7.0 mmol) in 20.0 mL of anhydrous THF, then slowly drop n-butyl lithium (670.0 mg, 10.5 mmol) at -78 °C, slowly drop anhydrous DMF (790.0 mg, 10.9 mmol) after reacting for 60 min, continue to react for 40 min to generate an orange liquid; quench with 1.0 M dilute hydrochloric acid, remove THF by rotary evaporation, adjust pH to 7 with ammonia water, then extract, filter, and purify the filtrate by silica gel column chromatography (eluent is n-hexane: ethyl acetate = 10:1, v:v) to obtain a white waxy solid, namely intermediate 3; S4, intermediate 3 (61.4 mg, 0.2 mmol) and (87.2 mg, 0.2 mmol) was dissolved in 10.0 mL of ethanol, refluxed at 90 °C for 12 h, and finally purified by silica gel column chromatography (eluent: dichloromethane: methanol = 20:1, v:v) to obtain a black solid powder, namely the fluorescent molecular rotor probe DA-5 (106.0 mg, 73.0%).

[0029] Example 6 A method for preparing a fluorescent molecular rotor probe with high quantum yield, the reaction equation is as follows: , The specific steps include: S1. CCNJ (210.0 mg, 0.6 mmol) prepared in Example 1 and tetrafluorophenol (139.5 mg, 0.8 mmol) were dissolved in 10.0 mL of dichloromethane, followed by addition of N,N'-dicyclohexylcarbodiimide (DCC, 173.3 mg, 0.8 mmol), and the mixture was reacted at room temperature for 12 h to generate a white suspension; the mixture was then filtered and purified by silica gel column chromatography (eluent: n-hexane: ethyl acetate = 9:1, v:v) to obtain an orange-red solid, namely, intermediate 4 (202.4 mg, 65.4%); S2. Intermediate 4 (120.0 mg, 0.2 mmol) and EuK (112.0 mg, 0.2 mmol) were dissolved in 10.0 mL of dichloromethane, followed by the addition of triethylamine (160.0 μL, 2.7 mmol), and the mixture was reacted at room temperature for 3 h. The mixture was then purified by silica gel column chromatography (eluent: dichloromethane: ethyl acetate = 2:1, v:v). The purified solution was mixed with 6 mL of a composite solution (trifluoroacetic acid: dichloromethane: triisopropylsilane = 1:1:0.5, v:v) at room temperature for 6 h. After purification by high performance liquid chromatography, an orange solid powder was obtained, namely the fluorescent molecular rotor probe CCNJ-EuK (12.4 mg, 6.4%).

[0030] Experimental Example 1 Optical Properties The commercial molecular rotors CCVJ and DCVJ (Shanghai Bid Pharmaceutical Technology Co., Ltd.) were used as controls, and the molecular optical properties were tested together with the fluorescent molecular rotor probes (CCNJ, DCNJ, CCNJ-FA, CCNJ-TPP and CCNJ-EuK) prepared in Examples 1-5. The ultraviolet absorption spectrum and molar extinction coefficient were measured using a Shimadzu UV-3600i UV-visible near-infrared spectrophotometer, the fluorescence excitation emission spectrum was measured using an Edinburgh FLS1000 steady-state / transient fluorescence spectrometer FLS1000, and the absolute quantum yield was measured using a Hamamatsu C11347 absolute quantum yield instrument. The results are shown in Table 1 and Figure 1-Figure 7 shown.

[0031] Table 1 Comparison of molecular optical properties

[0032] As can be seen from Table 1, the Stokes shift of CCNJ and DCNJ increased significantly, reaching 119nm for CCNJ and 105nm for DCNJ. This larger spectral separation significantly improved the detectability of the fluorescence signal and the signal-to-noise ratio. More importantly, the quantum yields of the CCNJ and DCNJ probes reached 36.5% and 36.4% in the non-polar solvent DCM, and 8.1% and 8.9% in the polar solvent PBS, respectively, showing higher fluorescence efficiency. In addition, the brightness values ​​of CCNJ and DCNJ are significantly better than those of CCVJ and DCVJ. For example, the brightness of DCNJ in DCM reaches 14768.2M -1 cm -1 , much higher than CCVJ's 212.0M -1 cm -1 .

