CN116693720B - D-pi-A red fluorescent marked chitosan and preparation method and application thereof - Google Patents
D-pi-A red fluorescent marked chitosan and preparation method and application thereof Download PDFInfo
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 61
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- JPJALAQPGMAKDF-UHFFFAOYSA-N selenium dioxide Chemical compound O=[Se]=O JPJALAQPGMAKDF-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000002243 precursor Substances 0.000 claims abstract description 13
- OJSCBKGRGMBEEW-UHFFFAOYSA-N 3-fluorobenzene-1,2-diamine Chemical compound NC1=CC=CC(F)=C1N OJSCBKGRGMBEEW-UHFFFAOYSA-N 0.000 claims abstract description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 14
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 7
- 229910017604 nitric acid Inorganic materials 0.000 claims description 7
- 238000010898 silica gel chromatography Methods 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 235000014655 lactic acid Nutrition 0.000 claims description 6
- 239000004310 lactic acid Substances 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 5
- 238000006482 condensation reaction Methods 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 238000000799 fluorescence microscopy Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 238000006396 nitration reaction Methods 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 239000000543 intermediate Substances 0.000 abstract description 12
- 239000000463 material Substances 0.000 abstract description 7
- 238000000746 purification Methods 0.000 abstract description 5
- 238000012984 biological imaging Methods 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 3
- BUOKUWQCVSZNCF-UHFFFAOYSA-N selenadiazole Chemical compound C1=C[se]N=N1 BUOKUWQCVSZNCF-UHFFFAOYSA-N 0.000 abstract description 3
- 125000003277 amino group Chemical group 0.000 abstract description 2
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 238000010534 nucleophilic substitution reaction Methods 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 description 16
- 238000002189 fluorescence spectrum Methods 0.000 description 6
- 239000007850 fluorescent dye Substances 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 238000000862 absorption spectrum Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 4
- 238000001215 fluorescent labelling Methods 0.000 description 4
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 3
- 239000011669 selenium Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 2
- 229940072056 alginate Drugs 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000006196 deacetylation Effects 0.000 description 2
- 238000003381 deacetylation reaction Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 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 description 2
- 235000010413 sodium alginate Nutrition 0.000 description 2
- 229940005550 sodium alginate Drugs 0.000 description 2
- 239000000661 sodium alginate Substances 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000004896 high resolution mass spectrometry Methods 0.000 description 1
- 239000012216 imaging agent Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000007709 nanocrystallization Methods 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention provides D-pi-A red fluorescent marked chitosan and a preparation method and application thereof, which belong to the fields of medical intermediates and material chemistry, wherein 2, 3-diaminofluorobenzene is used as a raw material, selenium dioxide is condensed to obtain a selenadiazole intermediate, the selenadiazole intermediate is further nitrified to obtain a fluorescent agent precursor SeFN, and finally nucleophilic substitution reaction is carried out with amino groups of the chitosan to obtain the red fluorescent marked chitosan. The preparation method is simple and easy to implement, the yield of the product is high, the separation and purification are easy, and the prepared red fluorescent-labeled chitosan can be used in the fields of biological imaging and the like.
Description
Technical Field
The invention belongs to the technical fields of medical intermediates and material chemistry, and particularly relates to D-pi-A red fluorescent marked chitosan and a preparation method and application thereof.
Background
In recent years, the development of fluorescent probes has become an urgent research hotspot, and biological fluorescent probes have been widely applied to life sciences such as diagnostics and molecular imaging. The natural high molecular polysaccharide such as chitosan has the characteristics of no toxicity, good biocompatibility and the like. At present, good matrixes of the high-molecular coupled fluorescent molecules are already applied to the fields of medicine, material science and the like. Many fluorescent-labeled natural chitosan derivatives are also present on the market. Such as: FITC-Chitosan green fluorescein green fluorescence labeling Chitosan, FITC-Alginate green fluorescein labeling sodium Alginate, rhodamine-Chitosan Rhodamine red fluorescence labeling Chitosan, rhodamine-Alginate Rhodamine labeling sodium Alginate, and the like. However, these derivatives still have some disadvantages in preparation or luminophores, mainly including: 1. the molecular weight of the luminophore is larger (> 360), which is not beneficial to the further preparation (such as nanocrystallization) of other chitosan material forms; 2. the synthesis step of the luminophore is long and the yield is low. 3. The luminophore needs to be activated to perform coupling reaction with chitosan, so that the reaction difficulty and steps are increased, and the product purification is complicated. 4. The emission wavelength of these luminophores is short, typically less than 600nm, and for biological imaging, the endogenous background interference is large.
