CN116589397A - Fluorescent probe for detecting glyoxal, and preparation method and application thereof - Google Patents
Fluorescent probe for detecting glyoxal, and preparation method and application thereof Download PDFInfo
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- CN116589397A CN116589397A CN202310552792.2A CN202310552792A CN116589397A CN 116589397 A CN116589397 A CN 116589397A CN 202310552792 A CN202310552792 A CN 202310552792A CN 116589397 A CN116589397 A CN 116589397A
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- glyoxal
- soluble salt
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- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 title claims abstract description 122
- 229940015043 glyoxal Drugs 0.000 title claims abstract description 61
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 47
- 150000001875 compounds Chemical class 0.000 claims description 41
- 238000006243 chemical reaction Methods 0.000 claims description 36
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 30
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 30
- 150000003839 salts Chemical class 0.000 claims description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 20
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- 239000003960 organic solvent Substances 0.000 claims description 17
- 238000007792 addition Methods 0.000 claims description 14
- 125000002091 cationic group Chemical group 0.000 claims description 12
- ILKWFRCNNILIJW-UHFFFAOYSA-N 4-fluoro-3-nitrobenzaldehyde Chemical compound [O-][N+](=O)C1=CC(C=O)=CC=C1F ILKWFRCNNILIJW-UHFFFAOYSA-N 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- FWPIDFUJEMBDLS-UHFFFAOYSA-L tin(II) chloride dihydrate Chemical compound O.O.Cl[Sn]Cl FWPIDFUJEMBDLS-UHFFFAOYSA-L 0.000 claims description 9
- 238000005349 anion exchange Methods 0.000 claims description 8
- 238000001953 recrystallisation Methods 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 7
- -1 tetrafluoroborate Chemical compound 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 238000006467 substitution reaction Methods 0.000 claims description 6
- 238000006482 condensation reaction Methods 0.000 claims description 5
- 150000001450 anions Chemical class 0.000 claims description 4
- 239000012472 biological sample Substances 0.000 claims description 4
- 206010012601 diabetes mellitus Diseases 0.000 claims description 4
- 238000004821 distillation Methods 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 2
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 claims description 2
- 125000003158 alcohol group Chemical group 0.000 claims description 2
- 150000002825 nitriles Chemical class 0.000 claims description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 2
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims description 2
- 238000006722 reduction reaction Methods 0.000 claims description 2
- 159000000000 sodium salts Chemical class 0.000 claims description 2
- 125000006318 tert-butyl amino group Chemical group [H]N(*)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 2
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 claims 1
- 239000000523 sample Substances 0.000 abstract description 41
- 238000001514 detection method Methods 0.000 abstract description 9
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 230000004044 response Effects 0.000 abstract description 5
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 230000003013 cytotoxicity Effects 0.000 abstract description 2
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 2
- 239000012528 membrane Substances 0.000 abstract description 2
- 230000035699 permeability Effects 0.000 abstract description 2
- 230000003287 optical effect Effects 0.000 abstract 1
- 239000012043 crude product Substances 0.000 description 22
- 238000005481 NMR spectroscopy Methods 0.000 description 20
- 238000003384 imaging method Methods 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 238000000799 fluorescence microscopy Methods 0.000 description 12
- 239000007787 solid Substances 0.000 description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 239000007864 aqueous solution Substances 0.000 description 10
- 239000011541 reaction mixture Substances 0.000 description 10
- 229940126062 Compound A Drugs 0.000 description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- 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 description 8
- 239000003795 chemical substances by application Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 6
- 230000035484 reaction time Effects 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 230000007935 neutral effect Effects 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 239000012044 organic layer Substances 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- AIJULSRZWUXGPQ-UHFFFAOYSA-N Methylglyoxal Chemical compound CC(=O)C=O AIJULSRZWUXGPQ-UHFFFAOYSA-N 0.000 description 4
- HAMNKKUPIHEESI-UHFFFAOYSA-N aminoguanidine Chemical compound NNC(N)=N HAMNKKUPIHEESI-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000004448 titration Methods 0.000 description 3
- 108010005094 Advanced Glycation End Products Proteins 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 208000037157 Azotemia Diseases 0.000 description 2
- XDLFNDNDKKUTBN-UHFFFAOYSA-N CCN1C2=CC=CC=C2C=CC1C.I Chemical compound CCN1C2=CC=CC=C2C=CC1C.I XDLFNDNDKKUTBN-UHFFFAOYSA-N 0.000 description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108091005461 Nucleic proteins Proteins 0.000 description 2
- 239000008364 bulk solution Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 208000009852 uremia Diseases 0.000 description 2
- ZBHTYKLLIUCNEF-UHFFFAOYSA-N 1,4-dimethyl-2h-quinoline Chemical compound C1=CC=C2N(C)CC=C(C)C2=C1 ZBHTYKLLIUCNEF-UHFFFAOYSA-N 0.000 description 1
- OISVCGZHLKNMSJ-UHFFFAOYSA-N 2,6-Lutidine Substances CC1=CC=CC(C)=N1 OISVCGZHLKNMSJ-UHFFFAOYSA-N 0.000 description 1
- POXIZPBFFUKMEQ-UHFFFAOYSA-N 2-cyanoethenylideneazanide Chemical group [N-]=C=[C+]C#N POXIZPBFFUKMEQ-UHFFFAOYSA-N 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000002249 Diabetes Complications Diseases 0.000 description 1
- 206010012655 Diabetic complications Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 208000037340 Rare genetic disease Diseases 0.000 description 1
- 208000017442 Retinal disease Diseases 0.000 description 1
- 206010038923 Retinopathy Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical compound [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000005882 aldol condensation reaction Methods 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000006701 autoxidation reaction Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940006460 bromide ion Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000002041 carbon nanotube Substances 0.000 description 1
- 229910021393 carbon nanotube Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 235000019439 ethyl acetate Nutrition 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 231100000024 genotoxic Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000003402 intramolecular cyclocondensation reaction Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 201000001119 neuropathy Diseases 0.000 description 1
- 230000007823 neuropathy Effects 0.000 description 1
- 238000010525 oxidative degradation reaction Methods 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 208000033808 peripheral neuropathy Diseases 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 150000003217 pyrazoles Chemical class 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 229910052761 rare earth metal Inorganic materials 0.000 description 1
- 150000002910 rare earth metals Chemical class 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- YBRBMKDOPFTVDT-UHFFFAOYSA-N tert-butylamine Chemical compound CC(C)(C)N YBRBMKDOPFTVDT-UHFFFAOYSA-N 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D213/36—Radicals substituted by singly-bound nitrogen atoms
- C07D213/38—Radicals substituted by singly-bound nitrogen atoms having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
- C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
- C07D215/12—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/68—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- 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
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- 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
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
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- 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
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Optics & Photonics (AREA)
- Engineering & Computer Science (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The application discloses a fluorescent probe for detecting glyoxal, a preparation method and application thereof, and belongs to the technical field of small organic molecule fluorescent probes. The application synthesizes a series of fluorescent probes with different electron donor-pi-acceptor (D-pi-A) groups and with o-phenylenediamine as a recognition unit, which has the following general formulaThe synthetic route has wide applicability. The probe of the application has ideal linear response to glyoxal, and has good sensitivity to glyoxalThe method has specific response, high sensitivity and good optical stability; and has good biological membrane permeability and lower cytotoxicity; can realize the detection of endogenous glyoxal in cells. Meanwhile, the preparation method of the probe provided by the application is simple and feasible, low in cost and obvious in economic and technical effects.
