CN115448928B - Semi-cyanine rhodamine fluorescent probe and preparation method and application thereof - Google Patents
Semi-cyanine rhodamine fluorescent probe and preparation method and application thereof Download PDFInfo
- Publication number
- CN115448928B CN115448928B CN202210947457.8A CN202210947457A CN115448928B CN 115448928 B CN115448928 B CN 115448928B CN 202210947457 A CN202210947457 A CN 202210947457A CN 115448928 B CN115448928 B CN 115448928B
- Authority
- CN
- China
- Prior art keywords
- solution
- reaction
- compound
- hemicyanine
- probe
- 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.)
- Active
Links
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 34
- 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 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Chemical compound C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims description 76
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 63
- 238000006243 chemical reaction Methods 0.000 claims description 49
- 150000001875 compounds Chemical class 0.000 claims description 31
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 27
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 24
- 238000003756 stirring Methods 0.000 claims description 24
- 239000005457 ice water Substances 0.000 claims description 19
- 239000002904 solvent Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 18
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 16
- 238000004440 column chromatography Methods 0.000 claims description 15
- 239000012043 crude product Substances 0.000 claims description 12
- 239000012299 nitrogen atmosphere Substances 0.000 claims description 12
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 11
- IUNJCFABHJZSKB-UHFFFAOYSA-N 2,4-dihydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C(O)=C1 IUNJCFABHJZSKB-UHFFFAOYSA-N 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 10
- 238000002390 rotary evaporation Methods 0.000 claims description 10
- 229940126062 Compound A Drugs 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- NLDMNSXOCDLTTB-UHFFFAOYSA-N Heterophylliin A Natural products O1C2COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)C(O)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 NLDMNSXOCDLTTB-UHFFFAOYSA-N 0.000 claims description 9
- 239000012265 solid product Substances 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 7
- 238000012123 point-of-care testing Methods 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
- -1 sodium triacetoxyborohydride Chemical compound 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- HUUPVABNAQUEJW-UHFFFAOYSA-N 1-methylpiperidin-4-one Chemical compound CN1CCC(=O)CC1 HUUPVABNAQUEJW-UHFFFAOYSA-N 0.000 claims description 5
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 5
- 229960000583 acetic acid Drugs 0.000 claims description 5
- 230000007613 environmental effect Effects 0.000 claims description 5
- 239000012362 glacial acetic acid Substances 0.000 claims description 5
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 claims description 5
- 239000012071 phase Substances 0.000 claims description 5
- 239000011541 reaction mixture Substances 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- 239000012047 saturated solution Substances 0.000 claims description 5
- 239000001632 sodium acetate Substances 0.000 claims description 5
- 235000017281 sodium acetate Nutrition 0.000 claims description 5
- 239000012321 sodium triacetoxyborohydride Substances 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000008346 aqueous phase Substances 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 230000000171 quenching effect Effects 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 2
- 238000010791 quenching Methods 0.000 claims description 2
- 238000010025 steaming Methods 0.000 claims description 2
- 239000000523 sample Substances 0.000 abstract description 63
- 238000001514 detection method Methods 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 10
- 230000004044 response Effects 0.000 abstract description 10
- 230000005588 protonation Effects 0.000 abstract description 9
- 125000004433 nitrogen atom Chemical group N* 0.000 abstract description 8
- 230000005595 deprotonation Effects 0.000 abstract description 6
- 238000010537 deprotonation reaction Methods 0.000 abstract description 6
- 150000003512 tertiary amines Chemical group 0.000 abstract description 4
- 230000002441 reversible effect Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 23
- 239000002953 phosphate buffered saline Substances 0.000 description 15
- 210000003712 lysosome Anatomy 0.000 description 14
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 13
- 238000003384 imaging method Methods 0.000 description 12
- 230000001868 lysosomic effect Effects 0.000 description 12
- 230000008859 change Effects 0.000 description 11
- 238000002189 fluorescence spectrum Methods 0.000 description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 7
- 230000002378 acidificating effect Effects 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 6
- 238000005160 1H NMR spectroscopy Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 229960003180 glutathione Drugs 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 229930182827 D-tryptophan Natural products 0.000 description 2
- QIVBCDIJIAJPQS-SECBINFHSA-N D-tryptophane Chemical compound C1=CC=C2C(C[C@@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-SECBINFHSA-N 0.000 description 2
- 235000019454 L-leucine Nutrition 0.000 description 2
- 239000004395 L-leucine Substances 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 229960003136 leucine Drugs 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 210000003463 organelle Anatomy 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 238000006862 quantum yield reaction Methods 0.000 description 2
- 239000010453 quartz Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- OKJPEAGHQZHRQV-UHFFFAOYSA-N Triiodomethane Natural products IC(I)I OKJPEAGHQZHRQV-UHFFFAOYSA-N 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- JNGZXGGOCLZBFB-IVCQMTBJSA-N compound E Chemical compound N([C@@H](C)C(=O)N[C@@H]1C(N(C)C2=CC=CC=C2C(C=2C=CC=CC=2)=N1)=O)C(=O)CC1=CC(F)=CC(F)=C1 JNGZXGGOCLZBFB-IVCQMTBJSA-N 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 230000001320 lysogenic effect Effects 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000004798 organs belonging to the digestive system Anatomy 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/04—Ortho-condensed systems
- C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
- C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered
-
- 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
- A61K49/0032—Methine dyes, e.g. cyanine dyes
-
- C—CHEMISTRY; METALLURGY
- 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"
-
- 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"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
-
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
-
- C—CHEMISTRY; METALLURGY
- 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
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
Abstract
The invention belongs to the technical field of fluorescent probes, and particularly relates to a hemicyanine rhodamine fluorescent probe, and a preparation method and application thereof. In order to solve the problems that the application of the probe in a living sample is limited, a complex preparation method is adopted, photobleaching is easy and the like in the prior art, the invention adds phenolic hydroxyl and tertiary amine groups as hydrophilic groups on the basis of the structure of hemicyanine. And the reversible protonation and deprotonation effects of the phenolic hydroxyl and tertiary N atoms enable the probe molecule to have pH detection reversibility. The N on the recognition group morpholine provides one site for recognition of pH, and the fluorescent response is rapid and significant in different pH environments. The probe molecules absorb in the near infrared to facilitate detection of different pH changes.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to a hemicyanine rhodamine fluorescent probe, and a preparation method and application thereof.