Disclosure of Invention
In order to solve the technical problems, the invention provides D-pi-A red fluorescent marked chitosan and a preparation method and application thereof.
In order to achieve the aim, the invention provides D-pi-A red fluorescent marked chitosan, which has the following structural formula:
wherein, the formula (I) is D-pi-A type red fluorescent marked O-carboxymethyl chitosan, the formula (II) is D-pi-A type red fluorescent marked chitosan, and the formula (III) is D-pi-A type red fluorescent marked chitosan oligosaccharide lactic acid.
The invention synthesizes a small molecular fluorescent molecular precursor, which directly reacts with the nucleophilic center of chitosan to directly obtain the conjugate of the luminophore and the polymer. The small molecular fluorescent molecular precursor is easy to synthesize, high in yield, and the coupling reaction with chitosan can be completed at room temperature, and the fluorescent labeled chitosan can be obtained by simple treatment. In addition, the fluorescent light has longer emission wavelength, is in a red light range, has smaller endogenous background than fluorescent molecules such as fluorescein and the like, and has stronger practicability.
The preparation method of the D-pi-A red fluorescent marked chitosan comprises the following steps:
step 1: adding 2, 3-diaminofluorobenzene and selenium dioxide into ethanol for condensation reaction, and separating by silica gel column chromatography to obtain an intermediate SeF;
step 2: adding the intermediate SeF into mixed acid of concentrated sulfuric acid and concentrated nitric acid for nitration, adding the mixture into pure water after the reaction is finished, filtering, and separating by silica gel column chromatography to obtain a fluorescent molecular precursor SeFN;
step 3: respectively dissolving the fluorescent molecular precursor SeFN and chitosan in respective solvents, dripping the fluorescent molecular precursor SeFN solution into the chitosan solution, adding the solution into cold pure water with 3-5 times of volume after reaction, filtering, washing with the cold pure water, and drying to obtain the D-pi-A red fluorescent marked chitosan.
The invention takes 2, 3-diaminofluorobenzene as a raw material, obtains a selenadiazole intermediate by condensation with selenium dioxide, further nitrifies to obtain a fluorescent agent precursor SeFN, and finally obtains red fluorescence-marked chitosan by nucleophilic substitution reaction with amino groups of chitosan. The preparation method is simple and easy to implement, the yield of the product is high, the separation and purification are easy, and the prepared red fluorescent-labeled chitosan can be used in the fields of biological imaging and the like.
Further, in the step 1, the mass ratio of the 2, 3-diaminofluorobenzene to the selenium dioxide substance is 1:1-2;
the temperature of the condensation reaction is 25-80 ℃ and the time is 6-24 h;
the mobile phase of the silica gel column chromatography is dichloromethane and methanol, and the volume ratio is 30:1.
Further, in the step 2, the volume ratio of the mass of the intermediate SeF to the volume of the concentrated sulfuric acid and the volume ratio of the concentrated nitric acid are 10mmol to 1.5-3 mL to 3-9 mL;
the volume of the pure water is 5-10 times of the volume of the mixed acid.
Further, in the step 3, the chitosan is common chitosan, O-carboxymethyl chitosan or chitosan oligosaccharide lactic acid;
the solvent of the fluorescent molecule precursor SeFN is acetonitrile or dimethyl sulfoxide (DMSO);
the solvent of chitosan is DMSO or pure water.
The application of the D-pi-A red fluorescent marked chitosan in cell fluorescence imaging.
Compared with the prior art, the invention has the following advantages and technical effects:
the method for synthesizing the D-pi-A red fluorescent-labeled chitosan is simple and convenient, has high yield and longer luminophor wavelength, and overcomes the defects of multiple preparation steps, low yield, troublesome purification, short emission wavelength and the like of a plurality of fluorescent-labeled chitosan in the past. The fluorescence group emission wavelength of the D-pi-A red fluorescence marked chitosan is more than 593nm, the maximum emission wavelength is 620nm, and the Stokes shift is 137nm, so that the red fluorescence marked chitosan can be used as a good fluorescence imaging agent.
The D-pi-A red fluorescent marked chitosan can be used in cell fluorescent imaging, has longer emission wavelength and longer Stokes displacement, can be effectively applied to subcellular imaging positioning, and has a certain medical application prospect. In addition, the preparation method of the D-pi-A red fluorescent marked chitosan is simple and easy to implement, has low equipment requirements, is easy to separate and purify target compounds, and is convenient to popularize and use.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 is an ultraviolet absorption spectrum (UV-Vis) and a fluorescence emission spectrum (Flu) of a D-pi-A red fluorescent-labeled O-carboxymethyl chitosan obtained in example 1 of the present invention;
FIG. 2 shows ultraviolet absorption spectrum (UV-Vis) and fluorescence emission spectrum (Flu) of D-pi-A type red fluorescence-labeled chitosan obtained in example II of the present invention;
FIG. 3 shows the ultraviolet absorption spectrum (UV-Vis) and fluorescence emission spectrum (Flu) of D-pi-A red fluorescent-labeled chitosan oligosaccharide lactic acid obtained in example III of the invention;
FIG. 4 is a fluorescent image of D-pi-A red fluorescent-labeled O-carboxymethyl chitosan obtained in example 1 in A549 cells, wherein Blank is bright field, O-CMC-Se is D-pi-A red fluorescent-labeled O-carboxymethyl chitosan obtained in example 1, and Merge is a fluorescent image obtained by integrating and superposing bright field and fluorescent-labeled chitosan.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, 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. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The raw materials in the examples of the present invention were all obtained by purchasing them through a commercial route.
The room temperature in the examples of the present invention refers to 25.+ -. 2 ℃.
In the embodiment of the invention, the concentration of the concentrated nitric acid is 16mol/L, and the concentration of the concentrated nitric acid is 18mol/L.
Example 1
A D-pi-A red fluorescent-labeled O-carboxymethyl chitosan, which has a molecular structure shown in a formula (I):
the preparation method comprises the following steps:
2, 3-diaminofluorobenzene (10 mmol) and selenium dioxide (11 mmol) were added to ethanol (80 mL), heated to 80 ℃ for reaction for 10h, the solvent was removed, and purification was performed by silica gel column chromatography with a mobile phase of dichloromethane: methanol=30:1 (v: v) to give intermediate SeF. Then, intermediate SeF (8 mmol) was added to mixed acid (1.2 mL of concentrated nitric acid+3 mL of concentrated sulfuric acid) for nitration reaction for 30min, cooled, added to 25mL of cold pure water, stirred, filtered, chromatographed on silica gel column, and recrystallized from acetonitrile/dichloromethane (v: v=1:2) as mobile phase to obtain fluorescent molecular precursor SeFN. Characterization data of SeFN: 1 H NMR(400MHz,DMSO-d 6 )δ:8.49(d,J＝9.35Hz,1H,Ar-H),6.56(d,J＝9.35Hz,1H,Ar-H). 13 C NMR(100MHz,DMSO-d 6 )δ:153.83,152.59,149.51,133.10,129.38,99.43.HR-MS(ESI)m/z:Calcd for C 6 H 3 FN 3 O 2 Se[M+H] + 247.9375,found247.9377。
SeFN (1 mmol) was added to 6mL of acetonitrile, 100mL of pure water solution (dropwise with stirring) containing 1.120g of carboxymethyl chitosan (molecular weight 3 ten thousand, degree of deacetylation 90%) was added to NaHCO 3 (1 mmol), reacting at room temperature for 24h, filtering, washing with cold water once, and drying to obtain the compound (O-CMC-Se) shown in the formula (I).
