CN118047787A - 2,1, 3-Benzothiadiazole and spiropyran conjugated fluorescent compound and application thereof in lipid drop imaging - Google Patents

2,1, 3-Benzothiadiazole and spiropyran conjugated fluorescent compound and application thereof in lipid drop imaging Download PDF

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CN118047787A
CN118047787A CN202410148403.4A CN202410148403A CN118047787A CN 118047787 A CN118047787 A CN 118047787A CN 202410148403 A CN202410148403 A CN 202410148403A CN 118047787 A CN118047787 A CN 118047787A
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spiropyran
compound
benzothiadiazole
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fluorescent compound
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牛广乐
朱晓铭
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Shandong University
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Abstract

The invention belongs to the field of organic light fluorescent dyes, and relates to a preparation method of a2, 1, 3-benzothiadiazole-spiropyran conjugated fluorescent compound, a photophysical property research and application thereof in lipid drop imaging. The chemical structure of the compound is shown as formula I: Wherein R 1 is selected from a C 1-C6 linear or branched hydrocarbyl, an ether group, or a C 1-C6 alkyl substituted acyl; r 2 is hydrogen, methyl, substituted alkyl, fluoro, chloro, bromo, iodo, cyano, trihalomethyl, nitro or dimethylamino. The 2,1, 3-benzothiadiazole-spiropyran conjugated fluorescent compound provided by the invention not only has excellent reversible photochromic property, but also has acid-induced color and strong solution and solid state fluorescence. Importantly, the probe has low toxicity and good biocompatibility, can precisely target cell lipid drops, and realizes high-contrast fluorescence imaging, which has important significance for further applying the probe to research on diseases caused by lipid lesions such as obesity, atherosclerosis and the like.

Description

2,1, 3-Benzothiadiazole and spiropyran conjugated fluorescent compound and application thereof in lipid drop imaging
Technical Field
The invention belongs to the field of organic light fluorescent dyes, and relates to a2, 1, 3-benzothiadiazole and spiropyran conjugated fluorescent compound and application thereof in lipid drop imaging.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
At present, the spiropyran is used as a very promising photochromic dye and has wide application in the aspects of anti-counterfeiting intelligent materials, biosensing and the like. When it is stimulated by external environment (such as ultraviolet-visible light, acid/base, polarity, etc.), the molecule is converted from lipophilic spiropyran Guan Huanjie structure to open-loop structure with pi extending positive charge. More importantly, the two isomers generally exhibit distinct physicochemical properties. Thus, researchers have conducted extensive research on the mechanism and nature of their switching loops over the years. However, to date, fluorescent probes designed for the spiropyran backbone have been less studied and have some significant drawbacks. For example, modifications to spiropyran derivatives are mostly present at the benzopyran and indole nitrogen sites, whereas modifications to the right indole benzene ring are less. In addition, the low solid state fluorescence quantum yield of the spiropyran derivatives greatly limits the application of the spiropyran derivatives in the fields of organic light emitting diodes, biological imaging and the like.
Fluorescent probes can be used for lipid droplet imaging and dynamic tracking, however, most lipid fluorescent probes can not only target lipid droplets but also other lipid structures in cells, such as endoplasmic reticulum and the like, due to insufficient imaging contrast. Meanwhile, some key imaging parameters of the current lipid drop fluorescent probe, such as fluorescence quantum yield, imaging contrast and the like, are still to be further improved and perfected.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a2, 1, 3-benzothiadiazole and spiropyran conjugated fluorescent compound and application thereof in lipid drop imaging. Because of the characteristics of the rigid donor-acceptor type structure, the fluorescent dye has strong solution and solid dual-state fluorescence, has the advantages of high permeability, low toxicity and the like, and can realize the specific and high-contrast visual imaging of intracellular lipid droplets.
In a first aspect of the invention, there is provided a fluorescent compound conjugated and coupled with 2,1, 3-benzothiadiazole and spiropyran, the chemical structure of which is shown in formula I:
Wherein R 1 is selected from a C 1-C6 linear or branched hydrocarbyl, an ether group, or a C 1-C6 alkyl substituted acyl; r 2 is hydrogen, methyl, substituted alkyl, fluoro, chloro, bromo, iodo, cyano, trihalomethyl, nitro or dimethylamino.
