CN111233715A - Two-photon fluorescent probe, preparation method and application in formaldehyde detection - Google Patents
Two-photon fluorescent probe, preparation method and application in formaldehyde detection Download PDFInfo
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 51
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title abstract description 11
- KLIIGADLHUOXPS-UHFFFAOYSA-N 2-(1,2,2-triphenylethenyl)phenol Chemical group OC1=CC=CC=C1C(C=1C=CC=CC=1)=C(C=1C=CC=CC=1)C1=CC=CC=C1 KLIIGADLHUOXPS-UHFFFAOYSA-N 0.000 claims abstract description 11
- TZRQZPMQUXEZMC-UHFFFAOYSA-N tert-butyl n-(2-bromoethyl)carbamate Chemical compound CC(C)(C)OC(=O)NCCBr TZRQZPMQUXEZMC-UHFFFAOYSA-N 0.000 claims abstract description 11
- 125000000066 S-methyl group Chemical group [H]C([H])([H])S* 0.000 claims abstract description 7
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000000523 sample Substances 0.000 claims description 40
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 30
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- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 18
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 16
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- 229940125904 compound 1 Drugs 0.000 claims description 12
- 229940125782 compound 2 Drugs 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 11
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- 238000010992 reflux Methods 0.000 claims description 9
- XEZNGIUYQVAUSS-UHFFFAOYSA-N 18-crown-6 Chemical compound C1COCCOCCOCCOCCOCCO1 XEZNGIUYQVAUSS-UHFFFAOYSA-N 0.000 claims description 8
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- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 8
- 238000003384 imaging method Methods 0.000 claims description 7
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- 238000003756 stirring Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 3
- WPQZSUSLLBSFIH-UHFFFAOYSA-N [amino(hydroxy)methylidene]azanium;iodide Chemical compound I.NC(N)=O WPQZSUSLLBSFIH-UHFFFAOYSA-N 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
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- 230000004044 response Effects 0.000 abstract description 12
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- -1 aminoguanidino Chemical group 0.000 abstract description 2
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- 238000010186 staining Methods 0.000 description 9
- 230000005284 excitation Effects 0.000 description 7
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 6
- AIJULSRZWUXGPQ-UHFFFAOYSA-N Methylglyoxal Chemical compound CC(=O)C=O AIJULSRZWUXGPQ-UHFFFAOYSA-N 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 4
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- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- HGINCPLSRVDWNT-UHFFFAOYSA-N Acrolein Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 description 2
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- 206010061218 Inflammation Diseases 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical compound [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 description 1
- HAMNKKUPIHEESI-UHFFFAOYSA-N aminoguanidine Chemical group NNC(N)=N HAMNKKUPIHEESI-UHFFFAOYSA-N 0.000 description 1
- 238000006157 aza-Cope rearrangement reaction Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 150000001728 carbonyl compounds Chemical class 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
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- 208000019622 heart disease Diseases 0.000 description 1
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- 125000000717 hydrazino group Chemical group [H]N([*])N([H])[H] 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C281/00—Derivatives of carbonic acid containing functional groups covered by groups C07C269/00 - C07C279/00 in which at least one nitrogen atom of these functional groups is further bound to another nitrogen atom not being part of a nitro or nitroso group
- C07C281/16—Compounds containing any of the groups, e.g. aminoguanidine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
Abstract
The invention belongs to the technical field of formaldehyde detection, and particularly relates to a two-photon fluorescent probe, a preparation method and application in the aspect of formaldehyde detection. The invention provides a two-photon fluorescent probe, which adopts aminoguanidino as a formaldehyde activity recognition group, has the characteristics of high sensitivity, good selectivity and higher response speed, and can be applied to in-vivo detection. The invention also provides a preparation method of the two-photon fluorescent probe, which comprises the steps of reacting hydroxy tetraphenylethylene with N-Boc-bromoethylamine, removing Boc, and adding S-methyl isothioaminourea hydroiodide to generate a product, namely the fluorescent probe for detecting formaldehyde.
