CN117186140A - Novel fluorescent dye platform and preparation method and application thereof - Google Patents
Novel fluorescent dye platform and preparation method and application thereof Download PDFInfo
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims abstract description 35
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 64
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 45
- 238000003756 stirring Methods 0.000 claims description 42
- 150000001875 compounds Chemical class 0.000 claims description 37
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 36
- 238000004440 column chromatography Methods 0.000 claims description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 23
- -1 oxalyl chloride monoethyl ester Chemical class 0.000 claims description 19
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 238000000746 purification Methods 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 238000000926 separation method Methods 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 12
- KZMGYPLQYOPHEL-UHFFFAOYSA-N Boron trifluoride etherate Chemical compound FB(F)F.CCOCC KZMGYPLQYOPHEL-UHFFFAOYSA-N 0.000 claims description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- PCLIMKBDDGJMGD-UHFFFAOYSA-N N-bromosuccinimide Chemical compound BrN1C(=O)CCC1=O PCLIMKBDDGJMGD-UHFFFAOYSA-N 0.000 claims description 10
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 238000004821 distillation Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 229940126214 compound 3 Drugs 0.000 claims description 8
- 239000012044 organic layer Substances 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 7
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- NLQMSBJFLQPLIJ-UHFFFAOYSA-N (3-methyloxetan-3-yl)methanol Chemical compound OCC1(C)COC1 NLQMSBJFLQPLIJ-UHFFFAOYSA-N 0.000 claims description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 5
- 229940125898 compound 5 Drugs 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 claims description 5
- UBJFKNSINUCEAL-UHFFFAOYSA-N lithium;2-methylpropane Chemical compound [Li+].C[C-](C)C UBJFKNSINUCEAL-UHFFFAOYSA-N 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- CTSLXHKWHWQRSH-UHFFFAOYSA-N oxalyl chloride Substances ClC(=O)C(Cl)=O CTSLXHKWHWQRSH-UHFFFAOYSA-N 0.000 claims description 5
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 5
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 3
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical class [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- 230000020477 pH reduction Effects 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- XVMSFILGAMDHEY-UHFFFAOYSA-N 6-(4-aminophenyl)sulfonylpyridin-3-amine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)C1=CC=C(N)C=N1 XVMSFILGAMDHEY-UHFFFAOYSA-N 0.000 claims description 2
- 239000012295 chemical reaction liquid Substances 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims 1
- 239000000975 dye Substances 0.000 abstract description 27
- 230000005281 excited state Effects 0.000 abstract description 19
- 125000004185 ester group Chemical group 0.000 abstract description 17
- 230000007246 mechanism Effects 0.000 abstract description 8
- 150000002148 esters Chemical group 0.000 abstract description 7
- 229910052710 silicon Inorganic materials 0.000 abstract description 7
- 239000010703 silicon Substances 0.000 abstract description 6
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 4
- 230000005284 excitation Effects 0.000 abstract description 4
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 abstract description 2
- 230000005283 ground state Effects 0.000 description 7
- 238000000862 absorption spectrum Methods 0.000 description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 238000003775 Density Functional Theory Methods 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000000295 emission spectrum Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 4
- 238000004768 lowest unoccupied molecular orbital Methods 0.000 description 4
- 239000001022 rhodamine dye Substances 0.000 description 4
- 238000004770 highest occupied molecular orbital Methods 0.000 description 3
- 238000006862 quantum yield reaction Methods 0.000 description 3
- 230000008707 rearrangement Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 230000007704 transition Effects 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N DMSO Substances CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000004776 molecular orbital Methods 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 150000003376 silicon Chemical class 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229940125904 compound 1 Drugs 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 229960000956 coumarin Drugs 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- HFCSXCKLARAMIQ-UHFFFAOYSA-L disodium;sulfate;hydrate Chemical compound O.[Na+].[Na+].[O-]S([O-])(=O)=O HFCSXCKLARAMIQ-UHFFFAOYSA-L 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000005865 ionizing radiation Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003077 quantum chemistry computational method Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Abstract
The invention belongs to the field of fluorescent probes, and particularly relates to a novel fluorescent dye platform and a preparation method and application thereof. In order to develop a dye platform with better performance, the invention successfully applies the excited state pi-conjugation mechanism participated by the central ester group to the silicon rhodamine, and develops a novel fluorescent dye platform SiR-COOM, and a fluorescent probe constructed based on the dye platform and combined with an ester group-carboxyl group conversion strategy has the advantages of excitation and emission wavelength from far infrared to Near Infrared (NIR), good biocompatibility, large fluorescence off-on ratio of the ester group-carboxyl group and the like, so that the fluorescent probe has potential application value.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to a novel fluorescent dye platform and a preparation method and application thereof.