In a second aspect, there is provided a method for synthesizing the above-described 2,1, 3-benzothiadiazole and spiropyran conjugated fluorescent compound, comprising the step of obtaining a compound of formula I according to the following reaction scheme;
The compound 1 generates indole salt through simple alkylation reaction, then the indole salt and the compound 3 are subjected to Knoevenagel condensation reaction to obtain a spiropyran derivative 4, and finally the compound 4 and the compound 5 are subjected to simple Suzuki coupling reaction to obtain a final compound I; wherein R 1 is selected from a C 1-C6 linear or branched hydrocarbyl, an ether group, or a C 1-C6 alkyl substituted acyl; r 2 is hydrogen, methyl, substituted alkyl, fluoro, chloro, bromo, iodo, cyano, trihalomethyl, nitro or dimethylamino.
The invention is based on commercial materials, can realize the synthesis of the spiropyran derivative through simple three-step reaction, and has simple process and low reaction condition requirement.
In a third aspect, the use of a fluorescent compound conjugated and coupled with 2,1, 3-benzothiadiazole and spiropyran as described above in a fluorescent probe.
The 2,1, 3-benzothiadiazole and spiropyran conjugated fluorescent compound provided by the invention has strong two-state fluorescence in a nonpolar solvent and under a solid; meanwhile, the fluorescent dye has the characteristic of photochromism.
In a fourth aspect, a composition comprises a fluorescent compound conjugated and coupled with 2,1, 3-benzothiadiazole and spiropyran as described above, or a pharmaceutically acceptable salt thereof.
In a fifth aspect, a formulation comprises an active ingredient which is a fluorescent compound or composition of conjugated 2,1, 3-benzothiadiazole and spiropyran described above and a pharmaceutically acceptable carrier.
In a sixth aspect, the use of a fluorescent compound, composition or formulation conjugated and coupled with 2,1, 3-benzothiadiazole and spiropyran as described above in lipid-specific fluorescence imaging.
In a seventh aspect, a lipid droplet detection kit comprises a fluorescent compound, composition or formulation conjugated and coupled with 2,1, 3-benzothiadiazole and spiropyran as described above, and a solvent or diluent.
The beneficial results of the invention are:
The derivative obtained by introducing the strong acceptor 2,1, 3-benzothiadiazole into the spiropyran skeleton through conjugated coupling has the advantages of acid color change, strong solution and solid fluorescence while maintaining excellent reversible photochromic characteristics. Importantly, the probe has low toxicity and good biocompatibility, can precisely target cell lipid drops, and realizes high-contrast fluorescence imaging, which has important significance for further applying the probe to research on diseases caused by lipid lesions, such as obesity, atherosclerosis and the like.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 shows the single crystal structure of BT-SP-NO 2 and BT-SP-NMe 2 viewed from different directions. C: grey; h: white; n: blue; o: red and S: yellow.
FIG. 2A is a normalized absorption spectrum of BT-SP-NO 2 and BT-SP-NMe 2 in n-hexane solution. FIGS. 2B and 2C are fluorescence emission spectra of BT-SP-NO 2 and BT-SP-NMe 2, respectively, in different solvents. FIG. 2D shows fluorescence quantum yields of BT-SP-NO 2 and BT-SP-NMe 2 in different solvents. Concentration: 10. Mu.M.
FIGS. 3A and 3B are fluorescence emission spectra of BT-SP-NO 2 and BT-SP-NMe 2 in different DMSO/water mixtures, respectively, with different f w values. FIG. 3C is a graph of fluorescence intensity I/I 0 versus DMSO/water mixture composition. FIG. 3D is a normalized fluorescence spectrum in the solid state of BT-SP-NO 2 and BT-SP-NMe 2. Insert: solid state fluorescence photographs of BT-SP-NO 2 and BT-SP-NMe 2 taken under 365nm ultraviolet lamp and their solid state fluorescence Quantum Yields (QY). Concentration: 10. Mu.M.
FIGS. 4A and 4B are Dynamic Light Scattering (DLS) data for BT-SP-NO 2 and BT-SP-NMe 2, respectively, in a DMSO/water mixture containing 30% DMSO.
FIGS. 5A and 5B are spectra of the response of BT-SP-NO 2 and BT-SP-NMe 2, respectively, to trifluoroacetic acid (TFA) in THF. Concentration: 10. Mu.M.