Description
Technical Field
The invention belongs to the technical field of formaldehyde detection, and particularly relates to a two-photon fluorescent probe adopting an aminoguanidine group as a novel formaldehyde recognition group, a preparation method of the two-photon fluorescent probe and application of the two-photon fluorescent probe in the field of formaldehyde fluorescence detection in living tissues and cells.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Active carbonyl compounds have been receiving wide attention from human beings due to their carcinogenic toxicity, and their kinds mainly include formaldehyde, glyoxal, malonaldehyde, methylglyoxal, acrolein, and the like. Among them, formaldehyde exists in various parts of the human body, and when the concentration of formaldehyde is abnormal, various diseases such as cancer, heart disease, neurodegenerative disease and the like can be caused. Therefore, it is important to design and develop a technology capable of rapidly, highly selectively and highly sensitively detecting formaldehyde in a living body.
In recent years, fluorescent probes have unique advantages in the detection of formaldehyde in cells and living tissues due to the advantages of simple operation, high sensitivity, good selectivity, real-time detection and the like. Several representative fluorescent probes for the detection of formaldehyde are: fluorescent probes constructed with the Aza-Cope rearrangement strategy (Brewer T F, Chang C J. an Aza-type reactivity-based Fluorescent probes for imaging for Formaldehyde in Living cells [ J ]. am. chem. Soc.2015,137,10886-10889), and Fluorescent probes with hydrazino as Formaldehyde recognition group (Tang Y, Kong X, Xu A, et al. development of a Two-Photon Fluorescent probes for imaging for Formaldehyde in Living Tissues [ J ]. Angew. chem. int. Ed.2016,55, 3356-3359). However, the two types of probes still have some defects, such as low detection sensitivity, overlong response time and the like. Therefore, the development of a novel fluorescent probe capable of rapidly responding to formaldehyde with high selectivity and high sensitivity is still of great significance.
Disclosure of Invention
Aiming at the research background, the invention provides a fluorescent probe for detecting formaldehyde in cells and a preparation method thereof, wherein the fluorescent probe takes aminoguanidino as a recognition group of formaldehyde, has two-photon performance, is proved to have higher sensitivity and response speed for formaldehyde detection, and can be applied to a fluorescent detection mode of formaldehyde in a living sample.
In a first aspect of the present invention, a two-photon fluorescent probe is provided, which has a structure as shown in the following formula:
in a second aspect of the present invention, there is provided a method for preparing the two-photon fluorescent probe of the first aspect, the method comprising the steps of: reacting hydroxyl tetraphenylethylene with N-Boc-bromoethylamine, removing Boc, and adding S-methyl isothioaminourea hydroiodide to generate a product, namely the two-photon fluorescent probe.
Preferably, the preparation method comprises the following reaction processes:
preferably, the preparation method comprises the following steps:
(1) adding potassium carbonate and 18-crown ether-6 into a solvent system of hydroxyl tetraphenylethylene and N-Boc-bromoethylamine, and heating and refluxing to obtain a compound 1;
(2) removing Boc from the compound 1 to obtain a compound 2;
(3) and adding S-methyl isothioamino urea hydroiodide and triethylamine into the solvent system of the compound 2 to obtain the fluorescent probe for detecting formaldehyde.
Further preferably, the specific steps of step (1) are as follows: dissolving hydroxyl tetraphenylethylene and N-Boc-bromoethylamine in acetonitrile, adding potassium carbonate and 18-crown ether-6, heating and refluxing for 10-14 h, evaporating the solvent to dryness, and eluting and separating by using a chromatographic column to obtain a compound 1.
In some embodiments, the elution is performed by a single elution stepThe agent is a mixed solution of dichloromethane and petroleum ether with a volume ratio VMethylene dichloride：VPetroleum ether＝1-5：1。
In some embodiments, the hydroxy tetraphenylethylene, N-Boc-bromoethylamine, potassium carbonate, 18-crown-6 and acetonitrile are used in a ratio of 0.14 to 0.17 g: 0.14-0.17 mL: 0.11-0.14 g: 0.10-0.13 g: 4-5 mL.