Background
In biomedical aspect, biological research has proved that intracellular pH plays an important role in maintaining normal physiological functions and metabolic processes, lysosomes play a key role in various physiological processes as important digestive organs in cells, the lysosomes serve as acidic organelles in cells, the normal pH range is 4.5-5.5, fluctuation of the lysosomes pH can reduce the digestive enzyme activity in the lysosomes to cause cell dysfunction, and various diseases such as cancers, renal failure, alzheimer's disease and the like can be caused by serious fluctuation of the lysosomes pH, so that the diseases such as cancers and the like can be judged and prevented by monitoring the pH change of the lysosomes in living cells, and the functions, physiology and pathological processes of the cells can be studied.
Therefore, the research on substances capable of simultaneously detecting the environment and the pH in the organism is needed, the existing pH detection methods include Nuclear Magnetic Resonance (NMR), electrochemical, chemical titration, fluorescent probe and the like, but fluorescent probes in the methods are widely focused by researchers with the unique advantages, on one hand, near infrared fluorescent probes are easy to synthesize and convenient to use, can sensitively and rapidly respond to target detection and accurately detect the target detection, and can be used for real-time imaging; on the other hand, in biological applications, it has the advantages of low autofluorescence background, lower biological damage, deeper tissue penetration, etc. To date, many excellent performance lysosomal pH fluorescent probes have been reported. However, these probes have limited applications in living samples due to low fluorescence quantum yields, small Stocks shifts, etc., and complicated preparation methods, easy photobleaching, etc., have also been improved. Ratio imaging techniques can eliminate interference from environmental changes, probe concentration fluctuations, and instrument performance, thereby providing more accurate analysis. Therefore, development of a ratiometric fluorescent probe with large Stocks displacement, high sensitivity and high fluorescence quantum yield has important scientific significance for tumor cell lysosome pH imaging.
Disclosure of Invention
The invention adds phenolic hydroxyl and tertiary amine groups as hydrophilic groups based on the structure of hemicyanine. And the reversible protonation and deprotonation effects of the phenolic hydroxyl and tertiary N atoms enable the probe molecule to have pH detection reversibility. The N on the recognition group morpholine provides one site for recognition of pH, and the fluorescent response is rapid and significant in different pH environments. The probe molecules absorb in the near infrared to facilitate detection of different pH changes.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a hemicyanine rhodamine fluorescent probe has the following structural formula:
。
a preparation method of a hemicyanine rhodamine fluorescent probe comprises the following steps:
step 1, under the condition of ice water bath in nitrogen atmosphere, dissolving N, N-dimethylformamide in dichloromethane, uniformly stirring to obtain solution 1, and carrying out POCl (point of care testing) 3 Dissolving in dichloromethane, stirring uniformly to obtain a solution 2, slowly dripping the solution 2 into the solution 1 for reaction, slowly dripping the 1-methyl-4-piperidone solution, removing ice water bath, heating, refluxing condensed water, stirring for reaction, airing the reaction solution to room temperature after the reaction is finished, dropwise adding ice water into the reaction mixture for reaction under the ice water bath condition, vacuumizing to dry the solvent, and washing the crude product with diethyl ether to obtain a compound A;
step 2, methyl iodide and 2, 3-trimethyl indole are dissolved in acetonitrile, heated, condensed water is refluxed, stirred and reacted, after the reaction is finished, the reaction solution is cooled to room temperature, the solvent is removed by rotary evaporation, and the mixture is washed by cold anhydrous diethyl ether to obtain a pink solid product compound B;
step 3, dissolving the compound A and the compound B in absolute ethyl alcohol in a nitrogen atmosphere, stirring until the compound A and the compound B are dissolved, adding sodium acetate, heating, stirring for reaction, removing the solvent by rotary evaporation, and separating by column chromatography to obtain a blue solid product compound C;
step 4, dissolving 2, 4-dihydroxybenzaldehyde, morpholine and glacial acetic acid in methylene dichloride in a nitrogen atmosphere, stirring to dissolve the materials, reacting at room temperature, adding sodium triacetoxyborohydride under an ice water bath, removing the ice water bath, reacting at room temperature overnight, quenching the reaction with secondary distilled water after the reaction is finished, and then using NaHCO 3 The saturated solution was adjusted to pH, the aqueous phase was extracted with dichloromethane, and the dichloromethane phase was dried over anhydrous MgSO 4 Drying and rotary steaming to remove the solvent, and separating the obtained crude product by column chromatography to obtain a white solid product compound D;
and 5, in a nitrogen atmosphere, stirring and reacting the compound C and an anhydrous potassium carbonate solution acetonitrile at room temperature to obtain a solution 3, dissolving the compound D in acetonitrile, stirring until the solution is dissolved to obtain a solution 4, slowly adding the solution 4 into the solution 3, heating, reacting overnight, after the reaction is finished, airing the reaction solution to room temperature, removing the solvent by rotary evaporation, and separating a crude product by column chromatography to obtain a blue solid product, namely a compound Lyso-M-pH, namely the hemicyanine rhodamine fluorescent probe (compound E).