FIGS. 6A and 6B show normalized absorption spectra of BT-SP-NO 2 and BT-SP-NMe 2, respectively, after different ultraviolet irradiation in tetrahydrofuran solution. Fig. 6C and 6D are fluorescence spectra obtained by excitation with different excitation light after the same irradiation time of BT-SP-NO 2 in tetrahydrofuran solution, respectively. FIG. 6E is a schematic diagram of the isomerization transition between BT-SP-NO 2 and BT-MC-NO 2. Concentration: 10. Mu.M.
FIG. 7 shows fluorescence intensity values of BT-SP-NO 2 in tetrahydrofuran solution after 6 cycles of UV-Vis alternating irradiation. UV:30s; vis for 3min; concentration: 10. Mu.M.
FIG. 8A is a spatial electron distribution of HOMO and LUMO in an optimized ground state for BT-SP-NO 2 and BT-SP-NMe 2. FIG. 8B shows molecular packing of BT-SP-NO 2 and BT-SP-NMe 2 crystals in different directions.
FIGS. 9A and 9B are schematic views of the intramolecular dihedral angles of BT-SP-NO 2 and BT-SP-NMe 2, respectively.
FIGS. 10A and 10B are the distances between the spiro carbon atoms on one molecule and the spiro carbon atoms on the remaining three molecules in the BT-SP-NO 2 and BT-SP-NMe 2 unit cells, respectively. Dashed line unit:
FIG. 11 is an in situ spectrum of BT-SP-NO 2 and BT-SP-NMe 2 in HeLa cells.
FIG. 12 is a CLSM image of HeLa cells incubated with BT-SP-NO 2 or BT-SP-NMe 2 and Lipi-Deep Red. Scale bar: 10 μm. Probe concentration: BT-SP-NO 2(500nM),BT-SP-NMe2 (1. Mu.M), lipi-Deep Red (200 nM).
FIG. 13 shows cytotoxicity of BT-SP-NO 2 and BT-SP-NMe 2 in HeLa cells at various concentrations.
FIG. 14 is a hydrogen spectrum of compound 2 prepared in example 1 of the present invention in deuterated DMSO.
FIG. 15 is a hydrogen spectrum of compound 4 prepared in example 2 of the present invention in deuterated DMSO.
FIG. 16 is a hydrogen spectrum of compound 7 prepared in example 2 of the present invention in deuterated DMSO.
FIG. 17 is a hydrogen spectrum of BT-SP-NO 2 prepared in example 3 of the present invention in deuterated DMSO.
FIG. 18 is a hydrogen spectrum of BT-SP-NMe 2 prepared in example 3 of the present invention in deuterated chloroform.
FIG. 19 is a carbon spectrum of BT-SP-NO 2 prepared in example 3 of the present invention in deuterated chloroform.
FIG. 20 is a carbon spectrum of BT-SP-NMe 2 prepared in example 3 of the present invention in deuterated chloroform.
FIG. 21 is a high resolution mass spectrum of BT-SP-NO 2 prepared in example 3 of the present invention.
FIG. 22 is a high resolution mass spectrum of BT-SP-NMe 2 prepared in example 3 of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In view of the limitations of the existing spiropyran derivative probe design strategies, the limited solid fluorescence quantum yield and other problems, the invention provides a2, 1, 3-benzothiadiazole and spiropyran conjugated fluorescent compound and application thereof in lipid drop imaging.
In an exemplary embodiment of the invention, a fluorescent compound conjugated and coupled by 2,1, 3-benzothiadiazole and spiropyran is provided, and the chemical structure of the fluorescent compound is shown in formula I:
Wherein R 1 is selected from a C 1-C6 linear or branched hydrocarbyl, an ether group, or a C 1-C6 alkyl substituted acyl; r 2 is hydrogen, methyl, substituted alkyl, fluoro, chloro, bromo, iodo, cyano, trihalomethyl, nitro or dimethylamino.