Description
Technical Field
The application relates to the technical field of organic micromolecular fluorescent probes, and discloses an organic micromolecular fluorescent probe which has special responsiveness to glyoxal and high sensitivity and can detect endogenous glyoxal of cells by regulating and controlling fluorescent groups.
Background
Glyoxal (GL) is an alpha-ketoaldehyde, a physiological metabolite formed in vivo by lipid peroxidation, ascorbic acid autoxidation, oxidative degradation of glucose and degradation of glycosylated proteins. Because of its ability to form irreversible adducts with nucleic acids and proteins, for example, during the formation of advanced glycation end products (AGEs), a range of cytotoxic and genotoxic reactions can be elicited, including apoptosis and cell growth arrest. Modifications of GL to nucleic acids and proteins are associated with the development of diabetic complications such as retinopathy, neuropathy, nephropathy, and diseases such as atherosclerosis, aging and uremia, which are characterized by a state of high oxidative stress. In rare genetic diseases with a high cancer tendency, such as Vanconi anemia and Willner's syndrome, GL levels in the plasma of patients are found to be higher than normal, and it has been confirmed that the concentration of GL in the serum of patients with diabetes, uremia and peritoneal dialysis is also elevated. However, currently little is known about the toxicity of GL to cells and the pathogenesis of related diseases. Therefore, it is of great importance to monitor the progression of related diseases and elucidate the underlying pathological mechanisms of organisms by detecting GL.
Although HPLC (high performance liquid chromatography), LC-MS (liquid chromatography) and GC-MS (gas chromatography) have been used for high sensitivity determination of GL, these methods are complex, time consuming, most importantly incompatible with biological systems, so further development of these methods is limited. In contrast, fluorescent probe technology has attracted great attention in detection and imaging of various biological species in organisms due to the advantages of high sensitivity, noninvasive property, real-time monitoring and the like, wherein the excellent reproducibility of the organic small molecular fluorescent probe compared with other fluorescent probes such as carbon nanotubes, quantum dots, rare earth nanoparticles and the like makes the organic small molecular fluorescent probe have certain advantages in preparation and application. In recent years, some small organic molecule fluorescent probes have been used in tissue imaging, such as detecting changes in formaldehyde, acetaldehyde, methylglyoxal, etc. levels in organisms, but few small organic molecule fluorescent probes have been used to date to detect GL and its fluctuations in cancer cells. Therefore, the application has important significance that the fluorescent probe can detect exogenous GL and endogenous GL of cells simply and effectively.
Disclosure of Invention
The primary object of the present application is to provide a fluorescent probe for detecting glyoxal, as follows.
The second application aims to provide a preparation method of the fluorescent probe.
A third object of the present application is to provide the use of such fluorescent probes.
The application has at least the following beneficial effects:
the fluorescent probe provided by the application is used for specifically recognizing glyoxal, and the fluorescent probe can increase the absorption of the probe from 440nm to 570nm, increase the maximum emission wavelength from 570nm to 630nm and increase the Stokes shift from 60nm to 130nm by regulating and controlling the fluorescent group, so that the fluorescent probe is very effective in eliminating the background interference during imaging. And the probes have high sensitivity to glyoxal response (the detection limit can reach 10 -8 M/L) and good light stability (stable light emission intensity under continuous irradiation of laser light for 30 minutes). The probe has good biological membrane permeability and lower cytotoxicity; the detection of endogenous glyoxal in cells can be realized, and the probe also has the mitochondrial targeting positioning capability.
The preparation method provided by the application is simple and feasible, low in cost, high in yield and obvious in economic and technical effects.
