CN115043818B - Succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier and preparation method and application thereof - Google Patents
Succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier and preparation method and application thereof Download PDFInfo
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- CN115043818B CN115043818B CN202210636913.7A CN202210636913A CN115043818B CN 115043818 B CN115043818 B CN 115043818B CN 202210636913 A CN202210636913 A CN 202210636913A CN 115043818 B CN115043818 B CN 115043818B
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- China
- Prior art keywords
- piperazine
- succinimidyl
- heptamethine cyanine
- succinyl
- fluorescent carrier
- Prior art date
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- -1 Succinimidyl succinyl piperazine heptamethine cyanine salt Chemical class 0.000 title claims abstract description 90
- GLUUGHFHXGJENI-UHFFFAOYSA-N diethylenediamine Natural products C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 title claims abstract description 90
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 21
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical group ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims abstract description 19
- 230000000269 nucleophilic effect Effects 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 238000001215 fluorescent labelling Methods 0.000 claims abstract description 11
- 125000003277 amino group Chemical group 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 8
- 230000009471 action Effects 0.000 claims abstract description 6
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000005977 Ethylene Substances 0.000 claims abstract description 3
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims abstract description 3
- 229920002554 vinyl polymer Polymers 0.000 claims abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 28
- 239000003795 chemical substances by application Substances 0.000 claims description 19
- 238000006243 chemical reaction Methods 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 15
- 239000003960 organic solvent Substances 0.000 claims description 14
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims description 13
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 13
- 229940014800 succinic anhydride Drugs 0.000 claims description 13
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 12
- 229920001184 polypeptide Polymers 0.000 claims description 12
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 11
- 150000003384 small molecules Chemical class 0.000 claims description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- 239000011550 stock solution Substances 0.000 claims description 8
- 239000003513 alkali Substances 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 6
- 229920001577 copolymer Polymers 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 5
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 4
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 4
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- 238000003745 diagnosis Methods 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 4
- 239000002585 base Substances 0.000 claims description 3
- 238000011161 development Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 3
- FJDQFPXHSGXQBY-UHFFFAOYSA-L caesium carbonate Chemical compound [Cs+].[Cs+].[O-]C([O-])=O FJDQFPXHSGXQBY-UHFFFAOYSA-L 0.000 claims description 2
- 229910000024 caesium carbonate Inorganic materials 0.000 claims description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 claims description 2
- 230000010355 oscillation Effects 0.000 claims description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 229960002317 succinimide Drugs 0.000 claims description 2
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 abstract description 8
- 239000003937 drug carrier Substances 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 abstract description 4
- 238000006073 displacement reaction Methods 0.000 abstract description 2
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 27
- GDIYMWAMJKRXRE-UHFFFAOYSA-N (2z)-2-[(2e)-2-[2-chloro-3-[(z)-2-(1,3,3-trimethylindol-1-ium-2-yl)ethenyl]cyclohex-2-en-1-ylidene]ethylidene]-1,3,3-trimethylindole Chemical compound CC1(C)C2=CC=CC=C2N(C)C1=CC=C1C(Cl)=C(C=CC=2C(C3=CC=CC=C3[N+]=2C)(C)C)CCC1 GDIYMWAMJKRXRE-UHFFFAOYSA-N 0.000 description 26
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 21
- 230000000052 comparative effect Effects 0.000 description 15
- 238000012360 testing method Methods 0.000 description 11
- 238000010521 absorption reaction Methods 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- WYXRRDDCDPLQGO-UHFFFAOYSA-N 1-piperazin-1-ylpyrrolidine-2,5-dione Chemical compound O=C1CCC(=O)N1N1CCNCC1 WYXRRDDCDPLQGO-UHFFFAOYSA-N 0.000 description 6
- 238000009739 binding Methods 0.000 description 6
- 229940098773 bovine serum albumin Drugs 0.000 description 6
- 238000000862 absorption spectrum Methods 0.000 description 5
- 238000002189 fluorescence spectrum Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000000975 dye Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 239000012467 final product Substances 0.000 description 3
- 239000002798 polar solvent Substances 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000007810 chemical reaction solvent Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 description 2