Further, in the step 1, 1-methyl-4-piperidone and POCl 3 And the molar ratio of N, N-dimethylformamide is 1:4:5.3.
further, in the step 1, the solution 2 is slowly dripped into the solution 1 for reaction for 5min, the slowly dripping process is 10min, the heating is carried out to 70 ℃, the stirring reaction is carried out for 3.5h, the ice water is dropwise added into the reaction mixture for reaction for 15min, the dropwise adding process is 10min, and the washing times are 2 times.
Further, the molar ratio of methyl iodide to 2, 3-trimethylindole in the step 2 is 2:1, a step of; the temperature is increased to 85 ℃, and the stirring reaction is carried out for 24 hours.
Further, the molar ratio of compound a, compound B and sodium acetate in step 3 is 1:4.5:9, a step of performing the process; heating to 70 ℃, stirring and reacting for 4 hours, wherein the volume ratio of anhydrous methanol and dichloromethane of the developing agent for column chromatography separation is 40:1.
further, the molar ratio of 2, 4-dihydroxybenzaldehyde, morpholine, glacial acetic acid and sodium triacetoxyborohydride in the step 4 is 1:1.05:1.2:1.5.
further, the reaction is carried out for 2 hours at room temperature in the step 4, and the NaHCO is prepared 3 The pH value of the saturated solution is regulated to 6-7, the water phase is extracted by methylene dichloride for 3-5 times, and the volume ratio of the petroleum ether and the ethyl acetate serving as the developing agents for column chromatography separation is 3:1.
further, the molar ratio of compound C, anhydrous potassium carbonate and compound D in step 5 is 1:2:2; the temperature is raised to 50 ℃, and the volume of anhydrous methanol and methylene dichloride used as developing agents for column chromatography separation is 20:1.
the application of the hemicyanine rhodamine fluorescent probe is used for preparing a reagent for detecting the environment and the pH change in cells.
The invention utilizes the reaction of photochromic molecules and rhodamine to synthesize a fluorescent probe Lyso-M-pH. Compared with the existing fluorescent probe, the fluorescent probe has the advantages that:
(1) The introduction of two pH-sensitive N atoms on the basis of the phenolic hydroxyl groups of the molecule increases the response range of the pH.
(2) Due to phenolic hydroxyl groups and tertiary N atoms with the environmental H + The reaction is rapid, which allows the probe molecules to rapidly and sensitively sense the environmental pH.
(3) Reversible protonation and deprotonation effects of phenolic hydroxyl groups and tertiary N atoms provide the probe molecule with pH detection reversibility to monitor pH change in real time.
(4) The phenolic hydroxyl and tertiary amine are used as hydrophilic groups, so that the water solubility of molecules is increased, the probe can be directly dissolved in water to detect the pH value of the environment, and the quenching effect caused by aggregation of molecules due to hydrophobic molecules can be prevented during cell imaging.
(5) Tertiary amine groups as weakly basic groups, because their protonation selectively aggregates in an acidic environment, living cell lysosomes happen to be an acidic subcellular organelle, and thus the probe molecule Lyso-M-pH can be selectively targeted to lysosomes in a cellular environment.
(6) The probe has good biocompatibility and is expected to be applied to living bodies.
Drawings
FIG. 1 is a 1H-NMR chart of Compound C;
FIG. 2 is a 14C-NMR chart of compound C;
FIG. 3 is the HRMS spectra of Compound C;
FIG. 4 is a 1H NMR chart of compound D;
FIG. 5 is a 1H-NMR spectrum of the compound Lyso-M-pH;
FIG. 6 HRMS spectra of Compound Lyso-M-pH;
FIG. 7 is a pH response mechanism of probe Lyso-M-pH;
FIG. 8 is a graph showing the color of PBS solutions at different pH's for Lyso-M-pH;
FIG. 9 is an ultraviolet-visible absorption spectrum of a probe at different pH conditions;
in FIG. 10, a) is the ultraviolet-visible absorption spectrum of the probe under acidic pH conditions; b) Is the ultraviolet-visible absorption spectrum of the probe under the alkaline pH condition;
in FIG. 11, a) is the fluorescence emission spectrum of probe Lyso-M-pH response to different pH; b) The probe Lyso-M-pH is a graph of the relationship between the fluorescence intensity and the pH value of response of the probe Lyso-M-pH to different pH values, and the inset is a graph of the linear relationship between the fluorescence intensity and the pH value, wherein the pH value is in the range of 3.84-10.95;
fig. 12 shows the change in fluorescence intensity of the probe in PBS solution at ph=5.20 in the presence of different interferents;
in fig. 13, a) is a reversibility study line graph of the probe at ph=3.5 and 8.0; b) Solution color change profiles were studied for reversibility of the probe between ph=3.5 and 8.0;
FIG. 14 is a graph showing the change of fluorescence intensity of a probe with time at pH 5.20;
in FIG. 15, a) is a 588 nm (DND-99) cell imaging map; b) Imaging of 633 nm (Lyso-M-pH) cells; c) An overlaid image of 588 nm (DND-99) and 633 nm (Lyso-M-pH) cells; d) Imaging the DIC cells; e) Is a graph of fluorescence intensity relationship between DND-99 and Lyso-M-pH.