Further preferably, the specific steps of step (2) are as follows: dissolving the compound 1 in dichloromethane, and dropwise adding trifluoroacetic acid while stirring for reaction for 1.5-2.5 h.
In some embodiments, the ratio of compound 1, dichloromethane, and trifluoroacetic acid is 0.16 to 0.20 g: 4-5 mL: 1.2-1.5 mL. The feeding proportion is beneficial to improving the conversion rate of raw materials and the yield and the purity of the compound 2.
The step (2) can control the reaction end time by tracking the reaction progress by means of TLC spot plate.
Further preferably, the specific operation of step (3) is as follows: dissolving a compound 2 and S-methyl isothioaminourea hydroiodide in absolute ethyl alcohol, adding triethylamine, refluxing for 3.5-4.5 h at 75-85 ℃, drying in a spinning mode, and purifying residues through a chromatographic column to obtain the two-photon fluorescent probe.
In a third aspect of the invention, an application of a two-photon fluorescent probe in the field of formaldehyde detection is provided.
Preferably, the formaldehyde detection includes formaldehyde detection in living samples, including living individuals, living tissues and cells. More specifically, the fluorescent probe can be applied to the development of formaldehyde-related disease diagnostic reagents and kits, or the detection of formaldehyde residues in food raw materials, such as water-borne food and the like.
In a fourth aspect of the present invention, there is provided a method for detecting formaldehyde in a sample, the method comprising the steps of: adding the buffer solution of the two-photon fluorescent probe into a sample to be tested for incubation, washing off redundant probes after incubation is finished, and shooting and measuring the fluorescence intensity in the sample to be tested through microscopic imaging.
Compared with the prior art, the invention has the beneficial effects that:
1. the fluorescent probe provided by the invention has two-photon performance, high sensitivity to formaldehyde detection, high response speed, small damage to a sample when being applied to living tissue detection, strong penetrating power and clear imaging effect. In addition, the two-photon fluorescent probe has high response speed, and can carry out detection without long-time incubation.
2. The research and verification of the invention prove that the two-photon fluorescent probe has higher selectivity, and the detection of the two-photon fluorescent probe is not interfered under the condition that other bioactive molecules exist.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a graph showing fluorescence response spectra of the probe and formaldehyde in example 3.
FIG. 2 is a graph showing the linear relationship between the fluorescence intensity of the probe at 480nm and the concentration of formaldehyde in example 3.
FIG. 3 is a graph showing the kinetics of the response of the probe to formaldehyde in example 3.
FIG. 4 shows the results of experiments on the selectivity of the probe for formaldehyde in example 3.
FIG. 5 is a graph of the staining of HepG2 cells with the probe of example 3;
wherein, FIG. 5(1) is a photograph showing fluorescent staining of cells;
FIG. 5a is a graph of cell staining with sodium bisulfite scavenging endogenous formaldehyde;
FIG. 5b is a staining pattern of a blank cell;
FIG. 5c is a staining pattern of cells in the formalin-treated group;
FIG. 5d is a staining pattern of cells in the formalin-treated group;
FIG. 5(2) is a graph showing the average fluorescence intensity output in HepG2 cells.
FIG. 6 is a photograph of a fluorescent photograph of leg tissue of a mouse in example 3;
wherein, fig. 6A is normal leg tissue; FIG. 6B shows the tissues of the leg that were stimulated by LPS to produce an inflammatory response.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As introduced in the background art, aiming at the defects in the prior art, the invention provides a two-photon fluorescence probe, a preparation method of the probe and application of the probe in the field of formaldehyde detection in living tissues.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Example 1
In this embodiment, a preparation scheme of a two-photon fluorescent probe is provided:
(1) hydroxytetraphenylethylene (348mg) and N-Boc-bromoethylamine (344. mu.L) were dissolved in acetonitrile (10mL), and potassium carbonate (276mg) and 18-crown-6 (264mg) were added to the solution, which was heated under reflux for 12h, dried by spinning, and separated by a column chromatography with an eluent ratio of VMethylene dichloride：VPetroleum ether1: and 1, spin-drying to obtain the compound 1.