In some embodiments, the compound is selected from the group consisting of:
In a second embodiment of the present invention, there is provided a method for synthesizing the above-mentioned 2,1, 3-benzothiadiazole and spiropyran conjugated fluorescent compound, comprising the steps of obtaining the compound of formula I according to the following reaction scheme;
The compound 1 generates indole salt (compound 2) through alkylation reaction, then the indole salt and the compound 3 are subjected to Knoevenagel condensation reaction to obtain a spiropyran derivative 4, and finally the compounds 4 and 5 are subjected to simple Suzuki coupling reaction to obtain a final compound I; wherein R 1 is selected from a C 1-C6 linear or branched hydrocarbyl, an ether group, or a C 1-C6 alkyl substituted acyl; r 2 is hydrogen, methyl, substituted alkyl, fluoro, chloro, bromo, iodo, cyano, trihalomethyl, nitro or dimethylamino.
In some embodiments, the reaction conditions for preparing compound 2 from compound 1 are: the temperature is 60-80 ℃, and the reaction time is 5-7 h. Acetonitrile is used as a solvent in the reaction system.
In some embodiments, the reaction conditions for Knoevenagel condensation are: the temperature is 60-80 ℃ and the reaction time is 4-6 h. Specifically, piperidine is added as a catalyst during the reaction. Specifically, ethanol is used as a solvent in the reaction system.
In some embodiments, the reaction conditions for the Suzuki coupling reaction are: the temperature is 95-120 ℃, and the reaction time is 23-25 h. Specifically, tetraphenylphosphine palladium is added as a catalyst in the reaction process. Specifically, toluene and water are used as solvents in the reaction system.
In a third embodiment of the invention, the application of the 2,1, 3-benzothiadiazole and spiropyran conjugated fluorescent compound in a fluorescent probe is provided.
In a fourth embodiment of the present invention, there is provided a composition comprising the above-described 2,1, 3-benzothiadiazole and spiropyran conjugated fluorescent compound or a pharmaceutically acceptable salt thereof.
Pharmaceutically acceptable salts of the invention include hydrochloride, sulfate, acetate, oxalate, citrate, and the like.
In a fifth embodiment of the present invention, there is provided a formulation comprising an active ingredient which is a fluorescent compound or composition conjugated and coupled with 2,1, 3-benzothiadiazole and spiropyran as described above, and a pharmaceutically acceptable carrier.
The medicinal carrier comprises physiological saline, buffer solution and the like.
In a sixth embodiment of the present invention, there is provided the use of a fluorescent compound, composition or formulation conjugated and coupled with 2,1, 3-benzothiadiazole and spiropyran as described above in lipid-specific fluorescent imaging.
Specifically, a fluorescent compound, composition or formulation of conjugated coupling of 2,1, 3-benzothiadiazole and spiropyran is prepared as a lipid-specific fluorescent imaging reagent, followed by lipid-specific fluorescent imaging.
In a seventh embodiment of the present invention, there is provided a lipid droplet detection kit comprising the above-described fluorescent compound, composition or formulation of conjugated 2,1, 3-benzothiadiazole and spiropyran, and a solvent or diluent.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail below with reference to specific examples and comparative examples.
In the examples below, all chemicals were used as such without any purification unless otherwise indicated. Anhydrous solvents were used for fluorescence property studies. Deionized water was used throughout the study. Commercial lipid droplet probes Lipi-Deep Red are purchased from Dojindo Molecular Technologies, inc. 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were recorded on a Bruker AVANCE III spectrometer using tetramethylsilane as an internal reference. High Resolution Mass Spectrometry (HRMS) was recorded on Agilent Technologies 6510Q-TOF LC/MS instrument operating using ESI mode. UV-Vis absorption and fluorescence spectra were obtained using a Hitachi U-4100 spectrophotometer and Horiba FluoroMax-4 fluorescence photometer, respectively. The absolute fluorescence quantum yield was determined by means of an integrating sphere.
Example 1: synthesis of Compound 2
Compound 1 (2.4 g,10 mmol) was added sequentially to a Schlenk tube in anhydrous acetonitrile (15 mL), then CH 3 I (0.6 mL,10 mmol) was added to the solution and the mixture refluxed at 80 ℃ for 6 hours. After cooling to room temperature, the precipitate was collected and washed with acetonitrile, then dried under vacuum to give compound 2 as a white solid (2.76 g, 73%).
1H NMR(400MHz,DMSO-d6),δ(ppm):8.17(s,1H),7.87(t,J＝8.8Hz,2H),3.94(s,3H),2.74(s,3H),1.53(s,6H).. As shown in fig. 14.
