CN110317199B - Alpha-pinene-based nuclear fluorescent probe and preparation method and application thereof - Google Patents
Alpha-pinene-based nuclear fluorescent probe and preparation method and application thereof Download PDFInfo
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- CN110317199B CN110317199B CN201910675545.5A CN201910675545A CN110317199B CN 110317199 B CN110317199 B CN 110317199B CN 201910675545 A CN201910675545 A CN 201910675545A CN 110317199 B CN110317199 B CN 110317199B
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- GRWFGVWFFZKLTI-UHFFFAOYSA-N α-pinene Chemical compound CC1=CCC2C(C)(C)C1C2 GRWFGVWFFZKLTI-UHFFFAOYSA-N 0.000 title claims abstract description 91
- GRWFGVWFFZKLTI-IUCAKERBSA-N 1S,5S-(-)-alpha-Pinene Natural products CC1=CC[C@@H]2C(C)(C)[C@H]1C2 GRWFGVWFFZKLTI-IUCAKERBSA-N 0.000 title claims abstract description 45
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 43
- MVNCAPSFBDBCGF-UHFFFAOYSA-N alpha-pinene Natural products CC1=CCC23C1CC2C3(C)C MVNCAPSFBDBCGF-UHFFFAOYSA-N 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims abstract description 19
- 210000003855 cell nucleus Anatomy 0.000 claims abstract description 15
- KMRMUZKLFIEVAO-UHFFFAOYSA-N 7,7-dimethylbicyclo[3.1.1]hept-3-ene-4-carbaldehyde Chemical compound C1C2C(C)(C)C1CC=C2C=O KMRMUZKLFIEVAO-UHFFFAOYSA-N 0.000 claims abstract description 9
- KMRMUZKLFIEVAO-RKDXNWHRSA-N Myrtenal Natural products C1[C@H]2C(C)(C)[C@@H]1CC=C2C=O KMRMUZKLFIEVAO-RKDXNWHRSA-N 0.000 claims abstract description 9
- FOCAUTSVDIKZOP-UHFFFAOYSA-N chloroacetic acid Chemical compound OC(=O)CCl FOCAUTSVDIKZOP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229940106681 chloroacetic acid Drugs 0.000 claims abstract description 8
- 210000004940 nucleus Anatomy 0.000 claims abstract description 8
- PTVZQOAHCSKAAS-UHFFFAOYSA-N 4-methyl-3-thiosemicarbazide Chemical compound CNC(=S)NN PTVZQOAHCSKAAS-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000003384 imaging method Methods 0.000 claims abstract description 5
- 238000011065 in-situ storage Methods 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 24
- 239000012074 organic phase Substances 0.000 claims description 15
- 238000010992 reflux Methods 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 239000012043 crude product Substances 0.000 claims description 14
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
- 239000001632 sodium acetate Substances 0.000 claims description 9
- 235000017281 sodium acetate Nutrition 0.000 claims description 9
- 125000000814 indol-3-yl group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C([*])C2=C1[H] 0.000 claims description 8
- OLNJUISKUQQNIM-UHFFFAOYSA-N indole-3-carbaldehyde Chemical compound C1=CC=C2C(C=O)=CNC2=C1 OLNJUISKUQQNIM-UHFFFAOYSA-N 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- -1 1H-indole-3-yl Chemical group 0.000 claims description 4
- 239000013078 crystal Substances 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000007795 chemical reaction product Substances 0.000 claims description 3
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 claims description 3
- 239000003054 catalyst Substances 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 230000001413 cellular effect Effects 0.000 claims 1
- 239000000376 reactant Substances 0.000 claims 1
- 239000002994 raw material Substances 0.000 abstract description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 abstract 3
- SRVJKTDHMYAMHA-WUXMJOGZSA-N thioacetazone Chemical compound CC(=O)NC1=CC=C(\C=N\NC(N)=S)C=C1 SRVJKTDHMYAMHA-WUXMJOGZSA-N 0.000 abstract 2
- SLYRGJDSFOCAAI-UHFFFAOYSA-N 1,3-thiazolidin-2-one Chemical compound O=C1NCCS1 SLYRGJDSFOCAAI-UHFFFAOYSA-N 0.000 abstract 1
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 7
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 6
- 239000000523 sample Substances 0.000 description 5
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000012758 nuclear staining Methods 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010034972 Photosensitivity reaction Diseases 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 125000003447 alpha-pinene group Chemical group 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 238000010226 confocal imaging Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000004963 pathophysiological condition Effects 0.000 description 1
- 230000036211 photosensitivity Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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Abstract
The invention discloses an alpha-pinene-based cell nucleus fluorescent probe and a preparation method and application thereof. The invention uses natural renewable resource alpha-pinene derivative myrtenal as raw material, which is condensed with 4-methyl thiosemicarbazide to generate alpha-pinene thiosemicarbazone; cyclizing the alpha-pinene thiosemicarbazone with chloroacetic acid to obtain alpha-pinene thiazolidinone; the alpha-pinene thiazolidone is condensed with 3-indole formaldehyde to obtain the alpha-pinene nucleus fluorescent probe, the fluorescent probe can carry out specific in-situ imaging on the nucleus of a living cell, and emits green fluorescence under 390nm ultraviolet irradiation, and the fluorescent probe has good application prospect as a novel nucleus fluorescent probe.