Background
The fluorescent probe technology is used as a non-ionizing radiation method, has remarkable advantages of cell permeability, visualization, non-invasiveness, high space-time resolution, real-time and in-situ monitoring and the like, is one of the most effective cell biological tools, and plays an important role in revealing the distribution and functions of bioactive molecules in cells, tissues and living bodies. Two key factors should be considered in constructing an effective fluorescent probe: firstly, selecting fluorescent dye with long excitation and emission wavelength, high fluorescent brightness and strong light stability to improve imaging space-time resolution; the other is to define a fluorescence control mechanism to ensure that the probe has a distinct fluorescence response upon interaction with the target substrate. Over several decades of effort, various fluorescent dyes suitable for bioimaging have been developed, such as coumarin dyes, BODIPY dyes, rhodamine dyes, and cyanine dyes, and derivatives thereof. In contrast, the reported fluorescence control mechanisms are limited to a few, and commonly used include photo-induced electron transfer (PeT), fluorescence Resonance Energy Transfer (FRET), and Intramolecular Charge Transfer (ICT), etc. Therefore, there is an urgent need to develop novel fluorescence control mechanisms for developing various novel fluorescent probes to meet various biomedical application studies. It was found that the centrally-located carboxy-substituted Bodipys dye is a strong fluorescent material, whereas the corresponding centrally-located ester-substituted Bodipys dye has little fluorescence, the non-fluorescent property being mainly due to the non-radiative transition promoted by the excited pi-conjugation between the ester group and the Bodipy backbone, and therefore, the conversion of the "ester- & gtcarboxy" group at the center of the Bodipys dye causes a significant "off- & gton" change in fluorescence. However, the above mechanism is currently only applied in Bodipy dyes, it is not clear whether it is equally applicable in other dyes.
Disclosure of Invention
Aiming at the problems, the invention synthesizes a central ester group substituted silicon rhodamine dye SiR-COOM and a central carboxyl anion substituted silicon rhodamine dye SiR-COO, takes SiR-COOM and SiR-COO as model molecules, and verifies the feasibility of constructing a fluorescent probe by taking SiR-COOM as a dye platform.
Because of the strong electron-withdrawing effect of the ester group, an excited state pi-conjugation effect exists between the ester group in SiR-COOM and the silicon rhodamine skeleton, and the effect can cause obvious configuration change of the dye conjugation skeleton in the excited state, so that a heat radiation path is increased, and weak fluorescence of the dye is finally caused; in contrast, siR-COO excited state configuration shows small configuration change compared with the ground state configuration thereof due to weak electron withdrawing ability of carboxyl anions, and finally, strong fluorescence of the dye is caused. Therefore, siR-COOM can be used as a novel fluorescent dye platform to construct a fluorescent probe based on an ester-carboxyl conversion strategy.