Drawings
FIG. 1 shows a probe Z-GL according to an embodiment of the application 5 Is the fluorescence titration spectrum (emission wavelength on the abscissa and fluorescence intensity on the ordinate);
FIG. 2 shows a probe Z-GL according to an embodiment of the application 5 Is checked by (1)A limit spectrum (glyoxal concentration on the abscissa and fluorescence intensity difference on the ordinate);
FIG. 3 shows a probe Z-GL according to an embodiment of the application 5 Cell imaging of exogenous GL;
FIG. 4 shows a probe Z-GL according to an embodiment of the application 5 Detecting a cell imaging map of endogenous GL;
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular forms also include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present application will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The application provides a fluorescent probe for detecting glyoxal, which has a general formula shown in formula I:
in the general formula: r is R 1 Is isopropylamine (-NHCH (CH) 3 ) 2 ) Or tert-butylamino (-NHC (CH) 3 ) 3 )；R 2 The structure is as follows:
in particular, when cationic groups are involved, the anions of the soluble salts used are selected from iodide ions (I - ) Fluoride ion (F) - ) Chloride ion (Cl) - ) Bromide ion (Br) - ) Tetrafluoroborate ion (BF) 4 - ) And hexafluorophosphate ion (PF) 6 - )。
The probe of the embodiment of the application constructs a series of fluorescent probes taking o-phenylenediamine as an identification unit based on a PET (photo-electron transfer) mechanism, adopts a structural model of an electron Donor (Donor) -pi-Acceptor (accepter), and obtains a series of fluorescent probes which have good fluorescence enhancement response to glyoxal through aldol condensation reaction, and have high sensitivity, good selectivity and low detection limit. The application introduces the o-phenylenediamine structure which has specific recognition to glyoxal on the benzene ring which is taken as an electron acceptor, and the o-phenylenediamine group has a function of protecting against fluorescent group R 2 Has strong PET quenching effect, and leads to non-fluorescence of the probe. However, when the probe and glyoxal are condensed, intramolecular cyclization, dehydration and other reactions produce cyclized pyrazoles, the PET effect is blocked, so that the fluorescence of the probe is recovered and enhanced, and the fluorescence detection of glyoxal is realized.
The application also relates to a preparation method of the probe, which at least comprises the following steps:
s1, 4-fluoro-3-nitrobenzaldehyde and compound R 1 Carrying out substitution reaction in an organic solvent to obtain a compound shown in a general formula A;
s2, compounds R 2 And the compound shown in the general formula A is subjected to condensation reaction in an organic solvent under alkaline conditions, and a soluble salt raw material is added to perform anion exchange reaction to obtain a soluble salt of the compound shown in the general formula B or a cationic group;
the soluble salt raw material is selected from potassium salt or sodium salt of anions of the soluble salt;
s3, performing a nitro reduction reaction on the soluble salt of the compound shown in the general formula B or the cationic group to obtain the soluble salt of the compound shown in the general formula I or the cationic group.
In one embodiment of the present application, in S1, the organic solvent is selected from nitrile organic solvents, preferably acetonitrile.
As an embodiment of the present application, 4-fluoro-3-nitrobenzaldehyde is reacted with the compound R 1 The molar ratio of (2) is 1:1.8 to 2.2, preferably 1:2; if the compound R 1 If the addition ratio of (2) is too small, the reaction time will be long, if the compound R 1 If the ratio of the addition of (2) is too large, the reaction cost increases.
As an embodiment of the present application, 4-fluoro-3-nitrobenzaldehyde is reacted with the compound R 1 The weight ratio of the total weight of (2) to acetonitrile solvent is 1:13 to 15.
In one embodiment of the present application, in S1, the time of the substitution reaction is 0.5 to 1.5 hours, and the temperature of the substitution reaction is preferably 15 to 25 ℃.
As one embodiment of the present application, the step of distillation under reduced pressure and purification are further included in S1, and the purification is more preferably recrystallisation from methanol and n-hexane.
In S2, as one embodiment of the present application, the alkaline condition is to add piperidine to the reaction system; the organic solvent is an alcohol organic solvent, preferably ethanol.
As an embodiment of the present application, compound R 2 The molar ratio of the compound shown in the general formula A to the soluble salt raw material is 1:1.05 to 1.15:2 to 2.2, preferably 1:1.1:2; if the addition ratio of the compound represented by the general formula a is too small, the reaction time will be long, and if the addition ratio of the compound represented by the general formula a is too large, the reaction cost will be increased. If the addition ratio of the soluble salt raw material is too small, the reaction time will be long, and if the addition ratio of the soluble salt raw material is too large, the reaction cost will be increased. As an embodiment of the present application, compound R 2 The molar ratio of the piperidine to the piperidine is 1:0.02 to 0.03; preferably 1:0.02; if the addition ratio of piperidine is too small, the pH is low, and if the addition ratio of piperidine is too large, the pH is too high. As an embodiment of the present application, compound R 2 And the weight ratio of the total amount of the compounds represented by the general formula A to the organic solvent is 1:10 to 12. If the amount of the organic solvent added is too large, the reaction cost increases. If the amount of the organic solvent added is too small, the reaction time may be too long. In one embodiment of the present application, in S2, the temperature of the condensation reaction is 85 to 95 ℃ and the time of the condensation reaction is 18 to 30 hours.
As one embodiment of the application, the reaction condition of the anion exchange reaction is light shielding, the temperature of the anion exchange reaction is reflux temperature, and the time of the anion exchange reaction is 1-3 h.
As an embodiment of the present application, the step of distillation under reduced pressure and purification, more preferably ethanol recrystallization, are further included in S2.
In one embodiment of the present application, in S3, tin dichloride dihydrate and concentrated hydrochloric acid are added to perform a nitroreduction reaction. The concentration of the concentrated hydrochloric acid is 36%.
As one embodiment of the present application, the molar ratio of the compound of formula B or the soluble salt of a cationic group, concentrated hydrochloric acid, tin dichloride dihydrate is 1:0.03 to 0.04: 12-14; preferably 1:0.04:12; if the addition ratio of tin dichloride dihydrate is too small, the reaction time is long, and if the addition ratio of tin dichloride dihydrate is too large, the reaction cost is increased. If the addition ratio of the concentrated hydrochloric acid is too small, the reaction yield decreases, and if the addition ratio of the concentrated hydrochloric acid is too large, the reaction cost increases.
As one embodiment of the present application, the weight ratio of the total weight of the compound represented by the general formula B or the soluble salt of a cationic group, concentrated hydrochloric acid and tin dichloride dihydrate to ethanol is 1:10 to 12; if the amount of ethanol added is too large, the reaction cost increases. If the amount of ethanol added is too small, the reaction time may be too long. As one embodiment of the present application, the temperature of the nitroreduction reaction is a reflux temperature, and the time of the nitroreduction reaction is 10 to 15 hours.