Description
Technical Field
The invention belongs to the technical field of fine organic synthesis, and relates to a novel alpha-pinene-based nuclear fluorescent probe, and a preparation method and application thereof.
Background
The nucleus plays a key role in various cell activities such as metabolism, reproduction and heredity, so the nuclear stain is an important means for researching the form and function of the nucleus. The nucleolus is an important sub-nuclear structure, and the dynamic form of the nucleolus reflects the pathophysiological condition. Existing nuclear stains such as DAPI and Hoechst suffer from photobleaching and self-quenching phenomena, limit applicability in long-term observation, and are expensive. Therefore, the development of a new generation of nuclear fluorescent probe with low cytotoxicity and high photosensitivity is highly regarded.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects in the prior art, the invention aims to provide a novel nuclear fluorescent probe which can be used for nuclear staining, can emit good fluorescence under ultraviolet irradiation and is used for long-time nuclear staining. Another object of the present invention is to provide a method for preparing the nuclear fluorescent probe having the above function. The invention also aims to provide an application of the nuclear fluorescent probe.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the technical scheme that:
an alpha-pinene-based cell nucleus fluorescent probe, which has a structural formula as follows:
the preparation method of the alpha-pinene-based cell nucleus fluorescent probe comprises the following steps:
1) the myrtenal is condensed with 4-methyl thiosemicarbazide to obtain alpha-pinene-4-methyl thiosemicarbazone.
2) Cyclizing the alpha-pinene-4-methyl thiosemicarbazone and chloroacetic acid to obtain the alpha-pinene-thiazoline-4-ketone.
3) The alpha-pinene-based thiazoline-4-ketone and indole-3-formaldehyde are condensed to obtain the alpha-pinene-based nuclear fluorescent probe 5- (1H-indole-3-yl) methylene-2- (2- (6, 6-dimethylbicyclo [3.1.1] hept-2-ene-2-yl) methylene) hydrazino-3-methylthiazoline-4-ketone. The reaction formula is as follows:
in the step 1), the myrtenal and the 4-methyl thiosemicarbazide are condensed to obtain the alpha-pinenyl-4-methyl thiosemicarbazone, and the specific preparation method comprises the following steps:
(1) adding 10mmol of myrtenal, 9-12 mmol of 4-methyl thiosemicarbazide, 3mL of 10% hydrochloric acid aqueous solution and 10mL of ethanol into a three-neck flask with a stirrer, a thermometer and a reflux condenser in sequence, and reacting at 80-90 ℃.
(2) Extracting the reaction product with 60mL ethyl acetate for 3 times, combining organic phases, and washing the organic phases with saturated saline solution to be neutral; drying the organic phase by anhydrous sodium sulfate, filtering, concentrating and recovering the solvent to obtain a crude product of the alpha-pinene-4-methyl thiosemicarbazone, and washing and drying the crude product to obtain the alpha-pinene-4-methyl thiosemicarbazone.