The invention adopts the following technical scheme to achieve the aim:
a novel fluorescent dye platform has a structural formula:
the preparation method of the compound SiR-COO and the compound SiR-COOM comprises the following steps:
step 1, dissolving 3-hydroxymethyl-3-methyl oxetane (compound 1) and pyridine in methylene chloride at the temperature of 0 ℃, slowly dropwise adding oxalyl chloride monoethyl ester (compound 2) at the temperature, stirring at room temperature, diluting a reaction solution with water, extracting with methylene chloride, combining organic layers, washing with saturated copper sulfate, drying with anhydrous magnesium sulfate, filtering and evaporating to obtain a crude product, and separating and purifying the crude product by column chromatography to obtain a compound 3 as a colorless oily liquid;
step 2, at N 2 Dissolving the compound 3 in dichloromethane at the temperature of 0 ℃, gradually dropwise adding boron trifluoride diethyl etherate, then heating to room temperature, stirring for reaction, adding triethylamine, continuing stirring for reaction, and carrying out reduced pressure distillation and column chromatography separation and purification on the reaction liquid to obtain a compound 4 as a white solid;
step 3, at N 2 In the environment, compound 5 is dissolved in anhydrous acetonitrile, cooled to 0 ℃, then N-bromosuccinimide is gradually added, stirring reaction is carried out at the temperature, dichloromethane is added, and the organic phase is respectively washed by saturated sodium bicarbonate solution and water and is free of waterDrying sodium sulfate hydrate, distilling under reduced pressure, and separating and purifying by column chromatography to obtain a compound 6 as a white solid;
step 4, at N 2 In the environment, compound 6 is dissolved in anhydrous tetrahydrofuran, cooled to-78 ℃, tertiary butyl lithium is slowly added dropwise, then the temperature is raised after stirring reaction at the temperature, then tetrahydrofuran solution of compound 4 is slowly added dropwise, the temperature is gradually raised to room temperature and stirring reaction is continued overnight, and saturated NH is then added 4 Cl solution and water, extracting with ethyl acetate, combining organic layers, drying the organic layers by anhydrous sodium sulfate, evaporating the solvent under reduced pressure, dissolving the obtained residue in methanol, adding hydrochloric acid solution, stirring at room temperature for reaction, removing the solvent under vacuum, and separating and purifying by column chromatography to obtain a 2, 2-bis (hydroxymethyl) propyl ester intermediate;
step 5, dissolving the 2, 2-bis (hydroxymethyl) propyl ester intermediate in MeOH, adding sodium hydroxide solution, stirring for reaction, and obtaining a compound SiR-COO after acidification of acetic acid, reduced pressure distillation and column chromatography separation and purification, wherein the compound SiR-COO is a dark blue solid;
step 6, dissolving a compound SiR-COO in anhydrous acetonitrile, sequentially adding methyl iodide and anhydrous potassium carbonate into the mixture, stirring the mixture for reaction, and carrying out reduced pressure distillation and column chromatography separation and purification to obtain the compound SiR-COOM which is a dark blue solid;
further, the molar ratio of 3-hydroxymethyl-3-methyloxetane, pyridine and oxalyl chloride monoethyl ester in step 1 was 1.5:2:1, stirring and reacting for 18h, wherein the developing agent for column chromatography separation and purification is 0-50% ethyl acetate/hexane (v/v).
Further, the molar ratio of the compound 3 to the boron trifluoride etherate in the step 2 is 1:0.25, stirring and reacting for 18h, continuously stirring and reacting for 10min, and separating and purifying by column chromatography with developing agent of 10-20% ethyl acetate/hexane, v/v, and containing l% triethylamine.
Further, the molar ratio of compound 5 to N-bromosuccinimide in step 3 is 1:1.1, stirring and reacting for 2h, wherein the developing agent for column chromatography separation and purification is 10-20% ethyl acetate/n-hexane (v/v).
Further, the molar ratio of compound 6, t-butyllithium and compound 4 in step 4 was 0.5:2.2:1.1, stirring and reacting for 0.5h, heating to-20 ℃ after stirring and reacting, wherein the concentration of hydrochloric acid solution is 1M, stirring and reacting at room temperature for 1h, and the developing agent for separating and purifying by column chromatography is dichloromethane/methanol=10/1 (v/v).