(2) Dissolving the compound 1(200mg) in dichloromethane (5mL), dropwise adding trifluoroacetic acid (1.5mL) with stirring, tracking the reaction progress through a TLC spot plate, completing the reaction for about 2h, and then directly carrying out spin drying to obtain a compound 2;
(3) compound 2(100mg), S-methylisothiazoleSemicarbazide hydroiodide (120mg) was dissolved in absolute ethanol (5mL) and triethylamine (72. mu.L) was added, after which time it was refluxed for 4h at 80 ℃ and dried by spinning, and the residue was purified by column chromatography with eluent ratio VMethylene dichloride：VMethanol5: 1. and obtaining the TPE-AM for detecting the two-photon fluorescent probe.
Example 2
In this example, another preparation scheme of the two-photon fluorescent probe is provided:
(1) hydroxytetraphenylethylene (174mg) and N-Boc-bromoethylamine (172. mu.L) were dissolved in acetonitrile (5mL), and potassium carbonate (138mg) and 18-crown-6 (132mg) were added to the solution, which was heated under reflux for 12h, dried by spinning, and separated by a column chromatography with an eluent ratio of VMethylene dichloride：VPetroleum ether5: and 1, spin-drying to obtain the compound 1.
(2) Compound 1(100mg) was dissolved in dichloromethane (2.5mL), trifluoroacetic acid (0.75mL) was added dropwise with stirring, and the progress of the reaction was followed by TLC spot plate for about 2 h. Then directly carrying out spin drying to obtain a compound 2;
(3) compound 2(50mg), S-methylisothiamidourea hydroiodide (60mg) was dissolved in absolute ethanol (2.5mL) and triethylamine (36. mu.L) was added, after which time reflux at 80 ℃ for 4h, spin-dried, and the residue was purified by column chromatography with eluent ratio VMethylene dichloride：VMethanol10: and 1, obtaining the two-photon fluorescent probe TPE-AM.
Example 3 two-photon fluorescent probe structural characterization and Performance testing
1. Nuclear magnetic and mass spectrum characterization of intermediates and probes
Compound 1:1H NMR(400MHz,CDCl3):δ7.14-7.05(m,9H),7.05-6.98(m,6H),6.93(d,J＝8.8Hz,2H),6.62(d,J＝8.8Hz,2H),4.96(br s,1H),3.93(t,J＝5.0Hz,2H),3.52-3.44(m,2H),1.45(s,9H)ppm.13C NMR(100MHz,CDCl3):δ157.03,155.85,143.96,143.91,143.90,140.37,140.23,136.54,132.59,131.35,131.33,131.31,127.73,127.61,127.59,126.37,126.29,126.25,113.53,77.22,66.96,29.71,28.40ppm.HRMS(ESI):calculatedfor C33H33NO3Na+(M+Na)+514.2353,found 514.2354.
compound 2: HRMS (ESI) calculated for C28H26NO+(M+H)+392.2009,found392.1989.
And (3) a probe TPE-AM:1H NMR(400MHz,DMSO-d6):δ7.19-7.07(m,9H),7.01-6.92(m,6H),6.87(dd,J＝8.8,3.5Hz,2H),6.71(dd,J＝8.8,3.5Hz,2H),4.74(br s,1H),3.97(t,J＝5.0Hz,1H),3.92(t,J＝5.4Hz,1H),3.51(br s,1H),2.96(t,J＝5.4Hz,1H)ppm.13C NMR(100MHz,CDCl3):δ156.45,143.87,143.82,143.79,140.41,140.24,137.02,132.80,132.60,131.29,127.79,127.74,127.69,127.64,127.61,126.41,126.34,126.30,113.66,65.57,40.08ppm.HRMS(ESI):calculated for C29H29N4O+(M+H)+449.2336,found449.2350.