Description of the embodiments
Examples
A hemicyanine rhodamine fluorescent probe has the following structural formula:
。
the preparation method of the hemicyanine rhodamine fluorescent probe comprises the following steps:
1. synthesis of Compound A
1.8 mL (23.40 mmol) of N, N-Dimethylformamide (DMF) and 1.8 mL of Dichloromethane (DCM) were added to a Schlenk flask 1 equipped with a stirrer and a reflux condenser under nitrogen atmosphere and stirred uniformly to obtain a solution 1; in another Schlenk flask 2 equipped with a stirrer was added 1.63. 1.63 mL (17.5 mmol) POCl 3 Dissolving in 1.8 mLDCM, stirring to obtain solution 2. Solution 2 (procedure 10 min) was slowly dropped into Schlenk flask 1, and after 5 minutes of reaction, 0.54. 0.54 mL (4.40 mol) of 1-methyl-4-piperidone solution was slowly dropped into Schlenk flask 1. Removing ice water bath, heating to 70deg.C, refluxing condensed water,the reaction was stirred 3.5. 3.5 h. And after the reaction is finished, airing the reaction solution to room temperature, dropwise adding ice water (the process is 10 min) into the reaction mixture under the ice water bath condition, reacting for 15min, vacuum pumping the solvent, and washing the crude product twice with a small amount of diethyl ether to obtain the compound A.
2. Synthesis of Compound B
Into a round bottom flask equipped with a stirrer and a reflux condenser was added 4.4 g (25.12 mmol) iodomethane 4.29 g (30.22 mmol) 2, 3-trimethylindole and 60 mL acetonitrile, heated to 85 ℃, condensed water refluxed, and stirred for reaction 24h. After the reaction is finished, cooling the reaction solution to room temperature, removing the solvent by rotary evaporation to obtain a crude product of the compound B, and washing the crude product with cold anhydrous diethyl ether for 2-3 times to obtain a pink solid product compound B.
3. Synthesis of Compound C
28.5 (mg) (0.15 mmol) of compound A, 200.3 (mg) (0.68 mmol) of compound B and an appropriate amount of absolute ethanol as solvents were added to a Schlenk flask equipped with a stirrer under nitrogen atmosphere, stirred until dissolved, then 108 (mg) (1.316 mmol) of sodium acetate was added, the temperature was raised to 70 ℃, the reaction was stirred for 4 (h), the solvents were removed by rotary evaporation, and the crude product was isolated by column chromatography (absolute methanol: dichloromethane=40:1 v/v) to give pure compound C as a blue solid.
1H NMR (600 MHz, Chloroform-d) δ 7.54 (d, J = 2.8 Hz, 3H), 7.45 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 7.8 Hz, 2H), 7.21 (d, J = 7.3 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 5.93 (d, J = 14.3 Hz, 2H), 4.27 (s, 4H), 3.32 (s, 3H), 3.12 (s, 3H), 1.66 (s, 6H), 1.26 (s, 6H), 0.91 – 0.81 (m, 3H).
13C NMR (151 MHz, Chloroform-d) δ 174.05, 146.50, 143.99, 142.68, 141.34, 128.78, 125.63, 122.11, 110.92, 102.98, 53.41, 52.80, 49.39, 42.33, 33.60, 29.68, 28.09. [C 32 H 37 ClN 3 ] + 499.2734, found: 499.2738.
4. synthesis of Compound D
1. 1 g (7.24 mmol) of 2, 4-dihydroxybenzaldehyde, 0.67 mL (7.60 mmol) of morpholine, 0.71 mL (8.69 mmol) of glacial acetic acid and a proper amount of solvent DCM are added to a Schlenk flask equipped with a stirrer under nitrogen atmosphere and stirred to dissolve, after two hours of reaction at room temperature, 2.3 g (10.86 mmol) of sodium triacetoxyborohydride are added under ice water bath, the ice water bath is removed and the reaction is carried out at room temperature overnight. After the reaction is completed, the reaction is quenched by a proper amount of secondary distilled water and then NaHCO is used 3 The saturated solution is used for regulating the pH value of the solution to 6-7, the aqueous phase is extracted for 3-5 times by using DCM, and the DCM phase is used for anhydrous MgSO 4 After drying, the solvent was removed by rotary evaporation and the crude product was isolated by column chromatography (petroleum ether: ethyl acetate=3:1 v/v) to give pure compound D as a white solid.
1H NMR (600 MHz, Chloroform-d) δ 6.85 (d, J = 8.1 Hz, 1H), 6.35 (d, J = 2.5 Hz, 1H), 6.30 (dd, J = 8.1, 2.6 Hz, 1H), 3.77 (s, 4H), 3.66 (s, 2H), 2.58 (s, 4H).
