CN115925667A - Ratio type near-infrared fluorescent probe molecule and preparation method and application thereof - Google Patents
Ratio type near-infrared fluorescent probe molecule and preparation method and application thereof Download PDFInfo
- Publication number
- CN115925667A CN115925667A CN202211280811.2A CN202211280811A CN115925667A CN 115925667 A CN115925667 A CN 115925667A CN 202211280811 A CN202211280811 A CN 202211280811A CN 115925667 A CN115925667 A CN 115925667A
- Authority
- CN
- China
- Prior art keywords
- benzyloxy
- synthesis
- reacting
- fluorescent probe
- compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 238000003384 imaging method Methods 0.000 claims abstract description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 39
- 238000003786 synthesis reaction Methods 0.000 claims description 39
- JHIVVAPYMSGYDF-UHFFFAOYSA-N cyclohexanone Chemical compound O=C1CCCCC1 JHIVVAPYMSGYDF-UHFFFAOYSA-N 0.000 claims description 36
- 238000001816 cooling Methods 0.000 claims description 31
- 239000013067 intermediate product Substances 0.000 claims description 26
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 24
- WFDIJRYMOXRFFG-UHFFFAOYSA-N acetic acid anhydride Natural products CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 24
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 18
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 18
- 238000004440 column chromatography Methods 0.000 claims description 18
- 238000001914 filtration Methods 0.000 claims description 18
- -1 o-carboxyphenyl Chemical group 0.000 claims description 18
- 239000012074 organic phase Substances 0.000 claims description 18
- 239000007787 solid Substances 0.000 claims description 18
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 16
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 15
- 229910052760 oxygen Inorganic materials 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000005457 ice water Substances 0.000 claims description 12
- 239000012046 mixed solvent Substances 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 12
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 claims description 11
- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 claims description 10
- VYXSBFYARXAAKO-WTKGSRSZSA-N chembl402140 Chemical class Cl.C1=2C=C(C)C(NCC)=CC=2OC2=C\C(=N/CC)C(C)=CC2=C1C1=CC=CC=C1C(=O)OCC VYXSBFYARXAAKO-WTKGSRSZSA-N 0.000 claims description 8
- 229940125904 compound 1 Drugs 0.000 claims description 8
- 229940125782 compound 2 Drugs 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 7
- UIOAMIIUJLFKNB-UHFFFAOYSA-N N-phenylaniline propanedial hydrochloride Chemical compound Cl.O=CCC=O.C=1C=CC=CC=1NC1=CC=CC=C1 UIOAMIIUJLFKNB-UHFFFAOYSA-N 0.000 claims description 6
- 150000001263 acyl chlorides Chemical class 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 238000010791 quenching Methods 0.000 claims description 6
- 230000000171 quenching effect Effects 0.000 claims description 6
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 229910020366 ClO 4 Inorganic materials 0.000 claims description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 claims description 4
- 229940126214 compound 3 Drugs 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 229910052739 hydrogen Inorganic materials 0.000 claims description 4
- 150000007530 organic bases Chemical class 0.000 claims description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 229910052717 sulfur Inorganic materials 0.000 claims description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-diisopropylethylamine Substances CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 3
- 125000000896 monocarboxylic acid group Chemical group 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 1
- 239000000758 substrate Substances 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 2
- 239000012620 biological material Substances 0.000 abstract description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 16
- 206010028980 Neoplasm Diseases 0.000 description 11
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 11
- 239000001301 oxygen Substances 0.000 description 11
- 230000004044 response Effects 0.000 description 11
- 102000004459 Nitroreductase Human genes 0.000 description 9
- 108020001162 nitroreductase Proteins 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 230000008859 change Effects 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- MHSGOABISYIYKP-UHFFFAOYSA-N (4-nitrophenyl)methyl carbonochloridate Chemical compound [O-][N+](=O)C1=CC=C(COC(Cl)=O)C=C1 MHSGOABISYIYKP-UHFFFAOYSA-N 0.000 description 4
- 206010067125 Liver injury Diseases 0.000 description 4
- HSDAJNMJOMSNEV-UHFFFAOYSA-N benzyl chloroformate Chemical compound ClC(=O)OCC1=CC=CC=C1 HSDAJNMJOMSNEV-UHFFFAOYSA-N 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- 239000012467 final product Substances 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 231100000753 hepatic injury Toxicity 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000000693 micelle Substances 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000001678 irradiating effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000011155 quantitative monitoring Methods 0.000 description 2
- 239000007845 reactive nitrogen species Substances 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 229910000530 Gallium indium arsenide Inorganic materials 0.000 description 1
- 229920002565 Polyethylene Glycol 400 Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention belongs to the technical field of biological materials, and particularly relates to a ratio type near-infrared fluorescent probe molecule and a preparation method and application thereof. The general structural formula of the fluorescent probe molecule is shown as follows; the structural molecule can construct a ratio type near infrared second window fluorescent probe selectively activated by a specific substrate, and is used for imaging of deep tissues of organisms and biosensing analysis.