Further, the molar ratio of the 2, 2-bis (hydroxymethyl) propyl ester intermediate to NaOH in step 5 is 1:8, stirring the reaction at 60 ℃ for 18h, and separating and purifying the developing agent by column chromatography to obtain dichloromethane/methanol=10/1 (v/v).
Further, the molar ratio of the compound SiR-COO, methyl iodide and anhydrous potassium carbonate in the step 6 is 0.5:2:1.5, stirring and reacting at 60 ℃ for 2 hours, wherein the developing agent for separating and purifying by column chromatography is CH 2 Cl 2 /MeOH＝10:1(v/v)。
The application of the novel fluorescent dye platform is characterized in that SiR-COO/SiR-COOM is used as a basic molecular model, and quantum chemical calculation and in-vitro experiments are utilized to verify the feasibility of constructing a fluorescent probe by taking SiR-COOM as a dye platform.
Compared with the prior art, the invention has the following advantages:
at present, the fluorescent probe constructed based on the excited-state pi-conjugation participated by the central ester group is limited to the Bodipy dye, and the mechanism is successfully applied to the silicon rhodamine (SiR), and a novel fluorescent dye platform SiR-COOM is developed, so that the application range of the mechanism is further widened. In addition, the fluorescent probe constructed based on the SiR-COOM dye platform and combined with the ester group-carboxyl group conversion strategy has the advantages of excitation and emission wavelength from far infrared to Near Infrared (NIR), good biocompatibility, large fluorescence off-on ratio of the ester group-carboxyl group and the like.
Drawings
FIG. 1 is NMR and HRMS of Compound 4;
FIG. 2 is an NMR and HRMS plot of Compound 6;
FIG. 3 is an NMR and HRMS of the compound SiR-COOM;
FIG. 4 is an NMR and HRMS of the compound SiR-COO;
FIG. 5 shows the ground state (S) of SiR-COOM calculated by density functional theory 0 Left) and excited states (S 1 Right) front molecular orbital and optimized molecular structure;
FIG. 6 shows the ground state (S) 0 Left) and excited states (S 1 Right) front molecular orbital and optimized molecular structure;
in FIG. 7, (A) and (B) are normalized absorption spectra and emission spectra of SiR-COO and SiR-COOM (2. Mu.M) in PBS, respectively; lambda (lambda) ex ＝633nm；Slits：5/10nm；voltage：700V；
In FIG. 8, (A) and (B) are SiR-COO, siR-COOM (2. Mu.M) in CH, respectively 2 Cl 2 An absorption spectrum and an emission spectrum of medium normalization; lambda (lambda) ex ＝610nm；
In FIG. 9, (A) and (B) are SiR-COO, siR-COOM (2. Mu.M) in CH 3 Normalized absorption spectrum and emission spectrum in CN; lambda (lambda) ex ＝610nm。
Detailed Description
In order to further illustrate the technical scheme of the invention, the invention is further illustrated by the following examples.
Example 1
A novel fluorescent dye platform has a structural formula:
the preparation method comprises the following steps:
step 1, 3-hydroxymethyl-3-methyl oxetane (compound 1,2.74mL,27.5 mmol) and pyridine (2.96 mL,36.6 mmol) were dissolved in dichloromethane (35 mL) at 0deg.C (ice bath), and oxalyl chloride monoethyl ester (compound 2,2.05mL,18.3 mmol) was slowly added dropwise at this temperature, the ice bath was removed, followed by stirring at room temperature for 18 hours, the reaction solution was diluted with water and extracted with dichloromethane, the combined organic layers were washed with saturated copper sulfate, dried over anhydrous magnesium sulfate, filtered and evaporated to give crude product, which was purified (0-50% ethyl acetate/hexane, v/v) to give compound 3 (3.22 g, 87%) as colorless oily liquid by column chromatography;
step 2, at N 2 Compound 3 (5.7 g,28.2 mmol) was dissolved in dichloromethane (30 mL) at 0deg.C, then boron trifluoride diethyl etherate (l.0 g,0.25 eq) was gradually added dropwise, then warmed to room temperature and stirred for 18 hours, triethylamine (10 mL) was added, and after stirring the reaction solution continued for 10 minutes, the reaction solution was purified by distillation under reduced pressure and column chromatography (10-20% ethyl acetate/hexane, v/v, containing l% triethylamine) to give compound 4 (2.63 g, 46%) as a white solid;
1 H NMR(600MHz,CDCl 3 )δ4.31(q,J＝7.2Hz,2H),4.04(s,6H),1.34(t,J＝7.2Hz,3H),0.86(s,3H). 13 C NMR(150MHz,DMSO-d 6 )δ163.0,102.9,72.6,62.1,30.4,14.2,13.7；ESI-MS[M+Na] + :calcd for225.0739,Found 225.0735.