As an embodiment of the present application, a purification step is further included in S3, preferably recrystallization using methylene chloride and n-hexane or methanol and petroleum ether.
In vitro spectral response of fluorescent probe to glyoxal and cell imaging:
1) Probe fluorescence titration spectrum
The fluorescent probe prepared above was prepared into 5X 10 in dimethyl sulfoxide (DMSO) -3 M mother liquor is used;
mu.L of 5X 10 is taken -3 A standard mother solution of mol/L was prepared as a bulk solution at a concentration of 5. Mu.M in 3mL of the detection system. Different volumes of guest solution are added to the host solution, so that the concentration ratio of glyoxal to the probe in the sample bottle is increased in a gradient manner. Shaking uniformly, balancing for 3 hours, and measuring the fluorescence intensity of the solution.
2) Cell imaging of exogenous GL by probe
HeLa cells of appropriate density were seeded into 6-well dishes containing 5% CO at 37 ℃C 2 Is cultured in an incubator; after cell attachment, the first group: after living HeLa cells are cultured for 2 hours, the dead cells are washed by PBS buffer solution, and then the cells are placed under a confocal laser fluorescence microscope for fluorescence imaging; second group: performing fluorescence imaging after GL is added into HeLa cells for 2 hours; third group: after adding probes into HeLa cells for co-culture for 2 hours, washing probe molecules which do not enter the cells by using PBS buffer solution, and then performing fluorescence imaging; fourth group: after adding the probe and GL to HeLa cells and co-culturing for 2 hours, probe molecules which did not enter the cells were washed with PBS buffer solution, and then fluorescence imaging was performed.
3) Cell imaging of endogenous GL by probes
The first group was subjected to fluorescence imaging after half an hour of incubation with glyoxal inhibitor aminoguanidine (AG, aminoguandine, 20 mM) in HeLa cells; the second group is cultured in HeLa cells for half an hour by adding probes, and fluorescence imaging is carried out; the third group was subjected to fluorescence imaging after half an hour of incubation with glyoxal inhibitor aminoguanidine (AG, aminoguladine, 20 mM) and probe; the fourth group was incubated with aminoguanidine (AG, aminoguanidine,20 mM), a glyoxal inhibitor, for half an hour in HeLa cells, washed with PBS buffer, and incubated with probe and GL for half an hour before fluorescence imaging.
The application also relates to the application of the fluorescent probe, which is used for detecting the concentration of glyoxal in a living biological sample; the living biological sample comprises living cells or living tissue, preferably living HeLa cells.
The application also relates to application of the fluorescent probe for detecting glyoxal in preparation of a preparation for detecting diabetes mellitus, and the fluorescent probe has potential application value in exploring pathogenesis of diabetes mellitus.
For a better understanding of the technical solution of the present application, the following is further described in detail by specific examples:
all reagents were analytically pure and purchased directly from the reagent company such as enokak. Nuclear magnetic resonance spectroscopy was performed using Bruker spectrometer (MHz); mass spectra were determined using an Agilent 6510Q-TOF LC/MS instrument (Agilent Technologies, palo Alto, CA) and fluorescence spectra were determined using a Hitachi F-4600 fluorescence spectrometer; cell imaging was determined using FV 1000.
Example 1:
probe Z-GL 1 Is synthesized by the following steps:
a100 mL round bottom flask was charged with 4-fluoro-3-nitrobenzaldehyde (1.7 g,10 mmol), and then 30mL of acetonitrile was added to dissolve all of the mixture, and isopropylamine (1.7 mL,20 mmol) was added (three additions) and stirred at room temperature for 1h. After the reaction is finished, the reaction solution is decompressed and distilled to remove the solvent to obtain a crude product, and the crude product is recrystallized by methanol and n-hexane to obtain purified orange solid A 1 (1.998 g) and yield 96%.
Into a 50mL round bottom flask was added Compound A 1 (416 mg,2 mmol), 1, 4-lutidine (R) 1 220mg,2 mmol), piperidine (20. Mu.L) and absolute ethanol (15 mL). The reaction mixture was washed with a nitrogen stream for 30min to remove oxygen and then stirred at 90 ℃ for 24h. The reaction solution was cooled and filtered to obtain a crude product, which was purified by recrystallization from ethanol (10 mL)Obtaining purified orange solid B 1 (400 mg), yield 67%; 288-290 ℃.
HRMS:[M] + ＝298.1555；Calad:298.1570； 1 H NMR(400MHz,DMSO-d 6 )(δ,
ppm)8.82(d,J＝6.4Hz,2H),8.42(s,1H),8.22(d,J＝8Hz,1H),8.14(d,J＝6.4Hz,2H),8.00(t,J＝10.8Hz,2H),7.40(d,J＝16.4Hz,1H),7.26(d,J＝9.2Hz,1H),4.24(s,3H),4.07(m,J＝6.6Hz,1H),1.30(d,J＝6.4Hz,6H)； 13 C NMR(100MHz,DMSO-d 6 )(δ,ppm)153.2,145.7,145.4,139.8,135.0,131.2,128.1,123.4,122.9,121.2,116.4,47.3,44.5,22.7.
Add B to a 50mL round bottom flask 1 (330 mg,1.1 mmol) was dissolved in absolute ethanol (20 mL) and SnCl was added 2 ·2H 2 O (1.5 g,6.6 mmol) and concentrated hydrochloric acid (40. Mu.L), and the mixture was refluxed for 12h. The reaction mixture was poured into stirring water (50 mL) and NaHCO was added 3 The aqueous solution adjusts the pH of the aqueous solution to neutral. Followed by CH 2 Cl 2 (30 mL. Times.3) extraction. The organic layer is treated by Na 2 SO 4 Drying and distilling off the solvent under reduced pressure to give the crude product. The crude product was recrystallized from methylene chloride and n-hexane to give purified green solid Z-GL 1 (140 mg), yield 40%; 202-206 ℃.