- 229960004657 indocyanine green Drugs 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- IHXWECHPYNPJRR-UHFFFAOYSA-N 3-hydroxycyclobut-2-en-1-one Chemical compound OC1=CC(=O)C1 IHXWECHPYNPJRR-UHFFFAOYSA-N 0.000 description 1
- 125000004042 4-aminobutyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H] 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000002583 angiography Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 150000001450 anions Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 150000001768 cations Chemical group 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 125000004093 cyano group Chemical group *C#N 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000000990 laser dye Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 125000002730 succinyl group Chemical group C(CCC(=O)*)(=O)* 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
-
- 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
-
- 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
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0052—Small organic molecules
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/0066—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
- C09B23/086—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines more than five >CH- groups
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- 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"
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- 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
-
- 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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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Physics & Mathematics (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Optics & Photonics (AREA)
- Materials Engineering (AREA)
- Indole Compounds (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to the technical field of drug carriers, and discloses a succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier, and a preparation method and application thereof. The N-hydroxysuccinimide structure is introduced on the basis of the near infrared cyanine fluorescent dye to obtain the carrier, and the chemical name of the carrier is 2- ((E) -2- ((E) -2- (4- (4- ((2, 5-dioxopyrrole-1-yl) oxygen) -4-oxo butyryl) piperazine-1-yl) -3- (2- ((E) -1-ethyl-3, 3-dimethyl indole-2-fork) ethylene) cyclohex-1-ene-1-yl) vinyl) -1-ethyl-3, 3-dimethyl-3H-indole-1-salt. The drug carrier provided by the invention has a stable structure and obvious Stokes displacement, can effectively reduce the interference of background fluorescence, and greatly improves the detection sensitivity; the fluorescent label can effectively covalently bond corresponding molecules with biomolecules containing nucleophilic groups such as amino groups, sulfhydryl groups and the like to the drug carrier of the invention through covalent action, and carries out fluorescent labeling on the corresponding molecules.
Description
Technical Field
The invention relates to the field of drug carriers, in particular to a succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier, a preparation method and application thereof.
Background
Near-infrared biological fluorescence imaging technology is receiving more and more attention as an intuitive and visual observation technology, and the emission wavelength range of the near-infrared fluorescent dye is 700-1100nm, and in the wavelength range, the penetrating power of fluorescence to biological tissues is strong; the autofluorescence of the biological tissue is weak, so that the interference of background fluorescence can be avoided, and the detection sensitivity can be improved; at the same time, the damage to biological tissues can be reduced, so that the method is suitable for living body imaging.
The near infrared fluorescent dye mainly comprises cyanines, BODIPY (BODIPY-METHOD) derivatives) rhodamine, squaraine, porphyrin, and the like. The cyanine fluorescent dye has the absorption wavelength of 600-800nm, has the advantages of high molar extinction coefficient, high fluorescence quantum yield, high light stability and the like, and is a near infrared fluorescent dye which is paid attention at present. The current cyanine fluorescent dye has been applied to nucleic acid staining or marking, derivatization or marking of amino acid, peptide and protein, infrared laser dye, nonlinear optical material, fluorescent probe, biosensing and the like.
The only cyanine dye currently approved for clinical use by the U.S. Food and Drug Administration (FDA) is indocyanine green (Indocyanine Green, ICG), which is used clinically to detect cardiac output and liver function, liver blood flow, and ophthalmic angiography. The maximum absorption wavelength and the maximum emission wavelength of ICG are about 780nm and 810nm, and the ICG is longer than the wavelength of most cyanine dyes and can penetrate deeper tissues. ICG, however, suffers from the disadvantages of poor stability, susceptibility to decomposition in polar solvents, relatively small stokes shift, and inability to bind other molecules.
In the prior art, a maleimide propionyl piperazine heptamethine cyanine salt fluorescent carrier is disclosed, and the fluorescent carrier contains a maleimide structure and can be used for marking biomolecules with free sulfhydryl groups. However, for other types of nucleophilic groups, the feasibility of labelling by the fluorescent carrier is not explicitly elucidated and demonstrated.