5. Synthesis of the Compound Lyso-M-pH
122.5 mg (0.2 mmol) of Compound C, 55.3 mg (0.4 mmol) of anhydrous potassium carbonate and an appropriate amount of solvent acetonitrile were placed in a Schlenk flask 1 provided with a stirrer under a nitrogen atmosphere, and the mixture was stirred at room temperature for 30 minutes; in another Schlenk flask 2 equipped with a stirrer, 83.7. 83.7 mg (0.4 mmol) of compound D and an appropriate amount of acetonitrile as solvents were added, and stirred until dissolved. The solution in Schlenk flask 2 was slowly added to Schlenk flask 1, and the temperature was raised to 50 ℃ and reacted overnight. After the reaction was completed, the reaction solution was dried to room temperature, the solvent was removed by rotary evaporation, and the crude product was separated by column chromatography (anhydrous methanol: dichloromethane=20:1 v/v) to give pure compound Lyso-M-pH as a blue solid.
1H NMR (600 MHz, chloro-d) δ8.49 (d, J=15.0 Hz, 1H), 7.48 (dt, J=16.4, 6.3 Hz, 4H), 7.41 (q, J=8.4, 7.5 Hz, 2H), 7.36 (d, J=7.9 Hz, 1H), 6.83 (d, J=5.9 Hz, 1H), 6.56 (d, J=14.9 Hz, 1H), 4.04 (d, J=23.3 Hz, 4H), 3.86 (s, 2H), 2.68 (d, J=37.0 Hz, 8H), 2.05 (ddt, J=19.3, 12.9, 7.1 Hz, 3H), 1.79 (s, 6H), 0.83 (t, J=2.5 Hz, 3H) [ HRMS (ESI) calculated values: [ C 31 H 36 N 3 O 3 ] + (YT) 498.6431, found: 498.6438.
examples
1. Preparation of Lyso-M-pH stock solution
Weighing 6.3 mg probe molecule Lyso-M-pH, dissolving in a proper amount of dimethyl sulfoxide (Dimethyl sulfoxide, DMSO), transferring the obtained solution into a 10 mL volumetric flask to fix the volume to a scale mark, obtaining a Lyso-M-pH stock solution with the concentration of 1.0 mM, sealing, avoiding light, and storing in a refrigerator. All probe Lyso-M-pH solutions in the examples were formulated by dilution of this stock solution.
2. Configuration and detection of Phosphate Buffered Saline (PBS) solutions
Weigh 14.33 g Na 2 HPO 4 ·12H 2 O is dissolved in 400 mL distilled water to prepare Na of 0.1M 2 HPO 4 A solution; weighing 5.44 g KH 2 PO 4 Dissolving in 400 mL distilled water to obtain KH 0.1M 2 PO 4 A solution. Mixing and stirring the two solutions uniformly, measuring the standard pH value of the two solutions by using a pH meter, regulating the pH value by using a NaOH solution of 0.2M and an HCl solution of 0.2M, preparing PBS buffer solutions with different pH values of pH=3-12, and sealing and preserving at room temperature.
3. Configuration and detection of interfering substance solutions
Will NaCl, KI, mgCl 2 、CaCl 2 、CuSO 4 、H 2 O 2 Glutathione(GSH) was dissolved in distilled water to prepare an ionic solution of 0.1. 0.1M. Dissolving L-leucine and D-tryptophan in secondary distilled water to prepare amino acid solution of 0.01-mM, and sealing and preserving at room temperature.
4. Cell experiment
At 5% CO 2 In a constant temperature and humidity environment at 37 ℃, hela cells (human cervical cancer cells) were cultured in a medium (DMEM medium) containing 10% fetal bovine serum and antibiotics.
Examples
Mechanism of the pH response of Lyso-M-pH
The structural formula of the probe molecule Lyso-M-pH can be divided into two parts, a hemicyanine structure and a rhodamine structure. The semi-cyanine structure is an electron-withdrawing structure, the rhodamine structure is an electron-donating structure, the probe molecule Lyso-M-pH has a typical push-pull electron system (D-pi-A system), the electron cloud distribution of phenolic hydroxyl O atoms and two pH sensitive tertiary N on the rhodamine structure is changed through protonation and deprotonation effects, so that the electron donating capacity of the rhodamine group is changed, the fluorescence spectrum is changed to respond to different pH values, as shown in figure 7, in an acidic condition, the phenolic hydroxyl has the protonation effect, two N atoms in the molecule also have the protonation under a strong acid condition, the electron donating capacity of the rhodamine structure is weakened, the ICT effect is weakened, the emission wavelength is blue-shifted, and the fluorescence emission intensity is weakened; the free hydroxyl ions are combined with phenolic hydroxyl protons in alkaline conditions, so that the phenolic hydroxyl is subjected to deprotonation effect to form alkoxy anions, the charge density of O atoms is increased, the electron donating capacity of the rhodamine structure is enhanced, the ICT effect is enhanced, the emission wavelength is red shifted, and the fluorescence emission intensity is enhanced.
2. Color change and water solubility analysis
The probe molecules Lyso-M-PBS solutions of different pH have different colors, as shown in fig. 8, the color change is evident from the acidic to basic color, the solution gradually changes from purple to blue to green. The solution appeared purple in acid, blue in neutral and green in alkaline. Therefore, when the probe is used for detecting the pH of the environment, the color change can be recognized by naked eyes, and the detected pH range can be judged. On the other hand, PBS solutions of different pH containing the probe Lyso-M-pH were homogeneous systems, and no precipitation occurred, indicating good water solubility.