Description
Technical Field
The invention belongs to the technical field of biological materials, and particularly relates to a ratio type near-infrared fluorescent probe molecule, and a preparation method and application thereof.
Background
In recent years, near-infrared second window (1000-1700 nm) in vivo fluorescence imaging has been rapidly developed in the biomedical field, which benefits from the civilization of InGaAs detectors (response range 900-1700 nm). The current fluorescent probes in the near infrared second window are mainly ' off-on ' type probes, the fluorescent probes can be selectively activated by specific substrates in a living body so as to give signals, but only can realize signal change with ' and ' without ', and qualitative visual monitoring of specific substrates in a tumor microenvironment, such as enzyme, reactive oxygen species/reactive nitrogen species (ROS/RNS), can be realized by using the ' off-on ' type fluorescent probes, but quantitative change monitoring cannot be realized.
In contrast, the ratiometric fluorescent probe can acquire signals through a plurality of channels and then correlate the signals of the channels, so that the ratiometric fluorescent probe has a self-correction function and can realize quantitative analysis and monitoring. At present, the near-infrared second window ratio fluorescent probe and the probe design strategy are less, the types of substrates capable of realizing detection are mainly strong oxidizing substances, and the detection requirements on biomolecules such as enzyme and the like cannot be met. In order to realize quantitative visualization of a tumor microenvironment, a near-infrared second window ratio type probe molecular platform is urgently needed to be constructed, and a reliable (semi-) quantitative analysis method under deep tissues is established.
Disclosure of Invention
The invention aims to provide a ratiometric near-infrared second window fluorescent probe molecule capable of performing visual and quantitative monitoring on a tumor microenvironment, and a preparation method and application thereof.
The invention provides a ratio type near infrared fluorescent probe molecule, which is marked as Rap, and the structural general formula of the compound is as follows:
wherein X is selected from O or S; y is selected from ClO 4 、PF 6 、BF 4 、Cl、Br、I、CF 3 COO、CF 3 SO 3 、CH 3 COO or CH 3 SO 3 ；R 1 Selected from H, OH, OCH 3 Or N [ (CH) 2 )nCH 3 ] 2 ；R 2 And R 3 Is H, o-carboxyphenyl or phenyl or o-methylphenyl, R 4 The substituted benzyloxy can be selected from 4-nitrobenzyloxy, 4-phosphorylbenzyloxy, 4-glutamyl transpeptidyl benzyloxy, 4-beta-galactosyl benzyloxy, azidobenzyloxy, 2, 4-dinitrobenzyloxy, 4-boric benzyloxy or 4-boric benzyloxy ester, and n is an integer of 0-6.
The invention also provides a preparation method of the ratio type near-infrared fluorescent probe molecule, and the synthetic route is as follows:
wherein X is selected from O or S; y is selected from ClO 4 、PF 6 、BF 4 、Cl、Br、I、CF 3 COO、CF 3 SO 3 、CH 3 COO or CH 3 SO 3 ；R 1 Selected from H, OH, OCH 3 Or N [ (CH) 2 )nCH 3 ] 2 ；R 2 And R 3 Is H, o-carboxyphenyl or phenyl or o-methylphenyl, R 4 Is substituted benzyloxy and can be selected from 4-nitrobenzyloxy, 4-phosphoryl benzyloxy, 4-glutamyl transpeptidyl benzyloxy, 4-beta-galactosyl benzyloxy, azido benzyloxy, 2, 4-dinitrobenzyloxy, 4-boric benzyloxy or 4-boric benzyloxy, and n is an integer of 0 to 6; the compound 1 is substituted rhodamine 6G, the compound 2 is substituted benzoyl, and the compound 3 is substituted acyl chloride.