Therefore, there is a need to design a novel near infrared fluorescent dye to solve the problems that the fluorescent carrier has poor stability, is easily decomposed in a polar solvent, has relatively small stokes shift, or cannot be combined with molecules of various nucleophilic groups.
Disclosure of Invention
The invention provides a novel near-infrared fluorescent dye succinimidyl piperazine heptamethine cyanine salt fluorescent carrier, which aims to overcome the defects that ICG in the prior art is poor in stability, easy to decompose in a polar solvent, relatively small in Stokes displacement or incapable of being combined with molecules of various nucleophilic groups.
The invention also aims to provide a preparation method of the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier.
The invention further aims at providing an application of the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier.
In order to solve the technical problems, the technical scheme of the invention is as follows:
A succinimide succinyl piperazine heptamethine cyanine salt fluorescent carrier has a structural formula shown in a formula (1).
The chemical name is as follows:
2- ((E) -2- (4- (4- ((2, 5-dioxopyrrol-1-yl) oxy) -4-oxobutanoyl) piperazin-1-yl) -3- (2- ((E) -1-ethyl-3, 3-dimethylindol-2-ylidene) ethylene) cyclohex-1-en-1-yl) vinyl) -1-ethyl-3, 3-dimethyl-3H-indol-1-salt.
Preferably, X - is any organic or inorganic acid radical anion which is chemically reasonable. The cation part in the structural formula is an important structure for determining the fluorescence property and the binding property of the molecule, X - is any organic acid radical or inorganic acid radical anion which is reasonable in chemistry, and the fluorescence property and the binding property of the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier are not influenced.
Preferably, X - is iodide.
A preparation method of a succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier comprises the following steps of:
s1, dissolving succinic anhydride in an organic solvent I, adding piperazine heptamethine cyanine salt, and reacting under the action of alkali to obtain an intermediate;
s2, dissolving the intermediate in an organic solvent II, adding N-hydroxysuccinimide, and reacting under the action of a condensing agent to obtain the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier.
Preferably, the base is an organic base or an inorganic base.
Specifically, the succinic anhydride has the structural formulaThe structural formula of the piperazine heptamethine cyanine salt isThe structural formula of the intermediate is/>The structural formula of the N-hydroxysuccinimide is/>
Further specifically, the intermediate is succinyl piperazine heptamethine cyanine salt. Preferably, the succinyl piperazine heptamethine cyanine salt is succinyl piperazine heptamethine cyanine iodide.
Preferably, the alkali in S1 is any one or a combination of a plurality of potassium carbonate, cesium carbonate, sodium carbonate, triethylamine, pyridine and 4-dimethylaminopyridine.
The first organic solvent in S1 is any one or a combination of a plurality of methylene dichloride, acetonitrile, chloroform and N, N-dimethylformamide.
The condensing agent in S2 is any one or a combination of a plurality of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-diisopropyl carbodiimide, N-dicyclohexylcarbodiimide and benzotriazol-1-yl-oxy-tripyrrolidinyl phosphorus hexafluorophosphate.
The second organic solvent in S2 is any one or the combination of a plurality of methylene dichloride, chloroform, dimethyl sulfoxide and N, N-dimethylformamide.
Preferably, S1 specifically includes: dissolving succinic anhydride in an organic solvent to obtain a succinic anhydride solution, dropwise adding piperazine heptamethine cyanine salt into the succinic anhydride solution, adding alkali, adjusting the reaction temperature to be 30-60 ℃, stirring for reaction, and purifying to obtain an intermediate after the reaction is completed.
Preferably, S2 specifically includes: dissolving the intermediate in an organic solvent II to obtain an intermediate solution, adding N-hydroxysuccinimide and a condensing agent into the intermediate solution, reacting at 20-60 ℃, and purifying to obtain the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier after the reaction is completed.
Preferably, the ratio of the amounts of the substances of piperazine heptamethine cyanine salt to succinic anhydride in S1 is 1 (1-5), and the ratio of the amounts of the substances of piperazine heptamethine cyanine salt to alkali is 1 (0.8-3).
Further preferably, the ratio of the amounts of the substances of piperazine heptamethine cyanine salt, succinic anhydride, and base is 1:2:1.5.