3. Ultraviolet-visible absorption spectra of probe Lyso-M-pH in PBS solutions of different pH
The probe Lyso-M-pH concentration of 0.02 to mM in PBS solutions of different pH were placed in a quartz cuvette and measured for UV-vis spectra at a wavelength range of 200 nm to 900 nm, as shown in fig. 9, where only a spectrum of 450 nm to 900 nm was studied, and the change in the absorption wave waveform in this range was complicated, amplified for analysis as shown in fig. 10, and two different waveforms were observed for probe solutions of different pH in this range, because Lyso-M-pH formed different molecular formulas due to protonation and deprotonation effects in acid and alkaline solutions. Under acidic conditions (except for superacidity ph=3.18), there is an absorption peak at 710 nm, which increases with increasing pH, increasing peak intensity, increasing absorbance of Lyso-M-pH; the absorption peak at 710 nm disappeared with increasing pH in alkaline conditions and a strong absorption peak appears at 678 nm, which decreases with increasing pH, strong peak and decreasing absorbance of Lyso-M-pH.
4. Fluorescence emission spectra of probe Lyso-M-pH responses to different pH
The probe Lyso-M-PBS solutions of different pH concentrations of 0.02-mM were placed in a quartz cuvette, the excitation wavelength was set at 650 nm, and the fluorescence emission spectra of the probe response to different pH in the range 665 nm-900 nm were measured, as shown in fig. 11 a). In strongly acidic solutions, the N atom is protonated, the fluorescence spectrum has a weaker peak in emission intensity at 729 nm, the protonation effect of the N atom decreases with increasing pH, the peak at 729 nm disappears, a strong emission peak appears at 696 nm, the peak increases in intensity with increasing pH, and is accompanied by a slight red shift. By plotting the relationship between the fluorescence emission intensities of the probe solutions at 696 and nm and fitting the curve as shown in b) of fig. 11, it can be seen that there is a linear relationship in the ph=3.84 to 10.95, and the linear equation of the fluorescence emission intensity and the pH value in the pH range is fitted to y= 22073x-83142, and the linear correlation coefficient R is as follows 2 0.9959, shows that the fluorescence emission intensity has a good linear relationship with pH value,the pH value of the solution can be accurately deduced by measuring the fluorescence emission intensity of the probe solution with known probe concentration. From FIG. 10, it can be seen that the linear pH response range of Lyso-M-pH to pH covers the physiological pH range of lysosomes (pH 3.8-5.0), and therefore it is expected to become a novel hemicyanine near infrared fluorescent probe for accurately measuring the pH value of the environment in living cell lysosomes.
5. Anti-interference ability of probe Lyso-M-pH
In order to judge whether various ions and biological metabolites exist in the nature and organisms and whether the substances can have interference influence on the pH detection of the probe Lyso-M-pH, the anti-interference capability of the probe Lyso-M-pH is studied by adding different interferents to a PBS solution with the pH=5.20 (normal pH value of lysosomes) of which the concentration of the probe Lyso-M-pH is 0.02 mM, detecting fluorescence emission spectra of the interferents, and comparing the fluorescence emission spectra with a blank group. The selected interferents and their concentrations are NaCl (0.25 mM), KI (0.25 mM), mgCl, respectively 2 （0.25 mM）、CaCl 2 （0.25 mM）、CuSO 4 （0.25 mM）、H 2 O 2 (0.25 mM), GSH (0.25 mM), L-leucine (1.0 x 10-4 mM) and D-tryptophan (1.0 x 10-4 mM). 696 The detection result of fluorescence emission intensity at nm has no significant influence on the detection capability of the Lyso-M-pH due to the presence of an interfering substance, for example, as shown in fig. 12, so that the influence thereof can be ignored, and the probe Lyso-M-pH can be used for the detection of the pH in the natural and lysosome environments.
6. Reversibility of the probe Lyso-M-pH
The reversibility was studied by adding 1M HCl solution or 1M NaOH solution to PBS solution having a ph=3.65 of Lyso-M-pH concentration of 0.02 mM, adjusting the pH back and forth, monitoring the pH with a pH meter to about 3.6 and about 8, and repeating the adjustment and measurement of fluorescence spectra of two pH vicinity probes at 696 nm wavelengths multiple times. As a result, FIG. 13 a), the fluorescence intensity at the same acid/alkali level was slightly lowered because the probe concentration was diluted by adding the acid/alkali solution during the pH adjustment and the pH value was lower each time than the previous pH value at the same acid/alkali level, but the total result showed that the probe Lyso-M-pH had good reversibility. While the color of the probe solution is changed by the color change of the probe solution in fig. 13 b), the color of the probe solution is also changed reversibly.
7. Sensitivity and stability of probe Lyso-M-pH
To determine whether a probe can exist stably in a natural/biological environment, its stability was studied. Dissolving a probe Lyso-M-pH in PBS (phosphate buffer solution) with pH=5.20 to obtain a concentration of 0.02 mM, respectively measuring fluorescence emission intensities at 0min, 5min, 10min, 20 min, 30 min and 60 min from the time of adding the probe, and fitting a fluorescence intensity-time curve, wherein the result is shown in FIG. 14, the fluorescence intensity is hardly changed with time from the time of adding the probe, on the one hand, the probe has high sensitivity to pH, and the probe can quickly and accurately respond to the environmental pH after the probe; on the other hand, it was shown to have good stability in PBS solution.