step 3, at N 2 In a dry flask, compound 5 (1.2 g,4.0 mmol) and anhydrous acetonitrile (30 mL) were added thereto, cooled to 0℃and then N-bromosuccinimide (783 mg,4.4 mmol) was gradually added thereto, stirred at that temperature for 2 hours, methylene chloride (50 mL) was further added thereto, and the organic phase was washed with a saturated sodium hydrogencarbonate solution and water, dried over anhydrous sodium sulfate, distilled under reduced pressure and purified by column chromatography, respectively (10-20% ethyl acetate/N-hexane, v/v) to give compound 6 (1.73 g, 94.8%) as a white solid;
1 H NMR(600Hz,CD 3 CN)δ7.38(d,J＝9.0Hz,2H),6.86(s,2H),6.62(s,2H),2.89(s,12H),0.77(s,6H)； 13 C NMR(150MHz,CD 3 CN)δ148.9,138.8,133.0,121.8,116.8,115.3,40.6,0.94；ESI-MS[M+H] + :calcd for 457.0133,Found457.0128.
step 4, at N 2 Compound 6 (169 mg,0.5 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL) under ambient conditions, cooled to-78deg.C, and tert-butyllithium (1.69 mL,2.2 mmol) was slowly added dropwise, followed by stirring at this temperature for 30 min and then warming to-20deg.C, followed by slow dropwise addition of compound 4Tetrahydrofuran solution (222 mg,1.1mmol,4 mL) was gradually warmed to room temperature and the reaction was continued to stir overnight, after which saturated NH was added thereto 4 Cl solution and water, and extracted with ethyl acetate, the combined organic layers were dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, the resulting residue was dissolved in methanol (10 mL), and hydrochloric acid solution (1 m,500 μl) was added, after stirring at room temperature for 1 hour, the solvent was removed under vacuum and purified by column chromatography (dichloromethane/methanol=10/1) to give 2, 2-bis (hydroxymethyl) propyl ester intermediate (212 mg, 90.0%);
step 5, 2-bis (hydroxymethyl) propyl ester intermediate (94.4 mg,0.2 mmol) was dissolved in MeOH, and sodium hydroxide solution (1 m,1.6ml,1.6mmol,8 eq.) was added and stirred at 60 ℃ for 18 hours to give compound SiR-COO (65 mg, 83.7%) as a dark blue solid after acidification of acetic acid (500 μl), distillation under reduced pressure and purification by column chromatography (dichloromethane/methanol=10/1);
1 H NMR(600Hz,CD 3 OD)δ7.88(d,J＝12.6Hz,2H),7.25(d,J＝14.4Hz,2H),6.92(s,2H),1.95(s,12H),0.51(s,6H)； 13 C NMR(150MHz,CD 3 OD)δ156.5,150.4,143.2,124.8,122.0,117.5,116.6,115.7,,41.3,-0.81.ESI-MS[M] + :calcd for 353.1680,Found 353.1684.
step 6, dissolving the compound SiR-COO (194 mg,0.5 mmol) in anhydrous acetonitrile (10 mL), and sequentially adding methyl iodide (284 mg,2.0 mmol) and anhydrous potassium carbonate (207 mg,1.5 mmol) thereto, stirring at 60 ℃ for 2 hours, and obtaining the compound SiR-COOM (150 mg, 75.0%) as a dark blue solid after reduced pressure distillation and column chromatography separation and purification (dichloromethane/methanol=10/1);
1 H NMR(600Hz,d-DMSO)δ7.46(s,2H),7.41(d,J＝9.0Hz,2H),7.00(dd,J＝1.8Hz,J＝9.6Hz,2H),4.04(s,3H),3.36(s,12H),0.54(s,6H)； 13 C NMR(150MHz,d-DMSO)δ169.7,156.5,155.2,147.9,139.8,123.7,123.4,116.4,54.6,41.9,1.3；ESI-MS[M] + :calcd for 367.1836,Found 367.1840.
EXAMPLE 2 Quantum chemistry computation
To verify whether or not SiR-COOM is also presentThere is a configuration change of excited pi-conjugate driving, and we calculate SiR-COOM ground state (S) by a Density Functional Theory (DFT) method and a Time Dependent Density Functional Theory (TDDFT) method respectively 0 ) And a first excited state (S 1 ) Is a precursor track of (c) and an optimized molecular configuration. As shown in FIG. 5, in the ground state configuration of the optimized SiR-COOM, the centrally substituted ester group is almost perpendicular to its conjugate plane (O 1 -C 2 -C 3 -C 4 Is 89 °); while the optimized configuration of the excitation state of SiR-COOM has two features: one is that the dihedral angle between the centrally substituted ester group and its conjugate plane is greatly reduced (θ=5°), and the other is that its conjugate plane is butterfly-curved along the axis of the central carbon atom and silicon atom (dihedral angle of planes on both sides of the axis: ω=140°). Therefore, siR-COOM exhibits a large conformational rearrangement in the excited state compared to the ground state configuration. Importantly, neither the central carbon atom nor the centrally substituted ester group of SiR-COOM is involved in the HOMO orbital, but rather is largely involved in the LUMO orbital, indicating that there is an excited pi-conjugation between the ester moiety of SiR-COOM and its backbone, which can be attributed to the strong Intramolecular Charge Transfer (ICT) effect from the dye backbone to the electron-deficient ester moiety in the excited state. Thus, a large conformational rearrangement in the SiR-COOM excited state can be explained as follows: the transition of electrons from the HOMO to LUMO orbitals in the excited state promotes pi-conjugation of the excited state between the ester group and its conjugate plane, and the strong resonance stability forces the ester group to rotate and its backbone into a planar configuration, which results in a large butterfly-like bend of its backbone to accommodate the increasing steric repulsion between the ester moiety and the adjacent aryl H atom. The above results indicate that SiR-COOM will have weak fluorescence, since the apparent conformational rearrangement of SiR-COOM in the excited state will inactivate it mainly in the form of non-radiative heat in the excited state, compared to the ground state.
Further, the front-line orbitals of SiR-COO and the optimized molecular configuration were calculated by DFT and TDDFT. As shown in FIG. 6, although the dihedral angle between the center-substituted carboxylic acid anion and the dye platform backbone in the dye platform is optimized S 0 State and optimized S 1 Some states haveThe parent structure of the dye platform remains nearly planar in both states, which may be due to the significantly reduced electron withdrawing capacity of the carboxylate anions compared to the ester groups, resulting in weak pi-electron interactions between the carboxylate anions and the dye platform parent. Thus, siR-COO will have strong fluorescence.
In general, theoretical calculations show that SiR-COOM is hopefully a novel fluorescent dye platform based on the ester- & gt carboxyl conversion strategy, and various fluorescent probes can be constructed based on the platform.