In the step 2), under the catalysis of sodium acetate, the alpha-pinene-4-methyl thiosemicarbazone reacts with chloroacetic acid to obtain the alpha-pinene-thiazoline-4-ketone, and the specific preparation method comprises the following steps:
(1) adding 5mmol of alpha-pinene-4-methyl thiosemicarbazone, 5-6 mmol of chloroacetic acid, 2-4 mol of sodium acetate and 20mL of ethanol into a three-neck flask with a stirrer, a thermometer and a reflux condenser in sequence, heating and refluxing for 12 hours, detecting by TCL tracking, and stopping the reaction until the conversion rate of the alpha-pinene-4-methyl thiosemicarbazone reaches 95%.
(2) The reaction was extracted 3 times with 300mL dichloromethane, the organic phases were combined and washed to neutrality with saturated brine; drying the organic phase by anhydrous sodium sulfate, filtering, concentrating and recovering the solvent to obtain the crude product of the alpha-pinene thiazoline-4-ketone.
(3) Recrystallizing the crude product of the alpha-pinene-thiazoline-4-ketone by using ethanol to obtain white powdery alpha-pinene-thiazoline-4-ketone.
In the step 3), sodium acetate is used as a catalyst, and the alpha-pinene thiazoline-4-one and indole-3-formaldehyde are condensed to obtain the alpha-pinene nucleus fluorescent probe 5- (1H-indole-3-yl) methylene-2- (2- (6, 6-dimethylbicyclo [3.1.1] hept-2-ene-2-yl) methylene) hydrazino-3-methylthiazoline-4-one yellow crystal. The preparation method comprises the following steps:
(1) sequentially adding 5mmol of alpha-pinene thiazoline-4-one, 4-5.5 mmol of indole-3-formaldehyde, 2-4 mol of acetic acid and 20-50 mL of ethanol into a three-neck flask with a stirrer, a thermometer and a reflux condenser, and heating and refluxing for 6-18 h.
(2) The reaction mass is cooled to room temperature, and a bright yellow powdery crude product of 5- (1H-indole-3-yl) methylene-2- (2- (6, 6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methylene) hydrazino-3-methylthiazoline-4-ketone is separated out.
(3) Recrystallizing the crude product of 5- (1H-indol-3-yl) methylene-2- (2- (6, 6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methylene) hydrazino-3-methylthiazoline-4-one in dichloromethane-methanol to obtain the alpha-pinene nuclear fluorescent probe 5- (1H-indol-3-yl) methylene-2- (2- (6, 6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methylene) hydrazino-3-methylthiazoline-4-one bright yellow crystal.
The alpha-pinene-based nuclear fluorescent probe can perform specific in-situ imaging on the nucleus of a living cell and emit green fluorescence under 390nm ultraviolet light.
Has the advantages that: compared with the prior art, the alpha-pinene cell nucleus fluorescent probe prepared by using the natural renewable resource alpha-pinene derivative myrtenal as the raw material can carry out specific in-situ imaging on the cell nucleus of a living cell and emit green fluorescence under the irradiation of 390nm ultraviolet light.
Drawings
FIG. 1 is a graph showing the effect of the concentration of α -pinene-based nuclear fluorescent probe on the survival rate of Hela cells.
FIG. 2 is a photograph of a fluorescent confocal image of Hela nuclei using an alpha-pinene-based nuclear fluorescence probe.
FIG. 3 is a photograph of fluorescence confocal images of Hela cell nucleus by hoechst33342, a commercial fluorescent probe for cell nucleus.
FIG. 4 is a superimposed image of fluorescent confocal imaging photographs of Hela nucleus by using the alpha-pinene-based nuclear fluorescent probe and hoechst33342 nuclear fluorescent probe.
Detailed Description
The present invention will be further described with reference to the following specific examples.