Example 2: synthesis of Compound 4
Compound 2 (379 mg,1 mmol), compound 3 (167 mg,1 mmol), piperidine (200. Mu.L, 2 mmol) were dissolved in EtOH (10 mL) and refluxed at 80℃for 4.5 hours under nitrogen. After cooling to room temperature, the precipitate was collected and washed with EtOH, then dried under vacuum to give compound 4 as a milky white solid (220 mg, 55%).
1H NMR(400MHz,DMSO-d6),δ(ppm):8.23(d,J＝2.76Hz,1H),8.01(dd,J＝8.96,2.80Hz,1H),7.33-7.27(m,2H),7.24(d,J＝10.4Hz,1H),6.91(d,J＝9.0Hz,1H),6.60(d,J＝8.2Hz,1H),5.99(d,J＝10.36Hz,1H),2.66(s,3H),1.21(s,3H),1.12(s,3H). As shown in fig. 15.
Example 3: synthesis of Compound 7
Compound 2 (379 mg,1 mmol), compound 6 (165 mg,1 mmol), piperidine (200. Mu.L, 2 mmol) were dissolved in EtOH (10 mL) and refluxed at 80℃for 4.5 hours under nitrogen. After cooling to room temperature, the precipitate was collected and washed with EtOH, then dried under vacuum to give compound 7 as a brown solid (238 mg, 60%).
1H NMR(400MHz,DMSO-d6),δ(ppm):7.26-7.21(m,,2H),6.97(m,J＝10.2Hz,1H),6.63-6.60(m,1H),6.56(d,J＝1.52Hz,2H),6.52-6.48(m,1H),5.71(d,J＝10.16Hz,1H),2.78(s,6H),2.61(s,3H),1.19(s,3H),1.08(s,3H). As shown in fig. 16.
Example 4: synthesis of Compound BT-SP-NO 2
Compound 5 (400 mg,1 mmol), compound 7 (262..09 mg,1 mmol), pd (PPh 3)4 (57.78 mg,0.05 mmol) and K 2CO3 (414.63 mg,3 mmol) were mixed in toluene/H 2 O (8 mL/2 mL) solution and refluxed at 100 ℃ under nitrogen for 24 hours, after cooling to room temperature, the solvent was removed by evaporation under reduced pressure, and the residue was purified by silica gel column chromatography using a mixture of n-hexane/dichloromethane (15:1 to 5:1, v/v) as eluent to give orange solid BT-SP-NO2(136mg,30％).1H NMR(400MHz,DMSO-d6),δ(ppm):8.25(d,J＝2.80Hz,1H),8.04-7.99(m,2H),7.87(dd,J＝8.12,1.8Hz,1H),7.83-7.75(m,3H),7.27(d,J＝10.36Hz,1H),6.97(d,J＝9.04Hz,1H),6.81(d,J＝8.20Hz,1H),6.06(d,J＝10.36Hz,1H),2.77(s,3H),1.30(s,3H),1.21(s,3H).13C NMR(100MHz,CDCl3),δ(ppm):159.80,155.88,153.81,148.22,141.15,136.71,135.07,129.91,129.57,129.17,128.54,126.65,126.06,122.85,121.51,119.53,118.74,115.64,107.16,106.52,,52.49,29.07,26.17,20.16.HRMS(ESI)m/z calcd for[C25H21N4O3S+]457.1329(([M+H]+)),found 457.1300. as shown in fig. 17, 19, 21 single crystal structure as shown in fig. 1, unit cell parameters and refinement data were shown in table 1.