EXAMPLE 3 photophysical Property Studies
First, photophysical properties of SiR-COO and SiR-COOM were studied in dichloromethane, acetonitrile and PBS (10 mm, ph=7.4), respectively, as shown in table 1:
TABLE 1 photophysical Properties of SiR-COOM and SiR-COO in dichloromethane, acetonitrile and PBS
As can be seen from Table 1, in CH 2 Cl 2 、CH 3 In CN and PBS, the fluorescence quantum yield of SiR-COOM is about 0.01, and the fluorescence quantum yields of SiR-COO are respectively as high as 0.38, 0.33 and 0.17. SiR-COOM exhibits significantly reduced fluorescence quantum yield (Φ) in each of the three solvents, as compared to SiR-COO, consistent with previous theoretical predictions.
FIG. 7 shows the absorption and emission spectra of SiR-COOM/SiR-COO in PBS (10 mM, pH=7.4), at CH 2 Cl 2 And CH (CH) 3 The absorption and emission spectra in CN are shown in fig. 8 and 9. As shown in FIG. 7A, with SiR-COO (lambda abs Compared with 638nm, the absorption spectrum of SiR-COOM has a significant red shift (lambda) abs =667 nm), possibly because the ester group with strong electron withdrawing preferentially stabilizes LUMO rather than HOMO, thereby reducing the HOMO-LUMO energy level difference. Importantly, as shown in FIG. 7 (B), siR-COO exhibits a strong fluorescence signal (lambda) when excited with 633nm (near the peak of maximum absorption of SiR-COO and matched to the confocal laser source) em =664 nm), whereas the fluorescence of SiR-COOM is negligible.
The above results indicate that the SiR-COOM to SiR-COO transition can generate a larger fluorescent "off- > on" response at 662nm, and thus, it is feasible to construct a fluorescent probe based on the SiR-COOM platform in combination with an "ester- > carboxyl" conversion strategy.
In summary, the "ester- > carboxyl" conversion strategy has proven to be a general strategy for constructing fluorescent probes, however, this strategy is currently only used in Bodipy dyes. The invention successfully applies the excited-state pi-conjugation mechanism participated by the central-position ester group to the silicon rhodamine dye (SiR), and develops a novel fluorescent dye platform SiR-COOM. Quantum chemistry and experiments prove that the conversion of the dye platform from an ester group to a carboxyl group can cause obvious fluorescence off-on response, because the conversion of the ester group to the carboxyl group effectively inhibits the pi-conjugation of the excited state between the ester group and the SiR dye parent, the configuration change of the dye framework in the excited state is greatly weakened, and the non-radiative heat inactivation process of the excited state is blocked. Thus, with the SiR-COOM dye platform, various fluorescent probes can be designed based on the "ester- > carboxyl" conversion strategy.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (9)
1. The novel fluorescent dye platform is characterized by comprising the following structural formula:
2. the method for preparing a novel fluorescent dye platform as claimed in claim 1, comprising the steps of:
step 1, dissolving 3-hydroxymethyl-3-methyl oxetane and pyridine in methylene dichloride at the temperature of 0 ℃, slowly dropwise adding oxalyl chloride monoethyl ester at the temperature, then stirring at room temperature for reaction, diluting with water, extracting with methylene dichloride after the reaction, combining organic layers, washing with saturated copper sulfate, drying with anhydrous magnesium sulfate, filtering and evaporating to obtain a crude product, and separating and purifying the crude product by column chromatography to obtain a compound 3 as colorless oily liquid;
step 2, at N 2 Dissolving the compound 3 in dichloromethane at the temperature of 0 ℃, gradually dropwise adding boron trifluoride diethyl etherate, then heating to room temperature, stirring for reaction, adding triethylamine, continuing stirring for reaction, and carrying out reduced pressure distillation and column chromatography separation and purification on the reaction liquid to obtain a compound 4 as a white solid;