HRMS:[M] + ＝268.1814；Calad:268.1822； 1 H NMR(400MHz,DMSO-d 6 )(δ,
ppm)8.63(d,J＝6.4Hz,2H),8.02(d,J＝6.4Hz,2H),7.78(d,J＝15.6Hz,1H),6.94(m,J＝7.3Hz,3H),6.50(d,J＝8.4Hz,1H),5.12(d,J＝5.6Hz,1H),4.83(s,2H),4.15(s,3H),3.70(m,J＝6.5Hz,1H),1.20(d,J＝6.4Hz,6H)； 13 C NMR(100MHz,DMSO-d 6 )(δ,ppm)153.9,144.6,143.5,139.4,135.5,123.5,122.6,122.3,116.5,112.5,109.7,46.7,43.7,22.9.
Example 2:
probe Z-GL 2 Is synthesized by the following steps:
compound A 1 The synthesis was as described in example 1.
Into a 50mL round bottom flask was added Compound A 1 (312 mg,1.5 mmol), 1, 4-dimethylQuinoline (R) 2 237mg,1.5 mmol), piperidine (20. Mu.L) and absolute ethanol (15 mL). The reaction mixture was washed with a nitrogen stream for 30min to remove oxygen and then stirred at 90 ℃ for 24h. The reaction solution is cooled and filtered to obtain a crude product, and the crude product is recrystallized by ethanol (10 mL) to purify to obtain a purified orange solid B 2 (308 mg) in 59% yield; 258-261 ℃.
HRMS:[M] + ＝348.1712；Calad:348.1749； 1 H NMR(400MHz,DMSO-d 6 )(δ,
ppm)9.29(d,J＝6.8Hz,1H),9.06(d,J＝8.4Hz,1H),8.57(d,J＝1.6Hz,1H),8.42(s,1H),8.40(d,J＝1.6Hz,1H),8.34(d,J＝9.2Hz,1H),8.26(t,J＝6.4Hz,2H),8.20(d,J＝2.4Hz,1H),8.02(t,J＝7.6Hz,1H),7.26(d,J＝9.2Hz,1H),4.51(s,3H),4.12(m,J＝6.6Hz,1H),3.33(s,1H),1.32(d,J＝6.4Hz,6H)； 13 C NMR(100MHz,DMSO-d 6 )(δ,ppm)158.0,152.9,150.6,147.1,144.0,140.5,140.1,136.1,134.2,134.0,131.8,131.3,128.2,124.4,122.4,120.9,120.6,49.8,49.3,27.4.
Add B to a 50mL round bottom flask 2 (200 mg,0.57 mmol), and then added with absolute ethanol (15 mL) to dissolve all the materials, and then added with SnCl 2 ·2H 2 O (1.5 g,6.6 mmol) and concentrated hydrochloric acid (40. Mu.L), and the mixture was refluxed for 12h. The reaction mixture was poured into stirring water (50 mL) and NaHCO was added 3 The aqueous solution adjusts the pH of the aqueous solution to neutral. Followed by CH 2 Cl 2 (30 mL. Times.3) extraction. The organic layer is treated by Na 2 SO 4 Drying and distilling off the solvent under reduced pressure to give the crude product. The crude product was recrystallized from methylene chloride and n-hexane to give a purified dark green solid Z-GL 2 (152mg),m.p.:200-204℃.
HRMS:[M] + ＝318.1970calad:318.1986； 1 H NMR(400MHz,DMSO-d 6 )(δ,
ppm)9.03(d,J＝6.8Hz,1H),8.89(d,J＝8.4Hz,1H),8.30(t,J＝4.6Hz,2H),8.18(t,J＝7.8Hz,1H),8.06(d,J＝15.2Hz,1H),7.95(t,J＝7.8Hz,1H),7.80(d,J＝15.6Hz,1H),7.23(t,J＝9.0Hz,2H),6.57(d,J＝8.4Hz,1H),5.40(d,J＝7.2Hz,1H),4.85(s,2H),4.41(s,3H),3.77(m,J＝6.3Hz,1H),1.22(d,J＝6.4Hz,6H)； 13 C NMR(100MHz,DMSO-d 6 )(δ,ppm)153.3,146.9,146.1,140.4,139.3,135.5,134.9,128.9,126.4,126.0,124.3,124.2,119.5,114.0,113.5,112.4,109.7,44.3,43.8,22.9.
Example 3:
probe Z-GL 3 Is synthesized by the following steps:
compound A 1 The synthesis was as described in example 1.
Into a 50mL round bottom flask was added Compound A 1 (312 mg,1.5 mmol), 1-ethyl-2-methyl-quinoline iodide (R) 3 450mg,1.5 mmol), piperidine (20. Mu.L) and absolute ethanol (15 mL). The reaction mixture was washed with a nitrogen stream for 30min to remove oxygen and then stirred at 90 ℃ for 24h. The reaction solution is cooled and filtered to obtain a crude product, and the crude product is recrystallized by ethanol (10 mL) to purify to obtain a purified orange solid B 3 (430 mg), yield 79%; 252-253 ℃.
HRMS:[M] + ＝362.1869；Calad:362.1879； 1 H NMR(400MHz,DMSO-d 6 )(δ,
ppm)9.00(d,J＝8.8Hz,1H),8.62(d,J＝1.6Hz,1H),8.55(t,J＝9.4Hz,2H),8.34(m,J＝5.1Hz,4H),8.17(m,J＝5.2Hz,1H),7.93(t,J＝6.0Hz,1H),7.70(d,J＝15.6Hz,1H),7.28(d,J＝9.6Hz,1H),5.15(d,J＝7.2Hz,2H),4.14(m,J＝6.7Hz,1H),1.56(s,3H),1.32(d,J＝6.4Hz,6H)； 13 C NMR(100MHz,DMSO-d 6 )(δ,ppm)155.9,147.3,146.3,144.1,138.6,135.9,135.5,131.3,130.8,130.4,129.3,128.4,122.7,121.3,119.3,116.2,115.7,46.9,44.6,22.6,14.6.