The specific synthesis steps are as follows:
(1) Synthesis of intermediate 1:
dissolving substituted rhodamine 6G (compound 1) in a mixed solution of sodium hydroxide and ethanol, and reacting for 36-48 hours at 150-170 ℃; after cooling, regulating the pH value to 3-4 by using a hydrochloric acid solution, separating out solids, and filtering to obtain an intermediate product 1;
(2) And (3) synthesis of an intermediate 2:
dissolving the intermediate 1 and cyclohexanone in concentrated sulfuric acid, reacting at 80-100 ℃ for 1-3 hours, cooling to room temperature, adding ice water, adding protonic acid, separating out solids, and filtering to obtain an intermediate 2;
wherein the protonic acid is selected from HClO 4 、HPF 6 、HBF 4 、HCl、HBr、HI、CF 3 COOH、CF 3 SO 3 H and CH 3 SO 3 H; wherein the feeding molar ratio of the intermediate product 1 to the cyclohexanone is 1 (1-3);
(3) Synthesis of intermediate 3
Dissolving substituted benzoyl (compound 2) in concentrated sulfuric acid, reacting at 80-100 ℃ for 1-3 hours, cooling to room temperature, adding ice water, adding protonic acid, separating out solid, and filtering to obtain an intermediate 3; the feeding molar ratio of the intermediate product 2 to the cyclohexanone is 1 (1-3);
wherein the protonic acid is selected from HClO 4 、HPF 6 、HBF 4 、HCl、HBr、HI、CF 3 COOH、CF 3 SO 3 H and CH 3 SO 3 H;
(4) And (3) synthesis of an intermediate 4:
dissolving the intermediate 3 and malonaldehyde diphenylamine hydrochloride in a mixed solvent of acetic acid and acetic anhydride, and reacting for 1-3 hours at 90-110 ℃ under the protection of nitrogen; cooling to room temperature, concentrating the organic phase and separating by column chromatography to obtain an intermediate 4;
(5) Synthesis of intermediate 5:
under the protection of nitrogen, adding the intermediate 2 and the intermediate 4 into a mixed solvent of toluene and n-butyl alcohol, and reacting for 3-6 hours at 80-120 ℃; cooling to room temperature, concentrating the organic phase, and separating by column chromatography to obtain intermediate 5; the feeding molar ratio of the intermediate 2 to the intermediate 4 is 1 (1-3);
(6) Synthesis of ratiometric near-infrared fluorescent probe molecule (product 1):
dissolving the intermediate 5 and substituted acyl chloride (compound 3) in dichloromethane, adding organic base, and reacting at room temperature for 12-48 h; after quenching reaction, concentrating an organic phase, and separating by using column chromatography to obtain a product 1; the feeding molar ratio of the intermediate 5 to the substituted acyl chloride is 1 (2-3).
Wherein the organic base is selected from one of triethylamine, N, N-diisopropylethylamine and pyridine.
The ratiometric near-infrared second window fluorescent probe molecule obtained by the invention generates a ratiometric fluorescent signal after reacting with enzyme and reactive oxygen species/reactive nitrogen species (ROS/RNS).
The ratiometric near-infrared second window fluorescent probe molecule provided by the invention has the advantages of large molar extinction coefficient, long absorption and emission wavelength, good chemical stability and light stability, and good stability in a large acid-base range. The platform has good molecule universality, can be used for constructing various enzyme responses and ratio type near-infrared fluorescent probes of active oxygen responses, and can realize (semi) quantitative visual monitoring of biological small molecules (active oxygen), large molecules (enzyme) and the like in a biological living body.
Furthermore, the ratio type near infrared fluorescent probe molecule (general formula 1) of the invention has the maximum absorption peak positioned at-800 nm and the maximum emission peak positioned at-950 nm in a dichloromethane solution.
Furthermore, the molar extinction coefficient of the ratio type near infrared fluorescent probe molecule (general formula 1) in a dichloromethane solution is 95000M -1 cm -1 。
The ratio-type near-infrared fluorescent probe molecule provided by the invention can be used for imaging deep tissues of organisms, biosensing analysis and the like, and can be prepared into reagents or micellar solutions and the like.
Drawings
FIG. 1 shows the absorption and fluorescence emission spectra of a ratiometric near infrared fluorescent probe Rap-N in PBS (PBS/glycerol/PEG 400) under excitation at 808nm (corresponding to example 1).
FIG. 2 shows the fluorescence ratio change spectrum of the response of the ratiometric near-infrared fluorescent probe Rap-N to nitroreductase excited at 808nm (corresponding to example 1).
FIG. 3 is a graph showing the fluorescence ratio change spectrum of the response of the ratiometric near-infrared fluorescent probe Rap-R to hydrogen peroxide under 808nm excitation (corresponding to example 2).
FIG. 4 shows the use of the Rap-N probe for in vivo tumor imaging (corresponding to example 4).