Preferably, the ratio of the amount of N-hydroxysuccinimide to the amount of the intermediate in S2 is 1 (0.1 to 1), and the ratio of the amount of N-hydroxysuccinimide to the amount of the condensing agent is 1 (0.5 to 2).
Further preferably, the ratio of the amounts of N-hydroxysuccinimide, intermediate and condensing agent is 1:0.5:1.5.
The application of the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier is that the succinimidyl piperazine heptamethine cyanine salt fluorescent carrier is applied to fluorescent labeling, detection analysis and development tracing of small molecules, polypeptides, proteins and other biomolecules containing amino groups for the purposes of diagnosis and treatment of non-diseases.
The application of the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier is that the succinimidyl piperazine heptamethine cyanine salt fluorescent carrier is used as a carrier to prepare a copolymer with small molecules, polypeptides, proteins and other biomolecules containing nucleophilic groups.
Preferably, the nucleophilic group includes any one of a double bond, a triple bond, a cyano group, a hydroxyl group, an ether bond, an amino group, and a mercapto group.
Preferably, a method for preparing a copolymer by using succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier comprises the following steps:
dissolving succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier in an organic solvent to prepare a fluorescent carrier stock solution; dissolving small molecules, polypeptides, proteins and other biomolecules containing nucleophilic groups in sodium bicarbonate solution to obtain a sample to be marked, adding fluorescent carrier stock solution into the sample to be marked, uniformly mixing, carrying out low-temperature light-shielding oscillation reaction for 12-24 hours, and purifying to obtain a copolymer marked by the fluorescent carrier.
Preferably, the small molecules, polypeptides, proteins and other biomolecules containing nucleophilic groups include Bovine Serum Albumin (BSA), pepsin, insulin, immunoglobulins.
Compared with the prior art, the technical scheme of the invention has the beneficial effects that:
1. According to the invention, the N-hydroxysuccinimide structure-based drug carrier with a brand new structure is introduced based on the near infrared fluorescent cyanine dye, so that the drug carrier can be effectively and covalently combined with organic small molecules, polypeptides, proteins or other biomolecules containing nucleophilic groups such as amino groups, sulfhydryl groups and the like through covalent actions to carry out fluorescence labeling on the corresponding molecules, thereby realizing in vitro analysis detection and in vivo development tracing on the organic small molecules, polypeptides, proteins or other biomolecules.
2. The succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier provided by the invention has a relatively stable structure, has a maximum emission wavelength of 780nm and a Stokes shift of 90nm when the excitation wavelength is 690nm, has remarkable Stokes shift, can effectively reduce interference of background fluorescence, greatly improves detection sensitivity, and provides an efficient and practical tool for analysis and detection of small organic molecules, polypeptides, proteins or other biomolecules.
Drawings
FIG. 1 is a nuclear magnetic pattern of succinimidyl succinyl piperazine heptamethine cyanine iodide;
FIG. 2 is a high resolution mass spectrum of succinimidyl succinyl piperazine heptamethine cyanine iodide;
FIG. 3 is an absorption spectrum of succinimidyl succinyl piperazine heptamethine cyanine iodide;
FIG. 4 is a fluorescence emission spectrum of succinimidyl succinyl piperazine heptamethine cyanine iodide;
FIG. 5 is an absorption and emission wavelength spectrum of Cy 7-N-hydroxysuccinimide ester;
FIG. 6 is a graph showing the UV absorbance spectrum after binding succinimidyl succinyl piperazine heptamethine cyanine iodide to bovine serum albumin;
FIG. 7 is a fluorescence emission spectrum of succinimidyl succinyl piperazine heptamethine cyanine iodide combined with bovine serum albumin.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the present application will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present application. It will be apparent that the described embodiments are some, but not all, embodiments of the application. All other embodiments, based on the described embodiments, which a person of ordinary skill in the art would obtain without inventive faculty, are within the scope of the application.
The invention will be further illustrated with reference to the drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1
A succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier has the synthetic route as follows:
Wherein X - is iodide.