[0033] from Figure 1-Figure 7 (E m is the emission spectrum, E x It can be seen from the excitation spectrum that the fluorescent molecular rotor probe prepared by the present invention exhibits significant superiority in optical properties, and both the absorption spectrum and the emission spectrum are red-shifted to the long-wave direction (the fluorescence excitation wavelength is 471-505nm, and the fluorescence emission wavelength is 584-610nm), which helps to avoid background fluorescence interference of biological samples in fluorescence imaging.

[0034] Experimental Example 2 Viscosity Responsiveness The viscosity responsiveness test was performed on the fluorescent molecular rotor probes (CCNJ, DCNJ, CCNJ-FA, CCNJ-TPP and CCNJ-EuK) prepared in Examples 1-5. All samples were prepared from ethylene glycol mother solutions containing 100 μM dry probe and 2.5 v% DMSO, and each sample was mixed according to Table 2 to a final concentration of 10 μM. All samples were vortexed for at least 30 minutes before being poured into the cuvette for scanning; each dye was dissolved in a 620.7 cP viscosity solvent and a preliminary fluorescence measurement was performed to determine the optimal excitation wavelength, peak emission wavelength and slit setting. Each sample was recorded after equilibration at 20°C for 10 minutes. The above steps were repeated twice for each dye, and the logarithm of the average peak intensity was plotted as a function of the logarithm of viscosity by linear regression.

[0035] Table 2 Solvent composition and viscosity

[0036] The results are as follows Figure 8-Figure 17As shown, the fluorescence intensity of the fluorescent molecular rotor probe prepared by the present invention responds to the solution viscosity. As the solution viscosity increases, the probe fluorescence intensity response increases and the fluorescence intensity increases significantly. Figure 13-Figure 17 As shown, at room temperature 25°C, under the conditions of the same concentration and excitation wavelength, the maximum fluorescence emission intensity of the probe shows obvious differences in fluorescence intensity in solutions of different viscosities.

[0037] The above results show that compared with the parent core CCNJ, the modified probes CCNJ-FA, CCNJ-TPP and CCNJ-EuK maintain or even slightly improve the optical properties (such as fluorescence wavelength, quantum yield, brightness, etc.), and can effectively respond to different viscosities.

[0038] Experimental Example 3 Fluorescence Imaging Performance The commercial molecular rotors CCVJ and DCVJ were used as controls, and the fluorescence imaging performance was tested together with the fluorescent molecular rotor probes (CCNJ and DCNJ) prepared in Examples 1 and 2. 1% fat emulsion was used as a simulated tissue because its scattering properties are similar to those of tissue. Capillaries containing 10 µM CCVJ, DCVJ, CCNJ and DCNJ were placed in a cylindrical culture dish and immersed in different volumes of 1% fat emulsion to simulate the depth of tissue. Images were acquired using the IVIS imaging system at the optimal excitation wavelength and emission wavelength of the probe. At the same time, an area of ​​the same size was marked in the blank area, and the fluorescence intensity of the area was set as the background signal. By comparing and analyzing the fluorescence imaging performance of the probes DCNJ, CCNJ, DCVJ and CCVJ at different depths in the scattering medium (1% fat emulsion), the fluorescence signal intensity, signal-to-noise ratio (SBR) and half-width at half maximum (FWHM) were evaluated. The results are shown as follows: Figure 18-Figure 22 shown.

[0039] The results showed that the imaging performance of DCVJ and CCVJ was significantly insufficient. Their fluorescence signals were extremely weak and almost undetectable even in the shallow layer (1 mm). They could not even penetrate the fat emulsion with a depth of 1 mm, and could not form effective signals at all as the depth increased ( Fig.18 ), indicating that they are not suitable for fluorescence imaging in scattering media. In comparison, the imaging performance of DCNJ and CCNJ is similar. The fluorescence signal of fat emulsion is still visible in the deeper layer (6-8 mm), and the fluorescence intensity and signal-to-noise ratio of DCNJ are slightly better than those of CCNJ. The fluorescence signal intensity of both weakens with increasing depth, and the signal-to-noise ratio also gradually decreases, but the signal attenuation of DCNJ in the deep layer is slightly slower ( Fig.19 In addition, as the depth increases, the FWHM (half maximum width) of both gradually increases ( Fig. 20 ), reflecting the greater diffusion or blurring of the signal at greater depths, but with little difference in spatial resolution ( Fig.21 and Fig. 22 ).