2. effect test
(1) Fluorescence response test
Fluorescence response tests were performed on 0,2,4,6,8,10,12,14,16 and 18. mu.M formaldehyde using 2.0. mu.M of the probe TPE-AM of examples 1 and 2, respectively, to obtain the relationship between the fluorescence intensity of the probe TPE-AM and the change in formaldehyde concentration. The experimental conditions are as follows: 50mMPBS, pH 7.4, incubated at room temperature for 5min, excitation wavelength 330nm, and fluorescence emission spectrum at 400-600nm was scanned. The abscissa is the wavelength (nm) and the ordinate is the fluorescence intensity. The excitation wavelength of the probe is 330nm, the maximum emission wavelength is 480nm, and the test effect is shown in figure 1, so that the fluorescence of the test solution is gradually enhanced along with the increase of the concentration of formaldehyde, which shows that the fluorescence intensity of the probe has good correlation with the concentration of formaldehyde in a system, the probe can be used for detecting formaldehyde, the incubation time is short, and the detection speed is high.
(2) Regression curves of fluorescence intensity and formaldehyde concentration at 480nm for probe TPE-AM in examples 1 and 2.
As a result, as shown in FIG. 2, the horizontal axis represents the concentration of formaldehyde, the vertical axis represents the fluorescence intensity at 480nm, and the excitation wavelength is 330 nm.
(3) Experimental kinetics of response of the TPE-AM probe of examples 1 and 2.
The experimental conditions are as follows: the probe concentration is 2.0 μ M, PBS is 50mM, pH is 7.4, the added formaldehyde concentration is 20 μ M (shown by a dotted line) and 50 μ M (shown by a solid line), the excitation wavelength is 330nm, the fluorescence intensity of the probe at 480nm is collected in real time, the test effect is shown in figure 3, and it can be seen that the fluorescence of the system after the formaldehyde is added shows a rapid rise at a time point of 30s and basically reaches a maximum value within the following 30s, which proves that the reaction of the probe on the recognition of the formaldehyde is basically completed within 30s, and shows that the probe has short incubation time for detecting the formaldehyde and fast response speed.
(4) Selective experiments with the TPE-AM probe of examples 1 and 2.
2.0. mu.M of the probe TPE-AM was mixed with different bioactive substances such as ClO-，·OH，ONOO-，H2O2，Cys，GSH，Ca2+，Mg2+Acetaldehyde, methylglyoxal, formaldehyde (corresponding to 27 in FIG. 4) and the like, and the change in fluorescence intensity at 480nm before and after the incubation was measured. The test results are shown in FIG. 4, which shows that the probe TPE-AM of the invention has high selectivity to formaldehyde, and other bioactive small molecules do not interfere with the formaldehyde.
(5) The TPE-AM probe of examples 1 and 2 was used for two-photon confocal microscopy imaging of human hepatoma cells (HepG 2).
The results are shown in FIG. 5, where FIG. 5a is a cell staining pattern of 100. mu.M sodium bisulfite to remove endogenous formaldehyde, and weak fluorescence is observed; FIG. 5b is a staining graph of blank cells with a slight increase in fluorescence compared to the cleared cells in FIG. 5a, indicating that the probe is capable of detecting endogenous formaldehyde in the cells; FIG. 5c is a 20 μ M formaldehyde-treated group staining pattern with significant increase in fluorescence compared to the cells in FIG. 5 b; FIG. 5d is a 50 μ M formaldehyde-treated group staining pattern showing a more pronounced fluorescence enhancement compared to the cells in FIG. 5 b.
The experimental conditions are as follows: the excitation wavelength is 720nm, and the collection range of the emission wavelength is 400-600 nm. The cells maintained good cell viability throughout the experiment. The experimental result shows that the fluorescent probe can have good response to formaldehyde in living cells, basically has no damage to living tissues, and can realize the detection of formaldehyde in cells by a fluorescent means.
(6) The probe TPE-AM of examples 1 and 2 was used for two-photon laser confocal microscopy of mouse leg tissue.