Example 1 preparation of alpha-pinene-based Nuclear fluorescent Probe
1) Preparation of alpha-pinenyl-4-methyl thiosemicarbazone:
sequentially adding 10mmol of myrtenal, 12mmol of p-4-methyl thiosemicarbazide, 3mL of 10% hydrochloric acid aqueous solution and 10mL of ethanol into a three-neck flask with a stirrer, a thermometer and a reflux condenser, and reacting at 85 ℃; extracting the reaction product with 60mL ethyl acetate for 3 times, combining organic phases, and washing the organic phases with saturated saline solution to be neutral; drying the organic phase by anhydrous sodium sulfate, filtering, concentrating and recovering the solvent to obtain a crude product of the alpha-pinene-4-methyl thiosemicarbazone, washing by water and drying to obtain the alpha-pinene-4-methyl thiosemicarbazone, wherein the yield is 80 percent and the purity is 99 percent. The characterization data of the product are as follows:1H NMR(400MHz，CDCl3)δ：10.42(s，1H)，7.63(s，1H)，7.32(s，1H)，6.02(s，1H)，3.21(d，J＝4.9Hz，3H)，2.87(t，J＝5.2Hz，1H)，2.50-2.39(m，3H)，2.15(s，1H)，1.34(s，3H)，1.12(d，J＝9.0Hz，1H)，0.78(s，3H).13C NMR(101MHz，CDCl3)δ：177.65，144.66，144.56，134.03，40.71，39.94，37.68，32.51，31.07，30.92，26.04，20.89.
2) preparation of alpha-pinene thiazoline-4-one:
sequentially adding 5mmol of alpha-pinene-4-methyl thiosemicarbazone, 6mmol of chloroacetic acid, 4mol of sodium acetate and 20mL of ethanol into a three-neck flask with a stirrer, a thermometer and a reflux condenser, heating and refluxing for reaction for 12 hours, tracking and detecting by TCL (thermal conductive liquid chromatography), and stopping the reaction until the conversion rate of the alpha-pinene-4-methyl thiosemicarbazone reaches 95%; the reaction was extracted 3 times with 300mL dichloromethane, the organic phases were combined and washed to neutrality with saturated brine; drying the organic phase by anhydrous sodium sulfate, filtering, concentrating and recovering the solvent to obtain a crude product of the alpha-pinene thiazoline-4-ketone, and recrystallizing by ethanol to obtain white powdery alpha-pinene thiazoline-4-ketone with the yield of 79.3 percent and the purity of 99.1 percent. The characterization data of the product are as follows:1H NMR(400MHz，Chloroform-d)δ：8.01(s，1H)，6.10(s，1H)，3.74(s，2H)，3.26(s，3H)，3.02(t，J＝5.7Hz，1H)，2.56-2.38(m，3H)，2.17(s，1H)，1.36(s，3H)，1.17(d，J＝9.0Hz，1H)，0.81(s，3H).13C NMR(101MHz，CDCl3)δ：172.09，162.86，159.45，146.21，135.25，40.72，40.24，37.64，32.72，32.38，31.17，29.70，26.08，20.92.
3) preparation of alpha-pinene-based nuclear fluorescent probe 5- (1H-indol-3-yl) methylene-2- (2- (6, 6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methylene) hydrazino-3-methylthiazoline-4-one:
adding 5mmol of alpha-pinene thiazoline-4-ketone, 5.5mmol of indole-3-formaldehyde, 2mmol of sodium acetate and 35mL of ethanol into a three-neck flask with a stirrer, a thermometer and a reflux condenser in sequence, heating and refluxing for 8 hours, then cooling to room temperature, separating out bright yellow powder, and then recrystallizing in dichloromethane-methanol to obtain the alpha-pinene nuclear fluorescent probe 5- (1H-indol-3-yl) methylene-2- (2- (6, 6-dimethyl bicyclo [ 3.1.1)]Hept-2-en-2-yl) methylene) hydrazino-3-methylthiazoline-4-one bright yellow crystal, the yield is 53.2 percent, and the purity is 99.6 percent. The characterization data of the product are as follows:1H NMR(700MHz，DMSO-d6)δ：11.98(s，1H)，8.15(s，1H)，7.95(s，1H)，7.87(d，J＝7.9Hz，1H)，7.80(d，J＝2.2Hz，1H)，7.52(d，J＝8.1Hz，1H)，7.25(t，J＝7.5Hz，1H)，7.19(t，J＝7.4Hz，1H)，6.30(s，1H)，3.30(s，3H)，3.04(t，J＝5.3Hz，1H)，2.54(dd，J＝5.5，3.1Hz，1H)，2.43(d，J＝19.6Hz，1H)，2.17(s，1H)，1.39(s，3H)，1.13(d，J＝8.9Hz，1H)，0.80(s，3H).13C NMR(176MHz，DMSO-d6)δ：166.47，159.47，158.79，145.79，136.53，136.28，128.67，127.21，123.35，122.49，121.24，118.74，115.39，112.78，111.08，40.62，37.73，32.71，31.14，30.01，26.43，21.32.