The preparation method comprises the following specific preparation steps:
S1: succinic anhydride (88 mg,2.0 eq) was added to a round-bottom flask, dissolved in 5mL of dichloromethane, piperazine heptamethine cyanine iodide (300 mg,1.0 eq) was dissolved in 5mL of dichloromethane, then added dropwise to the reaction system, pyridine (53 ul,1.5 eq) was added dropwise, the temperature was raised to 40 ℃ and reacted for 12 to 24 hours, after the reaction was detected to be complete by TLC (thin layer Chromatography ), the solvent was removed by rotary evaporation, and purified by using a dichloromethane/methanol system as a mobile phase silica gel column Chromatography to obtain an intermediate.
S2, dissolving the intermediate (500 mg,1 eq) in 10mL of dry dichloromethane, adding N-hydroxysuccinimide (147 mg,2.0 eq) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (365 mg,3 eq), reacting for 12-24 hours at room temperature, adding a large amount of dichloromethane into the reaction solution after TLC detection reaction is basically complete, washing for 5 times, spin-drying, removing most residual raw materials, and recrystallizing with dichloromethane/normal hexane to obtain the succinimidyl succinyl piperazine heptamethine cyanine fluorescent carrier.
Examples 2 to 6
The procedure for the preparation of the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier in examples 2 to 6 was the same as in example 1, except for the following process conditions. Specific process conditions are shown in Table 1, and the prepared succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier is subjected to relevant fluorescent labeling test.
TABLE 1
Comparative examples 1 to 3
The procedure of the preparation method of the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier in comparative examples 1 to 3 was the same as that of example 1, except for the following process conditions. Specific process conditions are shown in Table 2, and the prepared succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier is subjected to relevant fluorescent labeling test.
TABLE 2
Comparative example 4
Comparing the prior art molecules of the same type with the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier prepared by the present application, as shown in FIG. 5, the Cy 7-N-hydroxysuccinimide ester (CAS: 2408482-09-5) of the same type has absorption and emission wavelengths of 750nm and 773nm, respectively, and Stokes shift of 23nm (data source: lumiprobe Corporation functional network).
The succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier prepared by the invention is subjected to result characterization and fluorescent marking test by the following method.
(1) Characterization of results
1) Comparison of the yield and the yield of the product
As shown in FIGS. 1-2, the intermediate products obtained in examples 1-6 and comparative examples 1-3 were confirmed by mass spectrometry MS-ESI:(C42H53N4O3 +Calc 661.911Found 661.55),1H-NMR(400MHz,DMSO)δ7.72(d,J＝13.48Hz,2H),7.52(d,J＝7.36Hz,2H),7.36(t,J＝7.48Hz,2H),7.27(d,J＝7.88Hz,2H),7.16(t,J＝7.4Hz,2H),6.01(d,J＝13.6Hz,2H),4.11(dd,J＝13.72,6.72Hz,4H),3.77(s,4H),3.64(s,2H),3.58(s,2H),2.54-2.50(m,4H),2.42(s,4H),1.80-1.76(m,2H),1.62(s,12H),1.26(t,J＝7.0Hz,6H).
The final products obtained in examples 1 to 6 and comparative examples 1 to 2 were confirmed by mass spectrometry to be unsuccessful in obtaining the final product in comparative example 3 (HRMS:C46H56N5O5 +Calc758.4276Found 758.4278),1H-NMR(400MHz,CDCl3)δ7.76(d,J＝13.12Hz,2H),7.35(d,J＝9.12Hz,4H),7.19(t,J＝7.4Hz,2H),7.03(d,J＝7.88Hz,2H),5.94(d,J＝13.08Hz,2H),4.07(d,J＝7.0Hz,4H),3.91(s,4H),3.78(s,2H),3.62(s,2H),3.09-2.98(m,6H),2.69(s,2H),2.56(s,4H),1.90-1.87(m,2H),1.69(s,12H),1.43(t,J＝7.0Hz,6H)..
The quality and yield of the intermediate products and final products obtained in examples 1 to 6 and comparative examples 1 to 3 are shown in tables 3 to 4, respectively.
TABLE 3 Table 3
TABLE 4 Table 4
Analysis shows that:
as shown in Table 3, the final products of examples 1-6 were all successfully produced, and the yields were distributed between 32% and 90% under different process conditions; wherein example 1 is the most preferred example, the yield can reach 90%.