Example 5: synthesis of Compound BT-SP-NMe 2
Compound 7 (398.10 mg,1 mmol), compound 5 (262.09 mg,1 mmol), pd (PPh 3)4 (57.78 mg,0.05 mmol) and K 2CO3 (414.63 mg,3 mmol) were mixed in toluene/H 2 O (8 mL/2 mL) solution and refluxed at 100 ℃ under nitrogen for 24 hours, after cooling to room temperature, the solvent was removed by evaporation under reduced pressure, and the residue was purified by silica gel column chromatography using a mixture of n-hexane/ethyl acetate (from 60:1 to 20:1, v/v) as eluent to give yellow solid BT-SP-NMe2(204mg,45％).1H NMR(400MHz,CDCl3),δ(ppm):7.90(dt,J＝9.8,3.64Hz,1H),7.85(dd,J＝8.08,1.8Hz,1H),7.67-7.62(m,3H),6.85(d,J＝10.16Hz,1H),6.70-6.60(m,3H),6.52(d,J＝2.8Hz,1H),5.72(d,J＝10.12Hz,1H),2.86(s,6H),2.80(s,3H),1.39(s,3H),1.25(s,3H).13C NMR(100MHz,CDCl3),δ(ppm):155.98,153.95,148.94,146.96,145.44,137.61,135.50,130.08,129.94,129.38,128.24,126.37,122.79,119.78,119.16,118.97,115.81,115.44,112.06,106.73,103.89,51.70,41.98,29.12,26.18,20.56,20.56.HRMS(ESI)m/z calcd for[C27H27N4OS+]455.1900(([M+H]+)),found 455.1871. as shown in fig. 18, 20, 22 single crystal structures as shown in the unit cell parameters and refinement data of fig. 1 were shown in table 2.
TABLE 1 unit cell parameters and refinement data for BT-SP-NO 2
Table 2 BT-SP-NMe 2 unit cell parameters and refinement data
Example 5: basic photophysical property characterization
First, photophysical properties of BT-SP-NO 2 and BT-SP-NMe 2 in different organic solvents were studied. As can be seen from FIG. 2A, BT-SP-NO 2 shows a maximum absorption at 400nm in n-hexane, while BT-SP-NMe 2 is red shifted at 420nm than the former. Furthermore, according to fig. 2B, 2C and 2D, both have excellent luminescence in a nonpolar environment, and fluorescence emission peak intensities are significantly reduced and slightly red-shifted with increasing polarity of the solvent, exhibiting typical tic properties.
Second, their emission properties in DMSO/water mixtures of different water fractions (f w) were studied. As can be seen from fig. 3, they all show a slight decrease in water content of 0% -30% with a slight red shift in fluorescence spectrum. With further increases in water composition, BT-SP-NO 2 and BT-SP-NMe 2 showed maximum fluorescence intensities at f w =70%, λ em =613 nm and λ em =571 nm, respectively. The reason for the decrease in fluorescence intensity at high water content may be related to their different aggregate size and morphology. Furthermore, as shown in fig. 4, dynamic light scattering was used to demonstrate their polymer formation at high water content. The above data demonstrate that they are all AIE dyes. Most importantly, unlike conventional SP, BT-SP-NMe 2 exhibits significantly higher fluorescence quantum yields in the solid state of about 59.0%, much higher than BT-SP-NO 2, which may be related to different modes of stacking of molecules.
Then, the response of both compounds to trifluoroacetic acid (TFA) was tested. The two compounds were dispersed in tetrahydrofuran solution and subjected to titration experiments. As shown in fig. 5A and 5B, the emission peaks of both appear to be red shifted to different extents as the TFA concentration increases. In contrast, BT-SP-NMe 2 exhibited an increase in fluorescence intensity, while BT-SP-NO 2 exhibited a different trend. This phenomenon suggests that proton response is more likely to occur when electron donating groups are introduced into the spiropyran backbone species.
Finally, the response of both compounds to uv-vis was tested. The molecules dispersed in Tetrahydrofuran (THF) solution were alternately irradiated with a hand-held uv lamp and a fluorescent lamp, and the spectral changes at different irradiation times were recorded by absorption and emission spectra. From FIG. 6A, it can be seen that BT-SP-NO 2 has a sensitive response to ultraviolet light, and a new absorption peak appears after 5s irradiation, reaching saturation within 50 s. Furthermore, the molecule remained well isomerised under 6 rounds of alternating irradiation (figure 7). In contrast, no similar situation was observed for BT-SP-NMe 2 dispersed in THF (FIG. 6B). This phenomenon illustrates that the introduction of electron withdrawing groups in the spiropyran backbone makes the molecule more responsive to light.
Example 6: density functional theory calculation (DFT) and molecular packing analysis
The HOMO and LUMO electron cloud distributions of BT-SP-NO 2 and BT-SP-NMe 2 were calculated by Density Functional Theory (DFT) (FIG. 8A), electrons of the LUMO were substantially delocalized in the benzothiadiazole moiety, and a large difference occurred between the electron cloud distributions of the HOMO orbitals. For BT-SP-NO 2,NO2, electron repulsion is reduced by its strong electron-attracting ability, lowering the HOMO level. In contrast, NMe 2 increases HOMO energy levels by a stronger electron donating ability, with the electron cloud concentrated predominantly at the left benzopyran moiety of the molecule. The spatial electron distribution of HOMO and LUMO shows that both AIE groups show significant electron separation due to the strong Intramolecular Charge Transfer (ICT) effect.