step 3, at N 2 Under the environment, dissolving the compound 5 in anhydrous acetonitrile, cooling to 0 ℃, then gradually adding N-bromosuccinimide, stirring and reacting at the temperature, adding dichloromethane into the mixture, washing an organic phase with saturated sodium bicarbonate solution and water, drying with anhydrous sodium sulfate, decompressing and distilling, and separating and purifying by column chromatography to obtain a compound 6 as a white solid;
step 4, at N 2 In the environment, compound 6 is dissolved in anhydrous tetrahydrofuran, cooled to-78 ℃, tertiary butyl lithium is slowly added dropwise, then the temperature is raised after stirring reaction at the temperature, then tetrahydrofuran solution of compound 4 is slowly added dropwise, the temperature is gradually raised to room temperature and stirring reaction is continued overnight, and saturated NH is then added 4 Cl solution and water, extracting with ethyl acetate, combining organic layers, drying the organic layers by anhydrous sodium sulfate, evaporating the solvent under reduced pressure, dissolving the obtained residue in methanol, adding hydrochloric acid solution, stirring at room temperature for reaction, removing the solvent under vacuum, and separating and purifying by column chromatography to obtain a 2, 2-bis (hydroxymethyl) propyl ester intermediate;
step 5, dissolving the 2, 2-bis (hydroxymethyl) propyl ester intermediate in MeOH, adding sodium hydroxide solution, stirring for reaction, and obtaining a compound SiR-COO after acidification of acetic acid, reduced pressure distillation and column chromatography separation and purification, wherein the compound SiR-COO is a dark blue solid;
and 6, dissolving the compound SiR-COO in anhydrous acetonitrile, sequentially adding methyl iodide and anhydrous potassium carbonate into the mixture, stirring the mixture for reaction, and carrying out reduced pressure distillation and column chromatography separation and purification to obtain the compound SiR-COOM which is a dark blue solid.
3. The method for preparing a novel fluorescent dye platform according to claim 2, wherein the molar ratio of 3-hydroxymethyl-3-methyl oxetane, pyridine and oxalyl chloride monoethyl ester in the step 1 is 1.5:2:1, stirring and reacting for 18h, wherein the developing agent for column chromatography separation and purification is 0-50% ethyl acetate/hexane (v/v).
4. The preparation method of the novel fluorescent dye platform according to claim 2, wherein the molar ratio of the compound 3 to the boron trifluoride diethyl etherate in the step 2 is 1:0.25, stirring and reacting for 18h, continuously stirring and reacting for 10min, and separating and purifying by column chromatography with developing agent of 10-20% ethyl acetate/hexane, v/v, and containing l% triethylamine.
5. The method for preparing a novel fluorescent dye platform according to claim 2, wherein the molar ratio of the compound 5 to the N-bromosuccinimide in the step 3 is 1:1.1, stirring and reacting for 2h, wherein the developing agent for column chromatography separation and purification is 10-20% ethyl acetate/n-hexane (v/v).
6. The method for preparing a novel fluorescent dye platform according to claim 2, wherein the molar ratio of the compound 6, the tert-butyllithium and the compound 4 in the step 4 is 0.5:2.2:1.1, stirring and reacting for 0.5h, heating to-20 ℃ after stirring and reacting, wherein the concentration of hydrochloric acid solution is 1M, stirring and reacting at room temperature for 1h, and the developing agent for separating and purifying by column chromatography is dichloromethane/methanol=10/1 (v/v).
7. The method for preparing a novel fluorescent dye platform according to claim 2, wherein the molar ratio of the 2, 2-bis (hydroxymethyl) propyl ester intermediate to NaOH in step 5 is 1:8, stirring the reaction at 60 ℃ for 18h, and separating and purifying the developing agent by column chromatography to obtain dichloromethane/methanol=10/1 (v/v).
8. The method for preparing a novel fluorescent dye platform according to claim 2, wherein the molar ratio of the compound SiR-COO, methyl iodide and anhydrous potassium carbonate in the step 6 is 0.5:2:1.5, stirring reaction temperature is 60 ℃, time is 2h, and developing agent for column chromatography separation and purification is dichloromethane/methanol=10/1 (v/v).
9. Use of a novel fluorescent dye platform according to claim 1, for constructing fluorescent probes.
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