FIG. 5 shows the use of the Rap-R probe for imaging liver damage in vivo (corresponding to example 5).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described with the following embodiments, but the present invention is by no means limited to these embodiments. The following are only preferred embodiments of the present invention, which are merely illustrative and should not be construed as limiting the scope of the present invention. It should be understood that any modification, substitution or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Example 1:
preparing a Nitroreductase (NTR) response ratio type near infrared fluorescent probe (marked as Rap-N), wherein the structural formula of the compound is as follows:
the specific synthetic route is as follows:
the specific synthesis steps are as follows:
(1) Synthesis of intermediate 1
Dissolving rhodamine 6G (compound 1) in a mixed solution of sodium hydroxide and ethanol, and reacting for 36-48 hours at 150-170 ℃; after cooling, regulating the pH value to 3-4 by using a hydrochloric acid solution, separating out solids and filtering to obtain an intermediate product 1;
(2) Synthesis of intermediate 2
Dissolving the intermediate 1 and cyclohexanone in concentrated sulfuric acid, reacting at 80-100 ℃ for 1-3 hours, cooling to room temperature, adding ice water, adding HClO 4 (ii) a Separating out solid and filtering to obtain an intermediate 2; wherein the feeding molar ratio of the intermediate product 1 to the cyclohexanone is 1 (1-3);
(3) Synthesis of intermediate 3
Dissolving the compound 2 in concentrated sulfuric acid, reacting at 80-100 ℃ for 1-3 hours, cooling to room temperature, adding ice water, adding HClO 4 (ii) a Separating out solid and filtering to obtain an intermediate 3; wherein the feeding molar ratio of the intermediate product 2 to the cyclohexanone is 1 (1-3);
(4) Synthesis of intermediate 4
Dissolving the intermediate 3 and malonaldehyde diphenylamine hydrochloride in a mixed solvent of acetic acid and acetic anhydride, and reacting for 1-3 hours at 90-110 ℃ under the protection of nitrogen; after cooling to room temperature, the organic phase was concentrated and separated by column chromatography to give intermediate 4;
(5) Synthesis of intermediate 5
Under the protection of nitrogen, adding the intermediate 2 and the intermediate 4 into a mixed solvent of toluene and n-butyl alcohol, and reacting for 3-6 hours at 80-120 ℃; cooling to room temperature, concentrating the organic phase and separating by column chromatography to obtain an intermediate 5; wherein the feeding molar ratio of the intermediate product 2 to the intermediate product 4 is 1 (1-3);
(6) Synthesis of Rap-N
Dissolving the intermediate 5 and p-nitrobenzyl chloroformate in dichloromethane, adding triethylamine, and reacting at room temperature for 12-48 h; after quenching reaction, concentrating an organic phase and separating by using column chromatography to obtain a final product Rap-N; wherein the feeding molar ratio of the intermediate 5 to the p-nitrobenzyl chloroformate is 1 (2-3).
Specific examples are:
(1) Synthesis of intermediate 1
Dissolving rhodamine 6G (compound 1) in a mixed solution of sodium hydroxide and ethanol, and reacting for 36 hours at 170 ℃; after cooling, regulating the pH value to about 3-4 by using a hydrochloric acid solution, separating out solids, and filtering to obtain an intermediate product 1;
(2) Synthesis of intermediate 2
Dissolving the intermediate 1 and cyclohexanone in concentrated sulfuric acid, reacting at 90 deg.C for 2 hr, cooling to room temperature, adding ice water, adding HClO 4 (ii) a Separating out solid and filtering to obtain an intermediate 2; wherein the feeding molar ratio of the intermediate product 1 to the cyclohexanone is 1.2;
(3) Synthesis of intermediate 3
Dissolving compound 2 in concentrated sulfuric acid, reacting at 90 deg.C for 2 hr, cooling to room temperature, adding ice water, adding HClO 4 (ii) a Separating out solid and filtering to obtain an intermediate 3; whereinThe feeding molar ratio of the intermediate product 2 to the cyclohexanone is 1.2;
(4) Synthesis of intermediate 4
Dissolving the intermediate 3 and malonaldehyde diphenylamine hydrochloride in a mixed solvent of acetic acid and acetic anhydride, and reacting for 2 hours at 100 ℃ under the protection of nitrogen; after cooling to room temperature, the organic phase was concentrated and separated by column chromatography to give intermediate 4;
(5) Synthesis of intermediate 5
Under the protection of nitrogen, adding the intermediate 2 and the intermediate 4 into a mixed solvent of toluene and n-butyl alcohol, and reacting for 5 hours at 110 ℃; cooling to room temperature, concentrating the organic phase and separating by column chromatography to obtain an intermediate 5; wherein the feeding molar ratio of the intermediate product 2 to the intermediate product 4 is 1;
(6) Synthesis of Rap-N
Dissolving the intermediate 5 and p-nitrobenzyl chloroformate in dichloromethane, adding triethylamine, and reacting at room temperature for 24 hours; after quenching reaction, concentrating an organic phase and separating by using column chromatography to obtain a final product Rap-N; wherein the feeding molar ratio of the intermediate 5 to the p-nitrobenzyl chloroformate is 1.