To elucidate the interactions and packing of BT-SP-NO 2 and BT-SP-NMe 2 in the crystalline state, to illustrate their large differences in solid state fluorescence quantum yield (fig. 8B), the present example also analyzed their crystal structure. They all use an alternating antiparallel packing pattern in the lattice. NO 2 and NMe 2 significantly affect the crystal structure of both molecules, resulting in significant differences in molecular packing, which may be responsible for their different fluorescence quantum yields in the solid state. The presence of NMe 2 resulted in a reduction of about 10 ° in dihedral angle between benzopyran and indole moieties compared to the NO 2 substituent (fig. 9). This resulted in pi-pi stacking interactions of the BTD portion of BT-SP-NMe 2, which were not observed at the BTD position of BT-SP-NO 2 (fig. 8B). In addition, four SP molecules within the crystal unit cell were selected and connected to their four central helical carbon atoms, forming two parallel spatial quadrilateral planes (fig. 10). Comparing the length, width and diagonal length of these planes, it is noted that the NMe 2 substituent results in a decrease in width and an increase in length. This suggests a more compact molecular arrangement, effectively restricting molecular movement. This may be responsible for the high fluorescence quantum yield observed in the solid state for BT-SP-NMe 2 compared to BT-SP-NO 2.
Example 7: culture and imaging of HeLa cells
Cervical cancer (HeLa) cells were passaged in high-sugar (H-DMEM) medium containing 10% Fetal Bovine Serum (FBS) and 1% diabodies (penicillin and streptomycin), the environment in the incubator being 37℃and containing 5% CO 2.
Before imaging, hela cells with proper concentration are inoculated into a confocal culture dish, a proper prepared culture medium is added, the mixture is cultured in an incubator for 1 to 2 days, and after the cells are attached to the wall, a confocal imaging experiment can be performed. The dye synthesized was dissolved in dimethyl sulfoxide (DMSO) and configured to a suitable concentration of stock solution (1 mM) for later use in cell staining.
During staining, fresh culture medium and 0.5. Mu.L of BT-SP-NO 2 or 1. Mu.L of SP-NMe 2 mother solution are added into the centrifuge tube, and the mixture is shaken uniformly. Before imaging, the mixed solution was changed to the original medium in a confocal dish and cultured in an incubator containing 5% CO 2 at 37℃for 30min. After the staining is finished, the culture dish is washed 2 to 3 times by using Phosphate Buffer Solution (PBS), and 1mL of fresh culture medium is added to maintain the cell activity, so that the imaging can be directly performed under a Confocal Laser Scanning Microscope (CLSM).
Co-staining with Lipi-Deep Red, BT-SP-NO 2 (0.5. Mu.M) or BT-SP-NMe 2 (1. Mu.M) was incubated with 200nM Lipi-Deep Red for 30min. After staining was completed, residual dye was washed with PBS, 1mL fresh medium was added to maintain cell viability, and imaging was performed directly under Confocal Laser Scanning Microscopy (CLSM). The in situ fluorescence spectrum is similar to that of the low polarity solvent (fig. 11). The fluorescence channel of BT-SP-NO 2 or BT-SP-NMe 2 showed good overlap with the fluorescence channel of Lipi-Deep Red, with respective pearson coefficients of 0.84 and 0.89 calculated (FIG. 12). These data demonstrate that BT-SP-NO 2 and BT-SP-NMe 2 probes in HeLa cells stained lipid droplets in a wash-free manner with high signal-to-noise and selectivity.
Example 7: toxicity test of BT-SP-NO 2 and BT-SP-NMe 2
To test the effect of the dye on Hela cell viability, the standard thiazole blue (MTT) method was chosen for evaluation. The main principle of the method is that succinic dehydrogenase in cell mitochondria can reduce exogenous MTT into water-insoluble blue-violet crystalline formazan and deposit the blue-violet crystalline formazan in cells, and dead cells have no function.
First, heLa cells were inoculated in 96-well plates and cultured in an incubator at 37℃for 24 hours. Subsequently, the mixed solution of fresh medium and different concentrations of dye (0, 2, 4, 6, 8, 10. Mu.M) was changed and the cell culture was continued for 24h. Then, the medium was replaced again with a mixed solution of MTT (5 mg/mL) and medium (MTT/DMEM=1:5) just prepared, and the culture was continued for 3 hours. After completion, the medium in each well was replaced with DMSO, and after beating uniformly, absorbance at 490nm was measured using an enzyme-labeled instrument. Cell viability (Cell viability) was calculated according to the following disclosure:
Cell viability＝(A1-A0)/(A2-A0).
A 0 is absorbance of a blank without cells inoculated in a 96-well plate, a 1 is absorbance of an experimental group after 24 hours of dye treatment, and a 2 is absorbance of a control group cultured with only medium without dye treatment.
The results are shown in FIG. 13, cell viability of HeLa cells incubated with different concentrations of the compounds for 24 hours. The experimental results showed that after 24 hours incubation of cells with 10 μm probe, the cell viability was still as high as 85%, indicating a higher biocompatibility of the probe.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The 2,1, 3-benzothiadiazole and spiropyran conjugated fluorescent compound is characterized in that the chemical structure is shown in a formula I:
Wherein R 1 is selected from a C 1-C6 linear or branched hydrocarbyl, an ether group, or a C 1-C6 alkyl substituted acyl; r 2 is hydrogen, methyl, substituted alkyl, fluoro, chloro, bromo, iodo, cyano, trihalomethyl, nitro or dimethylamino.
2. The conjugated 2,1, 3-benzothiadiazole and spiropyran fluorescent compound of claim 1, selected from the group consisting of:
3. A process for the preparation of a conjugated 2,1, 3-benzothiadiazole and spiropyran fluorescent compound according to claim 1 or 2, characterized by comprising the step of obtaining a compound of formula I according to the following reaction scheme;
4. A process for the preparation of a fluorescent compound conjugated with 2,1, 3-benzothiadiazole and spiropyran as claimed in claim 3, wherein compound 1 is alkylated to form compound 2, compound 2 is then condensed with compound 3 by Knoevenagel to give spiropyran derivative 4, and finally compounds 4 and 5 are subjected to a simple Suzuki coupling reaction to give final compound I.
5. The method for preparing the 2,1, 3-benzothiadiazole and spiropyran conjugated fluorescent compound according to claim 4, wherein the reaction conditions for preparing the compound 2 from the compound 1 are as follows: the temperature is 60-80 ℃ and the reaction time is 5-7 h; or acetonitrile is used as a solvent in a reaction system;
Alternatively, the reaction conditions for Knoevenagel condensation are: the temperature is 60-80 ℃ and the reaction time is 4-6 h; or piperidine is added as a catalyst in the reaction process; or ethanol is used as a solvent in the reaction system;
or, the reaction conditions of the Suzuki coupling reaction are as follows: the temperature is 95-120 ℃, and the reaction time is 23-25 h; or adding tetraphenylphosphine palladium as a catalyst in the reaction process; or toluene and water are used as solvents in the reaction system.
6. Use of a fluorescent compound conjugated with 2,1, 3-benzothiadiazole and spiropyran according to claim 1 or 2 in a fluorescent probe.
7. A composition comprising a2, 1, 3-benzothiadiazole conjugated to a spiropyran conjugated fluorescent compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof.
8. A formulation comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a 2,1, 3-benzothiadiazole and spiropyran conjugated fluorescent compound according to claim 1 or 2 or a composition according to claim 7.
9. Use of a2, 1, 3-benzothiadiazole conjugated with spiropyran conjugated fluorescent compound of claim 1 or 2, a composition of claim 7 or a formulation of claim 8 in lipid-specific fluorescent imaging.
10. A lipid droplet detection kit comprising a2, 1, 3-benzothiadiazole conjugated to a spiropyran fluorescent compound according to claim 1 or 2, a composition according to claim 7 or a formulation according to claim 8, and a solvent or diluent.
CN202410148403.4A 2024-01-31 2024-01-31 2,1, 3-Benzothiadiazole and spiropyran conjugated fluorescent compound and application thereof in lipid drop imaging Pending CN118047787A (en)

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