As shown in table 4, in comparative examples, comparative examples 1 to 2 were 1:1.5 in the ratio of the amount of N-hydroxysuccinimide to the amount of the condensing agent in example 1, 1:0.2 in the ratio of the amount of N-hydroxysuccinimide to the amount of the condensing agent in comparative example 1, and 1:4 in the ratio of the amount of N-hydroxysuccinimide to the amount of the condensing agent in comparative example 2, respectively; the yield of the final product in comparative example 1 was only 10% and that in comparative example 2 was only 18% compared to example 1.
In the preparation method, the quantity of the condensing agent has a large influence on the yield of the product, and when the quantity of the condensing agent is too small, the reaction with the reactant is not complete enough, so that the yield of the reaction product is low; if the condensing agent is used in an excessive amount, side reactions increase, which in turn leads to a decrease in yield. Therefore, the amount of condensing agent is controlled to be: when the ratio of the amount of N-hydroxysuccinimide to the amount of the condensing agent in S2 is in the range of (0.5-2), the yield of the final product is considerable.
In comparative example 3, when the type of condensing agent in S2 was used as an independent variable and 1-hydroxybenzotriazole was used as the condensing agent, the reaction was carried out at room temperature for 12 to 24 hours, and the TLC detection reaction revealed that no new product was produced, and that no new product was produced after the reaction time was prolonged to 48 hours, indicating that the objective product could not be obtained by using 1-hydroxybenzotriazole. Therefore, in this production method, the objective product can be successfully produced by using any one or a combination of several of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-diisopropylcarbodiimide, N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate in examples 1 to 6.
2) Fluorescence absorption and emission spectra of succinimidyl succinyl piperazine heptamethine cyanine iodide
As the anion part of the succinimidyl succinyl piperazine heptamethine Cyanine salt has no essential influence on the fluorescence property of the carrier, the example is mainly represented by succinimidyl piperazine heptamethine Cyanine iodide (code CY-NHS, wherein CY represents Cyanine dye, cyanine, and NHS represents N-hydroxysuccinimide, N-hydroxysuccinimide).
A. Absorption spectrum of succinimidyl succinyl piperazine heptamethine cyanine iodide
The succinimidyl succinyl piperazine heptamethine cyanine iodide prepared in example 1 was dissolved in dimethyl sulfoxide to prepare a stock solution of 20mmol/L, diluted to 5. Mu. Mol/L, and scanned for ultraviolet absorbance spectra, as shown in FIG. 3. The maximum absorption wavelength of succinimidyl succinyl piperazine heptamethine cyanine iodide is 690nm, a shoulder is arranged at 756nm, obvious double absorption is displayed, and the wide absorption peak in the near infrared region means that the most suitable excitation wavelength can be selected.
B. Fluorescence emission spectrum of succinimidyl succinyl piperazine heptamethine cyanine iodide
Stock solutions of succinimidyl succinyl piperazine heptamethine cyanine iodide in a were diluted to 5 μmol/L for scanning fluorescence spectra. The excitation wavelength was 690nm, the detection wavelength was 730-900nm, and the fluorescence spectrum was measured as shown in FIG. 4. As can be seen from the graph, the maximum emission wavelength is 780nm, the Stokes shift is 90nm, and the Stokes shift is larger, while as shown in FIG. 5, the Cy 7-N-hydroxysuccinimide ester (CAS: 2408482-09-5) of the same type in comparative example 4, which has absorption and emission wavelengths of 750nm and 773nm respectively, has Stokes shift of 23nm and is far smaller than the Stokes shift of CY-NHS, which means that the CY-NHS can reduce the interference of background fluorescence more effectively and greatly improve the detection sensitivity compared with the Cy 7-N-hydroxysuccinimide ester.
(2) Fluorescent labelling test
Since the anionic part of the succinimidyl succinyl piperazine heptamethine cyanine salt has no essential effect on the fluorescence property of the carrier, the example is mainly represented by succinimidyl piperazine heptamethine cyanine iodide (code CY-NHS).