8. Cell co-localization fluorescence imaging experiment
Cultured Hela cells were seeded into glass dishes and co-localized fluorescence imaging was performed with the commercially available lysosome targeting fluorescent dye DND-99 and the fluorescent probe Lyso-M-pH. First, hela cells were cultured in DMEM medium containing DND-99 (DND-99 concentration 1 μm) at 37 ℃ for 10min, then the culture solution was removed, then DMEM medium containing Lyso-M-pH (Lyso-M-pH concentration 1 μm) was used to culture in 37 ℃ for 10min, the culture solution was removed again, the cells were washed three times with PBS solution having ph=7.4 to wash out the fluorescent background, then fresh DMEM medium was injected into the dish, the glass dish was placed on a laser confocal microscope for cell imaging, excitation light having wavelengths of 588-nm (DND-99, green light) and 633 nm (Lyso-M-pH, red light) was selected for fluorescent imaging, and a cell imaging map having excitation wavelengths of 588-nm, a cell imaging map having 633-nm, a 588-nm and 633-nm cell imaging overlay map, DIC cell imaging map and a fluorescent intensity relationship map of DND-99 and Lyso-M-pH, respectively, as in fig. 15, were collected, and the two probes were used for the detection of the two-pH in vivo conditions of the two-dimensional map, and the two probes were well-located in vivo to have the function of detecting the lysogenic probe.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The hemicyanine rhodamine fluorescent probe is characterized by comprising the following structural formula:
。
2. a method for preparing a hemicyanine rhodamine fluorescent probe as claimed in claim 1, comprising the steps of:
step 1, under the condition of ice water bath in nitrogen atmosphere, dissolving N, N-dimethylformamide in dichloromethane, uniformly stirring to obtain solution 1, and carrying out POCl (point of care testing) 3 Dissolving in dichloromethane, stirring uniformly to obtain a solution 2, slowly dripping the solution 2 into the solution 1 for reaction, slowly dripping the 1-methyl-4-piperidone solution, removing ice water bath, heating, refluxing condensed water, stirring for reaction, airing the reaction solution to room temperature after the reaction is finished, dropwise adding ice water into the reaction mixture for reaction under the ice water bath condition, vacuumizing to dry the solvent, and washing the crude product with diethyl ether to obtain a compound A;
step 2, methyl iodide and 2, 3-trimethyl indole are dissolved in acetonitrile, heated, condensed water is refluxed, stirred and reacted, after the reaction is finished, the reaction solution is cooled to room temperature, the solvent is removed by rotary evaporation, and the mixture is washed by cold anhydrous diethyl ether to obtain a pink solid product compound B;
step 3, dissolving the compound A and the compound B in absolute ethyl alcohol in a nitrogen atmosphere, stirring until the compound A and the compound B are dissolved, adding sodium acetate, heating, stirring for reaction, removing the solvent by rotary evaporation, and separating by column chromatography to obtain a blue solid product compound C;
step 4, dissolving 2, 4-dihydroxybenzaldehyde, morpholine and glacial acetic acid in methylene dichloride in a nitrogen atmosphere, stirring to dissolve the materials, reacting at room temperature, adding sodium triacetoxyborohydride under an ice water bath, removing the ice water bath, reacting at room temperature overnight, quenching the reaction with secondary distilled water after the reaction is finished, and thenWith NaHCO 3 The saturated solution was adjusted to pH, the aqueous phase was extracted with dichloromethane, and the dichloromethane phase was dried over anhydrous MgSO 4 Drying and rotary steaming to remove the solvent, and separating the obtained crude product by column chromatography to obtain a white solid product compound D;
and 5, in a nitrogen atmosphere, stirring and reacting the compound C and anhydrous potassium carbonate solution acetonitrile at room temperature to obtain a solution 3, dissolving the compound D in acetonitrile, stirring until the compound D is dissolved to obtain a solution 4, slowly adding the solution 4 into the solution 3, heating, reacting overnight, after the reaction is finished, airing the reaction solution to room temperature, removing the solvent by rotary evaporation, and separating a crude product by column chromatography to obtain a blue solid product compound Lyso-M-pH, namely the hemicyanine rhodamine fluorescent probe.
3. The method for preparing a fluorescent probe of hemicyanine rhodamine according to claim 2, wherein the method is characterized in that 1-methyl-4-piperidone and POCl in the step 1 3 And the molar ratio of N, N-dimethylformamide is 1:4:5.3.
4. the method for preparing a fluorescent probe of hemicyanine rhodamine according to claim 2, wherein in the step 1, the solution 2 is slowly dripped into the solution 1 for reaction for 5min, the slowly dripped process is 10min, the heating is performed to 70 ℃, the stirring is performed for 3.5h, the ice water is added dropwise into the reaction mixture for reaction for 15min, the dropwise added process is 10min, and the washing times are 2 times.
5. The method for preparing a fluorescent probe of hemicyanine rhodamine according to claim 2, wherein the molar ratio of methyl iodide to 2, 3-trimethylindole in step 2 is 2:1, a step of; the temperature is increased to 85 ℃, and the stirring reaction is carried out for 24 hours.
6. The method for preparing a hemicyanine rhodamine fluorescent probe as claimed in claim 2, wherein the molar ratio of the compound a to the compound B to the sodium acetate in the step 3 is 1:4.5:9, a step of performing the process; heating to 70 ℃, stirring and reacting for 4 hours, wherein the volume ratio of anhydrous methanol and dichloromethane of the developing agent for column chromatography separation is 40:1.
7. the method for preparing a fluorescent probe of hemicyanine rhodamine according to claim 2, wherein the molar ratio of 2, 4-dihydroxybenzaldehyde, morpholine, glacial acetic acid and sodium triacetoxyborohydride in step 4 is 1:1.05:1.2:1.5.
8. the method for preparing a fluorescent probe of hemicyanine rhodamine according to claim 2, wherein in the step 4, the reaction is performed for 2 hours at room temperature, and the NaHCO is prepared by 3 The pH value of the saturated solution is regulated to 6-7, the water phase is extracted by methylene dichloride for 3-5 times, and the volume ratio of the petroleum ether and the ethyl acetate serving as the developing agents for column chromatography separation is 3:1.
9. the method for preparing a hemicyanine rhodamine fluorescent probe as claimed in claim 2, wherein the molar ratio of the compound C, the anhydrous potassium carbonate and the compound D in the step 5 is 1:2:2; the temperature is raised to 50 ℃, and the volume of anhydrous methanol and methylene dichloride used as developing agents for column chromatography separation is 20:1.
10. use of a hemicyanine rhodamine fluorescent probe as claimed in claim 1, for the preparation of a reagent for detecting environmental and pH changes in cells, said use being for non-disease diagnostic purposes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210947457.8A CN115448928B (en) | 2022-08-09 | 2022-08-09 | Semi-cyanine rhodamine fluorescent probe and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210947457.8A CN115448928B (en) | 2022-08-09 | 2022-08-09 | Semi-cyanine rhodamine fluorescent probe and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115448928A CN115448928A (en) | 2022-12-09 |
| CN115448928B true CN115448928B (en) | 2024-04-02 |
Family
ID=84297219
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210947457.8A Active CN115448928B (en) | 2022-08-09 | 2022-08-09 | Semi-cyanine rhodamine fluorescent probe and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115448928B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109081836A (en) * | 2018-08-23 | 2018-12-25 | 许昌学院 | A kind of mercury ion near infrared fluorescent probe and its preparation method and application based on half flower cyanines structure |
-
2022
- 2022-08-09 CN CN202210947457.8A patent/CN115448928B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109081836A (en) * | 2018-08-23 | 2018-12-25 | 许昌学院 | A kind of mercury ion near infrared fluorescent probe and its preparation method and application based on half flower cyanines structure |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115448928A (en) | 2022-12-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Qiu et al. | An AIE and FRET-based BODIPY sensor with large Stoke shift: Novel pH probe exhibiting application in CO32− detection and living cell imaging | |
| Shen et al. | A rhodamine B-based probe for the detection of HOCl in lysosomes | |
| Ye et al. | Highly photostable, lysosome-targeted BODIPYs with green to near-infrared emission for lysosome imaging in living cells | |
| CN104086536B (en) | A kind of for fluorescent probe detecting pH value and preparation method thereof and dedicated test test kit | |
| CN102146284B (en) | Ratiometric fluorescent probe and application thereof | |
| WO2010126077A1 (en) | Near-infrared fluorescent compound | |
| CN112521413B (en) | Two-channel fluorescent probe for detecting viscosity and hydrogen peroxide as well as preparation and application thereof | |
| CN112159522B (en) | Water-soluble rhodamine-based fluorescent/colorimetric dual-mode probe and preparation method and application thereof | |
| Jin et al. | Novel near-infrared pH-sensitive cyanine-based fluorescent probes for intracellular pH monitoring | |
| Dong et al. | Two-photon fluorescence visualization of lysosomal pH changes during mitophagy and cell apoptosis | |
| Wu et al. | Highly photostable ketopyrrolyl-BODIPYs with red aggregation-induced emission characteristics for ultrafast wash-free mitochondria-targeted bioimaging | |
| CN103896928A (en) | pH fluorescent chemical sensor as well as synthetic method and application thereof | |
| CN111004246B (en) | Rhodamine pH fluorescent probe for monitoring mitochondrial autophagy, preparation and application thereof | |
| CN104744453A (en) | Hemicyanine compound for detecting polarity of mitochondria | |
| Havlík et al. | Versatile fluorophores for bioimaging applications: π-expanded naphthalimide derivatives with skeletal and appendage diversity | |
| CN116217512A (en) | Hemicyanine dye fluorescent probe for detecting viscosity, preparation method thereof, detection method and application thereof in solution and biological system | |
| Guo et al. | A novel two-channel ratio fluorescent probe for monitoring intracellular pH fluctuations | |
| CN103382189B (en) | One class cyanine compound, its preparation method and application | |
| KR102513508B1 (en) | FLUOROGENIC pH-SENSITIVE DYES, FILM AND KIT COMPRISING THE SAME | |
| Song et al. | Self-Assembled Micellar Nanosensor toward pH with high photo-stability and its application in living cells | |
| CN115448928B (en) | Semi-cyanine rhodamine fluorescent probe and preparation method and application thereof | |
| CN116178349A (en) | Golgi targeting near infrared fluorescent probe for detecting cysteine, preparation method and application thereof | |
| CN110669350B (en) | Piperidyl BODIPY red-light fluorescent dye and preparation method and application thereof | |
| CN112341453A (en) | Fluorescent probe based on coumarin and preparation method and application thereof | |
| CN113416196A (en) | benzothiadiazole-TB compound and synthesis method and application thereof |
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 |