The results are shown in FIG. 6. The experimental mouse is injected with 100 mu L sterile PBS subcutaneously at one leg of the experimental mouse, LPS (1.0mg/mL,100 mu L) is injected subcutaneously at the other leg of the experimental mouse to induce inflammation, a probe (50 mu M,100 mu L) is injected into the abdominal cavity after 12h, two-photon imaging is respectively carried out on the leg of the experimental mouse after 1h, the excitation wavelength is 720nm, and the collection range of the emission wavelength is 400-600 nm. FIG. 6A shows weak fluorescence in normal leg tissue, and FIG. 6B shows clear fluorescence in leg tissue stimulated by LPS to produce inflammatory response at excitation wavelength, demonstrating that the probe is practically applicable to formaldehyde detection in living tissue.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A two-photon fluorescent probe is characterized in that the structure of the fluorescent probe is shown as the following formula:
2. the method for preparing the two-photon fluorescent probe according to claim 1, which comprises the steps of: reacting hydroxyl tetraphenylethylene with N-Boc-bromoethylamine, removing Boc, and adding S-methyl isothioaminourea hydroiodide to generate a product, namely the two-photon fluorescent probe.
3. The method for preparing the two-photon fluorescent probe according to claim 2, wherein the reaction process of the method is as follows:
4. the method for preparing the two-photon fluorescent probe according to claim 3, comprising the following steps:
(1) adding potassium carbonate and 18-crown ether-6 into a solvent system of hydroxyl tetraphenylethylene and N-Boc-bromoethylamine, and heating and refluxing to obtain a compound 1;
(2) removing Boc from the compound 1 to obtain a compound 2;
(3) and adding S-methyl isothioamino urea hydroiodide and triethylamine into the solvent system of the compound 2 to obtain the fluorescent probe for detecting formaldehyde.
5. The method for preparing the two-photon fluorescent probe according to claim 4, wherein the specific steps of the step (1) are as follows: dissolving hydroxyl tetraphenylethylene and N-Boc-bromoethylamine in acetonitrile, adding potassium carbonate and 18-crown ether-6, heating and refluxing for 10-14 h, evaporating the solvent to dryness, and eluting and separating by using a chromatographic column to obtain a compound 1;
preferably, the eluent is a mixed solution of dichloromethane and petroleum ether, and the volume ratio of the mixed solution is VMethylene dichloride：VPetroleum ether＝1-5：1；
Preferably, the usage ratio of the hydroxyl tetraphenylethylene, the N-Boc-bromoethylamine, the potassium carbonate, the 18-crown-6 and the acetonitrile is 0.14-0.17 g: 0.14-0.17 mL: 0.11-0.14 g: 0.10-0.13 g: 4-5 mL.
6. The method for preparing the two-photon fluorescent probe according to claim 4, wherein the specific steps of the step (2) are as follows: dissolving the compound 1 in dichloromethane, and dropwise adding trifluoroacetic acid while stirring for reaction for 1.5-2.5 h;
preferably, the ratio of compound 1, dichloromethane and trifluoroacetic acid is 0.16-0.20 g: 4-5 mL: 1.2-1.5 mL.
7. The method for preparing a two-photon fluorescent probe according to claim 4, wherein the specific operation of the step (3) is as follows: dissolving a compound 2 and S-methyl isothioaminourea hydroiodide in absolute ethyl alcohol, adding triethylamine, refluxing for 3.5-4.5 h at 75-85 ℃, drying in a spinning mode, and purifying residues through a chromatographic column to obtain the two-photon fluorescent probe.
8. The use of the two-photon fluorescent probe of claim 1 in the field of formaldehyde detection.
9. The application of the two-photon fluorescent probe in the field of formaldehyde detection as claimed in claim 8, wherein the formaldehyde detection comprises formaldehyde detection in living body samples, including living individuals, living tissues, cells and the like.
10. A method for detecting formaldehyde in a sample is characterized by comprising the following steps: adding the buffer solution of the two-photon fluorescent probe into a sample to be tested for incubation, washing off redundant probes after incubation is finished, and shooting and measuring the fluorescence intensity in the sample to be tested through microscopic imaging.
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