The change of fluorescence intensity of the Rap-N ratiometric fluorescent probe in response to nitroreductase under the excitation of 808nm is shown in FIG. 2.
Example 2:
preparing an active oxygen response ratio type near infrared fluorescent probe (marked as Rap-R), wherein the structural formula of the compound is as follows:
the specific synthetic route is as follows:
the specific synthesis steps are as follows:
(1) Synthesis of intermediate 1
Dissolving rhodamine 6G (compound 1) in a mixed solution of sodium hydroxide and ethanol, and reacting for 36-48 hours at 150-170 ℃; after cooling, regulating the pH value to 3-4 by using a hydrochloric acid solution, separating out solids, and filtering to obtain an intermediate product 1;
(2) Synthesis of intermediate 2
Dissolving the intermediate 1 and cyclohexanone in concentrated sulfuric acid, reacting at 80-100 ℃ for 1-3 hours, cooling to room temperature, adding ice water, adding HClO 4 (ii) a Separating out solid and filtering to obtain an intermediate 2; wherein the feeding molar ratio of the intermediate product 1 to the cyclohexanone is 1 (1-3);
(3) Synthesis of intermediate 3
Dissolving the compound 2 in concentrated sulfuric acid, reacting at 80-100 ℃ for 1-3 hours, cooling to room temperature, adding ice water, adding HClO 4 (ii) a Separating out solid and filtering to obtain an intermediate 3; wherein the feeding molar ratio of the intermediate product 2 to the cyclohexanone is 1 (1-3);
(4) Synthesis of intermediate 4
Dissolving the intermediate 3 and malonaldehyde diphenylamine hydrochloride in a mixed solvent of acetic acid and acetic anhydride, and reacting for 1-3 hours at 90-110 ℃ under the protection of nitrogen; after cooling to room temperature, the organic phase was concentrated and separated by column chromatography to give intermediate 4;
(5) Synthesis of intermediate 5
Under the protection of nitrogen, adding the intermediate 2 and the intermediate 4 into a mixed solvent of toluene and n-butanol, and reacting for 3-6 hours at 80-120 ℃; cooling to room temperature, concentrating the organic phase and separating by column chromatography to obtain an intermediate 5; wherein the feeding molar ratio of the intermediate product 2 to the intermediate product 4 is 1 (1-3);
(6) Synthesis of Rap-R
Dissolving the intermediate 5 and 4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl chloroformate in dichloromethane, adding triethylamine, and reacting at room temperature for 12-48 h; after quenching reaction, concentrating an organic phase and separating by using column chromatography to obtain a final product Rap-R; wherein the feeding molar ratio of the intermediate 5 to the 4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl chloroformate is 1 (2-3).
Specific examples are as follows:
(1) Synthesis of intermediate 1
Dissolving rhodamine 6G (compound 1) in a mixed solution of sodium hydroxide and ethanol, and reacting for 48 hours at 160 ℃; after cooling, regulating the pH value to about 3-4 by using a hydrochloric acid solution, separating out solids, and filtering to obtain an intermediate product 1;
(2) Synthesis of intermediate 2
Dissolving the intermediate 1 and cyclohexanone in concentrated sulfuric acid, reacting at 95 ℃ for 1.5 hours, cooling to room temperature, adding ice water, adding HClO 4 (ii) a Separating out solid and filtering to obtain an intermediate 2; wherein the feeding molar ratio of the intermediate product 1 to the cyclohexanone is 1.3;
(3) Synthesis of intermediate 3
Dissolving compound 2 in concentrated sulfuric acid, reacting at 95 deg.C for 1.5 hr, cooling to room temperature, adding ice water, adding HClO 4 (ii) a Separating out solid and filtering to obtain an intermediate 3; wherein the feeding molar ratio of the intermediate product 2 to the cyclohexanone is 1.3;
(4) Synthesis of intermediate 4
Dissolving the intermediate 3 and malonaldehyde diphenylamine hydrochloride in a mixed solvent of acetic acid and acetic anhydride, and reacting for 2 hours at 110 ℃ under the protection of nitrogen; after cooling to room temperature, the organic phase was concentrated and separated by column chromatography to give intermediate 4;
(5) Synthesis of intermediate 5
Under the protection of nitrogen, adding the intermediate 2 and the intermediate 4 into a mixed solvent of toluene and n-butanol, and reacting for 6 hours at 100 ℃; cooling to room temperature, concentrating the organic phase and separating by column chromatography to obtain an intermediate 5; wherein the feeding molar ratio of the intermediate product 2 to the intermediate product 4 is 1;
(6) Synthesis of Rap-R
Dissolving the intermediate 5 and 4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl chloroformate in dichloromethane, adding triethylamine, and reacting at room temperature for 24 hours; after quenching reaction, concentrating an organic phase and separating by using column chromatography to obtain a final product Rap-R; wherein the feeding molar ratio of the intermediate 5 to the 4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzylchloroformate is 1.