example 2 application of alpha-pinene-based Nuclear fluorescent Probe
1) Collecting Hela cells in logarithmic growth phase, digesting with pancreatin, adjusting cell concentration with culture solution at a ratio of 1 × 10 per well5The amount of individual cells was seeded in 6-well plates and 1X 104The amount of each cell was seeded in a 96-well plate at 37 ℃ in 5% CO2And incubating under saturated humidity conditions.
2) After the cells grew adherent, the culture medium was removed, washed twice with PBS, and the drug diluted with complete medium was added to each well and incubated for a further period of time.
3) Add 10 μ LCCK-8 reagent to 96 well plates and continue incubation for 2h, OD was measured at 450 nm.
4) The culture medium was aspirated, adherent cells were washed 2 times with pre-cooled PBS, drugs not taken up by the cells were removed, and fixed with 4% paraformaldehyde for 30 min.
5) Adding a proper amount of commercial cell nucleus probe hoechst33342 staining solution, and staining for 30 min; the staining solution was discarded and washed 3 times with PBS.
6) And (3) placing the glass substrate on an objective table of a laser confocal microscope, determining an observation area and an observation layer, and collecting a fluorescence image.
The excitation wavelength used by the alpha-pinene-based cell nucleus fluorescent probe is 390nm, the emission wavelength is 487nm, and the magnification is 630 times; commercial cell nucleus fluorescent probe hoechst33342 uses an excitation wavelength of 350nm, an emission wavelength of 460nm and a magnification of 630 times.
The alpha-pinene-based nuclear fluorescent probe is adopted to act on Hela cells, and a CCK-8 reagent is used for detecting the cell survival rate, and the result is shown in figure 1. Under the action of 6.25 mu M of the medicine, the survival rate of the hela cells reaches more than 90 percent, which indicates that the toxicity of the alpha-pinene-based nuclear fluorescence probe to the cells is low.
Staining Hela cell nucleus by using an alpha-pinene-based cell nucleus fluorescent probe, exciting fluorescence in the cell by using 390nm exciting light, and collecting fluorescence imaging of a 487nm waveband, wherein the result is shown in figure 2.
Hela cell nucleus is stained by a commercial cell nucleus probe hoechst33342, fluorescence in the cell is excited by 350nm exciting light, and fluorescence imaging of a 460nm wave band is collected, and the result is shown in FIG. 3.
The results of superimposing FIG. 2 and FIG. 3 show that the α -pinene-based nuclear fluorescent probe has the same staining effect as the commercial nuclear probe hoechst33342, as shown in FIG. 4. And the alpha-pinene-based nuclear fluorescent probe has the characteristics of simple preparation process, low price and low toxicity.