The present test protocol selects Bovine Serum Albumin (BSA) containing free amino groups as the nucleophilic molecule, covalently bound to succinimidyl succinyl piperazine heptamethine cyanine iodide. Succinimidyl piperazine heptamethine cyanine iodide can react with amine groups (primary or secondary amine) on target biomolecules to generate stable amide bonds, and since free amine groups are common functional groups (derived from lysine side chains) on the surfaces of proteins, antibodies and polypeptides, succinimidyl piperazine heptamethine cyanine iodide can directly react with the proteins, antibodies and polypeptides. The experiment further demonstrates that the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier can be efficiently and effectively covalently connected with molecules containing nucleophilic groups so as to carry out fluorescent labeling.
The specific operation is as follows:
Test one: the succinimidyl succinyl piperazine heptamethine cyanine iodide prepared in example 1 was dissolved in dimethyl sulfoxide to prepare a stock solution of 20 mmol/L; BSA protein (3 mg,1 eq) was weighed and dissolved in 0.1mol/L NaHCO3 solution, then stock solution (18 uL,8 eq) of CY-NHS was added, and after mixing well, the mixture was placed on a shaking table and reacted overnight at 4℃in the absence of light, and experimental phenomena were observed. After the reaction was completed, the reaction mixture was purified by using a G-50 sephadex column to obtain a labeled conclusion 1.
The experimental procedure for the tests two and three was the same as that of the test one, except that the reaction solvent for the protein was the same as that of the test one, and the specific conditions and conclusions are shown in Table 5.
TABLE 5
As shown in table 5, in the fluorescent labeling test, when BSA protein was dissolved in PBS (sodium phosphate) buffer solution and succinimidyl succinyl piperazine heptamethine cyanine iodide was added to perform fluorescent labeling, hydrolysis reaction of succinimidyl piperazine heptamethine cyanine iodide occurred, demonstrating that the stability thereof in the buffer solution was affected, failing to label BSA protein successfully; when BSA protein is dissolved in Tris buffer solution and succinimidyl succinyl piperazine heptamethine cyanine iodide is added for fluorescence labeling, the succinimidyl piperazine heptamethine cyanine iodide directly reacts with the Tris buffer solution because the Tris buffer solution contains amino groups, so that the BSA protein cannot be labeled successfully. Only when NaHCO 3 solution was used as reaction solvent with BSA protein, BSA protein was finally labeled successfully.
To further verify the covalent binding reaction of BSA protein with succinimidyl succinyl piperazine heptamethine cyanine iodide, ultraviolet absorption and fluorescence emission tests were performed on the purified CY-NHS-labeled BSA protein, and the resulting maps are shown in FIGS. 6 and 7. As can be seen from FIG. 6, the BSA protein itself is not absorbed in the wavelength range of 600-900 nm, and the BSA protein marked by CY-NHS has an absorption similar to CY-NHS in the region, which indicates that the CY-NHS successfully carries out covalent binding reaction with the BSA protein; as can be seen from FIG. 7, the BSA protein itself was not fluorescent at a wavelength of 700 to 900nm, whereas the CY-NHS-labeled BSA protein had fluorescence emission similar to CY-NHS in this region, which also demonstrated that CY-NHS successfully reacted with BSA protein by covalent binding.
It is to be understood that the above examples of the present invention are provided by way of illustration only and not by way of limitation of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (9)
1. The succinimide succinyl piperazine heptamethine cyanine salt fluorescent carrier is characterized in that the structural formula is shown in the formula (1):
the chemical name is as follows:
2- ((E) -2- (4- (4- ((2, 5-dioxopyrrol-1-yl) oxy) -4-oxobutanoyl) piperazin-1-yl) -3- (2- ((E) -1-ethyl-3, 3-dimethylindol-2-ylidene) ethylene) cyclohex-1-en-1-yl) vinyl) -1-ethyl-3, 3-dimethyl-3H-indol-1-salt;
X - is any organic acid radical or inorganic acid radical anion which is reasonable in chemistry.