[0040] The above results show that DCVJ and CCVJ cannot meet the imaging requirements in scattering environments due to their weak fluorescence signals and inability to penetrate deep layers. As red fluorescent molecular rotors, CCNJ and DCNJ both show good imaging capabilities and are suitable for fluorescence imaging in scattering media. Although the performance of the two is similar, DCNJ has a slight advantage in deep imaging (6 mm and above) with better signal intensity and signal-to-noise ratio. Both CCNJ and DCNJ can effectively penetrate biological tissues and avoid interference from biological autoluminescence, and have in vivo application value.

[0041] Experimental Example 4 Targeting CCNJ-FA and CCNJ-TPP were used as examples to evaluate the targeting of the fluorescent molecular rotor probe prepared by the present invention. Specifically, 4T1 cells in the logarithmic growth phase were taken, the cells were digested and counted, and 2 mL of 2×10 5 The cell suspension was added to a 35 mm diameter confocal cell culture dish and cultured in a cell culture incubator at 37 °C and 5% CO for 24 h. The old culture medium was then aspirated and 1 mL of basal culture medium containing 1 μM probe sample was added for 15 min. The culture medium was discarded, washed three times with PBS, and DiO (10 μM) or MTR (200 nM) was added for 30 min, where DiO is a cell membrane marker and MTG is a mitochondrial marker. After washing three times with PBS, the fluorescence distribution of the probe was observed using an inverted laser confocal microscope, and the colocalization coefficient was calculated using ImageJ software.

[0042] like Fig.23 As shown, by merging the fluorescence images of CCNJ-FA (left) and the cell membrane marker DiO (middle) (right), it was found that the localization fluorescence images of the two almost overlapped, indicating that the probe CCNJ-FA obtained by coupling CCNJ and farnesol can specifically target the cell membrane and can achieve localization of the cell membrane. Fig.24 As shown, by merging the fluorescence images of CCNJ-TPP (left) and the mitochondrial marker MTG (middle) (right), it was found that the localization fluorescence images of the two almost overlapped, indicating that CCNJ-TPP obtained by coupling and modifying with triphenylphosphine can achieve efficient targeting to mitochondria in living cells. CCNJ-EuK obtained by coupling and modifying with the prostate cancer targeting group EuK can achieve efficient targeting to prostate tumor cells.

[0043] Experimental Example 5 Pharmacokinetic Properties CCNJ was used as an example to evaluate the pharmacokinetic properties of the fluorescent molecular rotor probe prepared by the present invention. Specifically, 1 nmol of CCNJ was intravenously injected into nude mice, and then metabolic observation and biodistribution study were performed using a PerkinElmer IVIS small animal imaging system at its optimal excitation wavelength and emission wavelength.

[0044] like Figure 25-27 As shown, CCNJ was rapidly excreted through the kidneys after intravenous injection, and the fluorescence signal in the bladder was high. The biodistribution results showed that CCNJ was mainly concentrated in the tail and intestine of mice. In addition, there were higher fluorescence signals in brown adipose tissue and lymph nodes, and high fluorescence signals in the stomach and intestine indicated that CCNJ was also excreted through the intestine.

[0045] In summary, compared with CCVJ and DCVJ, the fluorescent molecular rotor probes CCNJ and DCNJ prepared by the present invention show obvious improvements in quantum yield, Stokes shift, brightness and spectral position, and are particularly suitable for application scenarios requiring red-shifted emission, high efficiency and high signal-to-noise ratio in fluorescence imaging and spectral analysis. In addition, the probes CCNJ-FA, CCNJ-TPP and CCNJ-EuK have targeting properties on the basis of their original optical properties, and have broad application prospects.

[0046] Experimental Example 6 1. Taking DA-5 as an example, the optical properties of the fluorescent molecular rotor probe prepared by the present invention in the near-infrared region II (1000-1700nm) were evaluated. Specifically, the ultraviolet absorption wavelength of DA-5 was measured to be 847nm using the Shimadzu UV-3600i ultraviolet-visible near-infrared spectrophotometer, and the fluorescence excitation emission wavelength of DA-5 was measured to be 1088nm using the Edinburgh FLS1000 steady-state / transient fluorescence spectrometer FLS1000. Fig.28 ), the molecular wavelength conforms to the near-infrared second region fluorescence range (1000-1700nm).

[0047] 2. 1 nmol of DA-5 was intravenously injected into nude mice, and then in vivo studies were performed under 808 nm laser excitation using a near-infrared second-zone fluorescence imaging system and a 1500 nm filter.

[0048] The results are as follows Figure 29-Figure 31 As shown, DA-5 can clearly perform ultra-clear near-infrared zone II imaging of mouse systemic blood vessels, brain blood vessels and calf blood vessels, indicating that the fluorescent molecule DA-5 prepared by the present invention has optical properties in the near-infrared zone II, and is particularly suitable for use in fluorescence imaging with strong tissue penetration ability and spectral analysis in application scenarios that require red-shifted emission, high efficiency and high signal-to-noise ratio, and has broad application prospects.

Claims

1. A method for preparing a fluorescent molecular rotor probe with high quantum yield, characterized in that: The following steps are involved: S1, 1-bromo-3-methyl-2-butene, 6-bromo-2-aminonaphthalene and a catalyst are mixed in a molar ratio of 12-15:2.5-3.5:25-35, and then reacted for 2-4 hours under a protective atmosphere to obtain an intermediate 1; S2, intermediate 1 and acid solution are mixed in a ratio of 8-9 mmol: 15-20 mL, followed by reaction at -15--5°C for 1.5-2.5 h. After the reaction, the pH of the reaction solution is adjusted to neutral to obtain intermediate 2; S3, reacting the intermediate 2 and the alkaline substance at -75--80°C for 30-60 min, then adding DMF and continuing the reaction for 20-40 min to obtain the intermediate 3; the molar ratio of the intermediate 2, the alkaline substance and DMF is 5-10:8-13:9-14; S4, mixing the intermediate 3 and the electron acceptor in a molar ratio of 0.2-0.7:0.2-3.1, and then reflux reacting at 50-90° C. for 6-12 h to obtain a fluorescent molecular rotor probe with a high quantum yield; the electron acceptor is , , , or .

2. The method for preparing a fluorescent molecular rotor probe with high quantum yield according to claim 1, characterized in that: The catalyst is potassium iodide or triethylamine.

3. The method for preparing a fluorescent molecular rotor probe with high quantum yield according to claim 1, characterized in that: The acidic solution is a concentrated sulfuric acid solution.

4. The method for preparing a fluorescent molecular rotor probe with high quantum yield according to claim 1, characterized in that: The reagent used to adjust the pH of the reaction solution in S2 is sodium hydroxide solution, potassium hydroxide solution or ammonia water.

5. The method for preparing a fluorescent molecular rotor probe with high quantum yield according to claim 1, characterized in that: The following steps are also included: A1, mixing the fluorescent molecular rotor probe, tetrafluorophenol and activator at a molar ratio of 0.5-0.7:0.7-0.9:0.7-0.9 for 11-13 hours to obtain intermediate 4; A2. The intermediate 4, EuK and the catalyst are mixed and reacted for 2-4 hours, and then purified. The purified solution is mixed with the composite solution and reacted for 5-7 hours to obtain a fluorescent molecular rotor probe CCNJ-EuK with a high quantum yield; the ratio of the intermediate 4, EuK, the catalyst and the composite solution is 0.1-0.3 mmol: 0.1-0.3 mmol: 2.5-3 mmol: 5-7 mL.

6. The method for preparing a fluorescent molecular rotor probe with high quantum yield according to claim 5, characterized in that: The composite liquid is obtained by mixing trifluoroacetic acid, dichloromethane and triisopropylsilane in a volume ratio of 0.8-1.2:0.8-1.2:0.3-0.

7.

7. A high quantum yield fluorescent molecular rotor probe, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 6.

8. The high quantum yield fluorescent molecular rotor probe according to claim 7, characterized in that: The general structural formula of the fluorescent molecular rotor probe is: ， Wherein, R1 is an electron acceptor compound having electron-withdrawing property.

9. The high quantum yield fluorescent molecular rotor probe according to claim 8, characterized in that: The structural formula of R1 in the fluorescent molecular rotor probe is: 。 10. Use of the fluorescent molecular rotor probe with high quantum yield according to any one of claims 7 to 9 in fluorescence detection.

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