Claims (7)
1. An alpha-pinene-based cell nucleus fluorescent probe is characterized in that the structural formula is as follows:
2. the method for preparing an alpha-pinene-based nuclear fluorescent probe of claim 1, which comprises the following steps:
1) condensing myrtenal and 4-methyl thiosemicarbazone to obtain alpha-pinenyl-4-methyl thiosemicarbazone;
2) cyclizing the alpha-pinene-4-methyl thiosemicarbazone and chloroacetic acid to obtain alpha-pinene-thiazoline-4-one;
3) condensing the alpha-pinene thiazoline-4-ketone and indole-3-formaldehyde to obtain an alpha-pinene nucleus fluorescent probe 5- (1H-indole-3-yl) methylene-2- (2- (6, 6-dimethylbicyclo [3.1.1] hept-2-ene-2-yl) methylene) hydrazino-3-methylthiazoline-4-ketone; the reaction formula is as follows:
3. the method for preparing the alpha-pinene-based nuclear fluorescent probe according to claim 2, wherein in the step 1), the myrtenal and the p-4-methyl thiosemicarbazone are condensed to obtain the alpha-pinene-4-methyl thiosemicarbazone; the preparation process comprises the following steps:
(1) sequentially adding 10mmol of myrtenal, 9-12 mmol of 4-methyl thiosemicarbazide, 3mL of 10% hydrochloric acid aqueous solution and 10mL of ethanol into a three-neck flask with a stirrer, a thermometer and a reflux condenser, and reacting at 80-90 ℃;
(2) extracting the reaction product with 60mL ethyl acetate for 3 times, combining organic phases, and washing the organic phases with saturated saline solution to be neutral; drying the organic phase by anhydrous sodium sulfate, filtering, concentrating and recovering the solvent to obtain a crude product of the alpha-pinene-4-methyl thiosemicarbazone, and washing and drying the crude product to obtain the alpha-pinene-4-methyl thiosemicarbazone.
4. The method for preparing the alpha-pinene-based nuclear fluorescent probe according to claim 2, wherein in the step 2), under the catalysis of sodium acetate, the alpha-pinene-4-methyl thiosemicarbazone reacts with chloroacetic acid to obtain alpha-pinene-based thiazoline-4-one; the preparation process comprises the following steps:
(1) sequentially adding 5mmol of alpha-pinene-4-methyl thiosemicarbazone, 5-6 mmol of chloroacetic acid, 2-4 mol of sodium acetate and 20mL of ethanol into a three-neck flask with a stirrer, a thermometer and a reflux condenser, heating and refluxing for 12 hours, detecting by TCL tracking, and stopping the reaction until the conversion rate of the alpha-pinene-4-methyl thiosemicarbazone reaches 95%;
(2) the reaction was extracted 3 times with 300mL dichloromethane, the organic phases were combined and washed to neutrality with saturated brine; drying the organic phase by anhydrous sodium sulfate, filtering, concentrating and recovering the solvent to obtain a crude product of the alpha-pinene thiazoline-4-ketone;
(3) recrystallizing the crude product of the alpha-pinene-thiazoline-4-ketone by using ethanol to obtain white powder of the alpha-pinene-thiazoline-4-ketone.
5. The method for preparing the alpha-pinene-based nuclear fluorescent probe according to claim 2, wherein in the step 3), sodium acetate is used as a catalyst, and alpha-pinene-based thiazoline-4-one is condensed with indole-3-formaldehyde to obtain the alpha-pinene-based nuclear fluorescent probe 5- (1H-indol-3-yl) methylene-2- (2- (6, 6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methylene) hydrazino-3-methylthiazoline-4-one; the preparation process comprises the following steps:
(1) sequentially adding 5mmol of alpha-pinene thiazoline-4-one, 4-5.5 mmol of indole-3-formaldehyde, 2-4 mol of sodium acetate and 20-50 mL of ethanol into a three-neck flask with a stirrer, a thermometer and a reflux condenser, and heating and carrying out reflux reaction for 6-18 h;
(2) cooling the reactant to room temperature to separate out a bright yellow powdery crude product of 5- (1H-indol-3-yl) methylene-2- (2- (6, 6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methylene) hydrazino-3-methylthiazoline-4-one;
(3) recrystallizing the crude product of 5- (1H-indol-3-yl) methylene-2- (2- (6, 6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methylene) hydrazino-3-methylthiazoline-4-one in dichloromethane-methanol to obtain the alpha-pinene nuclear fluorescent probe 5- (1H-indol-3-yl) methylene-2- (2- (6, 6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) methylene) hydrazino-3-methylthiazoline-4-one bright yellow crystal.
6. The use of the α -pinene-based nuclear fluorescent probe of claim 1 in cellular imaging.
7. The use of claim 6, wherein the fluorescent probe is capable of specific in situ imaging of the nucleus of a living cell and emits green fluorescence under 390nm UV light.
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