2. A method for preparing the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier according to claim 1, which is characterized by comprising the following steps of:
s1, dissolving succinic anhydride in an organic solvent I, adding piperazine heptamethine cyanine salt, and reacting under the action of alkali to obtain an intermediate;
S2, dissolving the intermediate in an organic solvent II, adding N-hydroxysuccinimide, and reacting under the action of a condensing agent to obtain the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier;
S2, the ratio of the amount of the N-hydroxysuccinimide to the amount of the condensing agent is 1 (0.5-2);
The condensing agent in the S2 is any one or a combination of a plurality of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-diisopropylcarbodiimide, N-dicyclohexylcarbodiimide and benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate.
3. The preparation method of the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier according to claim 2, which is characterized in that: the alkali in the S1 is any one or a combination of a plurality of potassium carbonate, cesium carbonate, sodium carbonate, triethylamine, pyridine and 4-dimethylaminopyridine, and the organic solvent in the S1 is any one or a combination of a plurality of dichloromethane, acetonitrile, chloroform and N, N-dimethylformamide; the second organic solvent in the step S2 is any one or a combination of a plurality of dichloromethane, chloroform, dimethyl sulfoxide and N, N-dimethylformamide.
4. The method for preparing the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier according to claim 2, wherein step S1 comprises: dissolving the succinic anhydride in the organic solvent to obtain a succinic anhydride solution, dropwise adding the piperazine heptamethine cyanine salt into the succinic anhydride solution, adding the alkali, adjusting the reaction temperature to be 30-60 ℃, stirring for reaction, and purifying to obtain the intermediate after the reaction is completed; the step S2 comprises the following steps: and dissolving the intermediate in the second organic solvent to obtain an intermediate solution, adding the N-hydroxysuccinimide and the condensing agent into the intermediate solution, reacting at the temperature of 20-60 ℃, and purifying to obtain the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier after the reaction is completed.
5. The preparation method of the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier according to claim 2, wherein the ratio of the amounts of the substances of the piperazine heptamethine cyanine salt and the succinic anhydride in the S1 is 1 (1-5), and the ratio of the amounts of the substances of the piperazine heptamethine cyanine salt and the base is 1 (0.8-3); the ratio of the amount of the N-hydroxysuccinimide to the amount of the intermediate in the S2 is 1 (0.1 to 1).
6. The intermediate of a fluorescent carrier of succinimidyl succinyl piperazine heptamethine cyanine salt according to claim 1, wherein the intermediate is succinimidyl piperazine heptamethine cyanine salt with a structural general formula:
7. The use of the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier according to claim 1, wherein the succinimidyl piperazine heptamethine cyanine salt fluorescent carrier is applied to fluorescent labeling, detection analysis and development tracing of small molecules, polypeptides and proteins containing amino groups for the purposes of diagnosis and treatment of non-diseases.
8. Use of the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier according to claim 1, for non-disease diagnosis and treatment purposes, wherein the succinimidyl piperazine heptamethine cyanine salt fluorescent carrier is used as a carrier for preparing copolymers with small molecules, polypeptides and proteins containing nucleophilic groups.
9. A method for preparing a copolymer using the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier of claim 1, characterized by being applied for the purpose of diagnosis and treatment of non-diseases, comprising the following steps:
Dissolving the succinimidyl succinyl piperazine heptamethine cyanine salt fluorescent carrier in an organic solvent to prepare a fluorescent carrier stock solution; dissolving small molecules, polypeptides and proteins containing nucleophilic groups in sodium bicarbonate solution to obtain a sample to be marked, adding the fluorescent carrier stock solution into the sample to be marked, uniformly mixing, carrying out low-temperature light-shielding oscillation reaction for 12-24 hours, and purifying to obtain the copolymer marked by the fluorescent carrier.
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| CN105884748A (en) * | 2016-06-13 | 2016-08-24 | 煦普生物技术（珠海）有限公司 | Maleimide propionyl piperazine heptamethine cyanine salt fluorescence carrier and preparation method and application thereof |
| CN108033907A (en) * | 2017-11-14 | 2018-05-15 | 中国医学科学院生物医学工程研究所 | A kind of Heptamethine cyanines active fluoro probe and preparation method and application |
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| CN108033907A (en) * | 2017-11-14 | 2018-05-15 | 中国医学科学院生物医学工程研究所 | A kind of Heptamethine cyanines active fluoro probe and preparation method and application |
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