Characterization data: IR cm -1 :3502,3486,3434,3378,3338,3290,3228,2939,2833,3790,2715,2358,2337,1658,1567,1357,1108,1066,7475.
Example 2
A D-pi-a red fluorescent-labeled chitosan having a molecular structure represented by formula (II):
the preparation method comprises the following steps:
SeFN was synthesized first, and the procedure was as in example 1. Then SeFN (1 mmol) was added to 6mL of acetonitrile containing 0.830g of carboxymethyl shellTo a DMSO (30 mL) solution (dropwise added under stirring) of glycan (molecular weight 2 ten thousand, degree of deacetylation 90%) was added NaHCO 3 (1 mmol), reacting at room temperature for 24h, adding 100mL of pure water, filtering, washing once with cold pure water, and drying to obtain the compound shown in the formula (II). Characterization data: IR cm -1 :3517,3446,3429,3394,3371,3338,2804,2744,2208,1670,1633,1577,1392,1188,798.
Example 3
A D-pi-a red fluorescent-labeled chitosan oligosaccharide lactic acid having a molecular structure represented by formula (III):
the preparation method comprises the following steps:
SeFN was synthesized first, and the procedure was as in example 1. Then, seFN (130 mg) was added to 20mL of acetonitrile, added to an aqueous solution (50 mL) containing 850mg of chitosan oligosaccharide lactic acid (molecular weight 2000) (dropwise with stirring), and reacted at room temperature for 24 hours, and dried to obtain the compound of formula (III). Characterization data: IR cm -1 :3542,3500,3458,3438,3421,3394,3371,3355,3328,2831,2719,2360,2339,1621,1569,1369,1070,775.
1. Ultraviolet and fluorescence spectra
The compounds prepared in examples 1, 2 and 3 were dissolved in water or DMSO, transferred to a two-way cuvette, and their absorption spectra were scanned by an ultraviolet spectrophotometer to obtain the ultraviolet absorption spectrum of the compound. Meanwhile, the compounds prepared in examples 1 to 2 were dissolved and transferred to a four-way cuvette, and the fluorescence emission spectrum of the compounds was scanned by a fluorescence spectrophotometer. The excitation wavelength was designed as the maximum absorption wavelength for each compound and the slit width was designed to be 10nm. The ultraviolet and fluorescence spectra of the compounds of examples 1-2 are shown in FIGS. 1 and 2.
The maximum absorption wavelength (lambda) of FIG. 1 max ) Maximum emission wavelength (lambda) em ) And Stokes shift (Stokes shift) data to obtain table 1. As can be seen from Table 1, the maximum absorption wavelength of the compound obtained in example 1 was 495nm, the maximum emission wavelength was 619nm and the Stokes shift was 124nm. The compound prepared in example 2 has a maximum absorption wavelength of 456nm, a maximum emission wavelength of 593nm and a Stokes shift of 137nm. The compound prepared in example 3 had a maximum absorption wavelength of 489nm and a maximum emission wavelength of 620nm, and the Stokes shift reached 131nm. These results indicate that the compounds prepared in examples 1 to 3 of the present invention have the characteristics of longer emission wavelength and larger stokes shift.
Lambda of the compound of Table 1 max ,λ em And Stokes shift
| Formula (I) | Formula (II) | Formula (III) | |
| λ max (nm) | 495 | 456 | 489 |
| λ em (nm) | 619 | 593 | 620 |
| Stokes shift(nm) | 124 | 137 | 131 |
4. Cell fluorescence imaging
A549 cells were seeded in 2 cm glass petri dishes, the compound of example 1 (20 μm) was added after cell attachment, incubated for 4h at 37 ℃, then the supernatant was gently blotted, washed once with PBS, and 2mL PBS was added. The Blank is a bright field, the fluorescence signal of the cells is observed through a confocal laser scanning microscope, the Merge is a fluorescence diagram formed by integrating and overlapping the bright field and the fluorescent-labeled chitosan of the embodiment 1, the result is shown in fig. 4, the fluorescent signal of the fluorescent-labeled chitosan is obvious, and the red fluorescent signal is clearly visible in the cells. The synthesized fluorescence labeling chitosan has a better fluorescence labeling function on cells.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.
Claims (6)
1. A D-pi-A red fluorescent-labeled chitosan is characterized by having the following structural formula:
2. a method for preparing the D-pi-a red fluorescent-labeled chitosan according to claim 1, comprising the steps of:
step 1: adding 2, 3-diaminofluorobenzene and selenium dioxide into ethanol for condensation reaction, and separating by silica gel column chromatography to obtain an intermediate SeF;
step 2: adding the intermediate SeF into mixed acid of concentrated sulfuric acid and concentrated nitric acid for nitration, adding the mixture into pure water after the reaction is finished, filtering, and separating by silica gel column chromatography to obtain a fluorescent molecular precursor SeFN;
step 3: respectively dissolving the fluorescent molecular precursor SeFN and chitosan in respective solvents, dripping the fluorescent molecular precursor SeFN solution into the chitosan solution, adding the solution into cold pure water with 3-5 times of volume after reaction, filtering, washing with the cold pure water, and drying to obtain the D-pi-A red fluorescent marked chitosan.
3. The preparation method according to claim 2, wherein in the step 1, the mass ratio of the 2, 3-diaminofluorobenzene to the selenium dioxide substance is 1:1-2;
the temperature of the condensation reaction is 25-80 ℃ and the time is 6-24 h;
the mobile phase of the silica gel column chromatography is dichloromethane and methanol, and the volume ratio is 30:1.
4. The process according to claim 2, wherein in step 2, the volume ratio of the amount of the intermediate SeF substance to the volume of the concentrated sulfuric acid and the volume of the concentrated nitric acid is 10 mmol:1.5 to 3 mL:3 to 9mL;
the volume of the pure water is 5-10 times of the volume of the mixed acid.
5. The preparation method according to claim 2, wherein in step 3, the chitosan is ordinary chitosan, O-carboxymethyl chitosan or chitosan oligosaccharide lactic acid;
the solvent of the fluorescent molecule precursor SeFN is acetonitrile or dimethyl sulfoxide;
the solvent of chitosan is dimethyl sulfoxide or pure water.
6. Use of red fluorescent-labeled chitosan of the D-pi-a type of claim 1 in cell fluorescence imaging.
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| CN101838344A (en) * | 2010-05-12 | 2010-09-22 | 上海交通大学 | Chitosan nanoparticle with fluorescence labeling characteristic and method for preparing same |
| CN104292362A (en) * | 2014-07-23 | 2015-01-21 | 北京理工大学 | Fluorescent labeling method for chitosan |
| CN108117612A (en) * | 2017-12-15 | 2018-06-05 | 浙江大学 | A kind of preparation method with the water soluble chitosan-based aggregation-induced emission fluorescence probe for reducing response |
| CN108690058A (en) * | 2018-06-20 | 2018-10-23 | 咸阳师范学院 | A kind of lead ion fluorescent sensing material, preparation method and application method |
| EP3941582A1 (en) * | 2019-03-18 | 2022-01-26 | The University Court Of The University Of Edinburgh | Small molecule photosensitizers for photodynamic therapy |
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| CN101838344A (en) * | 2010-05-12 | 2010-09-22 | 上海交通大学 | Chitosan nanoparticle with fluorescence labeling characteristic and method for preparing same |
| CN104292362A (en) * | 2014-07-23 | 2015-01-21 | 北京理工大学 | Fluorescent labeling method for chitosan |
| CN108117612A (en) * | 2017-12-15 | 2018-06-05 | 浙江大学 | A kind of preparation method with the water soluble chitosan-based aggregation-induced emission fluorescence probe for reducing response |
| CN108690058A (en) * | 2018-06-20 | 2018-10-23 | 咸阳师范学院 | A kind of lead ion fluorescent sensing material, preparation method and application method |
| EP3941582A1 (en) * | 2019-03-18 | 2022-01-26 | The University Court Of The University Of Edinburgh | Small molecule photosensitizers for photodynamic therapy |
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