The change of fluorescence intensity of the Rap-R ratiometric fluorescent probe in response to hydrogen peroxide under the excitation of 808nm is shown in FIG. 3.
Example 3:
the preparation method of the micelle formed by the active oxygen response ratio type near infrared fluorescent probe (Rap-R) and phospholipid polyethylene glycol comprises the following specific steps:
fluorescent probes Rap-R and DOPE-PEG 2000 Dissolving the mixture in chloroform, stirring the mixture for 0.5 to 2 hours according to the mass ratio of 1 (20 to 100), spin-drying the solvent, vacuum-drying, heating to 50 to 80 ℃, adding deionized water to dissolve the solvent, performing ultrasonic treatment, cooling to room temperature, and performing ultrafiltration concentration through an ultrafiltration tube to finally obtain the Rap-R micelle solution.
Example 4:
nitroreductase responsive ratiometric near infrared fluorescent probes Rap-N are used to monitor nitroreductase in tumors. The method comprises the following specific steps:
injecting 200 μ L of 100 μ M Rap-N probe solution into the tumor of anesthetized mouse, and irradiating the tumor with 808nm external laser with power density of 200mW/cm 2 The fluorescence of 900-1700nm and 1000-1700nm is collected by using 900nm and 1000nm long pass filters respectively, the intensity ratio of the two is calculated, and the content of nitroreductase is monitored according to the fluorescence signal ratio values of different tumors, as shown in figure 4. In FIG. 4, the Rap-N probe is used to monitor the nitroreductase content in 4T1 tumor and CT26 tumor, and the nitroreductase content in tumor is determined according to the fluorescence signal ratio value.
Example 5:
the active oxygen response ratio type near infrared fluorescent probe Rap-R is used for detecting the content of active oxygen (hydrogen peroxide) in inflammatory tissues. The method comprises the following specific steps:
injecting 200 μ L of 100 μ M Rap-R probe micelle solution into tail vein of anesthetized mouse, irradiating abdomen of mouse with 808nm external laser with laser power density of 200mW/cm 2 The fluorescence of 900-1700nm and 1000-1700nm is collected by using 900nm and 1000nm long pass filters respectively, the intensity ratio of the two is calculated, and the content of active oxygen (hydrogen peroxide) in liver injury inflammation is monitored according to the ratio value of non-fluorescence signals, which is shown in figure 5. In the context of figure 5, it is shown,the Rap-R probe is used for monitoring the content of active oxygen (hydrogen peroxide) in the living liver injury inflammation, and the content of the active oxygen (hydrogen peroxide) is judged according to the fluorescence signal ratio value, wherein the content of the active oxygen (hydrogen peroxide) is respectively the content of active oxygen (hydrogen peroxide) in a control group, liver injury and after drug treatment.
Claims (5)
1. A ratio-type near-infrared fluorescent probe molecule is marked as Rap, and is characterized in that the structural general formula of the compound is as follows:
wherein X is selected from O or S; y is selected from ClO 4 、PF 6 、BF 4 、Cl、Br、I、CF 3 COO、CF 3 SO 3 、CH 3 COO or CH 3 SO 3 ；R 1 Selected from H, OH, OCH 3 Or N [ (CH) 2 )nCH 3 ] 2 ；R 2 And R 3 Is H, o-carboxyphenyl or phenyl or o-methylphenyl, R 4 The substituted benzyloxy can be selected from 4-nitrobenzyloxy, 4-phosphorylbenzyloxy, 4-glutamyl transpeptidyl benzyloxy, 4-beta-galactosyl benzyloxy, azidobenzyloxy, 2, 4-dinitrobenzyloxy, 4-boric benzyloxy or 4-boric benzyloxy ester, and n is an integer of 0-6.