Add B to a 50mL round bottom flask 3 (200 mg,0.55 mmol), and then added with absolute ethanol (15 mL) to dissolve the whole, and then added with SnCl 2 ·2H 2 O (1.5 g,6.6 mmol) and concentrated hydrochloric acid (40. Mu.L), and the mixture was refluxed for 12h. The reaction mixture was poured into stirring water (50 mL) and NaHCO was added 3 The aqueous solution adjusts the pH of the aqueous solution to neutral. Followed by CH 2 Cl 2 (30 mL. Times.3) extraction. The organic layer is treated by Na 2 SO 4 Drying and distilling off the solvent under reduced pressure to give the crude product. The crude product was recrystallized from methylene chloride and n-hexane to give a purified dark green solid Z-GL 3 (143 mg), yield 78%; 212-216 ℃.
HRMS:[M] + ＝332.2127；Calad:332.21332； 1 H NMR(400MHz,DMSO-d 6 )(δ,
ppm)8.72(d,J＝9.2Hz,1H),8.52(d,J＝9.2Hz,1H),8.38(d,J＝8.8Hz,2H),8.21(t,J＝7.2Hz,2H),8.06(t,J＝8.0Hz,1H),7.81(t,J＝7.4Hz,1H),7.26(d,J＝8.8Hz,2H),7.20(d,J＝8.0Hz,1H),6.63(m,J＝10.2Hz,1H),5.63(d,J＝6.4Hz,1H),4.99(m,J＝6.5Hz,2H),3.80(m,J＝6.1Hz,1H),3.36(m,J＝7.0Hz,1H),1.55(m,J＝7.2Hz,3H),1.23(d,J＝6.0Hz,6H)； 13 C NMR(100MHz,DMSO-d 6 )(δ,ppm)156.7,155.5,150.8,141.9,141.5,138.6,134.7,130.5,128.2,127.3,125.7,123.7,120.7,118.7,109.9,109.7,45.8,43.9,22.8,14.1.
Example 4:
probe Z-GL 4 Is synthesized by the following steps:
4-fluoro-3-nitrobenzaldehyde (3 g,10 mmol) was added to a 100mL round bottom flask, and then 30mL of acetonitrile was added to dissolve all of the mixture, and tert-butylamine (2.2 mL,20 mmol) was added thereto (added three times), followed by stirring at room temperature for 1h. After the reaction is finished, the reaction solution is decompressed and distilled to remove the solvent to obtain a crude product, and the crude product is recrystallized by methanol and n-hexane to obtain purified orange solid A 2 (2g) The yield was 92%.
Into a 50mL round bottom flask was added Compound A 2 (333 g,1.5 mmol), 1-ethyl-2-methyl-quinoline iodide (R) 3 450mg,1.5 mmol), piperidine (20. Mu.L) and absolute ethanol (15 mL). The reaction mixture was washed with a nitrogen stream for 30min to remove oxygen and then stirred at 90 ℃ for 24h. The reaction solution is cooled and filtered to obtain a crude product, and the crude product is recrystallized by ethanol (10 mL) to purify to obtain a purified orange solid B 4 (498 mg) in 88% yield; 255-256 ℃.
HRMS:[M] + ＝376.2025；Calad:376.2036； 1 H NMR(400MHz,DMSO-d 6 )(δ,
ppm)9.02(d,J＝8.8Hz,1H),8.66(s,1H),8.56(m,J＝4.6Hz,2H),8.35(t,J＝8.0Hz,2H),8.28(t,J＝4.6Hz,1H),8.18(m,J＝3.7Hz,1H),7.93(m,J＝4.9Hz,1H),7.72(m,J＝14.8Hz,1H),7.41(m,J＝10.9Hz,1H),5.15(d,J＝7.2Hz,2H),3.34(s,3H),3.17(s,1H),1.58(m,J＝11.3Hz,12H)； 13 C NMR(100MHz,DMSO-d 6 )(δ,ppm)155.8,147.2,146.4,144.2,138.6,135.5,135.4,132.1,130.8,129.3,128.4,122.6,121.3,119.3,117.3,115.9,53.0,51.8,29.5,25.8,14.6.
Add B to a 50mL round bottom flask 4 (200 mg,0.5 mmol), and then added with absolute ethanol (15 mL) to dissolve the whole, and then added with SnCl 2 ·2H 2 O (1.5 g,6.6 mmol) and concentrated hydrochloric acid (40. Mu.L), and the mixture was refluxed for 12h. The reaction mixture was poured into stirring water (50 mL) and NaHCO was added 3 The aqueous solution adjusts the pH of the aqueous solution to neutral. Followed by CH 2 Cl 2 (30 mL. Times.3) extraction. The organic layer is treated by Na 2 SO 4 Drying and distilling off the solvent under reduced pressure to give the crude product. The crude product was recrystallized from methylene chloride and n-hexane to give a purified dark green solid Z-GL 4 (113 mg), yield 65%; 218-220 ℃.
HRMS:[M] + ＝346.2283；Calad:346.2288； 1 H NMR(400MHz,DMSO-d 6 )(δ,
ppm)8.73(d,J＝9.2Hz,1H),8.51(d,J＝9.2Hz,1H),8.39(d,J＝4.8Hz,1H),8.21(t,J＝12.2Hz,2H),8.08(t,J＝7.4Hz,1H),7.83(t,J＝7.6Hz,1H),7.25(t,J＝7.6Hz,3H),6.86(d,J＝8.0Hz,1H),4.97(d,J＝6.8Hz,2H),2.52(s,3H),1.57(t,J＝7.2Hz,3H),1.43(s,9H)； 13 C NMR(100MHz,DMSO-d 6 )(δ,ppm)155.5,150.6,142.1,141.2,138.5,136.3,134.8,130.5,128.3,127.4,125.3,123.6,120.7,118.6,113.9,112.0,110.2,51.5,45.9,29.7,14.1.
