CN113264866A - Non-fluorescent organic small molecular compound, preparation method of pentamethyl cyanine dye and application of pentamethyl cyanine dye - Google Patents
Non-fluorescent organic small molecular compound, preparation method of pentamethyl cyanine dye and application of pentamethyl cyanine dye Download PDFInfo
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- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 title claims abstract description 35
- 150000001875 compounds Chemical class 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims description 8
- 238000000799 fluorescence microscopy Methods 0.000 claims abstract description 27
- 230000003834 intracellular effect Effects 0.000 claims abstract description 15
- 210000003712 lysosome Anatomy 0.000 claims abstract description 12
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- 238000003384 imaging method Methods 0.000 claims abstract description 10
- 210000004027 cell Anatomy 0.000 claims description 67
- -1 small molecule compound Chemical class 0.000 claims description 21
- 238000005286 illumination Methods 0.000 claims description 20
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- 150000002148 esters Chemical class 0.000 claims description 9
- 229910052739 hydrogen Inorganic materials 0.000 claims description 7
- 239000001257 hydrogen Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 6
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 6
- 201000010881 cervical cancer Diseases 0.000 claims description 6
- 229910052736 halogen Inorganic materials 0.000 claims description 5
- 150000002367 halogens Chemical class 0.000 claims description 5
- 230000006698 induction Effects 0.000 claims description 5
- 239000003960 organic solvent Substances 0.000 claims description 5
- GHAZCVNUKKZTLG-UHFFFAOYSA-N N-ethylsuccinimide Chemical compound CCN1C(=O)CCC1=O GHAZCVNUKKZTLG-UHFFFAOYSA-N 0.000 claims description 4
- 230000001464 adherent effect Effects 0.000 claims description 4
- 125000003118 aryl group Chemical group 0.000 claims description 4
- 238000011065 in-situ storage Methods 0.000 claims description 4
- 210000005229 liver cell Anatomy 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 229910052717 sulfur Inorganic materials 0.000 claims description 4
- OVJRWHNWCJFPBL-UHFFFAOYSA-N 3-benzylpyrrolidine-2,5-dione Chemical compound O=C1NC(=O)CC1CC1=CC=CC=C1 OVJRWHNWCJFPBL-UHFFFAOYSA-N 0.000 claims description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical group [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 3
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical compound C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 claims description 3
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 3
- 229910052794 bromium Inorganic materials 0.000 claims description 3
- 229910052801 chlorine Inorganic materials 0.000 claims description 3
- 229910052731 fluorine Inorganic materials 0.000 claims description 3
- 229910052740 iodine Inorganic materials 0.000 claims description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
- 229910052711 selenium Inorganic materials 0.000 claims description 3
- 239000007810 chemical reaction solvent Substances 0.000 claims description 2
- IQZPDFORWZTSKT-UHFFFAOYSA-N nitrosulphonic acid Chemical group OS(=O)(=O)[N+]([O-])=O IQZPDFORWZTSKT-UHFFFAOYSA-N 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 abstract description 4
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- 230000033228 biological regulation Effects 0.000 abstract description 3
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- 230000002194 synthesizing effect Effects 0.000 abstract 1
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 18
- 239000007850 fluorescent dye Substances 0.000 description 10
- 238000011534 incubation Methods 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000005160 1H NMR spectroscopy Methods 0.000 description 6
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 6
- 230000007935 neutral effect Effects 0.000 description 6
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- 238000001514 detection method Methods 0.000 description 2
- 150000002431 hydrogen Chemical class 0.000 description 2
- OKKJLVBELUTLKV-VMNATFBRSA-N methanol-d1 Chemical compound [2H]OC OKKJLVBELUTLKV-VMNATFBRSA-N 0.000 description 2
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- 229960002317 succinimide Drugs 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 125000002672 4-bromobenzoyl group Chemical group BrC1=CC=C(C(=O)*)C=C1 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- SIKJAQJRHWYJAI-UHFFFAOYSA-N benzopyrrole Natural products C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
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- HEDRZPFGACZZDS-MICDWDOJSA-N deuterated chloroform Substances [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
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- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
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- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
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- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
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- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
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- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
- C09B23/083—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines five >CH- groups
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Abstract
The invention discloses a non-fluorescent organic small molecular compound, a pentamethyl cyanine dye and application thereof. The non-fluorescent organic small molecular compound is induced by LED white light, a condensing agent and a photosensitizer are not used for directly synthesizing the pentamethine cyanine dye with wide biological application, and the non-fluorescent organic small molecular compound is changed to obtain the near-infrared fluorescent pentamethine cyanine functional dye with various structures. The synthesis method can be applied to intracellular near-infrared fluorescence imaging, realizes intracellular space-time controllable near-infrared fluorescence imaging by utilizing the regulation and control of visible light, and can be used for space-time resolution imaging of intracellular lysosomes.
Description
Technical Field
The invention relates to a micromolecule compound and application thereof, in particular to a preparation method and application of a non-fluorescent organic micromolecule compound and pentamethyl cyanine dye.
Background
In recent years, fluorescence imaging technology has been rapidly developed and widely used in the fields of chemistry, biology, biomedicine and microscopic imaging. Cyanine dye is a near-infrared fluorescent dye, and the synthesis method is complex. The development of a synthetic method different from the traditional pentamethine cyanine dye and the application of the synthetic method in the field of low-background and space-time controllable fluorescence imaging have not been researched by people. In order to perform real-time tracking, qualitative and quantitative detection on important signal molecules or chemical processes in cells, researchers design a large number of fluorescence imaging biological probes, so that chemical and biological processes at a cell level are better explained. At present, the design and development of fluorescence imaging systems are limited by sensitivity, stability, background fluorescence, space/time controllability, fluorescence turn-on controllability, and the like. Cyanine dyes (cyanine dyes) are widely applied to the fields of chemical analysis, biological detection, photosensitive materials and the like as a class of functional dyes, and have the advantages of high molar extinction coefficient, adjustable spectral range, high sensitivity and the like. However, the synthesis of cyanine dyes (such as pentamethyl cyanine dyes) usually requires higher reaction temperature, catalysis by strong acid/base, and participation of unsaturated condensing agents.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a non-fluorescent organic small molecular compound and a pentamethine cyanine dye synthesized by the organic small molecular compound under the condition of LED white light illumination.
The technical scheme is as follows: the invention provides a non-fluorescent organic small molecule compound, which has the following structure:
wherein X is C (CH)3)2O, S or Se;
y is F, Cl, Br or I;
z is hydrogen or an aromatic group;
R1is hydrogen, halogen, methyl, arylA fragrance group, a nitro group, a sulfonic group or a benzyl group;
R2is amino, hydroxyl or halogen, and m is 0-18.
Further, 1-propylamine-2, 3, 3-trimethylindole is used.
Furthermore, after the cyanine dye fluorescent molecule is incubated with adherent cells, cyanine dye fluorescent molecules can be generated in situ in the cells through induction of LED white light illumination, so that intracellular space-time controllable fluorescence imaging is realized, and the cyanine dye fluorescent molecule can be used for space-time resolution imaging of lysosomes in the cells. The cell state is observed through a laser scanning confocal microscope, and the practicability of the LED white light illumination method for realizing the space-controllable fluorescence imaging in the cell is determined through the change of the fluorescence intensity in the cell before and after illumination.
Further, the adherent cells are human cervical cancer cells or human normal liver cells. The red fluorescence generated in human cervical cancer cells (HeLa cells) or human normal liver cells (QSG cells) is mainly located in lysosomes in the cells and can be used for space-time resolution near-infrared fluorescence imaging of the lysosomes in the cells.
The application of the organic small molecule compound in the field of fluorescence imaging.
A preparation method of pentamethyl cyanine dye, which is obtained by mixing the non-fluorescent organic small molecular compound of claim 1 or 2 with 2-thiophene ethyl-succinimide active ester, benzyl-succinimide active ester or 3-indole ethyl-succinimide active ester, reacting under the condition of triethylamine, and inducing by LED white light illumination.
The structural general formula of the pentamethyl cyanine dye is as follows:
wherein A is O, S, Se or N, and X is C (CH)3)2O, S or Se, Y is F, Cl, Br or I, Z is hydrogen or an aromatic radical, R is a hydrogen atom1Is hydrogen, halogen, methyl, aryl, nitro, sulfonic acid or benzyl, R2Is formyl, 2-thiopheneacetyl, benzoyl, 4-bromobenzoyl, isonicotinyl, indoleacetyl, m is 0-18。
Further, the reaction solvent is an organic solvent.
The application of the preparation method of the pentamethine cyanine dye in the field of fluorescence imaging.
Has the advantages that: the invention designs a novel synthesis method of the pentamethine cyanine dye (the operation steps are simplified, and the green chemical principle is better met), applies the process to intracellular fluorescence imaging, and utilizes the regulation and control of visible light so as to realize the purpose of intracellular space-time controllable fluorescence imaging. The near-infrared fluorescent pentamethine cyanine dye with wide biological application can be directly synthesized by the induction of LED white light illumination without using a condensing agent. Compared with the traditional synthesis method, the method simplifies the operation steps, and adopts LED light illumination to induce reaction without using a condensing agent and a photosensitizer, thereby conforming to the green chemical principle. By changing the non-fluorescent organic small molecular compound, the near-infrared fluorescent pentamethine cyanine functional dye with various structures can be obtained. The fluorescent-free organic small molecular compound and human cervical cancer cells or human normal liver cells are incubated together, and the fluorescence enhancement in the cells can be realized after the LED white light illumination, but almost no fluorescence in the cells is not illuminated. The product obtained by the LED white light illumination induction method can be applied to intracellular fluorescence imaging, the intracellular space-time controllable fluorescence imaging is realized by utilizing the regulation and control of visible light, the red fluorescence generated in the cell is gradually increased along with the extension of illumination time, and the generated red fluorescence is mainly positioned in lysosomes in the cell and can be used for space-time resolution imaging of the lysosomes in the cell.
Drawings
FIG. 1 is a drawing showing the preparation of 2-thienylethyl-Cy 5 compound prepared in example 1 of the present invention1H NMR 400M nuclear magnetic map;
FIG. 2 is an MS mass spectrum of compound 2-thienylethyl-Cy 5 prepared in example 1 of the present invention;
FIG. 3 shows a scheme for preparing a compound of benzyl-Cy 5 according to the present invention in example 21H NMR 400M nuclear magnetic map;
FIG. 4 is an MS mass spectrum of a compound prepared in example 2 of the present invention, benzyl-Cy 5;
FIG. 5 shows the present inventionPreparation of Compound 3-indoleethane-Cy 5 prepared in example 31H NMR 400M nuclear magnetic map;
FIG. 6 is an MS mass spectrum of compound 3-indoleethane-Cy 5 prepared in example 3 of the present invention;
FIG. 7 is a confocal laser scanning microscopy image of light-induced HeLa intracellular time-controlled fluorescence imaging in example 4 of the present invention;
FIG. 8 is a confocal micrograph of time-controlled fluorescence imaging in the optically induced QSG hepatocytes of example 4 of the present invention;
FIG. 9 is a confocal micrograph of laser scanning with co-localization of light-induced three-dimensional fluorescence in HeLa cells in accordance with example 5 of the present invention;
FIG. 10 is a confocal laser scanning micrograph of fluorescence co-localization imaging of mitochondria and lysosomes in light-induced HeLa cells in example 5 of the present invention;
FIG. 11 is a confocal micrograph of laser scanning for light-induced HeLa intracellular space controllable fluorescence co-localization slice scan imaging in accordance with example 5 of the present invention;
FIG. 12 is a graph showing the intensity analysis of light-induced HeLa intracellular space-controlled fluorescence co-localization imaging in example 5 of the present invention;
FIG. 13 is a confocal laser scanning micrograph of light-induced intracellular time-controlled fluorescence imaging in example 6 of the present invention.
Detailed Description
Example 1
Preparing the pentamethine cyanine dye by photoinduction: 2-thiophene ethyl-Cy 5 indole compound with amino side chain structure
(1) 1-Propylamine-2, 3, 3-trimethylindole (75.6mg), 2-thiopheneethyl-succinimide active ester (23.9mg) was charged into a 25mL single-neck flask, followed by 5mL HCl356 μ L of triethylamine, the solid was dissolved completely by ultrasound, heated to 65 ℃, and the mixture was refluxed for 12h using a white LED lamp, then cooled to room temperature, the organic solvent was dried, dichloromethane and neutral alumina were added for dry sample mixing, and the product was further purified by neutral alumina chromatography (eluent ratio dichloromethane: methanol 50: 1). The reaction yield was 8%, yielding 2-thienylethyl-Cy 53.2 mg. FIG. 1 shows the fluorescent molecule of pentamethylcyanine dye (2-thienylethyl-Cy 5) synthesized in step (1) of example 11H-NMR spectrum.1HNMR(400MHz，CDCl3δ)：8.33(t，J＝5.2Hz，2H)，7.74(t，J＝13.0Hz，2H)，7.37-7.31(m，6H)，7.20(dd，J＝9.1，4.8Hz，5H)，7.04(d，J＝7.9Hz，2H)，6.54(d，J＝13.5Hz，2H)，4.09-3.99(m，4H)，3.79(s，4H)，3.48(dd，J＝11.7，5.7Hz，4H)，2.03(dt，J＝13.9，6.9Hz，4H)，1.66(s，12H).
FIG. 2 is a MS mass spectrum of the fluorescent molecule of pentamethylcyanine dye (2-thienylethyl-Cy 5) synthesized in step (1) of example 1.
Example 2
Preparing the pentamethine cyanine dye by photoinduction: benzyl-Cy 5
(1) 1-Propylamine-2, 3, 3-trimethylindole (75.6mg), benzyl-succinimide active ester (21.9mg) was charged into a 25mL single-neck flask, followed by 5mL HCl356 μ L of triethylamine, the solid was dissolved completely by ultrasound, heated to 65 ℃, and the mixture was refluxed for 12h using a white LED lamp, then cooled to room temperature, the organic solvent was dried, dichloromethane and neutral alumina were added for dry sample mixing, and the product was further purified by neutral alumina chromatography (eluent ratio dichloromethane: methanol 50: 1). The reaction yield was 6%, giving benzyl-Cy52.3mg. FIG. 3 is a diagram showing the fluorescent molecule (benzyl-Cy 5) of the pentamethylcyanine dye synthesized in the step (1) of example 21H-NMR spectrum.1H NMR (400MHz, MeOD. delta.): 8.19(t, J ═ 3.1Hz, 2H), 7.87-7.83(m, 4H), 7.58-7.54(m, 2H), 7.51-7.46(m, 6H), 7.38(dd, J ═ 6.5, 5.5Hz, 2H), 7.29(dd, J ═ 12.2, 7.7Hz, 4H), 6.22(t, J ═ 12.4Hz, 1H), 6.14(d, J ═ 13.7Hz, 2H), 4.23-4.16(m, 4H), 3.54(t, J ═ 7.0Hz, 4H), 2.14(dd, J ═ 14.3, 7.1Hz, 4H), 1.73(d, J ═ 13.8, 12H), fig. 4 shows the fluorescence spectrum of cyanine dye synthesized in example 2 (Cy — 25) in example 2 (Cy).
Example 3
Preparing the pentamethine cyanine dye by photoinduction: 3-indoleethane-Cy 5
(1) 1-propylamine-2, 3, 3-trimethylindole(75.6mg), 3-Indoleethyl-succinimide active ester (27.2mg) was charged into a 25mL single-neck flask, followed by 5mL of HCl356 μ L of triethylamine, the solid was dissolved completely by ultrasound, heated to 65 ℃, and the mixture was refluxed for 12h using a white LED lamp, then cooled to room temperature, the organic solvent was dried, dichloromethane and neutral alumina were added for dry sample mixing, and the product was further purified by neutral alumina chromatography (eluent ratio dichloromethane: methanol 50: 1). The reaction yield was 6%, and 3-indoleethane-Cy52.6mg was obtained. FIG. 5 shows the fluorescent molecule (3-indolylethyl-Cy 5) synthesized in step (1) of example 2 using a pentamethylcyanine dye1H-NMR spectrum.1H NMR (400MHz, MeOD. delta.): examples of dyes include cyanine dyes, dye, cyanine dyes, dye 16-8.03(m, 2H), 7.52(t, J, 7.9.9 Hz, 2H), 7.38-7.33(m, 2H), 7.27 (m, 8.0Hz, 2H), 7.27 (m, H), cyanine dyes, and dye molecules, and dye molecules are shown in fig. 16.16.16.16-8.03 (13.03, 7.3.52, 7.3.3.3.3.3.3.3.3.3.3.3.3.3.3.33, 7.33, 7.0-7.0.0-7.0.0.3.3.3.0 (7.3.3H), 7.0.0-7.3H), 7.0 (7.0H), 7.7.7.0H), 7.7.7.7.7.0H), 7.7.1 Hz, 7.7H), 7.1H), 7H), 7.01(d, 7.7.7.7.01 (d, 7.7.7.7.7.01 (d, 7.7H), 7H), 7.0H), 7H), 7.7.7.7.7.7.01 (d, 7H), 7.1H), 7.7.7.7.7.7.7.1H), 7.7.7.7.7.7.7.7.7.01 (d, 7.7.7.7.7.7.1H), 7.1H), 7.7.7.7.01 (d, 7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.7.1H), 7.7.7.7.7.7.7.7.7.7.7.1H), 7.7.7.7.7.7.7.7.7.7.7.7.7.15.
Example 4
Photoinduced intracellular time-controllable fluorescence imaging
Incubating raw material 1-propylamine-2, 3, 3-trimethylindole with human cervical carcinoma cell Hela cell or human normal hepatocyte QSG cell, wherein the incubation time of the raw material 1-propylamine-2, 3, 3-trimethylindole with the cell is 30min, after the incubation is finished, a fresh culture medium is replaced, then LED white light illumination is used, the incubation temperature is 37 ℃, and the cell state is observed through a laser scanning confocal microscope: the excitation wavelength is 633nm, and the receiving wavelength is 650 nm-750 nm. The fluorescence intensity in the cells is regulated and controlled by controlling different illumination time: 5min, 15min and 30 min. FIG. 7 is a picture obtained by observing the cell state of human cervical cancer cell Hela through a laser scanning confocal microscope with different illumination times controlled at 5min, 15min and 30min in example 4, FIG. 8 is a picture obtained by observing the cell state of human normal hepatocyte QSG through a laser scanning confocal microscope with different illumination times controlled at 5min, 15min and 30min in example 4,
as can be seen from FIGS. 7 and 8, 1-propylamine-2, 3, 3-trimethylindole was incubated with Hela cells or QSG cells, no fluorescence was observed in the Hela cells and QSG cells in the control group which were not illuminated, and then white light was applied to the Hela cells and QSG cells in the control group which were not illuminated, the incubation temperature was 37 ℃, red fluorescence appeared in the cells after illumination, and with the increase of illumination time, the fluorescence intensity in the cells was gradually increased, and the red fluorescence was more significant and was more significant compared with the cells in the control group which were not illuminated.
Example 5
Light-induced intracellular space controllable fluorescence imaging
Incubating raw material 1-propylamine-2, 3, 3-trimethylindole with human cervical carcinoma cell Hela cell for 30min, replacing a fresh culture medium after incubation is finished, then illuminating with LED white light for 30min at 37 ℃, and then incubating lysosome green fluorescent probe with Hela cell, or incubating with mitochondria green fluorescent probe after illumination is finished, or incubating with Golgi green fluorescent probe for 60min, and replacing the fresh culture medium after incubation is finished. Cell status was observed by laser scanning confocal microscopy: the Cy5 channel excitation wavelength is 633nm, the receiving wavelength is 650 nm-750 nm, the green fluorescent probe channel excitation wavelength is 488nm, and the receiving wavelength is 500 nm-600 nm. FIG. 9 is a photograph obtained by co-incubating light-induced cells with lysosomal fluorescent probes and observing the state of the cells by laser scanning confocal microscope in example 5. FIG. 10 is a photograph obtained by incubating light-induced cells with a mitochondrial fluorescent probe and a Golgi fluorescent probe and observing the state of the cells by a laser scanning confocal microscope in example 5. FIG. 11 is a photograph obtained by co-incubating light-induced cells and lysosomal fluorescent probe and observing the state of the cells by laser scanning confocal microscope layer-by-layer scanning in example 5. FIG. 12 is a photograph of intensity analysis of co-localized fluorescence imaging obtained by co-incubation of light-induced cells with lysosomal fluorescent probes and observation of the state of the cells by laser scanning confocal microscopy in a layered scan in example 5.
As can be seen from fig. 9, fig. 10, fig. 11 and fig. 12, the red fluorescence generated in Hela cells of human cervical cancer cells after light induction is mainly located in lysosomes in the cells, but not in cell nuclei, or mitochondria and golgi organelles, and the imaging method is proved to be applicable to the space-time resolution imaging of lysosomes in the cells.
Example 6
Stability of light-induced intracellular in-situ fluorescence imaging
Incubating raw material 1-propylamine-2, 3, 3-trimethylindole with human cervical carcinoma cells (Hela cells), wherein the incubation time of the raw material 1-propylamine-2, 3, 3-trimethylindole and the Hela cells is 30min, after the incubation is finished, replacing a fresh culture medium, illuminating by using LED white light, the incubation temperature is 37 ℃, and then observing the cell state by a laser scanning confocal microscope at 3h, 15h, 24h, 48h and 72 h: the excitation wavelength is 633nm, and the receiving wavelength is 650 nm-750 nm.
FIG. 13 is a photograph obtained by observing the state of cells with a laser scanning confocal microscope at 3h, 15h, 24h, 48h and 72h after 30min of light irradiation in example 6.
As can be seen from FIG. 13, the red fluorescence in the cells after 30min of illumination is not significantly reduced after 24h, and the red fluorescence can still be observed even after 72h, which indicates that the in situ fluorescence imaging method has good stability and can be used for long-term intracellular lysosome fluorescence imaging.
Claims (8)
1. A non-fluorescent organic small molecule compound has the following structure:
wherein X is C (CH)3)2O, S or Se;
y is F, Cl, Br or I;
z is hydrogen or an aromatic group;
R1hydrogen, halogen, methyl, aryl, nitro, sulfonic acid group or benzyl;
R2is amino, hydroxyl or halogen, and m is 0-18.
2. The non-fluorescent small organic molecule compound of claim 1, wherein: is 1-propylamine-2, 3, 3-trimethylindole.
3. The non-fluorescent small organic molecule compound of claim 1, wherein: after the cyanine dye fluorescent molecule is incubated with adherent cells, cyanine dye fluorescent molecules can be generated in situ in the cells through induction of LED white light illumination, so that intracellular space-time controllable fluorescence imaging is realized, and the cyanine dye fluorescent molecule can be used for space-time resolution imaging of intracellular lysosomes.
4. The non-fluorescent small organic molecule compound of claim 3, wherein: the adherent cells are human cervical cancer cells or human normal liver cells.
5. Use of the organic small molecule compound of claim 1 in the field of fluorescence imaging.
6. A preparation method of pentamethyl cyanine dye is characterized in that: the fluorescent-free organic small molecular compound as claimed in any one of claims 1 to 4 is mixed with 2-thiophene ethyl-succinimide active ester, benzyl-succinimide active ester or 3-indole ethyl-succinimide active ester, reacted under the condition of triethylamine, and then induced by LED white light illumination to obtain the fluorescent-free organic small molecular compound.
7. The method for preparing pentamethylcyanine dye according to claim 6, characterized in that: the reaction solvent is an organic solvent.
8. The use of the process for the preparation of a pentamethylcyanine dye as claimed in claim 6 in the field of fluorescence imaging.
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