2. The method for preparing the ratiometric near-infrared fluorescent probe molecule of claim 1, wherein the synthetic route is as follows:
wherein X is selected from O or S; y is selected from ClO 4 、PF 6 、BF 4 、Cl、Br、I、CF 3 COO、CF 3 SO 3 、CH 3 COO or CH 3 SO 3 ；R 1 Selected from H, OH, OCH 3 Or N [ (CH) 2 )nCH 3 ] 2 ；R 2 And R 3 Is H, o-carboxyphenyl or phenyl or o-methylphenyl, R 4 Is substituted benzyloxy and can be selected from 4-nitrobenzyloxy, 4-phosphoryl benzyloxy, 4-glutamyl transpeptidyl benzyloxy, 4-beta-galactosyl benzyloxy, azido benzyloxy, 2, 4-dinitrobenzyloxy, 4-boric benzyloxy or 4-boric benzyloxy, and n is an integer of 0 to 6; the compound 1 is substituted rhodamine 6G, the compound 2 is substituted benzoyl, and the compound 3 is substituted acyl chloride;
the preparation method comprises the following specific steps:
(1) Synthesis of intermediate 1:
dissolving a compound 1 substituted rhodamine 6G in a mixed solution of sodium hydroxide and ethanol, and reacting for 36-48 hours at 150-170 ℃; after cooling, regulating the pH value to 3-4 by using a hydrochloric acid solution, separating out solids, and filtering to obtain an intermediate product 1;
(2) Synthesis of intermediate 2:
dissolving the intermediate 1 and cyclohexanone in concentrated sulfuric acid, reacting at 80-100 ℃ for 1-3 hours, cooling to room temperature, adding ice water, adding protonic acid, separating out solids, and filtering to obtain an intermediate 2; the feeding molar ratio of the intermediate product 1 to the cyclohexanone is 1 (1-3);
(3) And (3) synthesis of an intermediate 3:
dissolving the compound 2 substituted benzoyl in concentrated sulfuric acid, reacting at 80-100 ℃ for 1-3 hours, cooling to room temperature, adding ice water, adding protonic acid, separating out solid, and filtering to obtain an intermediate 3; the feeding molar ratio of the intermediate product 2 to the cyclohexanone is 1 (1-3);
(4) Synthesis of intermediate 4:
dissolving the intermediate 3 and malonaldehyde diphenylamine hydrochloride in a mixed solvent of acetic acid and acetic anhydride, and reacting for 1-3 hours at 90-110 ℃ under the protection of nitrogen; cooling to room temperature, concentrating the organic phase and separating by column chromatography to obtain an intermediate 4;
(5) Synthesis of intermediate 5:
under the protection of nitrogen, adding the intermediate 2 and the intermediate 4 into a mixed solvent of toluene and n-butyl alcohol, and reacting for 3-6 hours at 80-120 ℃; cooling to room temperature, concentrating the organic phase, and separating by column chromatography to obtain intermediate 5; the feeding molar ratio of the intermediate 2 to the intermediate 4 is 1 (1-3);
(6) Synthesis of ratiometric near-infrared fluorescent probe molecule (product 1):
dissolving the intermediate 5 and the compound 3 substituted acyl chloride in dichloromethane, adding organic base, and reacting at room temperature for 12-48 h; after quenching reaction, concentrating the organic phase, and separating by column chromatography to obtain a product 1; the feeding molar ratio of the intermediate 5 to the substituted acyl chloride is 1 (2-3).
3. The method according to claim 2, wherein the protonic acid in the step (2) or the step (3) is HClO 4 、HPF 6 、HBF 4 、HCl、HBr、HI、CF 3 COOH、CF 3 SO 3 H and CH 3 SO 3 H.
4. The method according to claim 2, wherein the organic base in step (6) is one selected from triethylamine, N-diisopropylethylamine, and pyridine.