Example 5:
probe Z-GL 5 Is synthesized by the following steps:
1) Probe Z-GL 5 Is synthesized by the following steps:
compound A 1 The synthesis was as described in example 1.
Into a 100mL round bottom flask was added Compound A 1 (2 g,9.6 mmol), 2- (dicyanomethylene) -3-carbonitrile-4, 5-trimethylfuran (R) 4 2.3g,11.5 mmol), piperidine (20. Mu.L) and absolute ethanol (30 mL). The reaction mixture was washed with a nitrogen stream for 30min to remove oxygen and then stirred at 90 ℃ for 24h. The crude product was purified by column chromatography on silica gel (PE/EtOAc, 10/1v/v) And further purified by recrystallization from ethanol (10 mL) to give purified purple solid B 5 (2.5 g) in 68% yield; m.p.268-272 ℃.
HRMS:[M+H] + ＝390.1566；Calad:390.1580； 1 H NMR(400MHz,CDCl 3 )(δ,
ppm)8.52(s,1H),8.42(s,1H),7.79(d,J＝8.8Hz,1H),7.53(d,J＝16.4Hz,1H),6.99(d,J＝8.8Hz,1H),6.86(d,J＝16.4Hz,1H),3.97(m,J＝4.6Hz,1H),1.79(s,6H),1.40(d,J＝6.0Hz,6H)； 13 C NMR(100MHz,CDCl 3 )(δ,ppm)175.71,173.91,147.08,145.70,135.35,134.48,132.09,130.83,129.09,121.49,115.87,112.62,97.70,45.23,37.46,30.05,26.95,22.95,14.46.
Add B to a 50mL round bottom flask 5 (200 mg,0.5 mmol), and then added with absolute ethanol (15 mL) to dissolve the whole, and then added with SnCl 2 ·2H 2 O (1.5 g,6.6 mmol) and concentrated hydrochloric acid (40. Mu.L), and the mixture was refluxed for 12h. The reaction mixture was poured into stirring water (50 mL) and NaHCO was added 3 The aqueous solution adjusts the pH of the aqueous solution to neutral. Followed by CH 2 Cl 2 (30 mL. Times.3) extraction. The organic layer is treated by Na 2 SO 4 Drying and distilling off the solvent under reduced pressure to give the crude product. Recrystallizing the crude product by methanol and petroleum ether to obtain purified green solid Z-GL 5 (152 mg), yield 83%; m.p.242-244 ℃.
HRMS:m/z[M+H] + ＝360.1833；Calcd:360.1824. 1 HNMR(400MHz,CDCl 3 )
(δ,ppm)7.52(d,J＝16.0Hz,1H),7.21(d,J＝6.8Hz,1H),7.19(s,1H),6.90(d,J＝16.0Hz,1H),6.63(d,J＝8.4Hz,1H),3.74(m,J＝6.3Hz,1H),1.70(s,6H),1.26(d,J＝6.4Hz,6H)； 13 C NMR(100MHz,CDCl 3 )(δ,ppm)178.2,175.7,150.8,144.1,136.6,124.2,114.8,114.0,113.3,110.3,108.1,98.6,91.3,57.0,50.7,44.7,26.9,23.2,19.6.
2) Probe Z-GL 5 Fluorescence titration spectrum
The Z-GL prepared by the method 5 Dissolving in dimethyl sulfoxide (DMSO) and making into 5×10 -3 M mother liquor is used;
mu.L of 5X 10 is taken -3 A standard mother solution of mol/L was prepared as a bulk solution at a concentration of 5. Mu.M in 3mL of the detection system. Adding different volumes of guest solution into the host solution to enable glyoxal and probe Z-GL in the sample bottle 5 The concentration ratio of (2) is as follows: 0.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000. Shaking, balancing for 3h, and measuring fluorescence intensity (excitation wavelength 540nm, slit width 10 nm). The experimental results are shown in FIG. 1.
As can be seen from fig. 1, the fluorescence intensity gradually increased with increasing glyoxal concentration.
According to the detection limit formula lod=3σ/k (Dyes pigments.2018,148, 353-358), where k is the concentration of calibration sensitivity (fluorescence intensity difference=maximum fluorescence intensity-initial fluorescence intensity) to methylglyoxal for the change in fluorescence intensity, σ is the standard deviation (σ) of the blank signal (initial fluorescence intensity) obtained without glyoxal Z-GL5 = 0.0835), the detection limit of the final calculated probe is lower than 10 -7 M/L. The experimental results are shown in FIG. 2.
3) Probe Z-GL 5 Cell imaging of exogenous GL
HeLa cells of appropriate density were seeded into 6-well dishes containing 5% CO at 37 ℃C 2 Is cultured in an incubator; after the cells are attached, the probe Z-GL is used for 5 (1. Mu.M) and HeLa cells were incubated for 2 hours, then washed with PBS buffer solution, and then the cells were subjected to fluorescence imaging (excitation wavelength: 559nm, collection wavelength: 585-680 nm) under a confocal laser fluorescence microscope, and the experimental results are shown in FIG. 3.
As can be seen from FIG. 3, FIG. a) is a fluorescence image of living HeLa cells, which are non-fluorescent. Panel b) viable HeLa cells were treated with GL (500. Mu.M) and the cells were almost non-fluorescent. Panel c) Probe Z-GL for living HeLa cells 5 After (1. Mu.M) treatment, the cells showed weak fluorescence in the red channel. Panel d) Z-GL for living HeLa cells 5 After (1. Mu.M) and GL (500. Mu.M) treatments, the fluorescence signal in the cells was significantly enhanced. Map e, f, g, h) corresponds to map a, b, c, d), respectively, of imaging under bright field, map i, j, kL) are the corresponding superimposed images of red fluorescence channel patterns a, b, c, d) and bright field patterns e, f, g, h), respectively. The results show that the probe can be used for detecting GL exogenous to cells.
4) Probe Z-GL 5 Cell imaging of endogenous GL
A first group of living HeLa cells was incubated with glyoxal inhibitor AG (20 mM) for half an hour and fluorescence imaged (excitation wavelength 559nm, collection band 585-680 nm); the second group of probes Z-GL for living HeLa cells 5 (1. Mu.M) for half an hour, and performing fluorescence imaging; a third group of living HeLa cells was treated with glyoxal inhibitor AG (20 mM) and probe Z-GL 5 (1. Mu.M) each incubated for half an hour followed by fluorescence imaging; the fourth group of living HeLa cells was first cultured with glyoxal inhibitor AG (20 mM) for half an hour, and then probe Z-GL was added 5 Fluorescence imaging was performed after incubation of (1. Mu.M) and GL (500. Mu.M) for half an hour. The imaging results are shown in fig. 4.
The results show that AG largely inhibits GL production in HeLa cells, probe Z-GL 5 Endogenous GL can be detected in living cells.
While the application has been described in terms of the preferred embodiment, it is not intended to limit the scope of the claims, and any person skilled in the art can make many variations and modifications without departing from the spirit of the application, so that the scope of the application shall be defined by the claims.
Claims (10)
1. The fluorescent probe for detecting glyoxal is characterized by having a general formula (I):
in the general formula: r is R 1 Is isopropylamine (-NHCH (CH) 3 ) 2 ) Or tert-butylamino (-NHC (CH) 3 ) 3 )；R 2 Each of the following four structures:
2. a method for preparing a fluorescent probe for detecting glyoxal, which is shown in the following general formula:
the preparation method comprises the following specific steps:
s1, 4-fluoro-3-nitrobenzaldehyde and compound R 1 Carrying out substitution reaction in an organic solvent to obtain a compound shown in a general formula A;
s2, compounds R 2 And the compound shown in the general formula A is subjected to condensation reaction in an organic solvent under alkaline conditions, and a soluble salt raw material is added to perform anion exchange reaction to obtain a soluble salt of the compound shown in the general formula B or a cationic group;
the soluble salt raw material is preferably potassium salt or sodium salt of an anion of a soluble salt; the anions of the soluble salts are selected from iodide, fluoride, chloride, bromide, tetrafluoroborate or hexafluorophosphate;
s3, performing a nitro reduction reaction on the compound shown in the general formula B or the soluble salt of the cationic group to obtain the compound shown in the general formula I or the soluble salt of the cationic group.
3. The preparation method according to claim 2, wherein in S1, the organic solvent is selected from the group consisting of nitrile organic solvents, which are acetonitrile;
4-fluoro-3-nitrobenzaldehyde and compound R 1 The molar ratio of (2) is 1:1.8 to 2.2;
4-fluoro-3-nitrobenzaldehyde and compound R 1 The weight ratio of the total amount of (2) to acetonitrile solvent is 1:13 to 15.
4. The preparation method according to claim 1, wherein in S1, the time of the substitution reaction is 0.5 to 1.5 hours, and the temperature of the substitution reaction is preferably 15 to 25 ℃;
in S1, the steps of reduced pressure distillation and purification are also included, wherein methanol and n-hexane are adopted for recrystallization.
5. The preparation method according to claim 2, wherein in S2, the alkaline condition is addition of piperidine in the reaction system; the organic solvent is an alcohol organic solvent, and the alcohol organic solvent is ethanol;
compound R 2 The molar ratio of the compound shown in the general formula A to the soluble salt raw material is 1:1.05 to 1.15:2 to 2.2;
compounds of the general formula R 2 The molar ratio of the piperidine to the piperidine is 1:0.02 to 0.03;
compounds of the general formula R 2 And the weight ratio of the total weight of the compound shown in the general formula A to the organic solvent is 1:10 to 12.
6. The preparation method according to claim 2, wherein in S2, the condensation reaction is carried out at a temperature of 85 to 95 ℃ for 18 to 30 hours;
the reaction condition of the anion exchange reaction is light shading, the temperature of the anion exchange reaction is reflux temperature, and the time of the anion exchange reaction is 1-3 h;
in S2, the steps of distillation under reduced pressure and purification using ethanol recrystallization are also included.
7. The preparation method according to claim 2, wherein in S3, tin dichloride dihydrate and 36% concentrated hydrochloric acid are added to perform a nitroreduction reaction;
the molar ratio of the compound shown in the general formula B or the soluble salt of the cationic group, the concentrated hydrochloric acid and the tin dichloride dihydrate is 1:0.03 to 0.04: 12-14; preferably 1:0.04:12;
the weight ratio of the total weight of the compound shown in the general formula B or the soluble salt of the cationic group, the concentrated hydrochloric acid and the tin dichloride dihydrate to the ethanol is 1:10 to 12;
the temperature of the nitroreduction reaction is reflux temperature, and the time of the nitroreduction reaction is 10-15 h.
In S3, a purification step is also included, preferably recrystallisation from methylene chloride and n-hexane or methanol and petroleum ether.
8. The process according to claim 3, 5 or 7, wherein 4-fluoro-3-nitrobenzaldehyde is reacted with the compound R 1 The molar ratio of (2) is 1:2;
compound R 2 The molar ratio of the compound shown in the general formula A to the soluble salt raw material is 1:1.05 to 1.15:2 to 2.2, preferably 1:1.1:2; compounds of the general formula R 2 The molar ratio of the piperidine to the piperidine is 1:0.02 to 0.03; preferably 1:0.02;
the molar ratio of the compound shown in the general formula B or the soluble salt of the cationic group, concentrated hydrochloric acid and tin dichloride dihydrate is 1:0.03 to 0.04: 12-14; preferably 1:0.04:12.
9. the use of a fluorescent probe for detecting glyoxal according to claim 1, wherein said use is for detecting the concentration of glyoxal in a living biological sample;
the living biological sample comprises living cells or living tissue, preferably living HeLa cells.
10. Use of a fluorescent probe for detecting glyoxal according to claim 1 for preparing a preparation for detecting diabetes.
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