5. Use of the ratiometric near-infrared fluorescent probe molecule of claim 1 in the preparation of reagents for imaging deep tissues of organisms, biosensing assays or micellar solutions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211280811.2A CN115925667B (en) | 2022-10-19 | 2022-10-19 | Ratio type near infrared fluorescent probe molecule and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211280811.2A CN115925667B (en) | 2022-10-19 | 2022-10-19 | Ratio type near infrared fluorescent probe molecule and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115925667A true CN115925667A (en) | 2023-04-07 |
| CN115925667B CN115925667B (en) | 2024-02-02 |
Family
ID=86551323
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211280811.2A Active CN115925667B (en) | 2022-10-19 | 2022-10-19 | Ratio type near infrared fluorescent probe molecule and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115925667B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108997771A (en) * | 2018-06-12 | 2018-12-14 | 大连理工大学 | One kind has the strong dyestuff and the preparation method and application thereof absorbed with photo-thermal effect near infrared region |
| CN109400572A (en) * | 2018-11-03 | 2019-03-01 | 复旦大学 | The fluorescent dye and its preparation method and application of the second window of near-infrared transmitting |
| CN112225721A (en) * | 2020-10-21 | 2021-01-15 | 复旦大学 | Acid-responsive near-infrared lysosome organic small-molecule fluorescent probe and preparation method and application thereof |
-
2022
- 2022-10-19 CN CN202211280811.2A patent/CN115925667B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108997771A (en) * | 2018-06-12 | 2018-12-14 | 大连理工大学 | One kind has the strong dyestuff and the preparation method and application thereof absorbed with photo-thermal effect near infrared region |
| CN109400572A (en) * | 2018-11-03 | 2019-03-01 | 复旦大学 | The fluorescent dye and its preparation method and application of the second window of near-infrared transmitting |
| CN112225721A (en) * | 2020-10-21 | 2021-01-15 | 复旦大学 | Acid-responsive near-infrared lysosome organic small-molecule fluorescent probe and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| EMILY D. COSCO等: "Bright Chromenylium Polymethine Dyes Enable Fast, Four-Color In Vivo Imaging with Shortwave Infrared Detection", 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》, pages 6836 * |
| KATARZYNA GORSKA等: "DNA-templated release of functional molecules with an azide-reductiontriggered immolative linker", 《CHEMCOMM》, pages 4364 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115925667B (en) | 2024-02-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cao et al. | Fluorescent supramolecular self-assembly gels and their application as sensors: A review | |
| CN109111915B (en) | Amino benzopyran cyanine fluorescent dye and probe, and synthetic method and application thereof | |
| Chen et al. | Fluorescent chemodosimeters for fluoride ions via silicon-fluorine chemistry: 20 years of progress | |
| Xu et al. | A novel fiber‐optic pH sensor incorporating carboxy SNAFL‐2 and fluorescent wavelength‐ratiometric detection | |
| WO2019192163A1 (en) | Preparation and application of a smart supramolecular vesicle functionalized by near-infrared dye | |
| CN113292582B (en) | Synthesis and application of bifunctional fluorescent probe capable of distinguishing hydroxyl free radicals and hydrogen peroxide simultaneously | |
| US20110111515A1 (en) | Reagent for measurement of reactive oxygen | |
| CN112442056A (en) | Fluorescent probe for simultaneously detecting hypochlorous acid and peroxynitrite anions, and synthetic method and application thereof | |
| CN113264904A (en) | Detection of HOCl/ClO-Fluorescent probe, preparation method and application thereof | |
| CN113801105A (en) | Mitochondrion targeted peroxynitrite/bisulfite dual-response fluorescent probe | |
| CN113956265A (en) | Near-infrared molecular probe based on malondialdehyde response, preparation method and application thereof | |
| CN105693655A (en) | Aminobenzothiazole-modified column aromatic hydrocarbons and synthesis thereof, and application of column aromatic hydrocarbons in continuous fluorescent recognition of Fe<3+> and F<-> in water phase | |
| CN115925667B (en) | Ratio type near infrared fluorescent probe molecule and preparation method and application thereof | |
| CN106188102B (en) | A kind of water-soluble dendroid list imide compound fluorescence probe and its preparation method and application | |
| Tanaka et al. | Ratiometric multimodal chemosensors based on cubic silsesquioxanes for monitoring solvent polarity | |
| CN108342190A (en) | A kind of namo fluorescence probe and its preparation method and application of ratio type detection HNO | |
| Zhang et al. | A facile ratiometric near-infrared fluorescent probe using conjugated 1, 8-naphthalimide and dicyanoisophorone with a vinylene linker for detection and bioimaging of hypochlorite | |
| WO2004076562A1 (en) | Water-soluble, fluorescent perylenetetracarboxylic acid bisimides | |
| Wang et al. | Highly sensitive optical sensors using electrospun polymeric nanofibrous membranes | |
| WO2023040089A1 (en) | Fluorescent compound and probe | |
| CN115677554A (en) | 3,4-dithioether maleimide derivative fluorescent molecule based on aggregation-induced emission, and preparation method and application thereof | |
| CN110669350B (en) | Piperidyl BODIPY red-light fluorescent dye and preparation method and application thereof | |
| CN113717134A (en) | Ratiometric fluorescent probe for detecting fluorine ions and preparation method thereof | |
| CN114702534B (en) | Hypochlorous acid trigger activation type near infrared fluorescent probe and preparation method and application thereof | |
| KR100862236B1 (en) | 1,1'-oxalyldisodiumbenzoate derivates, preparing method thereof and chemiluminescent composition containing the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |